Journal articles on the topic 'Ca 4 B C -65 A D Medea'

One form of early mathematical recognition is to introduce the concept of geometric shapes. Geometry is an important scientific discipline for present and future life by developing various ways that fit 21st century skills. This study aims to overcome the problem of early mathematical recognition of early childhood on geometry, especially how to recognize geometric forms based on computer learning. A total of 24 children aged 4-5 years in kindergarten has to carrying out 2 research cycles with a total of 5 meetings. Treatment activities in each learning cycle include mentioning, grouping and imitating geometric shapes. There were only 7 children who were able to recognize the geometric shapes in the pre-research cycle (29.2%). An increase in the number of children who are able to do activities well in each research cycle includes: 1) The activities mentioned in the first cycle and 75% in the second cycle; 2) Classifying activities in the first cycle were 37.5% and 75% in the second cycle; 3) Imitation activities in the first cycle 54.2% and 79.2% in the second cycle. The results of data acquisition show that computer learning application can improve the ability to recognize geometric shapes, this is because computer learning provides software that has activities to recognize geometric shapes with the animation and visuals displayed. Keywords: Early Childhood Computer Learning, Geometry Forms, Early Math Skills Reference Alia, T., & Irwansyah. (2018). Pendampingan Orang Tua pada Anak Usia Dini dalam Penggunaan Teknologi Digital. A Journal of Language, Literature, Culture and Education, 14(1), 65– 78. https://doi.org/10.19166/pji.v14i1.639 Ameliola, S., & Nugraha, H. D. (2013). 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Penelitian ini bertujuan untuk mengetahui level penambahan kembang telang ditinjau dari bahan organik dan mineral. Penelitian ini dilaksanakan di Farm Sesetan Fakultas Peternakan Universitas Udayana, Jl. Raya Sesetan, Gang Markisa, Denpasar. Pengujian bahan organik dilaksanakan di Laboratorium Nutrisi dan Makanan Ternak Fakultas Peternakan Universitas Udayana dan pengujian mineral dilakukan di laboratorium Analitik Universitas Udayana. Rancangan penelitian yang digunakan adalah Rancangan Acak Lengkap (RAL) dengan 4 perlakuan dan 4 ulangan. Perlakuan A (65% batang pisang + 30% pollar + 5% (molasis + EM4)), B (55% batang pisang + 10% C. ternatea + 30% pollar + 5% (molasis + EM4)), C (45% batang pisang + 20% C. ternatea + 30% pollar + 5% (molasis + EM4)), D (35% batang pisang + 30% C. ternatea + 30% pollar + 5% (molasis + EM4)). Variabel yang diamati adalah bahan organik dan mineral (Ca, P, dan Zn). Hasil penelitian menunjukkan bahwa persentase bahan organik mengalami peningkatan secara nyata ( P<0,05). Mineral Ca, P, dan Zn mengalami peningkatan dan tertinggi pada perlakuan C masing – masing 32,58 ml/L, 0,75% dan 19,26 mg/kg. Dari hasil penelitian ini dapat disimpulkan bahwa penambahan kembang telang pada silase batang pisang dapat meningkatkan kandungan bahan organik dan mineral (Ca, P, dan Zn). Penambahan 30% kembang telang menghasilkan bahan organik tertinggi, sedangkan penambahan 20% kembang telang menghasilkan mineral (Ca, P, dan Zn) tertinggi pada silase batang pisang. Kata kunci: batang pisang, bahan organik, kembang telang , mineral Ca, P, Zn

Abstract An experimental investigation on the interference effect of lee-side balance housing bodies on the longitudinal aerodynamic characteristics of thin slab delta wings has been made using a special and novel thin three-component balance. This study has been conducted using wings with a t/c of 0.053 and sweep of 76°, 70° and 65° for three different lee-side bodies viz:Body (A), Body (B) and Body (C) of which Body (C) is the smallest. The testing was carried out at Mach number of 8·2, Reynolds number of 2·13 ˘ 106 and incidence of –4° to 12°. For the smallest body (Body (C)) tested, at the incidence at which this body first goes into the aerodynamic shadow of the wing (α = 6·5°), the difference in CN, CA, CM and L/D between the wing-body and the plain wing is around by 22%, 12%, 75% and 35% respectively. This difference decreases with increase in incidence and at the highest angle-of-attack up to which testing was done (α = 12°), the. corresponding values of CN, CA, CM and L/D are around 9%, 12%, 15% and 4% respectively. Contrary to the assumption in the literature pertinent to force measurements on thin slab delta wings, the interference of the lee-side bodies are found to be appreciable even after they come into the aerodynamic shadow of the wing.

e16054 Background: TGCT are the most frequent cancers in men aged 15-45. This population is increasingly exposed to CA that is illegal in France. Its consequences on treatment and prognosis remain unknown. Methods: The use and the quantity of CA were evaluated for all pts treated by C for a TGCT in the CLB from 01/2016 to 06/2018. Tobacco (T) and alcohol (A) co-addictions were also analyzed. All pts had before starting C pulmonary test with diffusing capacity of the lung for carbon monoxyde (DLCO) evaluation. We searched a link between the use of toxics, contraindication to Bleomycin (B) due to decreased DLCO and tumor characteristics: stage and IGCCCG prognostic groups. Results: One hundred and thirty two pts with a median age of 33.5 years (interval: 17-65) received C for TGCT mainly for NSGCT (n = 97, 73%). Nine pts (7%) were treated in the adjuvant setting (1: 6 or 2 BEP: 3). In metastatic pts, 79 (60%), 30 (23%) and 14 (10%) were considered as good, intermediate and poor prognosis (PG) according to IGCCCG respectively. BEP was given for 3 cycles to 63 pts and 4 to29 and 6 pts received a dose dense regimen according to GETUG13. Eleven pts didn’t received B (5: 4EP, 6: 4VIP) because of DLCO alteration before the beginning of C. Eleven pts treated with B switched to a regimen without B due to a decrease of the DLCO during treatment. CA use was frequent (n = 20, 15%) with 8 pts (6%) having a regular CA consumption (median 5 joints/d). Seventy eight pts (59%) were regular T smokers. Co-addictions for CA consumers were frequent (90% for T and 50% for A). T and CA smokers had more DLCO alteration leading to B contraindication (p = 0.01 and p = 0.04). A quarter (n = 5) of pts using CA didn’t received B, and only 1 pt who didn’t receive B was not a smoker (T and CA). The use of CA was associated with PG, OR 5.46 (CI 95%: 1.35-21.2; p = 008). We could not observe a relationship between relapse and the use of CA (p = 0.34) because of the small number of relapses (n = 8). Half (n = 10) of CA consumers were single men and 25% (n = 5) were unemployed. Conclusions: The use of CA is significantly associated with PG and with a reduction of B use due to adecreased DLCO. The impact of CA intoxication is negative on the quality of treatment (because of more contraindications to B). The association with PG remains unclear, but may be related to social isolation.

Abstract To optimize the use of dietary P by pigs, 5 feeding strategies were studied in a 3-phase feeding trial on 240 pigs (initial bodyweight (BW) of 31 kg): 1) C-C-C providing 100% of digestible phosphorus (Pdig, 4.3 g/kg STTD) and calcium (Ca, 9.7 g/kg) requirement to maximize bone mineralization, 2) L-L-L 60% of the Pdig and Ca requirements of C-C-C, 3) Phyt-Phyt-Phyt (phosphate-free, with phytase, 750, 686, 390 FTU/kg), providing 60% of Pdig and Ca requirements in phase 1, then 100%, 4) and 5) C in phases 1 and 3, and 60% of the need for Pdig in phase 2, associated with 65% of the requirements for Ca (N) or 80% (H), namely C-N-C and C-H-C. The BW and bone mineral content (BMC) were measured at the beginning and end of each phase. The BMC gain (gBMC), average daily gain (ADG) and average daily feed intake (ADFI) were calculated by phase. In phase 1, ADG was lower in the Phyt group than the C group (1.05 vs 1.10 kg/d, P &lt; 0.01) and the BMC of group C and gBMC were higher than those of the Phyt and B groups (P &lt; 0.05). In phase 2, C-C and Phyt-Phyt groups had similar BMC due to higher gBMC in the Phyt-Phyt (27.1 vs 18.4 g/d, P &lt; 0.01). At the end of phases 2 and 3, C-C-C, C-N-C and C-H-C groups had similar BMC. The Phyt and B groups showed an increased phosphorus-use efficiency during phases 1 and 2 (+20% vs C). Phosphorus retention was also higher in the C-N-C and C-H-C groups, during the depletion in phase 2 (+24% vs C, P&lt; 0.05). These results showed the potential of a depletion-repletion strategy including free phosphate diet to reduce phosphorus intake and excretion without affecting final growth performance and bone mineralization because of increased minerals utilization efficacies.

AbstractA new mineral majzlanite, ideally K2Na(ZnNa)Ca(SO4)4, was found in high-temperature exhalative mineral assemblages in the Yadovitaya fumarole, Second scoria cone of the Great Tolbachik Fissure Eruption (1975–1976), Tolbachik volcano, Kamchatka Peninsula, Russia. Majzlanite is associated closely with langbeinite and K-bearing thénardite. Majzlanite is grey with a bluish tint, has a white streak and vitreous lustre. The mineral is soluble in warm water. Majzlanite is monoclinic, C2/c, a = 16.007(2), b = 9.5239(11), c = 9.1182(10) Å, β = 94.828(7)°, V = 1385.2(3) Å3 and Z = 16. The eight strongest lines of the X-ray powder diffraction pattern are [d, Å (I, %)(hkl)]: 3.3721(40)($\bar{3}$12), 3.1473(56)($\bar{4}$02), 3.1062(65)($\bar{2}$22), 2.9495(50)($\bar{1}$31), 2.8736(100)($\bar{1}$13), 2.8350(70)(421), 2.8031(45)(511) and 2.6162(41)($\bar{5}$12). The following structural formula was obtained: K2Na(Zn0.88Na0.60Cu0.36Mg0.16)(Ca0.76Na0.24)(S0.98Al0.015Si0.005O4)4. The chemical composition determined by electron-microprobe analysis is (wt.%): Na2O 9.73, K2O 15.27, ZnO 11.20, CaO 7.03, CuO 4.26, MgO 1.07, Al2O3 0.47, SO3 51.34, SiO2 0.12, total 100.49. The empirical formula calculated on the basis of 16 O apfu is K1.99Na1.93Zn0.84Ca0.77Cu0.33Mg0.16(S3.94Al0.06Si0.01)O16 and the simplified formula is K2Na(Zn,Na,Cu,Mg)Σ2(Ca,Na)(SO4)4. No natural or synthetic compounds directly chemically and/or structurally related to majzlanite are known to date. The topology of the heteropolyhedral framework in majzlanite is complex. An interesting feature of the structure of majzlanite is an edge-sharing of ZnO6 octahedra with SO4 tetrahedra.

Background: Recent attempts have focused on identifying fewer magnitude of minimal residual disease (MRD) rather than exploring the biological and genetic features of the residual plasma cells (PCs). Interphase fluorescence in situ hybridization (iFISH) analyses in sequential samples provide a simple and reliable method to longitudinal track the dynamic changes in clonal architecture and speculate the possible evolutionary pattern. Here, we report for the first time the incidence and prognostic significance of cytogenetic abnormalities (CA) existed in the PCs of patients achieving at least partial remission. Methods: A cohort of 193 patients with at least one CA at diagnosis were analyzed using data from the prospective, non-randomized clinical trial (BDH 2008/02), and iFISH analyses were performed in patient-paired diagnostic and post-therapy samples. Results: Persistent CA in residual tumor cells were observed for the majority of patients (63%), even detectable in 28/63 (44%) patients with MRD negativity (<10-4). The absence of CA in residual PCs was associated with prolonged survival regardless of MRD status. It was noted that MRD-positive but FISH-negative patients experienced similar survival to MRD-negative patients (m-TTP 4.5 vs. 5.1 years, P=0.983). According to the change of the clonal size of specific CA, patients were clustered into five groups, reflecting five patterns of clone selection under therapy pressure. 1) Pattern A were observed in 36 (19%) patients where a minor subpopulation or undetectable subclone in the pre-treatment sample became dominant after therapy. 2) Pattern B was identified in 29 (15%) patients whose fractions of PCs harboring different CA were decreased with inconsistent extent. 3) Identical CA fraction in residual PCs were found in 22 (11%) patients as Pattern C. 4) Pattern D. The fractions of PCs harboring specific CA in 35 patients (18%) were uniformly declined. 5) 71 patients lost their abnormal cytogenetic clone after therapy (less than cut-off level) were classified as Pattern E. The cytogenetic dynamics of pattern A and B can be interpreted as a therapy-induced selection process with comparable inferior survival (m-TTP 1.2 vs. 1.6 years, respectively). Patients with pattern E experienced the most favorable outcome (m-TTP 5.0 years), following those with pattern D and C (m-TTP 3.5 and 2.5 years). Among the 65 patients with clonal selection, 24 underwent upfront auto-transplantation that experienced significantly improved survival. However, upfront transplant failed to completely reverse the inferior outcome caused by therapy-induced clonal selection. Longitudinal cytogenetic studies at relapse were available in 43 patients. The results suggested that sequential cytogenetic dynamics were observed in most patients, and the cytogenetic architecture of residual cells could to some extent predict the evolutional pattern at relapse. Conclusions: The repeat cytogenetic evaluation in residual cells could not only serves as a good complementary tool for MRD detection, but also provides a better understanding of clinical response and clonal evolution. Therapy-induced clonal selection was associated with inferior outcome regardless of the baseline cytogenetic profiles. The early identification of resistant clone may contribute to guide better tailored therapy strategies based on the feature of the residual tumor cells. Figure Disclosures Munshi: Celgene: Consultancy; Adaptive: Consultancy; Oncopep: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Abbvie: Consultancy.

Abstract Abstract 3421 Poster Board III-309 Introduction While the prognosis of patients with MM has been drastically improved with the introduction of novel agents into the treatment armamentarium, relapse management remains a difficult task, especially in the post-transplant and high-risk MM setting, as defined by the presence of cytogenetic abnormalities (CA) and gene expression profiling (GEP). Patients and Methods We have explored the efficacy and safety of SB in 95 patients with MM. The regimen comprised BCNU at 300mg/m2 on day 1, etoposide 200mg/m2 on days 1-4; cytarabine 400mg/m2 on days 1-5; melphalan 140mg/m2 on day 5 plus: bortezomib 1.0-1.3mg/m2 on days 1+4, thalidomide 100-200mg on days 1-5, dexamethasone 20-40mg days 1-5, cisplatin 10-12.5mg/m2/d by continuous infusion on days 1-5, rapamycin 3mg on day 1 and 1mg on days 2-5; followed by autotransplant (AT) on day 6. Results Patient characteristics included age >=65, 19%; LDH>ULN, 43%; CA, 67%; GEP high-risk, 44%; GEP MF, 15%; GEP delTP53, 22%; prior AT, 75% including 46% with 2AT and 12% with 3 AT. CR and near-CR status was documented in 50%; TRM occurred within 100 days in 6%. Three-year estimates of overall survival (OS) and event-free survival (EFS) were 34% and 18%; OS/EFS were superior: (a) without CA – 60%/50% versus 15%/5% with CA (p=0.003/0.0005); (b) with GEP low-risk – 55%/20% versus 10%/15% with high-risk MM (p=0.009/0.01); (c) with GEP Hyperdiploidy and Low Bone disease molecular subgroups – 70%/35% versus 20%/10% in the remainder (p=0,001/0.002); and (d) absence of progression prior to SB – 52%/35% versus 16%/5% for the remainder (p=0.002/0.0006). Univariately significant adverse variables for OS and EFS included low albumin, B2M>=3.5mg/L, CA, GEP high-risk and relapse just prior to SB, of which B2M, CA and pre-SB survived on multivariate analysis for the 78 patients with all variables; among the 57 with GEP data, OS was inferior with GEP high-risk MM (HR=3.55, p=0.007) whereas EFS was inferior with high LDH (HR=3.44, p=0.004), CA (HR=2.79, p=0.02) and pre-SB relapse (HR=2.94, p=0.035). Conclusions We conclude that SB is a safe salvage regimen that was well-tolerated for the majority in the outpatient setting. Although beneficial to patients lacking CA and GEP high-risk status, the poor outcome also with SB in the presence of these adverse features emphasize the urgent need to discover agents/strategies effective in this setting. Disclosures van Rhee: Genzyme Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

AbstractThe new mineral pansnerite, ideally K3Na3Fe3+6(AsO4)8, was found in the Arsenatnaya fumarole at the Second scoria cone of the Northern Breakthrough of the Great Tolbachik Fissure Eruption, Tolbachik volcano, Kamchatka, Russia. It is associated with aphthitalite, hematite, sanidine, badalovite, khrenovite, achyrophanite, arsenatrotitanite, ozerovaite, tilasite, calciojohillerite, johillerite, nickenichite, svabite, katiarsite, yurmarinite, anhydrite, rutile, cassiterite and pseudobrookite. Pansnerite forms tabular to lamellar (flattened on {010}), usually pseudo-hexagonal crystals up to 0.2 mm × 0.7 mm × 1 mm and crystal clusters up to 2 mm across. It is transparent to translucent, light green, pale greenish, yellowish–greenish or yellowish, with vitreous lustre. The mineral is brittle, with perfect {010} cleavage. The Mohs’ hardness is ca 3. Dcalc is 3.596 g cm–3. Pansnerite is optically biaxial (–), α = 1.702(4), β = 1.713(4), γ = 1.717(4), 2Vmeas = 45(10)° and 2Vcalc = 62°. Chemical composition (holotype, wt.%, electron microprobe data) is: Na2O 6.39, K2O 8.52, CaO 0.08, MgO 0.08, MnO 0.02, NiO 0.02, CuO 1.35, ZnO 0.34, Al2O3 7.35, Cr2O3 0.04, Fe2O3 16.72, SiO2 0.16, P2O5 0.22, V2O5 0.09, As2O5 57.76, SO3 0.04, total 99.20. The empirical formula based on 32 O apfu is K2.86Na3.26Ca0.02(Fe3+3.31Al2.28Cu0.27Zn0.07Mg0.03Cr0.01)Σ5.97(As7.95P0.05Si0.04V0.02S0.01)Σ8.06O32. Pansnerite is orthorhombic, Cmce, a = 10.7372(3), b = 20.8367(8), c = 6.47335(15) Å, V = 1448.27(7) Å3 and Z = 2. The strongest reflections of the X-ray powder diffraction pattern [d,Å(I)(hkl)] are: 10.49(100)(020), 5.380(88)(111), 4.793(65)(220), 3.105(46)(311, 002), 3.079(32)(112, 061), 2.932(35)(260), 2.783(65)(202) and 2.694(52)(400, 222). The crystal structure was solved from single-crystal X-ray diffraction data, R1 = 2.82%. The structure is based on heteropolyhedral layers formed by MO6 octahedra (M = Fe3+ and Al) sharing common vertices and connected by AsO4 tetrahedra. Na+ and K+ cations are located in the interlayer space. The mineral is named in honour of the German–Russian mineralogist and geographer Lavrentiy Ivanovich Pansner (1777–1851). Pansnerite forms a solid-solution series with the isotypic mineral ozerovaite, ideally KNa2Al3(AsO4)4.

AbstractWhiteite-(CaMnMn), CaMnMn2Al2[PO4]4(OH)2·8H2O, is a new hydrous phosphate of Ca, Mn and Al, which is closely related to both jahnsite-(CaMnMn) and the minerals of the whiteite group. It is monoclinic, P2/a, with a = 15.02(2), b = 6.95(1), c =10.13(3) Å, β = 111.6(1)°, V = 983.3(6) Å3, Z = 2 (from powder diffraction data) or a = 15.020(5), b = 6.959(2), c = 10.237(3) Å, β = 111.740(4)°, V = 984.3(5) Å3, Z = 2 (from single-crystal diffraction data). The mineral was found in the Hagendorf Süd granitic pegmatite (Germany) as small (up to 0.5 mm in size) crystals elongated on a and tabular on {010}. The crystals are either simply or polysynthetically twinned on {001}. They crystallize on the walls of voids within altered zwieselite crystals or form coronas (up to 1 mm in diameter) around cubic crystals of uraninite. The mineral is transparent, colourless to pale yellow (depending on Al–Fe3+ substitution), with a vitreous lustre and a white streak. The cleavage is perfect on {001}, the fracture is stepped and the Mohs hardness is 3½. In transmitted light, the mineral is colourless; dispersion was not observed. Whiteite-(CaMnMn) is biaxial (+), α = 1.589(2), β = 1.592(2), γ = 1.601(2) (589 nm), 2Vmeas = 60(10)°, 2Vcalc = 60.3°. The optical orientation is X = b, Z^a = 5°. The calculated and measured densities are Dcalc = 2.768 and Dmeas = 2.70(3) g cm–3, respectively. The mean chemical composition determined by electron microprobe is Na2O 0.53, MgO 0.88, Al2O3 11.66, P2O5 34.58, CaO 4.29, MnO 17.32, FeO 8.32, ZnO 2.60 wt.%, with H2O 19.50 wt.% (determined by the Penfield method), giving a total of 99.68 wt.%. The empirical formula calculated on the basis of four phosphorus atoms per formula unit, with ferric iron calculated to maintain charge balance, is (Ca0.63Zn0.26Na0.14)Σ1.03(Mn0.60Fe0.402+)Σ1.00(Mn1.40Fe0.372+Mg0.18Fe0.063+)Σ2.01(Al1.88Fe0.123+)Σ2.00[PO4]4(OH)2·7.89H2O. The simplified formula is CaMnMn2Al2[PO4]4(OH)2·8H2O. The mineral is easily soluble in 10% HCl at room temperature. The strongest X-ray powder-diffraction lines [listed as d in Å (I) (hkl)] are as follows: 9.443(65)(001), 5.596(25)(011), 4.929(80)(210), 4.719(47)(002), 3.494(46)(400), 2.7958(100)(022). The crystal structure of whiteite-(CaMnMn) was refined for a single crystal twinned on (001) to R1 = 0.068 on the basis of 5702 unique observed reflections. It is similar to the structures of other members of the whiteite group. The mineral is named for the chemical composition, in accordance with whiteite-group nomenclature.

The new mineral species carmeltazite, ideally ZrAl2Ti4O11, was discovered in pockets of trapped melt interstitial to, or included in, corundum xenocrysts from the Cretaceous Mt Carmel volcanics of northern Israel, associated with corundum, tistarite, anorthite, osbornite, an unnamed REE (Rare Earth Element) phase, in a Ca-Mg-Al-Si-O glass. In reflected light, carmeltazite is weakly to moderately bireflectant and weakly pleochroic from dark brown to dark green. Internal reflections are absent. Under crossed polars, the mineral is anisotropic, without characteristic rotation tints. Reflectance values for the four COM wavelengths (Rmin, Rmax (%) (λ in nm)) are: 21.8, 22.9 (471.1); 21.0, 21.6 (548.3), 19.9, 20.7 (586.6); and 18.5, 19.8 (652.3). Electron microprobe analysis (average of eight spot analyses) gave, on the basis of 11 oxygen atoms per formula unit and assuming all Ti and Sc as trivalent, the chemical formula (Ti3+3.60Al1.89Zr1.04Mg0.24Si0.13Sc0.06Ca0.05Y0.02Hf0.01)Σ=7.04O11. The simplified formula is ZrAl2Ti4O11, which requires ZrO2 24.03, Al2O3 19.88, and Ti2O3 56.09, totaling 100.00 wt %. The main diffraction lines, corresponding to multiple hkl indices, are (d in Å (relative visual intensity)): 5.04 (65), 4.09 (60), 2.961 (100), 2.885 (40), and 2.047 (60). The crystal structure study revealed carmeltazite to be orthorhombic, space group Pnma, with unit-cell parameters a = 14.0951 (9), b = 5.8123 (4), c = 10.0848 (7) Å, V = 826.2 (1) Å3, and Z = 4. The crystal structure was refined to a final R1 = 0.0216 for 1165 observed reflections with Fo > 4σ(Fo). Carmeltazite exhibits a structural arrangement similar to that observed in a defective spinel structure. The name carmeltazite derives from Mt Carmel (“CARMEL”) and from the dominant metals present in the mineral, i.e., Titanium, Aluminum and Zirconium (“TAZ”). The mineral and its name have been approved by the IMA Commission on New Minerals, Nomenclature and Classification (2018-103).

AbstractThe new mineral species braccoite, ideally NaMn2+5[Si5AsO17(OH)](OH), has been discovered in the Valletta mine dumps, in Maira Valley, Cuneo province, Piedmont, Italy. Its origin is probably related to the reaction between ore minerals and hydrothermal fluids. It occurs as subhedral crystals in brown-red coloured thin masses, with a pale-yellow streak and vitreous to resinous lustre. Braccoite is associated with tiragalloite, for which new data are provided, as well as gamagarite, hematite, manganberzeliite, palenzonaite, quartz, saneroite, tokyoite, unidentified Mn oxides, organic compounds, and Mn arsenates and silicates under study.Braccoite is biaxial positive with refractive indices α = 1.749(1), β = 1.750(1), γ = 1.760(1). It is triclinic, space group P1̄, with a = 9.7354(4), b = 9.9572(3), c = 9.0657(3) Å, α = 92.691(2), β = 117.057(4), γ = 105.323(3)°, V = 740.37(4) Å3 and Z = 2. Its calculated density is 3.56 g/cm3. The ten strongest diffraction lines of the observed powder X-ray diffraction (XRD) pattern are [d in Å, (I), (hkl)]: 3.055 (69)(22̄1), 3.042 (43)(102), 3.012 (65)(32̄1̄), 2.985 (55)(23̄1̄), 2.825 (100)(213̄), 2.708 (92)(220), 2.627 (43)(23̄2̄), 2.381 (58)(41̄1̄), 2.226 (25)(214̄) and 1.680 (433̄)(36). Chemical analyses by wavelength-dispersive spectroscopy electron microprobe gave (wt.%): Na2O 4.06, CaO 0.05, MnO 41.76, MgO 0.96, Al2O3 0.04, CuO 0.02, SiO239.73, As2O5 6.87, V2O5 1.43, SO3 0.01 and F 0.04. H2O 2.20 was calculated on the basis of 2OH groups p.f.u. Raman spectroscopy confirmed the presence of (SiO4)4–, (AsO4)3– and OH groups. The empirical formula, calculated on the basis of Σ cations-(Na,K) = 11 p.f.u., in agreement with the results of the crystal structure, is Na1.06(Mn2+4.46Mn3+0.32Mg0.19V3+0.01Al0.01Ca0.01)[Si5(As0.48Si0.37V5+0.15)O17(OH)](OH0.98F0.02); the simplified formula is Na(Mn,Mg,Al,Ca)5[Si5(As,V,Si)O17(OH)](OH,F).Single-crystal XRD allowed the structure to be solved by direct methods and revealed that braccoite is the As-dominant analogue of saneroite. The structure model was refined on the basis of 4389 observed reflections to R1 = 3.47%. Braccoite is named in honour of Dr Roberto Bracco (b. 1959), a systematic minerals collector with a special interest in manganese minerals. The new mineral was approved by the International Mineralogical Association Commission on New Minerals, Nomenclature and Classification (IMA 2013-093).

To improve online learning pedagogy within the field of paralegal education, this study investigated how paralegal students and paralegal instructors perceived the effectiveness of synchronous and asynchronous online paralegal courses. This study intended to inform paralegal instructors and course developers how to better design, deliver, and evaluate effective online course instruction in the field of paralegal studies.Survey results were analyzed using independent samples t-test and correlational analysis, and indicated that overall, paralegal students and paralegal instructors positively perceived synchronous and asynchronous online paralegal courses. Paralegal instructors reported statistically significant higher perceptions than paralegal students: (1) of instructional design and course content in synchronous online paralegal courses; and (2) of technical assistance, communication, and course content in asynchronous online paralegal courses. Instructors also reported higher perceptions of the effectiveness of universal design, online instructional design, and course content in synchronous online paralegal courses than in asynchronous online paralegal courses. Paralegal students reported higher perceptions of asynchronous online paralegal course effectiveness regarding universal design than paralegal instructors. No statistically significant differences existed between paralegal students’ perceptions of the effectiveness of synchronous and asynchronous online paralegal courses. A strong, negative relationship existed between paralegal students’ age and their perceptions of effective synchronous paralegal courses, which were statistically and practically significant. Lastly, this study provided practical applicability and opportunities for future research. Akyol, Z., & Garrison, D. R. (2008). 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New benchmarks in higher education: Student engagement in online learning. The Journal of Education for Business, 84(2), 101-109. Retrieved from http://anitacrawley.net/Resources/Articles/New%20Benchmarks%20in%20Higher%20Education.pdf Salkind, N. J. (2008). Statistics for people who think they hate statistics. Los Angeles, CA: Sage Publications. Santos, J. (1999, April). Cronbach's Alpha: A tool for assessing the reliability of scales. Journal of Extension, 37, 2. Retrieved from https://www.joe.org/joe/1999april/tt3.php Seok, S., DaCosta, B., Kinsell, C., & Tung, C. K. (2010). Comparison of instructors' and students' perceptions of the effectiveness of online courses. Quarterly Review of Distance Education, 11(1), 25. Retrieved from http://online.nuc.edu/ctl_en/wp-content/uploads/2015/08/Online-education-effectiviness.pdf Sheridan, K., & Kelly, M. A. (2010). The indicators of instructor presence that are important to students in online courses. 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Marketing Education Review, 20, 29-34. doi:10.2753/MER1052-8008200105 Stoel, L., & Hye Lee, K. (2003). Modeling the effect of experience on student acceptance of web-based courseware. Internet Research, 13(5), 364-374. http://www.emeraldinsight.com/loi/intr Taggart, G., & Bodle, J. H. (2003). Example of assessment of student outcomes data from on-line paralegal courses: Lessons learned. Journal of Paralegal Education & Practice, 19, 29-36. Tanner, J. R., Noser, T. C., & Totaro, M. W. (2009). Business faculty and undergraduate students' perceptions of online learning: A comparative study. Journal of Information Systems Education, 20, 29-40. http://jise.org/ Tung, C.K. (2007). Perceptions of students and instructors of online and web-enhanced course effectiveness in community colleges (Doctoral dissertation). Retrieved from ProQuest Dissertations and Theses database (Publication No. AAT 3284232). Vodanovich, S. J. & Piotrowski, C., & (2000). Are the reported barriers to Internet-based instruction warranted? A synthesis of recent research. Education, 121(1), 48-53. http://www.projectinnovation.com/education.html Ward, M. E., Peters, G., & Shelley, K. (2010). Student and faculty perceptions of the quality of online learning experiences. The International Review of Research in Open and Distributed Learning, 11, 57-77. Retrieved from http://www.irrodl.org/index.php/irrodl/article/view/867/1610? Wilkes, R. B., Simon, J. C., & Brooks, L. D. (2006). A comparison of faculty and undergraduate students' perceptions of online courses and degree programs. Journal of Information Systems Education, 17, 131-140. http://jise.org/

Abstract Abstract 1469 The Wilms Tumor 1 (WT1) gene was first described as a tumour suppressor gene, but its accurate role in leukemia development has not been completely elucidated. Some authors support the role of WT1 as a prognostic marker in acute myeloid leukemia (AML) based on the assessment of its expression at the mRNA level. However, the prognostic value of the main isoforms of WT1 has been less studied. The aim of this study was to develop a specific quantitative assay to estimate the ratio of expression of the four major WT1 isoforms (A, 5-/KTS-; B, 5+/KTS-; C, 5-/KTS+; D, 5+/KTS+) and to evaluate their prognostic impact. WT1 expression was analyzed in bone marrow samples from 108 patients with AML at diagnosis (65 male/46 female, median age: 61 yr, range: 17 – 91). Likewise, peripheral blood samples of 20 healthy donors and 6 samples of cord blood CD34+ cell selection were analyzed as normal controls. We performed a new method to quantify the ratios of the four major isoforms of WT1. Briefly, to amplify all isoforms within a PCR reaction, specific WT1 primers flanking exon 4 to exon 10 were used in cDNA samples, followed by capillary electrophoresis with laser-induced fluorescence analysis on an ABIPRISM 310 DNA Analyzer (Applied Biosystems, Foster City, CA) and lastly analyzed with the Gene Mapper 4.2 software (Applied Biosystems). The amount of each isoform was calculated by the area under the curve. Subsequent comparisons of isoform ratios were made by standardized calculation of percentage. All values are given as the mean of duplicate PCRs. In parallel, RQ-PCR for total WT1 detection was performed as previously described by Barragan et al. (Haematologica 2004; 89: 926–933). GUS gene was used as housekeeping gene. Eighteen patients (17%) did not express WT1, while 90 patients (83%) overexpressed WT1 above background levels. The median value of each WT1 isoform was: 18% (range: 2 – 73) for A isoform; 16% (range: 7 – 63) for B isoform; 24% (range: 2 – 52) for C isoform; and 33% (range: 3 – 55) for D isoform. None of healthy donors had detectable WT1 levels in peripheral blood. All samples of CD34+ cells expressed the four isoforms of WT1: 21% (range: 2 – 26) for A isoform; 16% (range: 1 – 64) for B isoform; 24% (range: 1 – 47) for C isoform; and 36% (range: 25 – 44) for D isoform. These data reveal that, in our series, the most predominant isoform was +5/+KTS, both in AML and in cord blood CD34+ cell selection samples. There were no significant differences when comparing the proportion of each isoform between the cord blood CD34+ cell selection samples and the cohort of AML patients. There was not significant correlation between the overexpression of total WT1 with the ratio of each isoform, and we were unable to demonstrate that the overexpression of WT1 is due to a particular isoform overexpression. A significant lower event-free survival (EFS) was observed in those patients overexpressing total WT1, taking a cut-off value of 3000 WT1 copies/ GUS copies × 104 (75th percentile, P =.001). However, when the same cut-off as well as the median value for each one of the isoforms was used, we found no significant differences in EFS and in overall survival. To sum up, none of the isoforms were correlated with overexpression of total WT1 or survival. We were unable to find differences between the expression of each isoform of WT1 in CD34+ cells from normal cord blood and in AML patients. Further studies including larger controls need to be carried out. Disclosures: No relevant conflicts of interest to declare.

The present paper focuses on the study of the communicative nature of self-disclosure as a technique of verbal influence on people. The phenomenon of self-disclosure is interpreted as the process of divulging private information to others. The aim of the study is to determine the specific features of self-disclosure in a public speaking context. The paper discusses the communicative conventions and parameters of the public speaking scenario as the factors that determine a certain manner of self-disclosing. The key assumption of the study is that public self-disclosure should be viewed as a tactic of self-presentation that promotes the speaker’s image building. In order to indicate the verbal forms that speakers use while disclosing private information in public the discourse and lexical-grammatical analyses of eight celebrity interview texts are conducted. The results of the research demonstrate that the speakers use recurrently certain verbal patterns which are identified as statements of self-description (it includes statements of self-confirmation, self-assurance, personal preferences and personal aspirations), self-narrative and attitude statements. The study also shows which types of the statements are used more frequently than the others, particularly, the statements of self-confirmation, personal aspirations and self-narratives are apparently preferred by the speakers as tactics of public self-disclosure. The paper highlights the fact that the self-disclosure tactics are employed by public speakers selectively and that their choice is motivated by the speakers’ strategic goal of impression management. References Altman, I., Taylor, D. A. (1973). Social Penetration: The Development of InterpersonalRelationship. New York, NY: Holl, Rinehart & Winston. Baumeister, R. F. (1982). A self-presentational view of social phenomena. PsychologicalBulletin, 91, 3–26. Carver, C. S., Scheier, M. R. (1998). On the Self-Regulation of Behavior. CambridgeUniversity Press. Cozby, P.C. (1973). Self-disclosure: A literature review. Psychological Bulletin, 79(2),73–91. Derlega, V. J., Metts, S., Petronio, S., Margulis, S. T. (1993). Self-Disclosure. NewburyPart, CA: Sage. Hargie, O. (2011). Skilled Interpersonal Interaction: Research, Theory and Practice.London: Reutledge. Johnson, J.A. (1981). The ‘self-disclosure’ and ‘self-presentation’ views of item responsedynamics and personality scale validity. Journal of Personality and Social Psychology,40(4), 761–769. Jourard, S. (1971). The Transparent Self. (2nd ed.).New York: Van Nostrand Reinhold. Leary, M. R. (1995). Self-Presentation: Impression Management and InterpersonalBehavior. Madison, WI: Brown & Benchmark. Leary, M. R. (1996). Self-Presentation: Iimpression Management and InterpersonalBehavior. Boulder, CO: Westview Press. Leary, M. R. (2012). Introduction to Behavioral Research Methods. (6nd ed.). Boston:Pearson. Leary, M. R., Kowalski, R. M. (1990). Impression management: A literature review andtwo-component model. Psychological Bulletin, 107(1), 34–47. Luft, J., Ingham, H. (1969). Of Human Interaction. Palo Alto, CA: National Press Books. Rosenfeld, L. B. (2014). Overview of the ways privacy, secrecy, and disclosure arebalanced in today’s society. In: Balancing the Secrets of Private Disclosure, (pp. 3 – 18).S. Petronio, (ed.). New York and London: Psychology Press. Schlenker, B. R. (1980). Impression management: the self-concept, social identity, andinterpersonal relations. Monterey, CA: Brooks/Cole. Schlenker, B. R. (1985). Identity and self-identification. In: The self and social life, (pp.65–99). B.R.Schlenker, (ed.). New York: McGrow-Hill. Schlenker, B. R. (2003). Self-presentation. In: Handbook of Self and Identity, (pp. 492–518). M. R.Leary, J. P.Tangney, (eds.). New York: Guilford. Sources J.K. Rolling meets Lauren Laverne, 2015. Oprah talks to Barack Obama, 2004. O, The Oprah magazine. Oprah talks to Daniel Pink, 2008. O, The Oprah Magazine. Oprah talks to Ellen De Generes, 2009. O, The Oprah Magazine. Oprah talks to Jay-Z, 2009. O, The Oprah Magazine. Oprah talks to Tine Fey, 2009, O, The Oprah Magazine. Oprah talks to Thich Nhat Hanh, 2010.8.The ultimate O interview: Oprah answers all your questions, 2010. O, the Oprah magazine.

Background:Strategies for early diagnosis of Giant Cell Arteritis (GCA) have been implemented in the last decade in order to reduce the occurrence of blindness, ischemic events and other manifestations of the disease. The Southend pretest probability score (SPTPS) 1 seem to be useful in stratifying and select the referral patients sent to the GCA fast-track clinics.Objectives:The aim of this study was to test the validity and utility of this score in our patients.Methods:Retrospective available data records of all the patients referred to our GCA fast-track clinic in the last three years were collected. The clinical and laboratory features at referral were evaluated, and the SPTPS was generated. All patients had ultrasound of cranial and large vessel examination (axillary, subclavian and carotid arteries). GCA diagnosis was confirmed by the doctor after at least six months of follow-up. Quartile frequencies, ROC curves and accuracy of the SPTPS was calculated.Results:261 patients (180 females) with suspicions of GCA were evaluated in our fast-track clinic. Mean age (±SD) was 76±9.2 years, and C-reactive protein 75.7±68.6 mg/l. The time since the first symptoms was less of 6, 6-12, 12-24 or >24 weeks in 37.5%, 19.9, 12.3 and 15.7 respectively. Mean SPTPS was 11.4±4.4, with values of 9.1±3.2 and 12.9±4.4. The area under the ROC curve was 0.761 (95% confidence interval: 0.703-0.819). PTPS overall showed a 50th percentile score of 11 and a 75th percentile score of 14. We, therefore, classified “Low Risk” (LR) as SPTPS <11, “Intermediate Risk” (IR) 11–14 and “High Risk” (HR) >14. Of 261 referrals, 156 had GCA with cases categorised as LR (119), IR (91) and HR (51). HR score showed a specificity of 96.2% and in IR the specificity ranged between 86.7 and 94.3% (Table 1). Score below 7 appears in 29 cases with only 8 cases of GCA with a probability of GCA < 5%, more of these cases was associated a large vessel vasculitis: 3 cranial arteritis (CA), 4 large vessel vasculitis (LVV) and 1 mixed pattern (MP). LR score included 72 of the 105 (68.6%) non-GCA cases but 47 of 156 (30.1%) GCA patients (20 CA, 11 LVV and 16 MP). IR risk score included 26 (24.8%) of the non-GCA cases and 65 (41.7%) of GCA patients (31 CA, 13 LVV and 21 MP). Finally HR score had only 7 non-GCA cases and 44 (28.2%) patients with GCA (20 CA, 11 LVV and 13 MP). Mean score in cranial forms was 13.1±4.5 and in large vessel vasculitis 12.8±5.0 (p=0.807).Table 1.Sensitivity and Specificity of SPTPS at different cut-off pointsCut-off pointSensitivity1-Specificity21.0001.0003.50.9940.9714.50.9870.9435.50.9870.8766.50.949 0.7907.50.8970.6768.50.8460.4769.50.7500.36210.50.6790.24811.50.5380.13312.50.4490.08613.50.3530.05714.50.2180.03815.50.1470.01916.50.1280.01917.50.0770.010190.0380,00020.50.0320,00021.50.0190,00022.50.0130,000240.0000,000Conclusion:The SPTPS is helpful to stratified patients sent to the fast-track clinic. Scores below of 7 points has a very low probability to have GCA. The diagnostic probability increases directly with the score. There were minor differences without statistical significance between cranial or large vessel vasculitis patterns in the score.References:[1]Sebastian A, Tomelleri A, Kayani A, Prieto-Pena D, Ranasinghe C, Dasgupta B. Probability-based algorithm using ultrasound and additional tests for suspected GCA in a fast-track clinic. RMD Open. 2020 Sep;6(3):e001297. doi: 10.1136/rmdopen-2020-001297. PMID: 32994361; PMCID: PMC7547539.Disclosure of Interests:Javier Ortega: None declared, Irene Monjo Speakers bureau: Roche, Novartis, UCB, Gedeon Richter, Consultant of: Roche, Elisa Fernández-Fernández: None declared, Eugenio de Miguel Speakers bureau: AbbVie, Novartis, Pfizer, MSD, BMS, UCB, Roche, Grunental, Janssen, Sanofi., Paid instructor for: Janssen, Novartis, Roche, Consultant of: AbbVie, Novartis, Pfizer, Galapagos, Grant/research support from: Abbvie, Novartis, Pfizer

Since the relaxation of federal sanctions on the planting of Ribes crops because of the development of white pine blister rust (WPBR) immune Ribes cultivars (3), a small industry for the production of Ribes fruit for fresh and processing markets was established in New York and surrounding New England states. The majority of Ribes acreage in the region is planted to a WPBR immune black currant R. nigrum cv. Titania. From 2008 to June 2011, symptoms resembling those caused by WPBR pathogen Cronartium ribicola were observed at a large (>12 ha) R. nigrum cv. Titania planting in Preston, CT. In 2008, infection was restricted to a single field (100% incidence), but in 2009, all fields suffered from premature defoliation by late July. In 2010 and 2011, there was considerable incidence (>85%), but premature defoliation was kept in check by chemical management. Symptoms began as chlorotic lesions (0.5 to 4.3 mm in diameter) on both sides of the leaf. These chlorotic lesions had margins delineated by leaf veins and many developed necrotic (0.3 to 0.9 mm in diameter) centers on the upper surface of leaves within 2 to 3 weeks. The undersides of lesions developed blisters containing orange uredinia (0.1 to 0.33 mm in diameter) with smooth peridia that broke with the production of yellow-orange urediniospores (30 × 15 to 25 μm). Symptoms and signs were consistent with published descriptions of C. ribicola (1) and with WPBR infections on highly susceptible R. nigrum cv. Ben Alder planted at the New York State Agricultural Experiment Station in Geneva. Additional confirmation was provided by sequencing the two internal transcribed spacer (ITS) regions and the 5.8S gene (GenBank Accession No. JN587805; 98% identity with No. DQ533975) in the nuclear ribosomal repeat using primers ITS1-F and ITS4 as described previously (2,4). Furthermore, an attempt was made to confirm pathogenicity in the greenhouse by inoculating shoots of potted nursery stock of R. nigrum cv. Titania. Shoots were inoculated by rubbing leaves with either an uninfected currant leaf or a currant leaf from Preston, CT sporulating with urediniospores. Plants were subsequently misted with dH20 and covered with plastic bags for 24 h. Plants were watered biweekly and kept in a greenhouse with 39 to 65% relative humidity at 21 to 26°C. Shoots were monitored for symptom development on a weekly basis. After 3 weeks, 2 of the 10 plants inoculated with infected leaves developed chlorotic lesions and uredinia identical to those on leaves from Preston, CT, while all others remained healthy. Although not easily reproducible in a greenhouse, the breakdown of immunity in R. nigrum cv. Titania was observed for the last 4 years in Connecticut. Given that WPBR immunity was one of the conditions for legalized planting of Ribes, the breakdown of immunity has potentially deleterious implications particularly for nearby states like Massachusetts and New York, in which the Ribes industries are expanding. Moreover, Ribes growers may need to rely on chemical management programs to manage WPBR in the future. References: (1) G. B. Cummins. Illustrated Genera of Rust Fungi. Burgess Publishing Company, Minneapolis, MN, 1959. (2) M. Gardes and T. D. Bruns. Mol. Ecol. 2:113, 1993. (3) S. McKay. Hortic. Technol. 10:562, 2000. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego, CA, 1990.

Background Multiple myeloma (MM) is the abnormal proliferation of plasma cells in the bone marrow often resulting in debilitating symptoms ranging from ostealgia to pathological fractures from bone destruction. According to American Cancer Society, MM accounts for 1-2% of cancers and approximately 17% of hematological malignancies in the United States each year (1). Fifty percent of patients with symptomatic MM have three or more primary care visits before they are referred to a specialist, which is greater than any other cancer (5). It has been shown that a delay in diagnosing multiple myeloma negatively impacts the clinical course of the disease and hence the outcome in patients (2). Patients with longer diagnostic intervals also experience shorter disease free survival and more complications from treatment (4). Herein, a retrospective analysis was performed to determine the average delay in diagnosis of MM. Methods This is a retrospective electronic chart review of all indexed newly diagnosed MM cases between 1/1/2014 through 12/31/2018 at New York-Presbyterian Brooklyn Methodist Hospital (NYP BHM). NYP BMH is a Weill Cornell Medical College-affiliated hospital in Brooklyn, NY whose patient population includes those with private insurance, uninsured and Medicare/Medicaid. Data abstraction from the electronic medical record (EMR) was uniform and involved baseline characteristics such as age, gender and race. International Classification of Diseases (ICD)-10-CM code (C90.00) was used for extraction of data which identified 492 patients. After excluding patients with MGUS or a prior diagnosis of multiple myeloma, 104 patients were included in the final study. We calculated the number of days between the date of first abnormal laboratory value seen on bloodwork for a myeloma related sign (at least 90 days prior to diagnosis) to the date of bone marrow biopsy that confirmed the diagnosis. The inclusion criteria were anemia defined as hemoglobin &lt;12gm/dl, Hypercalcemia defined by corrected calcium &gt;10, kidney dysfunction with a creatinine &gt;1.5 and total protein &gt;8. Results Of the 104 patients with newly diagnosed MM, 69 patients were diagnosed within 90 days of the first abnormal lab value recorded in our electronic medical record (EMR). Thirty-five patients (34%) had a delay in diagnosis at least 90 days with a mean delay of 38 months. Isolated anemia was the most common abnormal lab finding with 29/104 (28%) having documented anemia at least 90 days prior to diagnosis of myeloma. The mean delay in diagnosis for patients with anemia was 41 months. There were four patients with anemia and elevated creatinine with an average delay of 23 months. Five patients had anemia and elevated calcium with an average delay of 21 months. Nine patients had anemia and elevated total protein with an average delay of 38 months. Conclusion: In the current era where we have effective therapies for MM it is now more important than ever to avoid a delay in diagnosis. We demonstrate that 34% of patients receiving care at an Urban Teaching Hospital had at least a 90 day delay in their diagnosis of MM. Our cohort consisted of 64% African Americans, suggesting that minorities are more commonly affected by this. There is a need for more awareness amongst clinicians to consider the diagnosis of MM in the workup of anemia. References: 1. Kariyawasan, C. C., D. A. Hughes, M. M. Jayatillake, and A. B. Mehta. 2007. "Multiple Myeloma: Causes and Consequences of Delay in Diagnosis." QJM: Monthly Journal of the Association of Physicians 100 (10): 635-40. 2. Siegel, Rebecca L., Kimberly D. Miller, and Ahmedin Jemal. 2019. "Cancer Statistics, 2019." CA: A Cancer Journal for Clinicians. https://doi.org/10.3322/caac.21551. 3. Vélez R, Turesson I, Landgren O, Kristinsson SY, Cuzick J. Incidence of multiple myeloma in Great Britain, Sweden, and Malmö, Sweden: the impact of differences in case ascertainment on observed incidence trends. BMJ Open. 2016;6:e009584. 4. Kariyawasan CC, Hughes DA, Jayatillake MM, et al. Multiple myeloma: causes and consequences of delay in diagnosis. QJM 2007;100:635-40. 10.1093/qjmed/hcm077 5. Lyratzopoulos G, Neal RD, Barbiere JM, et al. Variation in number of general practitioner consultations before hospital referral for cancer: findings from the 2010 National Cancer Patient Experience Survey in England. Lancet Oncol 2012;13:353-65. 10.1016/S1470-2045(12)70041-4 Disclosures No relevant conflicts of interest to declare.

Hamsters were exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days. Equal amounts of proteins from urinary bladder or liver extracts of control and arsenic-treated hamsters were labeled with Cy3 and Cy5 dyes, respectively. After differential in gel electrophoresis and analysis by the DeCyder software, several protein spots were found to be down-regulated and several were up regulated. Our experiments indicated that in the bladder tissues of hamsters exposed to arsenite, transgelin was down-regulated and GST-pi was up-regulated. The loss of transgelin expression has been reported to be an important early event in tumor progression and a diagnostic marker for cancer development [29-32]. Down-regulation of transgelin expression may be associated with the carcinogenicity of inorganic arsenic in the urinary bladder. In the liver of arsenite-treated hamsters, ornithine aminotransferase was up-regulated, and senescence marker protein 30 and fatty acid binding protein were down-regulated. The volume ratio changes of these proteins in the bladder and liver of hamsters exposed to arsenite were significantly different than that of control hamsters. Introduction Chronic exposure to inorganic arsenic can cause cancer of the skin, lungs, urinary bladder, kidneys, and liver [1-6]. The molecular mechanisms of the carcinogenicity and toxicity of inorganic arsenic are not well understood [7-9). Humans chronically exposed to inorganic arsenic excrete MMA(V), DMA(V) and the more toxic +3 oxidation state arsenic biotransformants MMA(III) and DMA (III) in their urine [10, 11], which are carcinogen [12]· After injection of mice with sodium arsenate, the highest concentrations of the very toxic MMA(III) and DMA(III) were in the kidneys and urinary bladder tissue, respectively, as shown by experiments of Chowdhury et al [13]. Many mechanisms of arsenic toxicity and carcinogenicity have been suggested [1, 7, 14] including chromosome abnormalities [15], oxidative stress [16, 17], altered growth factors [18], cell proliferation [19], altered DNA repair [20], altered DNA methylation patterns [21], inhibition of several key enzymes [22], gene amplification [23] etc. Some of these mechanisms result in alterations in protein expression. Methods for analyzing multiple proteins have advanced greatly in the last several years. In particularly, mass spectrometry (MS) and tandem MS (MS/MS) are used to analyze peptides following protein isolation using two-dimensional (2-D) gel electrophoresis and proteolytic digestion [24]. In the present study, Differential In Gel Electrophoresis (DIGE) coupled with Mass Spectrometry (MS) has been used to study some of the proteomic changes in the urinary bladder and liver of hamsters exposed to sodium arsenite in their drinking water. Our results indicated that transgelin was down-regulated and GST-pi was up-regulated in the bladder tissues. In the liver tissues ornithine aminotransferase was up-regulated, and senescence marker protein 30, and fatty acid binding protein were down-regulated. Materials and Methods Chemicals Tris, Urea, IPG strips, IPG buffer, CHAPS, Dry Strip Cover Fluid, Bind Silane, lodoacetamide, Cy3 and Cy5 were from GE Healthcare (formally known as Amersham Biosciences, Uppsala, Sweden). Thiourea, glycerol, SDS, DTT, and APS were from Sigma-Aldrich (St. Louis, MO, USA). Glycine was from USB (Cleveland, OH, USA). Acrylamide Bis 40% was from Bio-Rad (Hercules, CA, USA). All other chemicals and biochemicals used were of analytical grade. All solutions were made with Milli-Q water. Animals Male hamsters (Golden Syrian), 4 weeks of age, were purchased from Harlan Sprague Dawley, USA. Upon arrival, hamsters were acclimated in the University of Arizona animal care facility for at least 1 week and maintained in an environmentally controlled animal facility operating on a 12-h dark/12-h light cycle and at 22-24°C. They were provided with Teklad (Indianapolis, IN) 4% Mouse/Rat Diet # 7001 and water, ad libitum, throughout the acclimation and experimentation periods. Sample preparation and labelling Hamsters were exposed to sodium arsenite (173 mg) in drinking water for 6 days and the control hamsters were given tap water. On the 6th day hamsters were decapitated rapidly by guillotine. Urinary bladder tissues and liver were removed, blotted on tissue papers (Kimtech Science, Precision Wipes), and weighed. Hamster urinary bladder or liver tissues were homogenized in lysis buffer (30mMTris, 2M thiourea, 7M urea, and 4% w/w CHAPS adjusted to pH 8.5 with dilute HCI), at 4°C using a glass homogenizer and a Teflon coated steel pestle; transferred to a 5 ml acid-washed polypropylene tube, placed on ice and sonicated 3 times for 15 seconds. The sonicate was centrifuged at 12,000 rpm for 10 minutes at 4°C. Small aliquots of the supernatants were stored at -80°C until use (generally within one week). Protein concentration was determined by the method of Bradford [25] using bovine serum albumin as a standard. Fifty micrograms of lysate protein was labeled with 400 pmol of Cy3 Dye (for control homogenate sample) and Cy5 Dye (for arsenic-treated urinary bladder or liver homogenate sample). The samples containing proteins and dyes were incubated for 30 min on ice in the dark. To stop the labeling reaction, 1uL of 10 mM lysine was added followed by incubation for 10 min on ice in the dark. To each of the appropriate dye-labeled protein samples, an additional 200 ug of urinary bladderor liver unlabeled protein from control hamster sample or arsenic-treated hamster sample was added to the appropriate sample. Differentially labeled samples were combined into a single Microfuge tube (total protein 500 ug); protein was mixed with an equal volume of 2x sample buffer [2M thiourea, 7M urea, pH 3-10 pharmalyte for isoelectric focusing 2% (v/v), DTT 2% (w/v), CHAPS 4% (w/v)]; and was incubated on ice in the dark for 10 min. The combined samples containing 500 ug of total protein were mixed with rehydration buffer [CHAPS 4% (w/v), 8M urea, 13mM DTT, IPG buffer (3-10) 1% (v/v) and trace amount of bromophenol blue]. The 450 ul sample containing rehydration buffer was slowly pipetted into the slot of the ImmobilinedryStripReswelling Tray and any large bubbles were removed. The IPG strip (linear pH 3-10, 24 cm) was placed (gel side down) into the slot, covered with drystrip cover fluid (Fig. 1), and the lid of the Reswelling Tray was closed. The ImmobillineDryStrip was allowed to rehydrate at room temperature for 24 hours. First dimension Isoelectric focusing (IEF) The labeled sample was loaded using the cup loading method on universal strip holder. IEF was then carried out on EttanIPGphor II using multistep protocol (6 hr @ 500 V, 6 hr @ 1000 V, 8 hr @ 8000 V). The focused IPG strip was equilibrated in two steps (reduction and alkylation) by equilibrating the strip for 10 min first in 10 ml of 50mM Tris (pH 8.8), 6M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 0.5% (w/v) DTT, followed by another 10 min in 10 ml of 50mM Tris (pH 8.8), 6M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 4.5% (w/v) iodoacetamide to prepare it for the second dimension electrophoresis. Second dimension SDS-PAGE The equilibrated IPG strip was used for protein separation by 2D-gel electrophoresis (DIGE). The strip was sealed at the top of the acrylamide gel for the second dimension (vertical) (12.5% polyacrylamide gel, 20x25 cm x 1.5 mm) with 0.5% (w/v) agarose in SDS running buffer [25 mMTris, 192 mM Glycine, and 0.1% (w/v) SDS]. Electrophoresis was performed in an Ettan DALT six electrophoresis unit (Amersham Biosciences) at 1.5 watts per gel, until the tracking dye reached the anodic end of the gel. Image analysis and post-staining The gel then was imaged directly between glass plates on the Typhoon 9410 variable mode imager (Sunnyvale, CA, USA) using optimal excitation/emission wavelength for each DIGE fluor: Cy3 (532/580 nm) and Cy5 (633/670 nm). The DIGE images were previewed and checked with Image Quant software (GE Healthcare) where all the two separate gel images could be viewed as a single gel image. DeCyde v.5.02 was used to analyze the DIGE images as described in the Ettan DIGE User Manual (GE Healthcare). The appropriate up-/down regulated spots were filtered based on an average volume ratio of ± over 1.2 fold. After image acquisition, the gel was fixed overnight in a solution containing 40% ethanol and 10% acetic acid. The fixed gel was stained with SyproRuby (BioRad) according to the manufacturer protocol (Bio-Rad Labs., 2000 Alfred Nobel Drive, Hercules, CA 94547). Identification of proteins by MS Protein spot picking and digestion Sypro Ruby stained gels were imaged using an Investigator ProPic and HT Analyzer software, both from Genomic Solutions (Ann Arbor, MI). Protein spots of interest that matched those imaged using the DIGE Cy3/Cy5 labels were picked robotically, digested using trypsin as described previously [24] and saved for mass spectrometry identification. Liquid chromatography (LC)- MS/MS analysis LC-MS/MS analyses were carried out using a 3D quadrupole ion trap massspectrometer (ThermoFinnigan LCQ DECA XP PLUS; ThermoFinnigan, San Jose, CA) equipped with a Michrom Paradigm MS4 HPLC (MichromBiosources, Auburn, CA) and a nanospray source, or with a linear quadrupole ion trap mass spectrometer (ThermoFinnigan LTQ), also equipped with a Michrom MS4 HPLC and a nanospray source. Peptides were eluted from a 15 cm pulled tip capillary column (100 um I.D. x 360 um O.D.; 3-5 um tip opening) packed with 7 cm Vydac C18 (Vydac, Hesperia, CA) material (5 µm, 300 Å pore size), using a gradient of 0-65% solvent B (98% methanol/2% water/0.5% formic acid/0.01% triflouroacetic acid) over a 60 min period at a flow rate of 350 nL/min. The ESI positive mode spray voltage was set at 1.6 kV, and the capillary temperature was set at 200°C. Dependent data scanning was performed by the Xcalibur v 1.3 software on the LCQ DECA XP+ or v 1.4 on the LTQ [27], with a default charge of 2, an isolation width of 1.5 amu, an activation amplitude of 35%, activation time of 50 msec, and a minimal signal of 10,000 ion counts (100 ion counts on the LTQ). Global dependent data settings were as follows: reject mass width of 1.5 amu, dynamic exclusion enabled, exclusion mass width of 1.5 amu, repeat count of 1, repeat duration of a min, and exclusion duration of 5 min. Scan event series were included one full scan with mass range of 350-2000 Da, followed by 3 dependent MS/MS scans of the most intense ion. Database searching Tandem MS spectra of peptides were analyzed with Turbo SEQUEST, version 3.1 (ThermoFinnigan), a program that allows the correlation of experimental tandem MS data with theoretical spectra generated from known protein sequences. All spectra were searched against the latest version of the non redundant protein database from the National Center for Biotechnology Information (NCBI 2006; at that time, the database contained 3,783,042 entries). Statistical analysis The means and standard error were calculated. The Student's t-test was used to analyze the significance of the difference between the control and arsenite exposed hamsters. P values less than 0.05 were considered significant. The reproducibility was confirmed in separate experiments. Results Analysis of proteins expression After DIGE (Fig. 1), the gel was scanned by a Typhoon Scanner and the relative amount of protein from sample 1 (treated hamster) as compared to sample 2 (control hamster) was determined (Figs. 2, 3). A green spot indicates that the amount of protein from sodium arsenite-treated hamster sample was less than that of the control sample. A red spot indicates that the amount of protein from the sodium arsenite-treated hamster sample was greater than that of the control sample. A yellow spot indicates sodium arsenite-treated hamster and control hamster each had the same amount of that protein. Several protein spots were up-regulated (red) or down-regulated (green) in the urinary bladder samples of hamsters exposed to sodium arsenite (173 mg As/L) for 6 days as compared with the urinary bladder of controls (Fig. 2). In the case of liver, several protein spots were also over-expressed (red) or under-expressed (green) for hamsters exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days (Fig. 3). The urinary bladder samples were collected from the first and second experiments in which hamsters were exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days and the controls were given tap water. The urinary bladder samples from the 1st and 2nd experiments were run 5 times in DIGE gels on different days. The protein expression is shown in Figure 2 and Table 1. The liver samples from the 1st and 2nd experiments were also run 3 times in DIGE gels on different days. The proteins expression were shown in Figure 3 and Table 2. The volume ratio changed of the protein spots in the urinary bladder and liver of hamsters exposed to arsenite were significantly differences than that of the control hamsters (Table 1 and 2). Protein spots identified by LC-MS/MS Bladder The spots of interest were removed from the gel, digested, and their identities were determined by LC-MS/MS (Fig. 2 and Table 1). The spots 1, 2, & 3 from the gel were analyzed and were repeated for the confirmation of the results (experiments; 173 mg As/L). The proteins for the spots 1, 2, and 3 were identified as transgelin, transgelin, and glutathione S-transferase Pi, respectively (Fig. 2). Liver We also identified some of the proteins in the liver samples of hamsters exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days (Fig. 3). The spots 4, 5, & 6 from the gels were analyzed and were repeated for the confirmation of the results. The proteins for the spots 4, 5, and 6 were identified as ornithine aminotransferase, senescence marker protein 30, and fatty acid binding protein, respectively (Fig. 3) Discussion The identification and functional assignment of proteins is helpful for understanding the molecular events involved in disease. Weexposed hamsters to sodium arsenite in drinking water. Controls were given tap water. DIGE coupled with LC-MS/MS was then used to study the proteomic change in arsenite-exposed hamsters. After electrophoresis DeCyder software indicated that several protein spots were down-regulated (green) and several were up-regulated (red). Our overall results as to changes and functions of the proteins we have studied are summarized in Table 3. Bladder In the case of the urinary bladder tissue of hamsters exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days, transgelin was down-regulated and GST-pi was up-regulated. This is the first evidence that transgelin is down-regulated in the bladders of animals exposed to sodium arsenite. Transgelin, which is identical to SM22 or WS3-10, is an actin cross linking/gelling protein found in fibroblasts and smooth muscle [28, 29]. It has been suggested that the loss of transgelin expression may be an important early event in tumor progression and a diagnostic marker for cancer development [30-33]. It may function as a tumor suppressor via inhibition of ARA54 (co-regulator of androgen receptor)-enhanced AR (androgen receptor) function. Loss of transgelin and its suppressor function in prostate cancer might contribute to the progression of prostate cancer [30]. Down-regulation of transgelin occurs in the urinary bladders of rats having bladder outlet obstruction [32]. Ras-dependent and Ras-independent mechanisms can cause the down regulation of transgelin in human breast and colon carcinoma cell lines and patient-derived tumorsamples [33]. Transgelin plays a role in contractility, possibly by affecting the actin content of filaments [34]. In our experiments loss of transgelin expression may be associated or preliminary to bladder cancer due to arsenic exposure. Arsenite is a carcinogen [1]. In our experiments, LC-MS/MS analysis showed that two spots (1 and 2) represent transgelin (Fig. 2 and Table 1). In human colonic neoplasms there is a loss of transgelin expression and the appearance of transgelin isoforms (31). GST-pi protein was up-regulated in the bladders of the hamsters exposed to sodium arsenite. GSTs are a large family of multifunctional enzymes involved in the phase II detoxification of foreign compounds [35]. The most abundant GSTS are the classes alpha, mu, and pi classes [36]. They participate in protection against oxidative stress [37]. GST-omega has arsenic reductase activity [38]. Over-expression of GST-pi has been found in colon cancer tissues [39]. Strong expression of GST-pi also has been found in gastric cancer [40], malignant melanoma [41], lung cancer [42], breast cancer [43] and a range of other human tumors [44]. GST-pi has been up-regulated in transitional cell carcinoma of human urinary bladder [45]. Up-regulation of glutathione – related genes and enzyme activities has been found in cultured human cells by sub lethal concentration of inorganic arsenic [46]. There is evidence that arsenic induces DNA damage via the production of ROS (reactive oxygen species) [47]. GST-pi may be over-expressed in the urinary bladder to protect cells against arsenic-induced oxidative stress. Liver In the livers of hamsters exposed to sodium arsenite, ornithine amino transferase was over-expressed, senescence marker protein 30 was under-expressed, and fatty acid binding protein was under-expressed. Ornithine amino transferase has been found in the mitochondria of many different mammalian tissues, especially liver, kidney, and small intestine [48]. Ornithine amino transferase knockdown inhuman cervical carcinoma and osteosarcoma cells by RNA interference blocks cell division and causes cell death [49]. It has been suggested that ornithine amino transferase has a role in regulating mitotic cell division and it is required for proper spindle assembly in human cancer cells [49]. Senescence marker protein-30 (SMP30) is a unique enzyme that hydrolyzes diisopropylphosphorofluoridate. SMP30, which is expressed mostly in the liver, protects cells against various injuries by stimulating membrane calcium-pump activity [50]. SMP30 acts to protect cells from apoptosis [51]. In addition it protects the liver from toxic agents [52]. The livers of SMP30 knockout mice accumulate phosphatidylethanolamine, cardiolipin, phosphatidyl-choline, phosphatidylserine, and sphingomyelin [53]. Liver fatty acid binding protein (L-FABP) also was down- regulated. Decreased liver fatty acid-binding capacity and altered liver lipid distribution hasbeen reported in mice lacking the L-FABP gene [54]. High levels of saturated, branched-chain fatty acids are deleterious to cells and animals, resulting in lipid accumulation and cytotoxicity. The expression of fatty acid binding proteins (including L-FABP) protected cells against branched-chain saturated fatty acid toxicity [55]. Limitations: we preferred to study the pronounced spots seen in DIGE gels. Other spots were visible but not as pronounced. Because of limited funds, we did not identify these others protein spots. In conclusion, urinary bladders of hamsters exposed to sodium arsenite had a decrease in the expression of transgelin and an increase in the expression of GST-pi protein. Under-expression of transgelin has been found in various cancer systems and may be associated with arsenic carcinogenicity [30-33). Inorganic arsenic exposure has resulted in bladder cancer as has been reported in the past [1]. Over-expression of GST-pi may protect cells against oxidative stress caused by arsenite. In the liver OAT was up regulated and SMP-30 and FABP were down regulated. These proteomic results may be of help to investigators studying arsenic carcinogenicity. The Superfund Basic Research Program NIEHS Grant Number ES 04940 from the National Institute of Environmental Health Sciences supported this work. Additional support for the mass spectrometry analyses was provided by grants from NIWHS ES06694, NCI CA023074 and the BIOS Institute of the University of Arizona. Acknowledgement The Author wants to dedicate this paper to the memory of his former supervisor Dr. H. VaskenAposhian who passed away in September 6, 2019. He was an emeritus professor of the Department of Molecular and Cellular Biology at the University of Arizona. 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Seven spot ladybird beetle, (Coccinella septempunctata) is a widely distributed natural enemy of soft-bodied insect pests especially aphids worldwide. Both the adult and larvae of this coccinellid beetle are voracious feeders and serve as a commercially available biological control agent around the globe. Different techniques are adopted to enhance the mass rearing and storage of this natural enemy by taking advantage of its natural ability to withstand under extremely low temperatures and entering diapause under unfavorable low temperature conditions. The key objective of this study was to develop a cost effective technique for enhancing the storage life and predatory potential of the larvae of C. septempunctata through cold storage in conjunction with the use of nuclear techniques, gamma radiations. Results showed that the host eating potential of larvae was enhanced as the cold storage duration was increased. Gamma irradiation further enhanced the feeding potential of larvae that were kept under cold storage. Different irradiation doses also affected the development time of C. septempuntata larvae significantly. Without cold storage, the lower radiation doses (10 and 25 GY) prolonged the developmental time as compared to un-irradiated larvae. Furthermore, the higher dose of radiation (50GY) increased the developmental time after removal from cold storage. This study first time paves the way to use radiation in conjunction with cold storage as an effective technique in implementation of different biological control approaches as a part of any IPM programs.Key wordGamma irradiations; cold storage, Coccinella septempunctata larvae; predatory potential; integrated pest management programme.INTRODUCTIONNuclear techniques such as gamma radiations have a vast application in different programmes of biological control including continuous supply of sterilized host and improved rearing techniques (Greany and Carpenter, 2000; Cai et al., 2017). Similarly irradiation can be used for sentinel-host eggs and larvae for monitoring survival and distribution of parasitoids (Jordão-paranhos et al., 2003; Hendrichs et al., 2009; Tunçbilek et al., 2009; Zapater et al., 2009; Van Lenteren, 2012). Also, at the production level, such technique may facilitate the management of host rearing, improve quality and expedite transport of product (Fatima et al., 2009; Hamed et al., 2009; Wang et al., 2009). Gamma irradiations can also be used to stop insect’s development to enhance host suitability for their use in different mass rearing programs (Celmer-Warda, 2004; Hendrichs et al., 2009; Seth et al., 2009). Development and survival of all insects have a direct connection with temperatures which in turn affect the physical, functional and behavioral adaptations (Ramløy, 2000). Many insects living in moderate regions can survive at low temperature by process of diapause. A temperature between 0 to 10oC may cause some insects to become sluggish and they only become active when the temperature is suitable. Such insects show greater adaptations to flexible temperature regimes for better survival. Many studies have reported this concept of cold-hardiness in insects in general (Bale, 2002; Danks, 2006) and specifically in coccinellid beetles over past years (Watanabe, 2002; Koch et al., 2004; Pervez and Omkar, 2006; Labrie et al., 2008; Berkvens et al., 2010). Using this cold hardiness phenomenon, many coccinellids have been studied for the effect of cold storage such as Coccinella undecimpunctata (Abdel‐Salam and Abdel‐Baky, 2000), Coleomegilla maculata (Gagné and Coderre, 2001) and Harmonia axyridis (Watanabe, 2002). This natural phenomenon, therefore, can be a helpful tool in developing low temperature stockpiling for improving mass-rearing procedures (Mousapour et al., 2014). It may provide a significant output in terms of providing natural enemies as and when required during pest infestation peaks (Venkatesan et al., 2000). Use of irradiation in conjunction with cold storage proves to be an effective technique in implementation of different biological control approaches as a part of any IPM programme. A study reported that the pupate of house fly, Musca domestica irradiated at dose of 500 Gy and can stored up to 2 months at 6°C for future use for a parasitoid wasp Spalangia endius rearing (Zapater et al., 2009). Similarly, when irradiated at 20 GY, parasitic wasps Cotesia flavipes were stored safely up to two months without deterioration of their parasitic potential (Fatima et al., 2009). Similarly, bio-control program of sugarcane shoot borer Chilo infescatellus proved successful through the use of irradiation combined with cold storage of its egg and larval parasitoids Trichogramma chilonis and C. flavipes (Fatima et al., 2009). Less mobile life stages such as larvae are of significance in any IPM strategy because they remain on target site for more time period as compared to adults. Therefore, use of predatory larvae is very promising in different biological control approaches because of their immediate attack on pests and more resistance to unfavorable environmental conditions than delicate egg stage. In addition, with their augmentation into fields, larval stage shows their presence for longer time than adult stage and their feeding potential is also satisfactory as that of adults. For the best utilization of these predators in the field and maximum impact of 3rd and 4th larval instars on prey, we should encourage late 2nd second instar larvae of predatory beetles in the fields as these instars have more feeding capacity due to increased size and ability to handle larger preys.In spite of higher significance, there is little information available about the effect of cold storage on the survival of larval instars of different ladybird beetles and its effect on their predatory potential. Very few studies report the use of cold storage for non-diapausing larval stage like for Semiadalia undecimnotata and only one study reported the short-term storage (up to two weeks) of 2nd and 3rd instar coccinellid, C. maculate, without any loss in feeding voracity of larvae after storage (Gagné and Coderre, 2001). The survival of 3rd and 4th larval instars of C. undecimpunctata for 7 days after storage at 5oC was reported in a study but the survival rate declined after 15-60 days of storage (Abdel‐Salam and Abdel‐Baky, 2000). As C. septempunctata is considered one of the voracious predators (Afroz, 2001; Jandial and Malik, 2006; Bilashini and Singh, 2009; Xia et al., 2018) and diapause is a prominent feature of this beetle and it may undergo facultative diapause under suitable laboratory conditions (Suleman, 2015). No information is available to date about the combined effect of cold storage and irradiation on the larval instars of this species.OBJECTIVES The objective of this study was to devise a cost effective technique for the cold storage and its effect on the subsequent predatory potential of the seven spotted ladybird beetle larvae in conjunction with the use of gamma radiations. Hypothesis of the study was that an optimum length of low temperature treatment for storage purpose would not affect the predation capacity of C. septempunctata larvae and their developmental parameters including survival and pupation will remain unaffected. Furthermore, use of gamma irradiation will have some additional effects on survival and feeding capacity of irradiated C. septempunctata larvae. Such techniques can be utilized in different biocontrol programs where short term storage is required. So these larvae can be successfully imparted in different IPM programs against sucking complex of insect pests as a component of biological control strategyMATERIALS AND METHODSPlant materials: Collection and rearing of C. septempunctata: Adult C. septempunctata were collected from the wheat crop (in NIAB vicinity and farm area) in the month of March during late winter and early in spring season 2016-2017. They were kept in plastic jars and were fed with brassica aphids. Under controlled laboratory conditions (25+2oC, 16h: 8h L:D and 65+5% R.H.), eggs of C. septempuctata were obtained and after hatching, larvae were also given brassica aphids as dietary source. Larvae of second instar were selected for this experiment (as the first instar is generally very weak and vulnerable to mortality under low temperatures). As the larvae approached second instar, they were separated for the experimentation. Irradiation of larvae at different doses: Irradiation of larvae was carried out by the irradiation source 137CS at Radiation laboratory, and the larvae were then brought back to the IPM laboratory, Plant Protection Division, Nuclear Institute for Agriculture and Biology (NIAB) Faisalabad. Radiation doses of 10 GY (Grey), 25 GY and 50 GY were used to treat the second instar larvae. There were three replicates for each treatment and five larvae per replicate were used. Control treatment was left un-irradiated.Cold storage of irradiated larvae: In present work, second instar C. septempunctata larvae were studied for storage at low temperature of 8oC. The larvae were kept at 8oC for 0, I and II weeks where week 0 depicts no cold treatment and this set of larvae was left under laboratory conditions for feeding and to complete their development. For larvae that were kept under cold storage for one week at 8°C, the term week I was devised. Similarly, week II denotes the larvae that remained under cold conditions (8°C) for two continuous weeks. Larvae were removed from cold storage in their respective week i.e., after week I and week II and were left under laboratory conditions to complete their development by feeding on aphids. Data collection: For recording the predatory potential of C. septempunctata larvae, 100 aphids were provided per larva per replicate on a daily basis until pupation as this number was more than their feeding capacity to make sure that they were not starved (personal observation). Observations were recorded for survival rate, developmental time and feeding potential. Data analysis: Data were statistically analysed by Statistical Software SPSS (Version 16.0). The data were subjected to normality check through the One-sample Kolmogorov-Smirnov test. Non normal data were transformed to normal data which were then used for all parametric variance tests. One-way and two-way analyses of variance were used. For comparison between variables, LSD test at α 0.05 was applied.RESULTSFeeding potential of irradiated larvae after removal from cold storage: Results showed an increase in the feeding potential of C. septempunctata larvae with increased cold storage duration. The feeding potential was significantly higher for the larvae that spent maximum length of time (week II) under cold storage conditions followed by week I and week 0. Gamma irradiations further enhanced the feeding potential of larvae that were kept under cold storage. When larvae were irradiated at 10 GY, the eating capacity of larvae increased significantly with the duration of cold storage. Similarly, larvae that were irradiated at 25 GY, showed increase in feeding potential on aphids as the time period of cold storage increased. The feeding potential of larvae that were irradiated at 50 GY, was again significantly increased with increase of cold storage duration. When different radiation doses were compared to week 0 of storage, there was a significant difference in feeding potential and larvae irradiated at 50 GY consumed the maximum numbers of aphids when no cold storage was done followed by larvae irradiated at 10 and 25 GY. With the other treatment, where larvae were kept under cold storage for one week (week I) the larvae irradiated at 50GY again showed the highest feeding potential. The feeding potential of irradiated larvae was again significantly higher than the un-irradiated larvae that were kept for two weeks (week II) under cold storage (table 1).Two-way ANOVA was performed to check the interaction between the different radiation doses and different lengths of storage durations for feeding potential of C. septempunctata larvae on aphids. The feeding potential of larvae irradiated at different doses and subjected to variable durations of cold storage were significantly different for both the radiation doses and cold storage intervals. Furthermore, the interaction between the radiation doses and storage duration was also significant meaning that the larvae irradiated at different doses with different length of cold storage were having significant variations in feeding levels (table 2).Developmental time of irradiated larvae after removal from cold storage: Significant difference was found in the development time of the larvae of C. septempunctata when irradiated at different doses at week 0 (without cold storage). The larvae irradiated at 10 GY took the maximum time for development and with the increase in irradiation dosage, from 25 to 50 GY, the time of development was shortened. The larvae irradiated at 50 GY had the same development time as the un-irradiated ones. When, the irradiated larvae were subjected to cold storage of one week duration (week I), their development time after removal from storage condition varied significantly. The larvae irradiated at 25 GY took the maximum time for development followed by larvae irradiated at 50 GY and 10 GY. There was an indication that the development time was extended for irradiated larvae as compared to un-irradiated larvae.Results also depicted a significant difference in the time taken by irradiated larvae to complete their development after taken out from cold storage of two weeks duration (week II). As the storage time of irradiated larvae increased, the development time was prolonged. Results showed that the larvae that were irradiated at 25 and 50 GY, took the maximum time to complete their development. With the prolonged duration of cold storage up to two weeks (week II), this difference of development time was less evident at lower doses (10 GY). The larvae irradiated at 10 GY showed a significant difference in their developmental duration after being taken out of cold storage conditions of the week 0, I and II. There was no difference in the developmental duration of larvae that were un-irradiated and subjected to different regimes of storage. Un-irradiated larvae were least affected by the duration of storage. With the increase in the storage time, a decrease in the developmental time was recorded. Larvae that were irradiated at 10 GY, took the maximum period to complete their development when no cold storage was done (week 0) followed by week I and II of cold storage. When the larvae irradiated at 25 GY were compared for their development time, there was again significant difference for week 0, I and II of storage duration. Maximum time was taken by the larvae for their complete development when removed from cold storage after one week (week I). With the increase in storage duration the time taken by larvae to complete their development after removal from cold storage reduced.When the larvae were removed after different lengths of cold storage duration i.e., week 0, week I and week II, there was a significant difference in the developmental time afterwards. Results have shown that the higher dose of radiation, increased the developmental time after removal from cold storage. The larvae irradiated at 50 GY took the longest time to complete their development after removal from cold storage (week I and week II) as compared the larvae that were not kept under cold storage conditions (week 0) (table 3).Interaction between the different radiation doses and different lengths of storage durations for development time of larvae were checked by two-way ANOVA. The development time of larvae irradiated at different doses and subjected to variable durations of cold storage were significantly different for both the doses and cold storage intervals. Furthermore, the interaction between the radiation doses and storage duration was also significant meaning that the larvae irradiated at different doses with different length of cold storage were having significant variations in development times (table 4). DISCUSSIONThe present research work indicates the possibility of keeping the larval instars of C. septempunctata under cold storage conditions of 8oC for a short duration of around 14 days without affecting its further development and feeding potential. Furthermore, irradiation can enhance the feeding potential and increase the development time of larval instars. This in turn could be a useful technique in mass rearing and field release programmes for biological control through larval instars. Usually temperature range of 8-10oC is an optimal selection of low temperature for storage as reported earlier for eggs two spotted ladybird beetle, Adalia bipunctata and the eggs of C. septempunctata (Hamalainen and Markkula, 1977), Trichogramma species (Jalali and Singh, 1992) and fairyfly, Gonatocerus ashmeadi (Hymenoptra; Mymaridae) (Leopold and Chen, 2007). However, a study reported more than 80% survival rate for the coccinellid beetle, Harmonia axyridis for up to 150 days at moderately low temperature of 3-6oC (Ruan et al., 2012). So there is great flexibility in coccinellid adults and larvae for tolerating low temperature conditions. After removal from cold storage, larvae showed better feeding potential with consumption of more aphids when compared to normal larvae that were not placed under low temperature conditions. This indicates that when the adult or immature insect stages are subjected to low temperature environment, they tend to reduce their metabolic activity for keeping them alive on the reserves of their body fats and sustain themselves for a substantial length of time under such cold environment. Hereafter, the larval instars that were in cold storage were behaving as if starved for a certain length of time and showed more hunger. This behavior of improved or higher feeding potential of stored larvae has been reported previously (Chapman, 1998). Hence, the feeding potential of C. septempunctata larvae significantly increased after cold storage. Gagné and Coderre (2001) reported higher predatory efficacy in larvae of C. maculata when stored at the same temperature as in the present study i.e., 8oC. Similarly, Ruan et al. (2012) showed that the multicolored Asian ladybug, H. axyridis, when stored under cold conditions, had more eating capacity towards aphids Aphis craccivora Koch than the individuals that were not stored. Such studies indicate that the higher feeding potential in insects after being subjected to low temperature environmental conditions could be due to the maintenance of their metabolism rate to a certain level while utilizing their energy reserves to the maximum extent (Watanabe, 2002).The individuals coming out from cold storage are therefore capable of consuming more pray as they were in a condition of starvation and they have to regain their energy loss through enhanced consumption. Furthermore, the starvation in C. septempunctata has previously been reported to affect their feeding potential (Suleman et al., 2017). In the present study, the larval development was delayed after returning to normal laboratory conditions. Cold storage affects the life cycle of many insects other than coccinellids. The cold storage of green bug aphid parasitoid, Lysiphlebus testaceipes Cresson (Hymenoptra; Braconidae) mummies increased the life cycle 3-4 times. Nevertheless, in current study the development process of stored larvae resumed quickly after taking them out and larvae completed their development up to adult stage. Similar kinds of results were reported for resumption of larval development after removal from cold storage conditions. Such studies only report satisfactory survival rates and development for a short duration of cold storage but as the length of storage is increased, it could become harmful to certain insects. Gagné and Coderre (2001) reported that cold storage for longer period (three weeks) proved fatal for almost 40% of larvae of C. maculata. Furthermore, in the same study, the feeding potential of C. maculata larvae was also affected beyond two weeks of cold storage due to the loss of mobility after a long storage period. Many studies have reported that longer durations of low temperature conditions can either damage the metabolic pathways of body cells or may increase the levels of toxins within the bodies of insects. Also, low temperature exposure for longer duration may cause specific interruptions in the insect body especially neuro-hormones responsible for insect development, which could be dangerous or even life threatening.Chen et al. (2004) also reported that the biological qualities of parasitized Bemisia tabaci pupae on population quality of Encarsia formosa were affected negatively with increase in cold storage duration. Similarly, the egg hatchability of green lacewing Chrysoperla carnea Stephen was lost completely beyond 18 days of cold storage (Sohail et al., 2019). However, in the present study the cold storage was done for maximum two weeks and it is to be regarded as a short term storage hence the survival rate was satisfactory. Longer periods of cold storage for larvae are not considered safe due to their vulnerable state as compared to adults which are hardier. Also 2nd instar larvae used in the present study for cold storage for being bigger in size and physical stronger than 1st instar. Abdel‐Salam and Abdel‐Baky (2000) reported that in C. undecimpunctata the cold storage of 3rd and 4th larval instars was higher and considered safer than early larval instars. The same study showed sharp decline in survival rate after two weeks and there was no survival beyond 30-60 days of cold storage. The present study showed that short term storage of the larvae of C. septempunctata could be done without any loss of their feeding potential or development so the quality of predator remained unaffected. Similar kind of work for many other insects had been reported previously where cold storage technique proved useful without deteriorating the fitness of stored insects. For example, the flight ability of reared codling moth Cydia pomonella Linnaeus remained unaffected after removal from cold storage (Matveev et al., 2017). Moreover, a sturdy reported that pupae of a parasitoid wasp Trichogramma nerudai (Hymenoptera; Trichogrammatidae) could be safely put in cold storage for above than 50 days (Tezze and Botto, 2004). Similarly, a technique of cold storage of non-diapausing eggs of black fly Simulium ornaturm Meigen was developed at 1oC. Another study reported safe storage of a predatory bug insidious flower bug Orius insidiosus for more than 10 days at 8°C (Bueno et al., 2014).In present study without cold storage, the lower doses of 10 and 25 GY prolonged the developmental time as compared to un-irradiated larvae and higher doses of irradiations in conjunction with cold storage again significantly prolonged the developmental time of larvae when returned to the laboratory conditions. Salem et al. (2014) also reported that Gamma irradiations significantly increased the duration of developmental stages (larvae and pupae) in cutworm, Agrotis ipsilon (Hufnagel). In another study, where endoparasitic wasps Glyptapanteles liparidis were evaluated with irradiated and non-irradiated gypsy moth Lymantria dispar larvae for oviposition, it was found that non-irradiated larvae had a shorter time to reach the adult stage as compared to irradiated larvae (Novotny et al., 2003). Both for higher doses with cold storage and lower doses without cold storage extended the larval duration of C. septempunctata. In another study when the parasitoid wasp Habrobracon hebetor was irradiated at the dose of 10 GY, it resulted in prolonged longevity (Genchev et al., 2008). In the same study, when another parasitoid Ventruria canescens was irradiated at lower doses of 4GY and 3 GY, it resulted in increased emergence from the host larvae, while gamma irradiations at the dose of 1 GY and 2 GY significantly stimulated the rate of parasitism (Genchev et al., 2008). The current study also indicated higher rates of predation in the form of increased feeding potential of larvae as a result of irradiations at lower doses.CONCLUSIONThe outcome of the current study shows that storage of 2nd instar C. septempunctata at low temperature of 8oC for a short duration of about 14 days is completely safe and could have broader application in different biocontrol programs. Such flexibility in storage duration can also assist in different mass rearing techniques and commercial uses. The combination of gamma radiation with low temperature cold storage could be a useful tool in developing different biological pest management programs against sucking insect pests. Incidence of periodic occurrence of both the target insect pests with their predatory ladybird beetles in synchrony is an important aspect that could be further strengthened by cold storage techniques. Therefore, short or long term bulk cold storage of useful commercial biocontrol agents and then reactivating them at appropriate time of pest infestation is a simple but an advantageous method in mass rearing programs. Increased feeding capacity of stored larvae is another edge and hence such larvae may prove more beneficial as compared to unstored larvae. Both cold storage and improved feeding of the C. septempuctata larvae can be utilized for implementation of IPM for many sucking insect pests of various crops, fruits and vegetables. Due to some constraints this study could not be continued beyond two weeks but for future directions, higher doses and longer duration periods could further elaborate the understanding and better application of such useful techniques in future IPM programmes on a wider scale. Also, some other predatory coccinellid beetle species can be tested with similar doses and cold storage treatments to see how effective this technique is on other species as well.ACKNOWLEDGMENTS We acknowledge the Sugarcane Research and Development Board for providing a research grant (No. SRDB/P/4/16) to carry out this research work. 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Background and Aims: Dukan diet, a popular diet with high content of protein and carbohydrate and fat restriction has been widely used for weight loss. We aimed to compare the effects of the Dukan diet with traditional low-calorie diet in nutritional, laboratory and vascular parameters in obese subjects. Methods and Results: Obese subjects classes I or II of both genders, aging 19 to 65 years were allocated into two groups: Traditional low-calorie diet (n=17) and Dukan Diet (n=17). Anthropometric, laboratory and vascular evaluations were performed at baseline, 3, 6 and 12 months. Body composition was evaluated by bioelectric impedance and endothelial function by flow-mediated dilation of the brachial artery, at same times. After 12 months, it was verified that Dukan diet was more effective (p<0.05) than traditional diet for: weight loss (-10.6 vs -2.9 kg), body mass index (-3.7 vs -1.1 kg/m2), waist circumference (-11.2 vs -2.1 cm), fat (-5.7 vs -2.0 kg) and lean mass (-4.8 vs 0.8 kg) and basal metabolic rate (-152 vs -28 cal). In Dukan diet group, improvement (p<0.05 vs baseline) was observed in triglyceride levels (172.40 to 111.90 mg/dL) and insulin resistance, based on HOMA-IR index (4.98 to 3.26). The glomerular filtration rate decreased in this group after 3 months (132.50 to 113.80 mL/min) and no changes in flow-mediated dilation were observed throughout the study with both diets. Conclusion: Dukan diet was more effective than traditional diet for weight loss and laboratory parameters and without changes in endothelial function, in the 12-months follow-up of obese subjects. Introduction Low-carbohydrate diets have been one of the most recently used dietary therapies in patients with diabetes and obesity in clinical studies(1). Among them, in addition to carbohydrate restriction, fat restriction and high protein concentration, as in the Diet Dukan, has been widely used by the general population, aiming at weight loss. The Dukan diet is designed to reduce carbohydrate and fat intake in the first phase of the diet, with exclusive intake of protein, followed by another Three phases, with progressive and slow reintroduction of other nutrients such as fiber, carbohydrates and fats. In recent years, there has been increasing interest in the effectiveness of very low carbohydrate diets, called ketogenic diets, in the effectiveness of weight loss in order to combat obesity and cardiovascular disease risk(2). In this diet, ketone bodies are formed and they are used as an alternative energy source in the absence of glucose. Ketogenic diet promotes weight loss reducing appetite, increasing satiety and thermogenesis, due to the high protein consumption(3) affect hormones that control appetite, such as ghrelin and leptin(4) reduces lipogenesis and increases lipolysis(5,6) and gluconeogenesis(7). Replacing carbohydrates by proteins in the diet have been the aim of several studies but with inconsistent results. High protein intake has positive effects on weight loss, acting on satiety, body composition, lipid profile and glucose homeostasis. Furthermore, it increases thermogenesis, energy expenditure(8) and the elevation in the amino acid level in the plasma acts on the satiety center, decreasing appetite, since amino acids also stimulate insulin secretion resulting in decreased or maintained blood glucose levels(9). Few studies have been published with Dukan diet. Freeman et al. were the first to publish an article with the Dukan Diet in 2014, describing adverse effects in one patient undergoing this diet(10). Nouvenne et al. reviewed studies about the influence of popular diets on kidney stone formation risk. In this article, the authors suggest that in the Dukan diet, due to the high consumption of animal protein, urinary calcium can increase and the citrate urinary excretion can decrease, increasing the risk of kidney stone formation(11). In 2015, Wyka et al. evaluated dietary consumption in women adopting the Dukan-diet, based on the menu consumed in each of 4 phases of diet. They observed weight loss of around 15 kg after 8 to 10 weeks of diet and higher intake of proteins, mainly of animal origin, high consumption of potassium, iron and vitamins A, D and B2 and reduced consumption of carbohydrates, vitamin C and folates. They suggest that this diet may be harmful to health if adopted for a long time, developing of kidney and liver disease, osteoporosis and cardiovascular disease(12). Considering that the Dukan Diet is widely disseminated and it is used by the population in general for weight loss and few scientific studies are found in the literature, we propose to evaluate the nutritional, laboratory parameters related to cardiovascular disease, comparing this diet with traditional hypocaloric diet in obese individuals. Methods Study design This study was a clinical trial with nutritional intervention, for one year. Patients were recruited from the Lipids, Atherosclerosis and Vascular Biology Division of the Universidade Federal de São Paulo (UNIFESP). The study conforms to the ethical guidelines and approval was obtained from the ethics committee and it was registered in the Brazilian Registry of Clinical Trials. All participants provided written informed consent and received no monetary incentive. A total of 40 subjects were initially recruited and the participants were followed up clinically by a cardiologist and nutritionist during the 12-month period with monthly visits. Of the 40 participants who started the study, 34 completed the 12-month follow-up, whose data are presented in this study. The inclusion criteria were: both genders, aging 19-65 years old, obesity grade I or II (body mass index between 30 kg/m² and 39.9 kg/m²), stable body weight in the previous 3 months and desire to lose weight. The main exclusion criteria were: patients in primary or secondary prevention of coronary heart disease with low-density lipoprotein cholesterol (LDL-C) levels greater than 190 mg/dL and triglycerides greater than 400 mg/dL; diabetes mellitus; untreated hypothyroidism; psychiatric and hepatic disease; chronic renal failure; cardiac and respiratory insufficiency; systemic infections; use of antidepressants, corticoids, diuretics and diabetes medications; bariatric surgery, cancer and failure to accept the conditions necessary to conduct the research. Two groups were constituted: Traditional low-calorie diet (TD): n=17, 14 females and 3 males, 45±11 years old, 90±11 Kg body weight and body mass index (BMI) 34±2Kg/m2; High protein/Low carbohydrate diet-Dukan Diet (DD): n=17, 10 females and 7 males, 38±11 years old, 95±9 Kg of body weight and BMI 34±2 Kg/m2. The TD group received orientations according to the Food Guideline for the Brazilian Population, with 1 500–1 800 calories/day. They were stimulated to improve healthy eating habits increasing the consumption of natural foods without preservatives, such as vegetables and fruits rich in fiber and antioxidants. Daily consumption of fruits and vegetables at meals was recommended; carry out the fractionation of the meals throughout the day, avoiding prolonged fasting. Hydration and regular physical activity were recommended, according to healthier life habits(13). The DD group followed the high-protein/low-carbohydrate diet as proposed by Dukan Diet, available at https://www.dietadukan.com.br and received an illustrated book about this diet(14). This diet is structured in four phases: two for weight loss (1st and 2nd phases) and two for weight maintenance (3rd and 4th phases): 1st stage - Attack: For 5 consecutive days, it is allowed to consume only proteins with lean meats, eggs, light cheese and milk, 1.5 tablespoons of oat bran per day and light physical activity for 20 minutes. 2nd stage - Cruise: This phase is maintained until the desired weight loss. The vegetables are introduced alternating with the pure protein day (first stage). It is recommended 2 tablespoons of oat bran per day and light physical activity for 30 minutes. 3rd phase - Consolidation: The time of this phase is equivalent to 10 days per kg of lost weight. In this stage carbohydrates and lipids are introduced by a controlled and moderate way, being divided in two parts: in the first part, corresponding to half of the period to be followed, is allowed: 1 fruit, 2 slices of bread (50 g) or 1 spoon of farinaceous per day and 1 gala dinner per week. In the second part, it is allowed 2 fruits, 4 slices of bread (100 g) or 2 spoons of farinaceous per day and 2 gala dinners per week. This phase has one rule: make one day of the week with pure protein (first stage) and it is recommended 2.5 tablespoons of oat bran per day and light physical activity for 35 minutes. 4th phase - Stabilization: In this phase, three rules must to be followed: one day a week it should follow up the pure protein diet, the daily consumption of 3 tablespoons of oat bran and at least 40 minutes of daily walking. From this phase, the participants followed up the low calorie diet. The adherence of the participants was monitored by the interview with the nutritionist and qualitative evaluation of ketone bodies in the urine, using Labtest UriAction 10 reagent strips. At baseline, 3, 6 and 12 months, the following evaluations were performed: nutritional assessment determining anthropometry, blood samples were collected for laboratory tests. Endothelial function was evaluated in fasting and 2-hours post prandial situations. In the periods between the predetermined visits, the participants were followed up by the nutritionist monthly and by telephone contact whenever requested and with medical attention whenever necessary. Nutritional evaluation Nutritional assessment was performed by anthropometric determinations of weight, height, BMI, abdominal circumference and bioelectric impedance (BIA). BIA was carried out using the Biodynamics Model 450 TBW® apparatus, with portable plethysmograph and patients were instructed according to the manufacturer's instruction(15). Laboratory parameters Peripheral blood samples were collected for dosages of total cholesterol and fractions, triglycerides, glucoses, insulin, iron, ferritin, ALT, AST, urea, creatinine, hemoglobin and hematocrit. Biochemical parameters were determined through the automated colorimetric enzymatic method in Cobas Mira® (Roche, Switzerland) and LDL-c was estimated by the Friedewald equation. Serum insulin concentration was determined by immunofluorometry and the insulin resistance calculated by the HOMA-IR – Homeostasis Model Assessment Insulin Resistance, and values ≥ 2.5 values were considered as presence of insulin resistance(16). Glomerular Filtration Rate (GFR) was estimated by the Cockroft-Gault equation adapted to obese patients(17). Endothelial function Endothelial function was assessed by Endothelial-dependent flow-mediated dilation (FMD) of the brachial artery(18), using an ultrasound system (Sonos5500; Hewlett-Packard-Phillips, Palo Alto, CA), equipped with vascular software for two-dimensional imaging, color and spectral Doppler ultrasound modes, internal electrocardiogram monitor and linear-array transducer with a frequency range from 7.5 to 12.0 MHz. FMD evaluation was performed in two stages: fasted at least 6 hours and 2 hours after the consumption of a small meal, according to each diet. These meals were consisted of 374.04 calories, 36g proteins, 16g carbohydrates and 18g lipids in the DD and in TD, it was composed by 361.20 calories, 24g of protein, 41g of carbohydrates and 11g of lipids. Statistical Analysis The variables were expressed as mean and standard deviation. The distribution of the date normality was analyzed by the Kolmogorov-Smirmov (KS) test. When they did not present normal distribution, a logarithm [log(Y)] transformation was performed prior to analysis. The comparison between the variables of two groups was performed using Student's t-test for independent numerical variables and Fisher's exact test for categorical variables. Comparisons between more than two groups were performed by analysis of variance (ANOVA) for repeated measures, followed by the Tukey test, if differences were found. For the sample power calculation, the Statistical Software, Statistica Ultimate Academic, version 12.7, Concurrent Network was used. Values of p ≤ 0.05 were considered for statistical significance and analysis was performed using the software [GraphPadPrism 4.0 (GraphPad Software, San Diego, CA, USA)]. Results Participants’ characteristics At the beginning of the study, the groups were matched for age, gender, weight and BMI. At 3 months, all participants of DD group (100%) were in phase 2; at 6 months, 13 participants (76.4%) were in phase 3 and 4 (23.5%) in phase 2; and at 12 months, all (100%) were already in phase 4. The TD group followed the same recommendation during the 12 months. The qualitative evaluation of the presence of ketone bodies in the urine of the DD group participants, which were still in phase 2, was positive in 94% at 3rd month and 80% at the 6th month. The following adverse effects have been reported during the course of the study: weakness, fatigue, dizziness, lack of concentration, irritability, constipation, ketone breath and social life impairment. These symptoms were of low intensity and transient, especially in the early stages of the DD diet. These adverse effects were not causes for withdrawal from the study. Anthropometry The changes in body weight, BMI, waist abdominal circumference and BMR were more effective in DD than TD group during all follow-up evaluations. The changes after 12 months in relation to baseline of the anthropometric parameters in the DD and DT groups respectively were: Weight loss (-10.6 Kg, p<0.0001 and – 2.9 Kg, p<0.0001), BMI (-3.7 Kg/m2, p<0.0001 and -1.1 Kg/m2, p<0.0001), waist abdominal circumference (-11.2 cm, p<0.0001 and -2.1 cm, p=0.0008) and BMR (-152 cal, p<0.0001 and -28 cal, p=0.0198). After 12 months, the participants of DD group reached the overweight level but the TD group was still within the obesity range. Reductions were observed in both groups, in fat mass (-5.7 Kg, p<0.0001 and -2.0 Kg, p<0.0001), and in lean mass (-4.8 Kg, p<0.0001 and -0.8 Kg, p=0.0196, in DD and DT group, respectively). Laboratory parameters and endothelial function In TD group, there was only hematocrit reduction after 6 months (p=0.0103) and glucose level after 3 months (p=0.0021) compared to baseline. In DD group, laboratory alterations occurred in relation to hemoglobin, hematocrit, triglycerides, insulin, HOMA-IR and GFR. It was observed an improvement in the triglycerides levels (172.40 ± 62.36 mg/dL and 111.90 ± 43.22 mg/dL, p=0.0001) and insulin resistance determined by HOMA-IR at all times of study (4.98 ± 3.03 and 3.26 ± 2.03, p=0.0008) at baseline and 12 months, respectively. GFR was reduced only after 3 months (132.50 ± 31.13 and 113.80 ± 24.25 mL/min, p=0.0063) in the DD group. No differences were observed in endothelial function in the two study groups, in both fasting and postprandial. Discussion This study demonstrated higher weight loss in the Dukan diet group, compared to the traditional low calorie diet. The effect of weight loss in the DD group was persistent and remained until 6th month, but in 12 months it was observed a gain around 3.41 ± 0.21 Kg. The DD is performed in phases, with severe restriction until the 3rd phase and at about the 6th month; carbohydrates and a gala meal are reintroduced, promoting a weight gain. Sacks et al. observed that regardless of the nutritional composition of the diet, obese participants that had a weight loss, after 12 months of treatment, they can gain weight, but with a reduction of approximately 11.4% of the initial weight(19). We observed that participants of TD group also presented significant weight reduction, suggesting the effectiveness of the close follow up with nutritionist and physician. Abdominal circumference is an indirect parameter of fat mass corresponding to visceral fat that is associated with a higher risk for cardiovascular diseases. In our data, we observed a reduction in waist circumference in both groups after 12 months. Moreno et al. comparing ketogenic diet with standard diet in a group of obese patients found an important reduction in abdominal circumference with partial recovery after 24 months(20). Although DEXA Scan is considered the gold standard for body composition determination, BIA is a non-invasive and relatively inexpensive method and widely used(21). A significant reduction in the relative values of body fat was observed at 3 and 6 months in the DD group and only after 3 months in the TD. Increase in percent of lean mass was observed in the DD group at 3 and 6 months, but this increase does not represent a gain of lean mass, since the relative increase is a result of the reduction of body weight, promoting a relative increase in the values of lean mass. The loss of lean mass in the DD group may be due to the low caloric intake of the diet, as Chaston et al. (2007) pointed out that diet with low-calorie diet promote marked weight loss, but there is a decline in lean mass resulting from this process(22) . In our study, in spite of consuming a large amount of protein, this nutrient alone is not enough to promote the maintenance of lean mass and exercise stimulation is still necessary, which did not happen in this study, since the participants were all sedentary. In obese individuals, weight gain after marked loss is common, with reduction in basal metabolic rate(23). Several studies have observed this phenomenon during rapid weight loss(24) and diets with low carbohydrate intake are among the factors that influence metabolic adaptation. Some studies suggest that low amounts of carbohydrate (<45%) decrease the basal metabolic rate during and after weight loss. This type of diet can promote fat mass loss and preservation of lean mass during weight loss, reducing the basal metabolic rate. Reduction in BMR was observed in both groups, but in the DD group, the reduction occurred at all times in relation to baseline whereas in TD group the reduction was greater only after 6 months of intervention. Improvement in insulin resistance and triglycerides were observed only in the DD group. Individuals with insulin resistance have greater difficulty to metabolize carbohydrates, diverting a greater amount of dietary carbohydrates to the liver, where much of it is converted to fat (lipogenesis), rather than being oxidized in energy in the skeletal muscle. For this reason, very low carbohydrate diets applied in obese individuals, in addition to leading to weight loss also improves glycemic and lipid control. The effects of the very prolonged ketogenic diet are still poorly investigated and for this reason this diet should only be used for a limited period (from 3 weeks to a few months) to stimulate fat loss, improve metabolism, and then adjusting a transition to a normal diet(25). No changes in levels of total cholesterol, HDL-c and LDL-c were observed in any group. However, only in the DD group there was a significant reduction in TG level. In general, diets with reduced carbohydrates and high levels of proteins and fats increase LDL-c and TG levels showing beneficial effects of the ketogenic diet on cardiovascular risk factors. Most studies show that reducing carbohydrates can bring significant benefits in reducing total cholesterol, increases in HDL-c and reduction of triglycerides in the blood. HMG-CoA reductase, a key enzyme in the synthesis of endogenous cholesterol is activated by insulin, so that a reduction in blood glucose and hence insulin levels, leads to lower cholesterol synthesis. Thus, a reduction in dietary carbohydrate associated with adequate cholesterol consumption leads to inhibition of cholesterol biosynthesis(26). When insulin is elevated, lipolysis is reduced and lipogenesis is increased, resulting in overproduction of VLDL containing TG, formation of small and dense LDL particles and reduction of HDL. Low concentrations of glucose and insulin also reduce the expression of the carbohydrate-sensitive response element binding protein (ChREBP) transcription factor, and expression of the binding protein of the sterol regulatory element (SREBP-1c), responsible for the synthesis of fatty acids, as well as their incorporation into triglycerides and phospholipids, activating the main lipogenic enzymes, reducing hepatic lipogenesis and VLDL production(27). When we evaluated the GFR, a reduction only in DD group was observed at 3 months of intervention, but still in normal reference levels. Our results did not show significant changes in serum creatinine levels, but GFR decrease in DD group. Carbohydrate-restricted diets have higher amounts of protein may affect glomerular filtration leading to progressive loss of renal function(28). In the study conducted by Brinkworfh et al. (2010), renal function was evaluated in 68 obese individuals without renal dysfunction who consumed two similar hypocaloric diets for one year, one with carbohydrate reduction and another with high carbohydrate content, and observed that creatinine serum levels and the GFR did not change in any of the dietary groups(29). In general, endothelial function improves after weight loss in obese individuals(30). However, associations between changes in endothelial function with anthropometric and biochemical parameters are still controversial(31). We observed that the endothelial function did not present a significant difference in the two study groups, both in fasting and in the 2 hours postprandial. Volek et al. (2009) observed that low-carbohydrate diet improves postprandial vascular function compared to a low-fat diet in overweight individuals with moderate hypertriglyceridemia(32). Low-carbohydrate diets, may improve vascular function in individuals with metabolic adaptations(32) and carbohydrate-restricted diets may induce benefits in endothelial function in the presence of insulin resistance, since impaired insulin action may be related to endothelial dysfunction. In our study, the meal offered for postprandial evaluation was not high in fat, but correspond to the diet proposed in each group. According to Nicholls et al. (2006), a single carbohydrate-restricted meal does not alter endothelial function(33) and this may be the reason we did not observe a change in endothelial function in the DD group in this study. Conclusion Comparing the nutritional and laboratory effects of traditional and hyper-protein diets with carbohydrate reduction, we can conclude that Dukan diet was more effective than traditional diet for weight loss, as well as for laboratory parameters and without changes in endothelial function, in the 12-months follow-up of obese subjects. Conflict of interest No conflict of interest. Acknowledgement Patricia Naomi Sakae had a scholarship from CAPES – Brazil. References Gogebakan O.; Kohl A.; Osterhoff MA.; van Baak MA.; Jebb SA.; Papadaki A.; et al. Effects of weight loss and long-term weight maintenance with diets varying in protein and glycemic index on cardiovascular risk factors: the diet, obesity, and genes (DiOGenes) study: a randomized, controlled trial. Circulation. 2011, 124(25), 2829-2838. Merino J.; Kones R.; Ferre R.; Plana N.; Girona J.; Aragones G.; et al. Low-carbohydrate, high-protein, high-fat diet alters small peripheral artery reactivity in metabolic syndrome patients. Clin Investig Arterioscler. 2014, 26(2), 58-65. Krieger JW.; Sitren HS.; Daniels MJ.; Langkamp-Henken B. Effects of variation in protein and carbohydrate intake on body mass and composition during energy restriction: a meta-regression 1. Am J Clin Nutr. 2006, 83(2), 260-274. Samaha FF.; Iqbal N.; Seshadri P.; Chicano KL.; Daily DA.; McGrory J.; et al. A low-carbohydrate as compared with a low-fat diet in severe obesity. N Engl J Med. 2003, 348(21), 2074-2081. Paoli A.; Rubini A.; Volek JS.; Grimaldi KA. Beyond weight loss: a review of the therapeutic uses of very-low-carbohydrate (ketogenic) diets. Eur J Clin Nutr. 2013, 67(8), 789-796. Nordmann AJ.; Nordmann A.; Briel M.; Keller U.; Yancy WS, Jr.; Brehm BJ.; et al. Effects of low-carbohydrate vs low-fat diets on weight loss and cardiovascular risk factors: a meta-analysis of randomized controlled trials. 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Introducción: La enfermedad renal crónica asociada a la obesidad (ERC-AO) es una enfermedad con aumento en la prevalencia en las últimas décadas. Se caracteriza por un exceso de desequilibrios hormonales adipocíticos (adipoquinas), desregulación del sistema de equilibrio energético y desequilibrios en la homeostasis metabólica. Propósito de la revisión: El objetivo de la revisión es delinear el papel de los diferentes mecanismos fisiopatológicos para el desarrollo de enfermedad renal funcional o anatómica en pacientes con obesidad. Buscamos reportes actualizados en donde se incluye los resultados de mejor supervivencia para los pacientes con ERC-AO. Recientes hallazgos: Actualmente sabemos la ERC-AO tiene un comportamiento pro inflamatorio crónico. La obesidad y sobrepeso se asocian alteraciones hemodinámicas, estructurales e histopatológicas en el riñón, así como alteraciones metabólicas y bioquímicas que predisponen a la enfermedad renal, aun cuando la función renal y las pruebas convencionales sean normales. Conclusiones: Clasificamos a la ERC-AO en Tipo 1: Obesidad y alteraciones funcionales potencialmente reversibles. Tipo 2: Obesidad y alteraciones estructurales histopatológicas potencialmente no reversibles (Incluye la Glomerulopatía asociada a obesidad y glomeruloesclerosis focal y segmentaria). Tipo 3: Obesidad en relacionada con enfermedades crónicas (Diabetes, Hipertensión, Hipertensión pulmonar. Insuficiencia Cardíaca). Tipo 4: Obesidad en el paciente con terapia sustitutiva de la función renal. Recibido: Agosto 03, 2022 Aceptado: Septiembre 30, 2022 Publicado: Septiembre 30, 2022 Editor: Dr. Franklin Mora Bravo. Introducción La obesidad es una enfermedad en crecimiento con un aumento en su prevalencia en las últimas décadas, asociándose a un elevada carga asistencial y económica para los sistemas sanitaros derivado de su relación con enfermedades cardiovasculares, endocrinas, psicológicas, renales entre otras [1, 2]. El incremento en las tasas de obesidad en distintos grupos etarios, desde niños hasta adultos jóvenes conlleva a asumir que en el futuro veremos más enfermedad renal relacionada con la obesidad (ERC-AO) en la población general, con implicaciones relevantes para los sistemas de atención [3]. Por ello el conocimiento y comprensión de esta interacción podría tener implicaciones en la prevención y tratamiento de las enfermedades renales. Dentro de la población general la obesidad se asocia a incremento en el riesgo de diversas condiciones patológicas, como la hipertensión arterial crónica (HTA), enfermedad renal crónica (ERC), artrosis, infecciones, síndrome de apnea hipopnea obstructiva del sueño (SAHOS) y diabetes mellitus (DM) entre otras [3]. No obstante, en el escenario de la ERC, la obesidad juega un rol dual y paralelo en el desarrollo de la enfermedad, tradicionalmente se ha denominado “paradoja de la obesidad”, donde por un lado actúa como un factor de riesgo modificable para el desarrollo de la enfermedad renal crónica (ERC) y por otro se ha asocia de manera consistente con mejores resultados de supervivencia en pacientes con enfermedad renal terminal [1]. Por lo anterior, en las próximas páginas describimos aspectos fisiopatológicos que involucran la obesidad en el desarrollo de la ERC. Definición y epidemiología La obesidad es una condición que se caracteriza por la acumulación anormal o excesiva de tejido adiposo con consecuencias patológicas adversas e incremento del riesgo cardiovascular [4]. Utilizando para su definición y diagnostico un indicador simple como es la relación entre el peso y la talla denominado índice de masa corporal (IMC), se calcula dividiendo el peso de una persona en kilos por el cuadrado de su talla en metros (kg/m2). Un IMC entre 18.5 y 25 kg/m2 es considerado por la Organización Mundial de la Salud (OMS) como peso normal, un IMC entre 25 y 30 kg/m2 como sobrepeso y un IMC > 30 kg/m2, como obesidad [5-7]. Además, la obesidad puede ser clasificada en tres niveles de severidad: clase I (IMC 30.0 – 34.9), clase II (IMC 35.0 – 39.9) y clase III (IMC > 40) [8]. Durante las últimas tres décadas, la prevalencia de adultos con sobrepeso y obesidad (IMC ≥ 25 kg/m2) en todo el mundo ha aumentado sustancialmente, convirtiendo a la obesidad en una epidemia y se prevé que su prevalencia crezca un 40% en la próxima década [6]. Actualmente, el problema de obesidad se ha visto en mayor aumento debido al incremento en la afectación en niños, lo que ocasiona una mayor prevalencia de patologías a edad temprana. En 2016, según las estimaciones de la OMS unos 41 millones de niños menores de cinco años tenían sobrepeso o eran obesos [7]. Esto afectando a todos los países, independiente de su nivel de ingresos [7]. La prevalencia del sobrepeso y la obesidad en niños y adolescentes (de 5 a 19 años) ha aumentado de forma espectacular, del 4% en 1975 a más del 18% en 2016. Este aumento ha sido similar en ambos sexos: un 18% de niñas y un 19% de niños con sobrepeso en 2016. Mientras que en 1975 había menos de un 1% de niños y adolescentes de 5 a 19 años con obesidad, en 2016 eran 124 millones (un 6% de las niñas y un 8% de los niños) [7]. La creciente prevalencia de la obesidad tiene implicaciones para las enfermedades cardiovasculares (ECV) y también para la ERC. Un IMC alto es uno de los factores de riesgo más fuertes para la ERC de nueva aparición [6]. Epidemiología de la enfermedad renal crónica asociada a obesidad (ERC-AO) La enfermedad renal crónica (ERC) es una condición de interés en salud pública, asociada a una elevada morbilidad y mortalidad a nivel mundial. Las guías KDIGO (Kidney Disease: Improving Global Outcomes), definen la ERC como la presencia de alteraciones en la estructura o función renal durante al menos tres meses y con implicaciones para la salud [9, 10]. Los principales elementos clasificatorios para definir la presencia de ERC son la tasa de filtración glomerular (TFG) estimada (G1 a G5) utilizando como umbral definitorio una TFG 60 ml/min/1,73m2 y la tasa de excreción de albúmina en orina (A1 a A3) según el cociente albúmina/creatinina en una muestra aislada de orina sea < 30, 30-300 o > 300 mg/g, respectivamente [9, 10]. Si bien inicialmente existía cierta controversia sobre el uso de la TFG para el diagnóstico de la ERC en fases iniciales, trabajos recientes han puesto en evidencia que tanto una TFG< 60 ml/min/1.73 m2 como un cociente albúmina/creatinina (CAC) ≥ 1.1 mg/mmol (10 mg/g) son predictores independientes del riesgo de mortalidad e insuficiencia renal terminal (IRT) en población general [11, 12]. En consecuencia, debido a estas categorías podemos determinar el pronóstico de cada paciente. Los datos globales sugieren que la prevalencia de la ERC se encuentra entre el 10 y el 16 %, pero la información sobre la prevalencia de la población por categoría de TFG y ACR es escasa [13]. La ERC es una afección asociada a una elevada carga de morbilidad, mortalidad y enfermedad cardiovascular (ECV). A medida que disminuye la función renal, surgen trastornos metabólicos y hemodinámicos que aumentan las tasas de hospitalización, ECV y muerte [4]. El conjunto de factores de riesgo conocidos para la progresión de la ERC es relativamente pequeño, y las terapias y estrategias efectivas para retrasar la progresión de la ERC son limitadas [14]. Por lo cual resulta necesario conocer y entender los diferentes factores de riesgo y su impacto en el daño renal, en aras de lograr minimizar la progresión del mismo, sobre todo en aquellos en los cuales se puede realizar intervenciones activas, evaluables, controlables y con seguimiento continuo como es la obesidad. A la fecha existe suficiente evidencia para asociar la obesidad con el desarrollo y progresión de la enfermedad renal crónica. Los datos granulares sobre la prevalencia de la obesidad en personas con ERC son limitados pero consistentes en todo el espectro de la enfermedad renal. En la Encuesta Nacional de Examen de Salud y Nutrición de 2011–2014, el 44.1 % de los pacientes con ERC en los Estados Unidos también tenían obesidad (21.9 % con obesidad de clase 1 y 11.1 % con clase 2 y obesidad clase 3, habiéndose incrementado el porcentaje global un 5% en los últimos 12 años [15]. La glomeruloesclerosis focal y segmentaria (GEFS) es el tipo de glomerulonefritis que se asocia con mayor frecuencia a la obesidad [16]. La enfermedad glomerular habitualmente asociada a la obesidad se denomina glomerulopatía relacionada con la obesidad (GRO). Esta condición suele presentarse con síndrome nefrótico y pérdida progresiva de la función renal. Con la epidemia mundial de obesidad, se produjo un aumento progresivo de la GRO del 0.2% entre 1986 y 1990 al 2% entre 1996 y 2000, y se ha convertido en un tema emergente en el ámbito de la nefrología [15]. Etiología y patogénesis de la ERC-AO La obesidad se caracteriza por un exceso de desequilibrios hormonales adipocíticos (adipoquinas), desregulación del sistema de equilibrio energético y desequilibrios en la homeostasis metabólica [12]. Hay dos tipos de tejido adiposo presentes en los humanos: tejido adiposo blanco (WAT) y tejido adiposo marrón (BAT) [17-19]. El depósito de grasa ectópica primariamente ocurre en lugares donde no se almacena fisiológicamente, como el hígado, el páncreas, el corazón y el músculo esquelético; secundariamente hay un cambio en la distribución del tejido adiposo visceral con almacenamiento de tejido adiposo en los espacios intraperitoneal y retroperitoneal; luego se presenta la desregulación inflamatoria y de adipoquinas; y por último la resistencia a la insulina [20]. Tejido adiposo blanco (WAT) El tejido adiposo blanco (WAT) se caracteriza por ser un tejido blanco o amarillo con menor vascularización e inervación que el tejido marrón. Las células grasas tienen un tamaño que oscila entre 20 y 200 µm y contienen una única vacuola lipídica (uniloculares). En dicha vacuola se almacenan lípidos para su uso cuando hay demanda energética. De la totalidad de los lípidos que abarca la vacuola lipídica del adipocito blanco, del 90 al 99% son triacilgliceroles. El tejido adiposo blanco genera una gran cantidad de adipocinas y lipocinas. Las adipocinas son péptidos que actúan como hormonas o mensajeros que regulan el metabolismo. El tejido adiposo blanco se localiza en el tejido omental, mesentérico, retroperitoneal, perirrenal, gonadal y pericárdico [19]. Este tejido al igual que el tejido adiposo de otros sitios, está compuesto por una variedad de células que incluyen macrófagos, neutrófilos, células T CD4 y CD8, células B, neutrófilos, mastocitos, células T reguladoras y células T asesinas naturales (NK) [21, 22]. El tejido adiposo es responsable de la secreción de muchas moléculas de señalización, incluidas adipocinas, hormonas, citocinas y factores de crecimiento, como leptina, adiponectina, resistina, factor de necrosis tumoral-α (TNF-α), interleucina 6 (IL-6), monocito, proteína quimioatrayente-1 (MCP-1), factor de crecimiento transformante-β (TGF-β) y angiotensina II [23]. Tejido adiposo marrón o pardo (BAT) La coloración marrón del tejido adiposo se debe a que está más vascularizado y tiene un alto contenido de mitocondrias, las células grasas que componen el tejido adiposo pardo son multiloculares o tienen varias vacuolas lipídicas. Estas células tienen forma poligonal y miden de 15 a 50 µm. A diferencia del tejido adiposo blanco, el tejido marrón no tiene la función de almacenar energía, sino que la disipa a través de la termogénesis. Para lograr la regulación de la temperatura corporal, el tejido adiposo pardo se localiza en sitios superficiales y profundos [18]. Clasificación de la ERC-AO Se ha establecido que la obesidad es una enfermedad con un comportamiento pro inflamatorio crónico con múltiples comorbilidades asociadas [19]. El tejido adiposo como se describió previamente funciona como un órgano con actividad endocrina y esta infiltrado por diferentes poblaciones celulares que incluyen macrófagos y otras células con actividad inmune como linfocitos T, B y células dendríticas [19]. La mayor parte de la grasa corporal total, se considera como un sistema de órganos endocrinos, la perturbación de este tejido tiene como resultado una respuesta patológica al balance calórico positivo en individuos susceptibles que directa e indirectamente contribuye a la enfermedad cardiovascular y metabólica, se tiene conocimiento de tres principales mecanismos de disfunción del tejido adiposo “adiposopatía” [20]. Estos mecanismos incluyen alteraciones hemodinámicas, metabólicas e inflamatorias, lo que es la base de la clasificación de la ERC-AO propuesta en esta revisión (Tabla 1). ERC-AO tipo 1 La obesidad produce un daño renal de forma directa a través de alteraciones hemodinámicas, inflamatorias, y desregulación de factores de crecimiento y adipocitoquinas, además de aumento de leptina y disminución de adiponectina, aun cuando la función renal y las pruebas convencionales sean normales [16]. La obesidad desencadena una serie de eventos, que incluyen resistencia a la insulina, intolerancia a la glucosa, hiperlipidemia, aterosclerosis e hipertensión, todos los cuales están asociados con un mayor riesgo cardiovascular [4, 16] (Figura 1). La obesidad conduce a un incremento en la reabsorción tubular de sodio, alterando la natriuresis y provocando una expansión de volumen extracelular debido a la activación del sistema nervioso simpático (SNS) y el sistema renina-angiotensina-aldosterona (SRAA)(16). El aumento en la reabsorción tubular de sodio y la consiguiente expansión de volumen extracelular es un evento central en el desarrollo de HTA en la obesidad [4, 16]. Algunos estudios sugieren que se produce un aumento de la reabsorción de sodio en algunos segmentos además del túbulo proximal, posiblemente en el asa de Henle. Además, hay un aumento del flujo sanguíneo renal, la tasa de filtración glomerular (TFG) y la fracción de filtración [16]. La hiperfiltración glomerular, asociada con el aumento de la presión arterial y otras alteraciones metabólicas como la resistencia a la insulina y la DM, finalmente resultan en daño renal y disminución del filtrado glomerular [16]. Por otro lado, la activación del SNS también contribuye a la hipertensión relacionada con la obesidad [4]. Hay evidencia de que la denervación renal reduce la retención de sodio y la hipertensión en la obesidad, lo que sugiere que la activación del SNS inducida por la obesidad aumenta la presión arterial principalmente debido al estímulo de retención de sodio, más que a la vasoconstricción [16]. Los mecanismos que conducen a la activación del SNS en la obesidad aún no se conocen por completo, pero se han propuesto varios factores como desencadenantes de este estímulo, entre ellos la hiperinsulinemia, la hiperleptinemia, el aumento de los niveles de ácidos grasos, los niveles de angiotensina II y las alteraciones del reflejo barorreceptor. El aumento de los niveles de leptina está asociado a la activación del SNS y su efecto sobre el aumento de los niveles de presión arterial incluye también la inhibición de la síntesis de óxido nítrico (potente vasodilatador) [16, 24, 25].También se ha descrito un aumento de la producción de endotelina-1 en sujetos obesos, lo que contribuye aún más a la elevación de los niveles de presión arterial y, en consecuencia, a la disfunción renal. Estudios recientes han demostrado que la endotelina-1 está aumentada en pacientes con hipertensión intradiálisis, lo que sugiere que esta sustancia juega un papel clave en la génesis de la hipertensión en pacientes con ERC y posiblemente esté asociada con la hipertensión en pacientes obesos [16, 25]. Por lo anterior, las alteraciones hemodinámicas en los pacientes con obesidad conllevan a progresión de la ERC e incremento del riesgo cardiovascular derivado del desarrollo de enfermedades adicionales como la HTA, potencialmente estos cambios son reversibles con el control de la obesidad. ERC-AO Tipo 2 Mantener el estado de obesidad más allá de los efectos renales funcionales produce cambios estructurales irreversibles a nivel glomerular [25]. El estudio de pacientes con ERC y obesidad ha permitido identificar la presencia de enfermedad glomerular asociada a la obesidad, denominada glomerulopatía relacionada con la obesidad (GRO). En esta condición la hipertrofia glomerular parece ser la lesión inicial que estimula el borramiento de los podocitos y desencadena la respuesta inflamatoria local [25, 26]. Es relevante mencionar que las señales profibrogénicas inducen la formación de depósitos en la matriz extracelular de las nefronas, que conduce al engrosamiento de la membrana basal glomeruloesclerosis y fibrosis tubulointersticial [26]. Dentro del curso patogénico de la enfermedad la expansión de la superficie glomerular conduce a que los podocitos sean incapaces de cubrirla, esto lleva a disfunción y borramiento de los mismos, generando ruptura de la barrera de filtración glomerular con sobrecarga de las células restantes, lo que finalmente conduce a hiperfiltración y proteinuria [25, 26]. No obstante, no todos los pacientes con obesidad o IMC aumentado desarrollan ERC, lo cual sugiere que el incremento del IMC por sí solo no genera aumento en la incidencia o progresión de la ERC, ameritando alteraciones metabólicas adicionales. En los siguientes apartados se describen algunas de estas vías fisiopatológicas comunes a todos los tipos de ERC-AO. ERC-AO Tipo 3 La obesidad produce daño renal de forma secundaria ya que aumenta el riesgo de diabetes mellitus, hipertensión y daño cardiovascular, estas patologías causan enfermedad renal diabética (ERD), nefroangioesclerosis, y glomerulopatía asociada a hipertensión pulmonar e insuficiencia cardíaca. La mortalidad no solo se ve afectada por la presencia de la obesidad sino por la presencia de diabetes tipo 2, hipertensión arterial, hipertensión pulmonar e insuficiencia cardíaca. Los peores resultados en supervivencia lo padecen los pacientes con falla cardíaca, obesidad e insuficiencia renal. ERC-AO Tipo 4 En pacientes en hemodiálisis los niveles más elevados de adiponectina se asocian paradójicamente con tres veces más riesgo de muerte [24]. La obesidad se asocia a niveles muy bajos adiponectina por lo que la obesidad en el grupo poblacional que se realiza hemodiálisis es un fuerte factor protector con mejores resultados de supervivencia a 3 años comparados con pacientes con índice de masa corporal normal o baja. Mecanismos fisiopatológicos comunes en la ERC-AO Lipotoxicidad derivada del tejido adiposo En pacientes obesos el exceso de energía conduce a un microambiente sometido a estrés crónico, lo cual resulta en hipertrofia del tejido adiposo hasta que los adipocitos alcanzan su límite de crecimiento [25]. En ese momento, el exceso de especies toxicas lipídicas se acumula ectópicamente en diferentes órganos, induciendo un efecto nocivo conocido como lipotoxicidad; especialmente a nivel renal [27]. La lipotoxicidad se asocia a cambios estructurales y funcionales de las células mesangiales, podocitos y células tubulares proximales [28]. En los podocitos, esto interferiría con la vía de la insulina, crítica para la supervivencia y el mantenimiento de la estructura de los podocitos, lo que conduciría a la apoptosis de los podocitos e induciría una respuesta hipertrófica compensatoria en los podocitos restantes [25]. En el riñón, los depósitos de lípidos ectópicos contribuyen tanto a la inflamación local como al estrés oxidativo [27]. En modelos de ERD, la dislipidemia puede favorecer la acumulación de lípidos ectópicos e intermediarios lipídicos, no solo en el riñón sino también en tejidos extrarrenales como hígado, páncreas y corazón [27]. La acumulación de lípidos en el parénquima renal, genera daño en varias poblaciones celulares, incluídos podocitos, células epiteliales tubulares proximales y el tejido tubulointersticial a través de distintos mecanismos descritos en las siguientes apartados, pudiendo general compromiso a largo plazo de la función renal [27]. El tejido adiposo es una fuente importante de producción de diferentes factores proteicos activos, conocidos como adipocitocinas, las cuales participan en diferentes procesos metabólicos. Alteraciones en la secreción y señalización de moléculas derivadas del tejido adiposo durante la obesidad en gran medida puede mediar en la patogenia de los trastornos metabólicos [25]. A continuaciones se describe el rol de las adipocinas en la patogenia de la ERC y obesidad. Adiponectina La adiponectina es una proteína secretada principalmente por los adipocitos WAT, las principales funciones biológicas de la adiponectina incluyen una mayor biosíntesis de ácidos grasos y la inhibición de la gluconeogénesis hepática [17]. Es probablemente la adipocina secretada más abundantemente, forma alrededor del 0.05 % de las proteínas séricas y mide de 3 a 30 mg/ml en humanos, para su activación utiliza dos isoformas del receptor (AdipoR1 y AdipoR2) son receptores de siete transmembranas y tienen una homología del 66.7 % en su estructura [17]. Sin embargo, AdipoR1 y AdipoR2 son estructural y funcionalmente distintos de los receptores acoplados a proteína G porque su terminal N es intracelular, mientras que el terminal C es extracelular [29, 30]. La señalización de adiponectina se basa principalmente en interacciones de tipo receptor-ligando, en las que la adiponectina se une a sus receptores afines e inicia la activación de varias cascadas de señalización intracelular a través de las vías AMPK, mTOR, NF-κB, STAT3 y JNK [17]. La adiponectina inicia la activación de la señalización de AMPK mediada por la proteína adaptadora APPL1, que se une al dominio intracelular de AdipoR. Eso produce la activación de la biosíntesis de moléculas, otras proteínas reguladoras e importantes factores de transcripción. AMPK es un regulador que participa principalmente en la proliferación celular [17]. Hay dos tipos de macrófagos, M1 participan en la estimulación de los factores pro inflamatorios e induce la resistencia a la insulina y M2 bloquean una respuesta inflamatoria y promueve el metabolismo oxidativo; En los macrófagos, la adiponectina promueve la diferenciación celular de monocitos a macrófagos M2 y suprime su diferenciación a macrófagos M1, lo que muestra efectos pro inflamatorios y antiinflamatorios. Además, también activa los factores antiinflamatorios IL-10 pero reduce las citoquinas pro inflamatorias como IFN-γ, IL-6 y TNF-α en los macrófagos humanos [17]. Los pacientes con ERC muestran niveles elevados de proteína C reactiva (PCR), IL-6 y TNF-α y tienen una activación aberrante de receptor tipo toll (TLR)-4 [25]; en un estudio realizado en el año 2005 en 29 pacientes con ERC no diabéticos en etapa 5 y 14 controles sanos, se identificó que los pacientes con ERC tenían una expresión elevada del gen y la proteína TLR4, la estimulación de TLR-4 in vitro indujo la activación de TNF-α y NF-κB en células C2C12. Esto sugiere indirectamente que la activación de TLR-4 podría promover la inflamación muscular de los pacientes con ERC [31]. Los niveles de adiponectina se consideran predictivos de ERC, dado que estos se encuentran aumentados en pacientes con etapa pre diálisis [17, 29, 32]. Adicionalmente, en un estudio prospectivo realizado en el año 2008 en pacientes con ERC primaria no diabética identificó niveles elevados de adiponectina como un predictor novedoso de progresión de la ERC en hombres [33]. En estudios realizados en animales (ratones) muestran que la deficiencia de adiponectina se relaciona con varias alteraciones histológicas, incluida la fusión segmentaria procesos podocitarios, albuminuria y aumento del estrés oxidativo en los riñones [34]. Por otro lado, en pacientes obesos la producción de adiponectina se encuentra disminuida por lo que se cree que puede generar una función protectora sobre el riñón [29]. No obstante, paradójicamente, algunos estudios muestran que los pacientes con ERC y enfermedad renal crónica en diálisis (ERCT) tienen altos niveles de adipocinas, las explicaciones a esta situación son controversiales, se ha planteado podrían corresponder a un mecanismo compensatorio, otras consideraciones sugieren una disminución de la sensibilidad a la adiponectina o una reducción en el aclaramiento de la misma [35]. Leptina En pacientes con ERC independiente de la presencia de obesidad o no, se asocian a niveles elevados de leptina sérica. La leptina es una proteína de 167 aminoácidos, con una masa molecular de aproximadamente 16 kDa que está codificada por el gen LEP [23] secretada principalmente por los adipocitos, es una adipocina pleiotrópica. La leptina circulante llega a los órganos diana, donde se une a receptores específicos (conocidos como ObR, LR o LEPR), se conocen cinco isoformas del receptor de leptina en humanos (ObRa, ObRb, ObRc, ObRd y ObRe), de estas solo la isoforma ObRb (isoforma larga) se considera un receptor completamente activo, ya que es capaz de transducir completamente una señal de activación en la célula. Esta isoforma se encuentra altamente expresada en el sistema nervioso central (SNC), especialmente en el hipotálamo, donde participa en la regulación de la actividad secretora de este órgano. Los efectos de la leptina están mediados por cinco vías principales de señalización. Estas vías incluyen las vías de señalización JAK-STAT, PI3K, MAPK, AMPK y mTOR [23]. Por esta razón la principal función fisiológica de la leptina es transmitir información al hipotálamo sobre la cantidad de energía almacenada, como la masa de tejido adiposo, e influir en el gasto de energía al reducir el apetito. Regula el metabolismo energético, tiene efecto sobre la ingesta de alimentos, procesos de coagulación, angiogénesis, funciones relacionadas con la insulina y la remodelación vascular, además funciona como un pro inflamatorio molecular [36]. La leptina tiene efectos sobre el apetito y se ha demostrado que la hiperleptinemia contribuye a la hipertensión asociada a la obesidad por sobre activación del sistema nervioso simpático [37]. En cuanto al curso de la ERC, la leptina puede modular diferentes vías de señalización en el riñón, debido a que las células endoteliales glomerulares y mesangiales expresan abundantes receptores de leptina [25]. La leptina inducirá un incremento en la expresión de genes profibróticos, como TGF-β1 y citocinas pro inflamatorias [25]. El aumento en la expresión de TGF-β1, también contribuirá al desarrollarlo de la fibrosis renal, al unirse a receptores específicos a nivel renal, estimulara la expresión de factores profibróticos en un ciclo de retroalimentación positiva. Además, TGF-β1 es un potente iniciador de proliferación de células mesangiales renales [25]. Debido a su tamaño relativamente pequeño, la leptina atraviesa libremente el filtro glomerular de los riñones y luego se reabsorbe en la parte proximal de los túbulos contorneados [23]. Por lo que el estado elevado de leptina puede indicar una función renal deficiente [36]. Promueve la inflamación y trastorno de los lípidos, que contribuyen al riesgo de ERC [36]; se considera como “toxina urémica”, estando implicada tanto en la progresión de la enfermedad renal a través de efectos pro-hipertensivos y profibróticos, como en el desarrollo de complicaciones relacionadas con la ERC (inflamación crónica, pérdida de proteínas) [38]. Como se mencionó previamente, la leptina estimula la proliferación de células endoteliales glomerulares renales y aumenta la expresión de TGF-β1, un mediador clave de la hidrogénesis en estas células, el aumento de los niveles de leptina también contribuye al aumento de la expresión de colágeno tipo IV en el riñón, induce la proliferación de células mesangiales glomerulares mediante la activación de la vía PI3K, la hipertrofia de las células mesangiales aumenta la cantidad de proteína filtrada y albúmina que llega a las células del túbulo proximal y, como resultado, activa las vías inflamatorias y la fibrosis [23]. Puede presentarse un aumento en la síntesis del receptor TGFβ-1 secretado por las células endoteliales, este actúa de manera parácrina sobre el mesangio uniéndose a su receptor y activando la síntesis de proteínas de la matriz extracelular (ECM), incluyendo colágeno, fibronectina, tenazina y proteoglicanos; consiguientemente, un aumento en el nivel de TGFβ-1 conduce a la acumulación de MEC y, en consecuencia, a fibrosis glomerular y glomeruloesclerosis. En los podocitos, la leptina contribuye a la disminución de la expresión de las proteínas responsables de la filtración glomerular adecuada, incluidas la podocina, la nefrina, la podoplanina y la podocalixina. En las células del túbulo contorneado proximal (PTC), la leptina reduce la actividad metabólica de las células al activar la vía de señalización de mTOR [23]. Por otro lado, la leptina inhibe el apetito y aumenta el gasto de energía conduciendo a anorexia y desnutrición en pacientes con ERC, particularmente en casos de hemodiálisis de mantenimiento [36]. Por ende, una elevación de la leptina no solo nos indicaría daño renal, sino que además nos indica mayor progresión de complicaciones secundarias [39]. La obesidad aumenta la carga sobre los riñones y es un factor de riesgo de lesión renal, además de contribuir en los trastornos metabólicos asociados. Por lo que, teniendo en cuenta los efectos inhibitorios de la leptina sobre la obesidad, se puede considerar que puede proteger contra la lesión renal [39, 40]. Un estudio experimental publicado en el año 2017 demostró que la leptina disminuyó la ingesta calórica y los niveles de glucosa en ratas diabéticas [41], ese mismo año se publicó un estudio retrospectivo donde demostraron que la metreleptina, una metionil leptina humana recombinante, reduce el peso corporal y la dosis diaria de insulina en la diabetes mellitus tipo 1 [42]. La metreleptina ejerce efectos terapéuticos en la lipodistrofia [43], lo que indica que es probable que la leptina se aplique en los trastornos metabólicos [36]. Otras adipocinas Las principales adipocinas corresponden a la adiponectina y leptina como se ha descrito previamente. Además de estas, se distinguen la actividad de la visfatina y resistina, las cuales muestran propiedades pro-inflamatorias y efectos aterogénicos [25]. La visfatina estimula la expresión de TGF-β1, inhibidor del activador del plasminógeno-1 (PAI-1) y colágeno tipo I, los cuales han demostrado un rol importante como agentes profibróticos. Por otro lado, la resistina estimula la producción de las moléculas de adhesión como la molécula de adhesión intracelular 1 (ICAM-1) y la proteína de adhesión celular vascular 1 (VCAM-1) y promueve la activación del sistema renal simpático. Los niveles de estas adipocinas están marcadamente elevados en la obesidad y ERC correlacionándose con parámetros proinflamatorios y disminución de la tasa de filtración glomerular (TFG) [25, 37]. Durante el curso de la obesidad se presenta una sobre activación del SRAA, el tejido adiposo también estaría involucrado en la producción o estimulación de algunos de los componentes del RAS. Por ello la sobre estimulación del SRAA en obesos, asociado a la glomerulomegalia y desregulación de la reabsorción de sodio/glucosa, generalmente conlleva a hipertensión glomerular e hiperfiltración [25]. Otra adipocina a considerar, es la actividad de la adipocina proinflamatoria lipocalina 2 (LCN2), también denominada lipocalina asociada con la gelatinasa de neutrófilo (NGAL), estudiada como biomarcador funcional tanto para la enfermedad renal aguda como ERC(25). LCN2 es conocido por su papel en la respuesta inmune innata a través de su unión a sideróforos derivados de una infección bacteriana. Sin embargo, LCN2 no es secretada únicamente por neutrófilos sino también por otros tejidos como hígado, pulmones y de interés para este artículo, a nivel renal [25]. Se han informado niveles elevados de LCN2 en suero y orina en la lesión renal, debido a una expresión aumentada de LCN2 en el túbulo distal renal y una reabsorción alterada en el túbulo proximal [44]. El tejido adiposo, también puede producir factores angiogénicos como el factor de crecimiento del endotelio vascular (VEGF). Este elemento podría inducir la formación de novo de capilares glomerulares en gran parte defectuosos dentro del riñón, lo que contribuye a la hipertrofia glomerular característica de GRO [25] (Figura 2). Conclusiones La obesidad y el sobrepeso se asocian a alteraciones hemodinámicas, estructurales e histopatológicas en el riñón, así como alteraciones metabólicas y bioquímicas que predisponen a la enfermedad renal, aun cuando la función renal y las pruebas convencionales sean normales. Por lo tanto, los efectos renales de la obesidad son estructurales y funcionales. Hay varios mecanismos actualmente descritos que involucran a la obesidad como generador de alteraciones renales. Teniendo en cuenta las bases fisiopatológicas, proponemos una clasificación de la ERC-AO basadas en 4 tipos. Abreviaturas ERC: enfermedad renal crónica. ERC-AO: enfermedad renal crónica-asociada a enfermedad. VEGF: factor de crecimiento del endotelio vascular. OR: Odds ratio. Información suplementaria Materiales suplementarios no han sido declarados. Agradecimientos No aplica. Contribuciones de los autores Jorge Rico-Fontalvo: Conceptualización, Curación de datos, Análisis formal, Adquisición de fondos, Investigación, Metodología, Administración de proyecto, Recursos, Software, Escritura – borrador original. Rodrigo Daza-Arnedo: Conceptualización, Supervisión, Validación, Visualización, Redacción: revisión y edición. Tomás Rodríguez-Yanez: Metodología, validación, supervisión, redacción: Revisión y edición. Washington Osorio: Conceptualización, Supervisión, Validación, Visualización, Redacción: revisión y edición. Beatriz Suarez-Romero: Conceptualización, Supervisión, Validación, Visualización, Redacción: revisión y edición. Oscar Soto: Conceptualización, Supervisión, Validación, Visualización, Redacción: revisión y edición. Juan Montejo-Hernandez: Conceptualización, Supervisión, Validación, Visualización, Redacción: revisión y edición. María Cardona-Blanco: Conceptualización, Supervisión, Validación, Visualización, Redacción: revisión y edición. Juan Camilo Gutiérrez: Conceptualización, Supervisión, Validación, Visualización, Redacción: revisión y edición. Todos los autores leyeron y aprobaron la versión final del manuscrito. Financiamiento Los autores proveyeron los gastos de la investigación. Disponibilidad de datos o materiales Los conjuntos de datos generados y analizados durante el estudio actual no están disponibles públicamente debido a la confidencialidad de los participantes, pero están disponibles a través del autor correspondiente a pedido académico razonable. Declaraciones Aprobación del comité de ética y consentimiento para participar No aplica para revisiones narrativas. Consentimiento para publicación No aplica cuando no se publican imágenes o fotografías del examen físico o radiografías/tomografías/resonancias de pacientes. Conflictos de interés Los autores reportan no tener conflictos de interés. 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Although the sodium hypochlorite (NaOCl) solution has been part of the endodontic arsenal for more than one century, current investigations have been unable to determine which NaOCl volume and concentration or which time of application are able to dissolve organic matter without weakening the dental structure during the phase of biomechanical preparation of the root canal. Thus, the objective of the present study was to conduct a systematic literature review with no restriction of publication year or language in order to resolve these questions. The search strategy included the following databases: PubMed, LILACS, Web of Science and ClinicalTrials.gov, and only in vivo human clinical trials were included in the final review. After the removal of duplicates, the systematic literature review yielded 3,717 articles. Of these, 3,685 were excluded after applying the exclusion criteria (ex vivo studies, animal studies, cell-culture studies, narrative review, and studies with no available full texts). A total of 32 full-text articles were assessed for eligibility. After evaluating the full text, all articles were excluded for different reasons. No studies fulfilled our inclusion criteria. This review was unable to answer what time of irrigation, concentration or volume of NaOCl solution can be of maximum effectiveness in endodontic treatment without producing significant changes in the mechanical properties of dentin. Thus, future human clinical studies are needed in order to resolve these questions.Descriptors: Endodontics; Sodium Hypochlorite; Review.ReferênciasSiqueira JF Jr, Guimarães-Pinto T, Rôças IN. Effects of chemomechanical preparation with 2.5% sodium hypochlorite and intracanal medication with calcium hydroxide on cultivable bacteria in infected root canals. 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Abstract Background Early childhood caries has been designated as a serious public health problem. The traditional restoration method is very challenging, especially in uncooperative patients. Non-invasive therapy, like remineralization agents, which have been developed to reverse the demineralization progress at the early stage of caries, may be a better choice. This study aimed to evaluate the remineralization efficacy of different concentrations of 45S5 bioactive glass (BAG) on artifical carious lesions of deciduous enamel. Methods 65 caries-like enamel lesions of the deciduous teeth were assigned to 5 groups (n = 13) and transported to a 14 days pH-cycling: Group A: 2%BAG, Group B: 4%BAG, Group C: 6%BAG, Group D: 8%BAG, and Group E: deionized water (DDW, negative control). 8 sound (Group F) and 8 demineralized teeth (Group G) were prepared for contrast. The recovery power of mechanical property was evaluated by Vickers hardness test through the recovery of enamel microhardness (%REMH). Surface morphology, mass fraction of Ca and P ions, and Ca/P atomic ratio were analyzed by scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (EDX). Moreover, Fourier transform infrared spectroscopy equipped with attenuated total reflectance was used to identify the chemical structure of newly formed compounds. Results % REMH were (42.65 ± 1.35), (52.59 ± 2.96), (57.40 ± 1.72), (52.91 ± 2.55), (12.46 ± 2.81) in 2%BAG, 4%BAG, 6%BAG, 8%BAG, and DDW groups respectively. Micro-spherical particles were deposited in all BAG groups and 6% BAG showed the densest and most uniform surface. EDX analysis identified significantly higher Ca(wt%) and P(wt%) in four BAG groups than in the demineralized group (p < 0.005), while 6% BAG showed the highest mineral gain efficacy. The infrared spectrum demonstrated that newly mineralized crystals were consisted of type-B hydroxycarbonate apatite. Conclusion BAG possessed a promising remineralization effect on artificial lesions in deciduous enamel by recovering enamel surface mechanical property, morphology and chemical elements. Among them, 6% BAG performed the greatest overall efficacy. Acting as a new caries-arresting biomaterial, 45S5 BAG has the potential to facilitate the adaptation of better carious prevention strategies in children.

This paper presents basic theoretical knowledge of critical thinking. It describes characteristics of critical thinking, which are used for the formation of critical thinking indicators. These indicators are needed for assessing students’ critical thinking levels and for designing lessons to develop critical thinking competence for students. The paper articulates arguments for highlighting the necessity of critical thinking education for students in Vietnam. The paper contributes to knowledge base of critical thinking education and supports further studies on critical thinking in order to enhance the effectiveness of education in Vietnam. Key words Critical thinking, competence, education, students References Arend, B. (2009). Encouraging critical thinking in online threaded discussions. The Journal of Educators Online, 6(1), doi: 10.1.1.412.1694Bacon, F. (1605). The Advancement of Learning. Edited by Joseph Devey, M.A. (New York: P.F. Collier and Son, 1901).Bailin, S. (2002). 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As reforçadas propriedades intrínsecas dos materiais cerâmicos, associados à adesão resinosa fundamentam a desenvoltura de técnicas restauradoras mais simplificadas, conservadoras e sem a necessidade de preparo dental prévio seja em dentição anterior ou posterior. Assim sendo, o trabalho tem como objetivo apresentar dois casos clínicos os quais abordaram a confecção de restaurações delgadas em dissilicato de lítio cimentadas sobre diferentes áreas dos arcos dentários, enfatizando o mesmo protocolo clínico, ou seja, ausência de preparos dentais e consequentemente a cimentação resinosa adesiva das restaurações. Caso Clínico 1: laminados cerâmicos nos elementos 13, 12, 11, 21, 22 e 23; Caso Clínico 2: facetas delgadas sobre os elementos 34, 35, 36 e 37. Ambos os casos clínicos com 1 ano de acompanhamento após a cimentação das restaurações. Conclui-se que houve a recuperação da estética e da função em ambos os casos apresentados e a confiabilidade e o sucesso em longo prazo do protocolo clínico descrito estão diretamente relacionados ao substrato dentário, aos fatores de adesividade dos materiais e as propriedades intrínsecas das cerâmicas.Descritores: Cerâmica; Estética Dentária; Facetas Dentárias; Cimentos de Resina.ReferênciasMurdoch-Kinch CA, McLean ME. Minimally invasive dentistry. J Am Dent Assoc. 2003;134(1):87-95.Ericson D. The concept of minimally invasive dentistry. Dent Update. 2007;34(1):9-10,12-4,17-8.Beier US, Kapferer I, Burtscher D, Dumfahrt H. Clinical performance of porcelain laminate veneers for up to 20 years. Int J Prosthodont. 2012;25(1):79-85.D'Arcangelo C, Vadini M, D'Amario M, Chiavaroli Z, De Angelis F. Protocol for a new concept of no-prep ultrathin ceramic veneers. J Esthet Restor Dent. 2018;30(3):173-79.Vadini M, D'Amario M, De Angelis F, Falco A, D'Arcangelo C. 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O uso de medicamentos fitoterápicos vem crescendo mundialmente entre os programas preventivos e curativos, e tem estimulado a avaliação dos extratos de plantas para o uso na odontologia como controle do biofilme dental e outras desordens bucais. As plantas do gênero Lavandula, pertencem à família Lamiaceae, e têm sido utilizadas através dos anos para uma variedade de propósitos cosméticos e terapêuticos. Sua utilização na odontologia é, na grande maioria dos estudos, devido ao seu potencial ansiolítico. Entretanto, a Lavandula angustifólia demonstra outros potencias farmacológicos, como sua atividade antimicrobiana, antifúngica, anti-inflamatória e antinociceptiva. O presente estudo teve como objetivo integrar os conhecimentos já existentes sobre os aspectos das propriedades farmacológicas da Lavandula angustifolia Miller e sua aplicação na Odontologia. Trata-se de uma revisão bibliográfica no qual foi realizada uma seleção de artigos científicos a partir das bases de dados: Lilacs, MEDLINE, BVS e Scielo, além de monografias que atenderam aos requisitos do tema abordado, no período 2008 a 2018 com exceção de artigos clássicos que se apresentaram imprescindíveis ao presente estudo. Obteve-se um total de 1.532 artigos. Foram selecionados 38 artigos como amostra, que apresentaram a temática elencada para a pesquisa e que foram divididos por sessões: aspectos botânicos da planta; aspectos bioquímicos da planta; potencial antimicrobano; potencial anti-inflamatório; potencial ansiolítico e; potencial antinociceptivo. Pode-se concluir que a Lavandula angustifóliaMiller apresenta-se como uma boa alternativa para utilização na odontologia. Entretanto, a falta de trabalhos que abordem sua utilidade na odontologia revela a necessidade de se intensificar as pesquisas sobre o assunto.Descritores: Plantas Medicinais; Odontologia; Lavandula.ReferênciasFrancisco KSF. Fitoterapia: uma opção para o tratamento odontológico. Rev Saúde. 2010;4(1):18-24.Silveira SM, Cunha Júnior A, Scheuermann GN, Secchi FL, Silvani V, Marisete K et al . Composição química e atividade antibacteriana dos óleos essenciais de Cymbopogon winterianus (citronela), Eucalyptus paniculata (eucalipto) e Lavandula angustifolia (lavanda). Rev Inst Adolfo Lutz. 2012;71(3):462-70.Koulivand PH, Ghadiri MK, Gorji A. Lavender and the nervous system. Evid Based Complement Alternat Med. 2013;2013:681304.Verma RS, Rahman LU, Chanotiya CS, Verma RK, Chauhan A, Yadav A et al. Essential oil composition of Lavandula angustifolia Mill. cultivated in the mid hills of Uttarakhand, India. J Serb Chem Soc. 2010;75(3):343-48.Biasi LA, Deschamps C. 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Malignant hyperthermia (MH) is a clinical response happened to patient who is sensitive with inhaled anesthesia drug that could cause suddently death. Many previous studies showed that malignant hyperthermia strongly related to genetic background of patients including RYR1, CACNA1S or STAC3 gene polymorphisms. With the development of high technology such as next generation sequencing, scientists found that 37 to 86 percents of MH cases had RYR1 mutations and approximately 1 percent of those had CACNA1S mutations. Gene analysis testing was recommended to apply for patient with MH medical history or MH patient’s family relations. Keywords Malignant hyperthermia, inhaled anesthesia, RYR1, CACNA1S, STAC3. References [1] G. Torri, Inhalation anesthetics: a review, Minerva Anestesiologica 76 (2010) 215–228. [2] N. Kassiri, S. Ardehali, F. Rashidi, S. Hashemian, Inhalational anesthetics agents: The pharmacokinetic, pharmacodynamics, and their effects on human body, Biomed. Biotechnol. Res. J. BBRJ 2 (2018) 173. https://doi.org/10.4103/bbrj.bbrj_6618.[3] H. Rosenberg, N. Sambuughin, S. Riazi, R. Dirksen, Malignant Hyperthermia Susceptibility, in: M.P. Adam, H.H. Ardinger, R.A. Pagon, S.E. Wallace, L.J. Bean, K. Stephens, A. Amemiya (Eds.), GeneReviews, University of Washington, Seattle, Seattle (WA), 19932020. http://www.ncbi.nlm.nih.gov/books/NBK1146/ (accessed February 2, 2020).[4] H. Rosenberg, N. Pollock, A. Schiemann, T. Bulger, K. Stowell, Malignant hyperthermia: a review, Orphanet J. Rare Dis 10 (2015) 93. https://doi.org/10.1186/s13023-015-0310-1.[5] D. Carpenter, C. Ringrose, V. Leo, A. Morris, R.L. Robinson, P.J. Halsall, P.M. Hopkins, M.-A. Shaw, The role of CACNA1S in predisposition to malignant hyperthermia, BMC Med. Genet 10 (2009) 104. https://doi.org/10.1186/1471-2350-10-104.[6] S. Riazi, N. Kraeva, P.M. Hopkins, Updated guide for the management of malignant hyperthermia, Can. J. Anaesth. J. Can. Anesth 65 (2018) 709–721. https://doi.org/10.1007/s12630-018-1108-0.[7] S. Riazi, N. Kraeva, P.M. Hopkins, Malignant Hyperthermia in the Post-Genomics Era: New Perspectives on an Old Concept, Anesthesiology 128 (2018) 168–180. https://doi.org/10.1097/ALN.0000000000001878.[8] [D.M. Miller, C. Daly, E.M. Aboelsaod, L. Gardner, S.J. Hobson, K. Riasat, S. Shepherd, R.L. Robinson, J.G. Bilmen, P.K. Gupta, M.-A. Shaw, P.M. Hopkins, Genetic epidemiology of malignant hyperthermia in the UK, BJA Br. J. Anaesth 121 (2018) 944–952. https://doi.org/10.1016/j.bja.2018.06.028.[9] T.A. Beam, E.F. Loudermilk, D.F. Kisor, Pharmacogenetics and pathophysiology of CACNA1S mutations in malignant hyperthermia, Physiol. Genomics 49 (2017) 81–87. https://doi.org/10.1152/physiolgenomics.00126.2016.[10] I.T. Zaharieva, A. Sarkozy, P. Munot, A. Manzur, G. O’Grady, J. Rendu, E. Malfatti, H. Amthor, L. Servais, J.A. Urtizberea, O.A. Neto, E. Zanoteli, S. Donkervoort, J. Taylor, J. Dixon, G. Poke, A.R. Foley, C. Holmes, G. Williams, M. Holder, S. Yum, L. Medne, S. Quijano-Roy, N.B. Romero, J. Fauré, L. Feng, L. Bastaki, M.R. Davis, R. Phadke, C.A. Sewry, C.G. Bönnemann, H. Jungbluth, C. Bachmann, S. Treves, F. Muntoni, STAC3 variants cause a congenital myopathy with distinctive dysmorphic features and malignant hyperthermia susceptibility, Hum. Mutat 39 (2018) 1980–1994. https://doi.org/10.1002/humu.23635.[11] A.F. Dulhunty, The voltage-activation of contraction in skeletal muscle, Prog. Biophys. Mol. Biol 57 (1992) 181–223. https://doi.org/10.1016/0079-6107(92)90024-Z.[12] C. Franzini-Armstrong, A.O. Jorgensen, Structure and Development of E-C Coupling Units in Skeletal Muscle, Annu. Rev. Physiol 56 (1994) 509–534. https://doi.org/10.1146/annurev.ph.56.030194.002453.[13] D.H. MacLennan, M. Abu-Abed, C. Kang, Structure-function relationships in Ca(2+) cycling proteins, J. Mol. Cell. Cardiol 34 (2002) 897–918. https://doi.org/10.1006/jmcc.2002.2031.[14] H. Rosenberg, M. Davis, D. James, N. Pollock, K. Stowell, Malignant hyperthermia, Orphanet J. Rare Dis 2 (2007) 21. https://doi.org/10.1186/1750-1172-2-21.[15] S.M. Karan, F. Crowl, S.M. Muldoon, Malignant hyperthermia masked by capnographic monitoring, Anesth. Analg 78 (1994) 590–592. https://doi.org/10.1213/00000539-199403000-00029.[16] M.G. Larach, G.A. Gronert, G.C. Allen, B.W. Brandom, E.B. Lehman, Clinical presentation, treatment, and complications of malignant hyperthermia in North America from 1987 to 2006, Anesth. Analg 110 (2010) 498–507. https://doi.org/10.1213/ANE.0b013e3181c6b9b2.[17] M.G. Larach, A.R. Localio, G.C. Allen, M.A. Denborough, F.R. Ellis, G.A. Gronert, R.F. Kaplan, S.M. Muldoon, T.E. Nelson, H. Ording, H. Rosenberg, B.E. Waud, D.J. Wedel, A Clinical Grading Scale to Predict Malignant Hyperthermia Susceptibility, Anesthesiology 80 (1994) 771–779. https://doi.org/10.1097/00000542-199404000-00008.[18] D. Schneiderbanger, S. Johannsen, N. Roewer, F. Schuster, Management of malignant hyperthermia: diagnosis and treatment, Ther. Clin. Risk Manag 10 (2014) 355–362. https://doi.org/10.2147/TCRM.S47632.[19] R. Robinson, D. Carpenter, M.-A. Shaw, J. Halsall, P. Hopkins, Mutations in RYR1 in malignant hyperthermia and central core disease, Hum. Mutat 27 (2006) 977–989. https://doi.org/10.1002/humu.20356.[20] M.L. Alvarellos, R.M. Krauss, R.A. Wilke, R.B. Altman, T.E. Klein, PharmGKB summary: very important pharmacogene information for RYR1, Pharmacogenet. Genomics 26 (2016) 138–144. https://doi.org/10.1097/FPC.0000000000000198.[21] A. Merritt, P. Booms, M.-A. Shaw, D.M. Miller, C. Daly, J.G. Bilmen, K.M. Stowell, P.D. Allen, D.S. Steele, P.M. Hopkins, Assessing the pathogenicity of RYR1 variants in malignant hyperthermia, BJA Br. J. Anaesth 118 (2017) 533–543. https://doi.org/10.1093/bja/aex042.[22] P.M. Hopkins, H. Rüffert, M.M. Snoeck, T. Girard, K.P.E. Glahn, F.R. Ellis, C.R. Müller, A. Urwyler, European Malignant Hyperthermia Group, European Malignant Hyperthermia Group guidelines for investigation of malignant hyperthermia susceptibility, Br. J. Anaesth 115 (2015) 531–539. https://doi.org/10.1093/bja/aev225.[23] N.T. Thuy, L.N. Thanh, N.T.T. Mau, N.H. Hoang, N.T.K. Lien, D.D. Long, N.T. Bình, D.A. Tien, N.C. Huu, N.T. Hieu, P.T.H. Nhung, V.T. Thom, Whole exome sequencing revealed a pathogenic variant in a gene related to malignant hyperthermia in a Vietnamese cardiac surgical patient: A case report, Ann. Med. Surg 48 (2019) 88–90. https://doi.org/10.1016/j.amsu.2019.10.030.[24] B. Neuhuber, U. Gerster, F. Döring, H. Glossmann, T. Tanabe, B.E. Flucher, Association of calcium channel α1S and β1a subunits is required for the targeting of β1a but not of α1S into skeletal muscle triads, Proc. Natl. Acad. Sci. U. S. A 95 (1998) 5015–5020. https://doi.org/10.1073/pnas.95.9.5015.[25] M. Whirl-Carrillo, E.M. McDonagh, J.M. Hebert, L. Gong, K. Sangkuhl, C.F. Thorn, R.B. Altman, T.E. Klein, Pharmacogenomics Knowledge for Personalized Medicine, Clin. Pharmacol. Ther 92 (2012) 414–417. https://doi.org/10.1038/clpt.2012.96.[26] N. Monnier, V. Procaccio, P. Stieglitz, J. Lunardi, Malignant-hyperthermia susceptibility is associated with a mutation of the alpha 1-subunit of the human dihydropyridine-sensitive L-type voltage-dependent calcium-channel receptor in skeletal muscle, Am. J. Hum. Genet 60 (1997) 1316–1325 . https://doi.org/10.1086/515454.[27] S.L. Stewart, K. Hogan, H. Rosenberg, J.E. Fletcher, Identification of the Arg1086His mutation in the alpha subunit of the voltage-dependent calcium channel (CACNA1S) in a North American family with malignant hyperthermia, Clin. Genet 59 (2001) 178–184. https://doi.org/10.1034/j.1399 0004.2001.590306.x.[28] P.J. Toppin, T.T. Chandy, A. Ghanekar, N. Kraeva, W.S. Beattie, S. Riazi, A report of fulminant malignant hyperthermia in a patient with a novel mutation of the CACNA1S gene, Can. J. Anaesth. J. Can. Anesth 57 (2010) 689–693. https://doi.org/10.1007/s12630-010-9314-4.[29] E.J. Horstick, J.W. Linsley, J.J. Dowling, M.A. Hauser, K.K. McDonald, A. Ashley-Koch, L. Saint-Amant, A. Satish, W.W. Cui, W. Zhou, S.M. Sprague, D.S. Stamm, C.M. Powell, M.C. Speer, C. Franzini-Armstrong, H. Hirata, J.Y. Kuwada, Stac3 is a component of the excitation-contraction coupling machinery and mutated in Native American myopathy, Nat. Commun 4 (2013) 1952. https://doi.org/10.1038/ncomms2952.[30] D.S. Stamm, A.S. Aylsworth, J.M. Stajich, S.G. Kahler, L.B. Thorne, M.C. Speer, C.M. Powell, Native American myopathy: Congenital myopathy with cleft palate, skeletal anomalies, and susceptibility to malignant hyperthermia, Am. J. Med. Genet. A 146A (2008) 1832–1841. https://doi.org/10.1002/ajmg.a.32370.[31] A. Polster, B.R. Nelson, S. Papadopoulos, E.N. Olson, K.G. Beam, Stac proteins associate with the critical domain for excitation–contraction coupling in the II–III loop of CaV1.1, J. Gen. 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Despite an increasing number of studies focusing on leadership at Vietnamese small and medium sized enteprises (SMEs), there is a lack of empirical research on collective leadership and development of effective top management team (TMT). The purpose of this paper is to review the literature with a view of characteristics forming an effective TMT. Taking the team effectiveness theory, upper echelons theory and behavioral theory of the firm, theoretical characteristics of effective TMT was created, and then an empirical research was conducted on a sample of 141 SMEs. The common characteristics of an effective TMT are identified, including shared vision, optimal team size, role clarity, age diversity, functional background diversity, regular communication, solidarity and collective decision making. The study makes several contributions to the existing literature on effective TMT development at SMEs. Keywords Collective leadership, top management team, effective top management team, SMEs References [1] G.S. Becker, A theoretical and empirical analysis, with special reference to education University of Chicago Press, Chicago, 3rd ed, 1993.[2] R.S. Peterson, D.B. Smith, P.V. Martorana, P.D. Owens, The impact of chief executive officer personality on top management team dynamics: One mechanism by which leadership affects organizational performance, Journal of Applied Psychology. 88 (2003) 795-808.[3] G. Yukl, Leadership and organizational learning: An evaluative essay, Leadership Quarterly. 20 (2009) 49-53.[4] R.M. Cyert, J.M. March, A Behavioral Theory of the Firm, CA, Prentice-Hall, Englewood Cliffs, 1963.[5] D.C. Hambrick, P.A. Mason, Upper echelons: The organization as a reflection of its top managers, Academy of Management Review. 9 (1984) 193-106.[6] C.L. Pearce, The future of leadership development: The importance of identity, multi-level approaches, self-leadership, physical fitness, shared leadership, networking, creativity, emotions, spirituality and on-boarding processes, Human Resource Management Review. 17 (2007) 355-359.[7] R. Silzer, A.H. Church, Identifying and assessing high-potential talent: Current organizational practices, Jossey-Bass, San Francisco, 2010.[8] Lê Quân, Nguyễn Quốc Khánh, Đánh giá năng lực giám đốc điều hành doanh nghiệp nhỏ Việt Nam qua mô hình ASK, Tạp chí Khoa học ĐHQGHN Chuyên san Kinh tế và Kinh doanh. 28 (2012) 29-35.[9] T. Porter-O’Grady, A different age for leadership, part 1: new context, new content, Journal of Nursing Administration. 33 (2003) 105-110.[10] P.G. Northouse, Leadership, Thousand Oaks, CA: Sage, 6th ed, 2013.[11] C.L. Pearce, J.A. Conger, Shared leadership: Reframing the hows and whys of leadership, Thousand Oaks, CA: Sage, 2003, pp. 1-18.[12] J. Hauschildt, E. Kirchmann, Teamwork for innovation - the “troika” of promotors, R&D Management. 31 (2001) số trang đầu và cuối. [13] A. Mackey, The effect of CEOs on firm performance, Strategic Management Journal, 29 (2008) 1357-1367.[14] J. O’Toole, J. Galbraith, E.E. Lawler, When two (or more) heads are better than one: The promise and pitfalls of shared leadership, California Management Review. 44 (2002) 65-83.[15] G.A. Yukl, Leadership in organizations, Upper Saddle River, NJ: Pearson/Prentice Hall, 6th ed, 2006.[16] S. Finkelstein, D.C. Hambrick, Cannella A. A.Jr, Strategic leadership: Theory and research on executives, top management teams, and boards, Oxford University Press, New York, 2009.[17] M. Jensen, W. Meckling, Theory of the firm: managerial behavior, agency costs and ownership structure, Journal of Financial Economics, Amsterdam. 3 (1976) 305-360. [18] B.D. Baysinger, H.N. Butler, Corporate Governance and the Board of Directors: Performance Effects in Board Composition, Journal of Law, Economics and Organization. 1 1985) 101-124.[19] J.A. Pearce, S.A. Zahra, The relative power of CEOs and boards of directors, Strategic Management Journal. 12 (1991) 135-5.[20] Lê Quân, Lãnh đạo doanh nghiệp Việt Nam, NXB Đại học Quốc gia Hà Nội, Hà Nội, 2015.[21] C.A. Bowers, J.A. Pharmer, E. Salas, When member homogeneity is needed in work teams - A metaanalysis, Small Group Research. 31 (2000) 305-327.[22] J. Andersen, Leadership, personality and effectiveness, The Journal of Socio-Economics. 35 (2006) 1078-1091.[23] D.C. Hambrick, P.A. Mason, Upper echelons: The organization as a reflection of its top managers, Academy of Management Review. 9 (1984) 193-106. [24] P.F. Drucker, The Effective Executive, HarperCollins, New York, 1967.[25] S.J. Zaccaro, A.L. Rittman, M.A. Marks, Team leadership, Leadership Quarterly. 12 (2001) 451-483.[26] M. Knockaert, E.S. Bjornali, T. Erikson, Joining forces: Top management team and board chair characteristics as antecedents of board service involvement, Journal of Business Venturing. 30 (2015) 420-435.[27] S.W.J. Kozlowski, B.S. Bell, Work groups and teams in organizations, Handbook of psychology: Industrial and organizational psychology. 12 (2003) 333-375.[28] S. Finkelstein, D.C. Hambrick, Cannella A. A.Jr. Strategic leadership: Theory and research on executives, top management teams, and boards, Oxford University Press, New York, 2009.[29] M. Jensen, W. Meckling, Theory of the firm: managerial behavior, agency costs and ownership structure, Journal of Financial Economics, Amsterdam. 3 (1976) 305-360.[30] J. Haleblian, S. Finkelstein, Top management team size, CEO dominance, and firm performance - The moderating roles of environmental turbulence and discretion, Academy of Management Journal. 36 (1993) 844-8633.[31] N.K. Alexander, K. Victoria, Board size and composition: The main tradeoffs, Corporate Board journal. 2(2006) 48-5.[32] E. Sundstrom, K.P. De Meuse, D. Futrell, Work Teams: Applications and Effectiveness, American Psychologist, vol. 45, no. 2, 120-133 (Firth-Cozens, 1998).[33] Tihanyi, L., A.E. Ellstrand and C.M. Daily. (2000), Composition of the top management team and firm international diversification, Journal of Management. 26 (1990) 1157-1177.[34] J.B. Shaw, E. Barrett-Power, The effects of diversity on small work group processes and Performance, Human Relations. 51 (1998) 1307-1325.[35] K.A. Bantel, S.E. Jackson, Top Management and innovations in Banking: Does the composition of Top Management make a difference?, Strategic Management Journal. 10 (1989) 107-124.[36] M.J. Gelfand, D.P. Bhawuk, L. Nishii, D. Bechtold, Individualism and collectivism, In R. J. House, P. J. Hanges, M. Javidan, P.W. Dorfman, and V. 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IntroductionAs the teaching staff working in a university postgraduate program—the Graduate Certificate of Creative Industries (Creative Practice) at Central Queensland University, Australia—an ongoing concern has been to ensure our students engage with the digital course content (delivered via the Moodle learning management system). This is an issue shared across the sector (La Pointe and Reisetter; Dargusch et al.) and, in our case, specifically in the area of students understanding how this online course content and tasks could benefit them in a program that is based around individual projects. As such, we are invested in enhancing student engagement both within the framework of this individual program and at an institution level. Like many institutions which now offer degrees which are either partially or fully online, the program in question offers a blended learning environment, with internal students also expected to engage with online materials (Rovai and Jordan; Colis and Moonen). The program was developed in 2011, first offered in 2012, and conducted two and sometimes three terms a year since then.Within the first year of delivery, low levels of student participation in online learning were identified as problematic. This issue was addressed using strategies that made use of characteristic strengths among our creative industries students, by developing and linking a peer-to-peer mentoring approach to our blended learning course design. Our challenge in this (as project facilitators and as teachers) has been to devise strategies to shift the students from reluctant to engaged online content users. A key strategy has evolved around introducing peer-mentoring as an intrinsic behaviour in the courses in the program. While not using a full case study approach, we do offer this singular instance for consideration as “much can be learned from a particular case” (Merriam 51). The below is based on our own observations, together with formal and informal student feedback gathered since 2012.Mentors and MentoringThe term mentor can have different meanings depending on the context in which the phrase is used. Ambrosetti and Dekkers note that “it is evident from the literature that there is no single definition for mentoring” (42). Drawing on an array of literature from a number of disciplines to qualify the definition of the term mentoring, Ambrosetti and Dekkers have identified a series of theorists whose definitions demonstrate the wide-ranging interpretation of what this act might be. Interestingly, they found that, even within the relatively narrow context of pre-service teacher research, words used to identify the term mentor varied from relatively collegial descriptors for the established teacher such as supporter, friend, collaborator, role model, and protector, to more formalised roles including trainer, teacher, assessor, and evaluator. The role to be played by a mentor—and how it is described—can also vary according to parameters around, and the purpose of, the mentoring relationship. That is, even though “mentoring, as described in literature, generally involves supporting and providing feedback to the mentee without judgment or criteria” (43), the dynamics of the mentor-mentee relationship may influence the perception and the nature of these roles. For example, the mentoring relationship between a teacher and pre-service teacher may be perceived as hierarchical whereby knowledge and feedback is “passed down” from mentor to mentee, that is, from a more authoritative, experienced figure to a less knowledgeable recipient. As such, this configuration implies a power imbalance between the roles.The relationships involved in peer-to-peer mentoring can be similarly defined. In fact, Colvin and Ashman describe the act of peer-mentoring as “a more experienced student helping a less experienced student improve overall academic performance”, and a relationship that “provides advice, support, and knowledge to the mentee” (122). Colvin and Ashman’s research also suggests that “if mentors and mentees do not have a clear sense of their roles and responsibilities, mentors will find it difficult to maintain any sort of self‐efficacy” (122)—a view that is held by others researchers in this field (see Hall et al.; Reid; Storrs, Putsche and Taylor). However, this collective view of peer-to-peer mentorship was not what we aimed to foster. Instead, we wanted our courses and program to both exhibit and inculcate practices and processes which we felt are more in line with our understanding of the creative industries, including a more organic, voluntary and non-hierarchical approach to peer-to-peer mentorship. This could use Ambrosetti and Dekker’s less hierarchical descriptors of supporter, friend, and collaborator listed above.Student CohortThe student cohort in this program regularly includes on-campus and distance education students in approximately equal ratios, with those studying by distance often geographically very widely dispersed across Australia, and sometimes internationally. The students in this program come from a diverse spectrum of creative industries’ art forms, including creative writing, digital media, film, music, and visual arts. Most enter the program with advanced skills, undergraduate or equivalent qualifications and/or considerable professional experience in their individual areas of creative practice and are seeking to add a postgraduate-level of understanding and scholarly extension to this practice (Kroll and Brien; Webb and Brien). Students also utilise a wide range of learning styles and approaches when developing and completing the creative works and research-informed reflective reports which comprise their assessment. All the students in the program’s courses utilise, and contribute to, a single online Moodle site each term. Some also wish to progress to research higher degree study in creative practice-led research projects (Barrett and Bolt) after completing the program.Applying Peer-to-Peer Mentoring in a Project-Based ProgramThe student cohort in this program is diverse, both geographically and in terms of the area of individual creative industries’ specialisation and the actual project that each student is working on. This diversity was a significant factor in the complexity of the challenge of how to make the course online site and its contents and tasks (required and optional) relevant and engaging for all students. We attempted to achieve this, in part, by always focusing on content and tasks directly related to the course learning outcomes and assessment tasks, so that their usefulness and authenticity in terms of the student learning journey was, we hoped, obvious to students. While this is a common practice in line with foundational conceptions of effective learning and teaching in higher education, we also proposed that we might be able to insure that course content was accessed and engaged with, and tasks completed, by linking the content and tasks in Moodle to the action of mentoring. In this, students were encouraged to discuss their projects in the online discussion forum throughout the term. This began with students offering brief descriptions of their projects as they worked through the project development stage, to reports on progress including challenges and problems as well as achievements. Staff input to these discussions offered guidance—both through example and (at times) gentle direction—on how students could also give collegial advice to other students on their projects. This was in terms of student knowledge and experience gained from previous work plus that learned during the program. In this, students reported on their own activities and how learning gained could potentially be used in other professional fields, as for example: “I specifically enjoyed the black out activity and found the online videos exceptional, inspiring and innovating. I really enjoyed this activity and it was something that I can take away and use within the classroom when educating” (‘Student 1’, week 8, Term 1 2015). Students also gave advice for others to follow: “I understand that this may not have been the original intended goal of Free Writing—but it is something I would highly recommend … students to try and see if it works for you” (‘Student 2’, week 5, Term 1 2015). As each term progressed, and trust built up—a key aspect of online collaboration (Holton) as well as a fruitful mentoring relationship (Allen and Poteet)—joint problem solving also began to take place in these discussions.As most of the students never interact face-to-face during the term, the relative impersonality of the online discussions in Moodle, although certainly not anonymous, seemed to provide a safe platform for peer-to-peer mentoring, even when this was offered by those who were also interacting in class as well. As facilitators of this process, we also sought to model best-practice interaction in this communication and ensure that any posts were responded to in an encouraging and timely manner (Aragon). As a result, the traffic within these forums generally increased each week so that, by the end of the term, every student (both external and internal) had contributed significantly to online discussions—even those who appeared to be more reluctant participants in the beginning weeks of the term. Strategies to Facilitate Peer-to-Peer MentoringSeeking to facilitate this process, we identified discrete points within the term’s course delivery at which we would encourage a greater level of engagement with the online resources and, through this, also encourage more discussion in the online discussion forum. One of the strategies we employed was to introduce specific interactions as compulsory components of the course but, at the same time, always ensuring that these mandated interactions related directly to assessment items. For example, a key assessment task requires students to write reflectively about their creative work and processes. We duly included information and examples of reflective writing as resources online. In order to further develop this skill for both internal and external students, we adopted an active and iterative learning approach to this task by asking students to write reflectively, each week, about the online resources provided to them. In asking students to do this, we reiterated that, at the end of term, a core part of the assessment item was that each student would be asked to describe, analyse and reflect on how they used these resources to facilitate their creative practice. At the end of the term, therefore, each student could collate his or her weekly responses, and use these as part of this assessment task. However, before this final reflection needed to be completed, these reflective musings were already being refined and extended as a result of the commentaries offered by other students responding to these weekly reflections. In this, these commenting students were, in fact, playing the role of peer-to-peer mentors, assisting each other to enhance their abilities in reflective thinking and writing.It should be stated that neither formal mentoring roles nor expectations of the process or its outcomes were pre-determined, defined or outlined to students by the teaching staff or communicated directly to them in any way (such as via the course materials). Instead, internal and distance students were encouraged to communicate with each other and offer guidance, help and support to each other (but which was never described as peer-to-peer mentorship) via their use of the Moodle learning managements system as both a group communication tool and a collaborative learning resource (Dixon, Dixon and Axmann). It is common for creative practitioners to collect data in the form of objects, resources, tools, and memories in order to progress their work and this habit has been termed that of the “bowerbird” (Brady). Knowing that it likely that many of our students are already proficient bowerbirds with many resources in their personal collections, we also facilitated a peer-to-peer mentoring activity in the form of an online competition. This competition asked students to post their favourite interactive resource onto the Moodle site, accompanied by a commentary explaining why and how it could be used. Many students engaged with these peer-posted resources and then, in turn, posted reflections on their usefulness, or not, for their own personal practice and learning. This, in turn, engendered more resources to be posted, shared, and discussed in terms of project problem-solving and, thus, became another ongoing activity that encouraged students to act as increasingly valued peer-mentors to each other.The Practical Application of Peer-to-Peer MentoringEach term, it is a course requirement that the student cohort, both internal and external, combine to create a group outcome—an exhibition of their creative work (Sturm, Beckton and Brien). For some students, the work exhibited is completed; for others, particularly part-time students, the work shown is frequently still in progress. Given that the work in the student exhibition regularly includes music and creative writing as well as visual art, this activity forces students to engage with their peers in ways that most of them have not previously encountered. This interaction includes communication across the internal and distance members of the cohort to determine what work will be included in the exhibition, and how work will be sent for display by external students, as well as liaising in relation to range of related considerations including: curatorial (what the exhibition will be named, and how work is to be displayed), cataloguing (how the works, and their contributors, are to be described), and the overall design of the catalogue and invitation (Sturm, Beckton and Brien). Students make these decisions, as a group, with guidance from staff mainly being offered in terms of practical information (such as what days and times the exhibition space can be accessed) and any limitations due to on-site health and safety considerations and other university-wide regulations.Student feedback has been very positive in relation to this aspect of the course (Sturm, Beckton and Brien), and its collective nature is often remarked on in both formal and informal feedback. We are also finding that some prospective students are applying to the program with a knowledge of this group exhibition and some information about how it is achieved. After graduation, students have reported that this experience of peer-to-peer working across the spectrum of creative industries’ art forms has given them a confidence that they were able to apply in real work situations and has, moreover been a factor that directly led to relevant employment. One student offered in unsolicited feedback: “It was a brilliant course that I gained a lot from. One year on, I have since released another single and work as an artist manager, independently running campaigns for other artists. The course also helped make me more employable as well, and I now work … as a casual admin and projects officer” (Student 3, 2015).Issues Arising from Peer-to-Peer MentoringAn intrinsic aspect of facilitating and encouraging this peer-to-peer mentoring was to allow a degree of latitude in relation to student online communication. The week-to-week reflection on the online resources was, for instance, the only mandated activity. Other participation was modeled and encouraged, but left to students as to how often and when they participated, as well as the length of their posts. In each term, we have found student involvement in discussions increased throughout the term, and tended to exceed our expectations in both quantity and quality of posts.We have also found that the level of intimate detail offered, and intimacy developed, in the communications was far greater than we had initially anticipated, and that there were occasions when students raised personal issues. Initially, we were apprehensive about this, particularly when one student discussed past mental health challenges. At the time, we discussed that the creative arts – whether in terms of its creation or appreciation – are highly personal practices (Sternberg), and that the tone taken by many of the creative individuals, theorists, and researchers whose materials we use as resources was often personally revealing (see, for example, Brien and Brady). By not interfering, other than ensuring that the tone students used with each other was always respectful and focused on the professional aspects of what was being discussed, we observed that this personal revelation translated into high levels of engagement in the discussions, and indeed, encouraged peer support and understanding. Thus, in terms of the student who revealed information about past health issues and who at one stage had considered withdrawing from the course, this student later related to staff—in an unsolicited communication—that these discussions led to him feeling well supported. This student has, moreover, continued to work on related creative practice projects after completing the program and, indeed, is now considering continuing onto Masters level studies.ConclusionIn relation to much of the literature of mentoring, this experience of student interaction with others through an online discussion board appears to offer a point of difference. While that literature reports on other examples of peer-to-peer mentoring, most of these follow the seemingly more usual vertical mentoring model (that is, one which is hierarchical), rather than what developed organically in our case as a more horizontal mode. This is, moreover, a mode which has many synergies with the community of practice and collaborative problem solving models which are central to the creative industries (Brien and Bruns).Collings, Swanson, and Watkins have reported on the positive impact of peer mentoring on student wellbeing, integration, and retention. In terms of effects and student outcomes, although we have not yet collected data on these aspects of this activity, our observations together with informal and University-solicited feedback suggests that this peer-to-peer mentoring was useful (in terms of their project work) and affirming and confidence-building (personally and professionally) for students who are both mentors and mentees. These peer-to-peer mentoring activities assisted in developing, and was encouraged by, an atmosphere in which students felt it was appropriate and safe to both offer support and critique of each others’ work and ideas, as well as encouragement when students felt discouraged or creatively blocked. Students, indeed, reported in class and online that this input assisted them in moving through their projects and, as program staff, we saw that that this online space created a place where collaborative problem-solving could be engaged in as the need arose—rather than in a more forced manner. As teachers, we also found these students became our post-graduate colleagues in the way more usually experienced in the doctoral supervisor-student relationship (Dibble and Loon).The above reports on a responsive learning and teaching strategy that grew out of our understanding of our students’ needs that was, moreover, in line with our institution’s imperatives. 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