Journal articles on the topic '333.78/309273 b'

Collagen, gelatine and total chromium recovery was achieved from the sequential upflow anaerobic sludge blanket digestion (UASB) reactor sludge and concentrate of forward osmosis (FO) treating raw leather industry wastewater. It is aimed to reduce the cost of raw materials in leather industry process by reusing the collagen, gelatine and total chromium from the leather industry process during tanning. The recoveries of collagen and gelatine, total chromium was 78%, 80% and 27% respectively from the waste sludge in UASB process while the recoveries of these chemicals were 95%, 96% and 99% in the concentrate of FO. The maximum 85% CODtotal, 94% CODdissolved, 89% total-N, 88% total-P, 87% color, 87% total chrome (Cr+3 + Cr+6), 86% TOC, 87% SO3-2, 90% percentage of exchangeable sodium, 87% sodium adsorption rate (SAR), 84% sodium carbonate residue (RSC), 92% Cl-1, 92% SO4-2, 93% total salt, 88% B+3, 94% NO3-, 94% NH4+, 99% fecal coliform, 94% TSS, 93% EC25x106 removal efficiencies were measured to output anaerobic UABS reactor in leather industry wastewater. The maximum 99% CODtotal, 99% CODdissolved, 99% total-N, 99% total-P, 99% color, 99% total chrome (Cr+3 + Cr+6), 99% TOC, 99% SO3-2, 99% percentage of exchangeable sodium, 99% sodium adsorption rate (SAR), 99% sodium carbonate residue (RSC), 99% Cl-1, 99% SO4-2, 99% total salt, 99% B+3, 99% NO3-, 99% NH4+, 99% fecal coliform, 99% TSS, 99% EC25x106 removal efficiencies were observed to output aerobic FO reactor in leather industry wastewater. The costs of anaerobic UASB and aerobic FO processes are 0.57 euro and 0.145 euro, respectively, to treat 1 m3 leather industry wastewater. The cost of the consecutive total system is 0.715 euro for the treatment of 1m3 leather industry wastewater. The methane gas obtained in the UABS reactor is electrical energy and the electrical energy income was calculated as 1.9 euro from 1 m3 anaerobic waste sludge. The income to be obtained from the recovery of total chromium, collagen and gelatine from UABS and FO process is 2.75 Euro/l. As a result, the total cost decreases from 1.55 Euro/l to 0.715 Euro/l after aerobic FO process. The cost of these consecutive treatment processes is reduced by the production of methane gas and is increased the recovery of economic chemicals.

SUMMARY Objective To compare pulsed-field gel electrophoresis (PFGE) patterns of Staphylococcus aureus from chickens in England, Belgium, Bulgaria, Argentina, and Japan, to assess the value of PFGE for discriminating strains, and to compare results obtained by PFGE with those obtained by biotyping and phage typing. Sample Population 78 S aureus isolates from diseased and healthy chickens. Procedure Chromosomal DNA of S aureus was digested with restriction endonuclease Sma l, and fragments were separated by PFGE in 1 % agarose gel. Results All 78 strains from 5 countries were classified as poultry ecovar according to a previously established biotyping system. Chromosomal DNA was cut by Sma l into 18 to 23 fragments ranging from about 3 to 685 kb. Seventy-eight strains produced 15 types, arbitrarily designated A to O, and 45 subtypes. Some differences were observed in PFGE patterns among countries. However, 10 fragments (333, 190, 110, 63, 55, 42, 34, 19, 10, and 3 kb) were highly conserved and were shared by almost all (> 78%) of the strains examined. The PFGE patterns were compared with those obtained by phage typing. All 29 strains belonging to avian phage-group II produced type A and 19 subtypes. Of the 15 strains belonging to phage-group I, 11 produced 8 types (B to H, O) and 5 subtypes that were different from those of type A. Conclusions Genomic DNA fingerprinting by PFGE is an effective technique for discriminating poultry S aureus strains and appears to be a useful method for subtyping strains of avian phage groups or the poultry-specific ecovar. (Am J Vet Res 1997;58:1412–1416)

Thomas, C. K. and B. H. Ross. Distinct patterns of motor unit behavior during muscle spasms in spinal cord injured subjects. J. Neurophysiol. 77: 2847–2850, 1997. Surface electromyograms (EMG) and force were recorded during repeated involuntary spasms of paralyzed triceps surae muscles of four men with chronic cervical spinal cord injury. The firing rates of 78 medial gastrocnemius (MG) motor units also were recorded intramuscularly with tungsten microelectrodes. Spasms typically involved a relatively rapid rise, then a more gradual fall in triceps surae EMG and torque. Motor unit firing rates either increased and then decreased with the spasm intensity (54%) or were relatively constant (26%), firing mainly at 2–10 Hz. The remaining units (20%) produced trains that included one or several doublets. Mean peak spasm firing rates were 18 ± 9 Hz (mean ± SD) for rate modulated units and 11 ± 10 Hz for units with little or no rate modulation. Some motor units fired at rates comparable with those recorded previously during maximum voluntary contractions performed by intact subjects. Others fired at rates below the minimum usually seen when normal units are first recruited (<6 Hz). Doublets (interspike interval <10 ms) often repeated every 123–333 ms, or were interspersed in trains firing at low steady rates (<11 Hz). This study shows that rate coding for many motor units appears to be similar whether descending motor input is intact or whether it has been reduced severely by spinal cord injury. In contrast, rate modulation in other units appears to depend mainly on voluntary motor commands.

Highlights Our project implementing a VAST produced these improved outcomes. There were 31% fewer IV devices. There were 22% fewer needle sticks. There were 49% reduction in delays. Abstract Background: Vascular access is essential in the care of hospitalized pediatric patients. Planning and device selection are important for optimal patient outcomes. Vessel preservation is critical for pediatric patients. Purpose: To determine and evaluate the impact of a vascular access team on pediatric patient care. Methods: A vascular access specialist team (VAST) was implemented on one medical unit at a tertiary children’s hospital, 16 hours a day for 4 weeks. Data Analysis: Descriptive statistics were used, including (a) first-stick success rates, (b) number of needle sticks and vascular access devices per hospital stay, (c) delays in therapy, and (d) frequency of topical analgesic use. Preintervention data were compared with postintervention data. Qualitatively, nursing and medical staff were surveyed to evaluate their perception of project experiences. Results: Compared with preintervention data, 56% fewer peripheral intravenous lines were initiated, and 31% fewer total vascular access devices were used during the hospital stay. There were 22% fewer needle sticks per peripheral intravenous line attempt. The first-stick success rate of the VAST was 71% compared with the bedside nurse rate of 52%. There was a 78% reduction in time between devices/1000 patient-days/week, representing reduced interruptions in therapy. The use of topical anesthetics for intravenous device placement increased 333%. Conclusion: This project provided clarity regarding the value of the VAST for pediatric hospitalized patients. The VAST model is evidence based, follows best-practice guidelines, is fiscally sound, and optimizes nursing practice and quality patient care.

Abstract Background We describe the decentralisation of antiretroviral therapy (ART) alongside Option B+ roll-out in public healthcare facilities in the Lablite project in Uganda. Lessons learned will inform programmes now implementing universal test and treat (UTT). Methods Routine data were retrospectively extracted from ART registers between October 2012 and March 2015 for all adults and children initiating ART at two primary care facilities (spokes) and their corresponding district hospitals (hubs) in northern and central Uganda. We describe ART initiation over time and retention and use of Cox models to explore risk factors for attrition due to mortality and loss to follow-up. Results from tracing of patients lost to follow-up were used to correct retention estimates. Results Of 2100 ART initiations, 1125 were in the north, including 944 (84%) at the hub and 181 (16%) at the spokes; children comprised 95 (10%) initiations at the hubs and 14 (8%) at the spokes. Corresponding numbers were 642 (66%) at the hub and 333 (34%) at the spokes in the central region (77 [12%] and 22 [7%], respectively, in children). Children &lt;3 y of age comprised the minority of initiations in children at all sites. Twenty-three percent of adult ART initiations at the north hub were Option B+ compared with 45% at the spokes (25% and 65%, respectively, in the central region). Proportions retained in care in the north hub at 6 and 12 mo were 92% (95% CI 90 to 93) and 89% (895% CI 7 to 91), respectively. Corresponding corrected estimates in the north spokes were 87% (95% CI 78 to 93) and 82% (95% CI 72 to 89), respectively. In the central hub, corrected estimates were 84% (95% CI 80 to 87) and 78% (95% CI 74 to 82), and were 89% (95% CI 77.9 to 95.1) and 83% (95% CI 64.1 to 92.9) at the spokes, respectively. Among adults newly initiating ART, being older was independently associated with a lower risk of attrition (adjusted hazard ratio [aHR] 0.93 per 5 y [95% CI 0.88 to 0.97]). Other independent risk factors included initiating with a tenofovir-based regimen vs zidovudine (aHR 0.60 [95% CI 0.46 to 0.77]), year of ART initiation (2013 aHR 1.55 [95% CI 1.21 to 1.97], ≥2014 aHR 1.41 [95% CI 1.06 to 1.87]) vs 2012, hub vs spoke (aHR 0.35 [95% CI 0.29 to 0.43]) and central vs north (aHR 2.28 [95% CI 1.86 to 2.81]). Independently, patient type was associated with retention. Conclusions After ART decentralisation, people living with human immunodeficiency virus (HIV) were willing to initiate ART in rural primary care facilities. Retention on ART was variable across facilities and attrition was higher among some groups, including younger adults and women initiating ART during pregnancy/breastfeeding. Interventions to support these groups are required to optimise benefits of expanded access to HIV services under UTT.

The objectives of this study were to evaluate the follicular dynamics (experiment 1) and the effects of the timing of insemination with sexed or nonsexed semen on pregnancy rates (experiment 2) of Jersey heifers detected in heat by a radiotelemetric estrus detection system. In experiment 1, 43 Jersey heifers, around 12 mo old and BCS of 2.68 ± 0.11 (1 to 5 scale) were used. The Heat Watch (HW) system was utilized to detect the onset of estrus and mounting behavior associated with estrus. Ultrasound examinations to monitor follicular dynamics occurred every 12 h from estrus onset for 48 h. Statistical analyses were performed using GLM and GLIMMIX procedure of SAS (SAS Institute, Cary, NC, USA). The results of experiment 1 indicated a mean ovulatory follicle diameter of 14.1 ± 0.3 mm, ovulation rate of 86.1% (37/43), and an interval of 31.2 ± 0.9 h from onset of heat to ovulation. In experiment 2, 753 Jersey heifers were allocated in a 2 × 4 factorial with semen (sexed and nonsexed) and AI period (0 to 6, 6 to 12, 12 to 18, and 18 to 24 h after heat onset) as parameters. Semen from 3 bulls was used, with ejaculates divided in 2 fractions: one fraction was submitted to the traditional freezing procedure and the other was submitted to the sexing process and then frozen. The statistical analysis was performed using GLIMMIX procedure of SAS. There was no interaction among the semen, bull, and AI period. There were effects on pregnancy rate by type of semen [sexed 49.5% (189/382) and nonsexed 64.2% (238/371); P = 0.001] and by bull [bull A 53.5% (107/200)b, bull B 50.0% (108/216)b and bull C 63.4% (211/333)a; P = 0.008]. Semen from bull C resulted in a greater pregnancy rate for both sexed and nonsexed semen. Within semen type, there were no differences in pregnancy rates by AI moment [sexed: 0 to 6 h 48.2% (41/85), 6 to 12 h 48.7% (54/111), 12 to 18 h 49.5% (49/99), 18 to 24h 52.4% (44/84) and nonsexed: 0 to 6h 62.8% (49/78), 6 to 12h 60.6% (63/104), 12 to 18h 68.0% (68/100), 18 to 24h 64.8% (57/88); P = 0.77]. We conclude that the use of sexed semen resulted in a lower pregnancy rate than nonsexed semen, and that AI timing does not affect conception rate in Jersey heifers identified in estrus by radiotelemetric estrus detection system. However, there was a bull effect on conception rate. The authors wish to thank Sexing Technologies and Dalhart Jersey Ranch.

BackgroundAnti-neutrophil cytoplasmatic antibody (ANCA) associated vasculitis (AAV) is a rare, life-threatening, systemic auto-immune disease.[1]Due to the low prevalence, multiple treating disciplines and poor registration, including ICD-10 classification, identifying AAV patients for (pre-)clinical studies, research and health care evaluation is challenging.[2, 3]Therefore, there is an urgent need to improve identifying these patients in health care organisations. Employing artificial intelligence (AI) – supported search engines are increasingly suggested to achieve this.ObjectivesReliably identify AAV patients in electronic health records (EHR) using an AI-based tool that incorporates text mining and Natural Language Processing (NLP).MethodsThe identification method consists of a search strategy combined with NLP-based exclusion. A search strategy to optimally identify AAV patients within a single center EHR system of an academic hospital was developed using an established AAV cohort (n=203) as a gold-standard reference set. Patient records, identified by the search strategy outside of the reference set, underwent manual review to confirm newly-identified AAV patients. Then, improvement in performance by adding NLP to the text-mining search strategy was investigated. Validation was performed on an independent EHR system of a non-academic hospital with an established AAV cohort available for reference (n=84).ResultsThe search strategy combined five queries based on disease description, laboratory measurements, medication and relevant specialisms. In the determination EHR, the search strategy identified 608 patients, including 197/203 (97.0%) AAV patients of the reference set and 149 newly-identified AAV cases confirmed by manual review. Employing NLP in the identification method improved the positive predictive value (PPV) from 57% (346/608 patients) to 78% (339/444 patients). These results were validated in an independent EHR system where the search strategy identified 333 patient, including 82/84 (97.6%) AAV patients of the reference set and 112 newly-identified AAV cases upon manual review. NLP improved PPV from 59% (194/333 patients) to 86% (192/223 patients). Negative predictive values and specificities were above 98% in all analyses.ConclusionWe present an AI based identification method to identify low-prevalent AAV patients in EHR systems. We demonstrated improved performance when adding NLP to the text-mining search strategy. Successful validation in an independent health organisation supports the applicability and transportability of this method which can be an important accelerator for research efforts and health care evaluation in AAV patients.References[1]Kitching AR, Anders HJ, Basu N, Brouwer E, Gordon J, Jayne DR, et al. ANCA-associated vasculitis. Nat Rev Dis Primers. 2020;6(1):71.[2]Quan H, Li B, Saunders LD, Parsons GA, Nilsson CI, Alibhai A, et al. Assessing validity of ICD-9-CM and ICD-10 administrative data in recording clinical conditions in a unique dually coded database. Health Serv Res. 2008;43(4):1424-41.[3]Spierings J BT, Dirikgil E, Moens HB. Overdaad aan ICD-coderingen hindert onderzoek. Medisch Contact. 2020;22:18-20.Acknowledgements:NIL.Disclosure of InterestsJolijn van Leeuwen: None declared, Erik Penne: None declared, Y.K. Onno Teng Consultant of: YKOT received an unrestricted research grant and consultancy fees from Vifor Pharma., Grant/research support from: The work of YKOT was supported by the Dutch Kidney Foundation (17OKG04) and by the Arthritis Research and Collaboration Hub Foundation. Arthritis Research and Collaboration Hub is funded by Dutch Arthritis Foundation (ReumaNederland).

Abstract Background: T cell lymphoma, comprising 15% of diagnosed US lymphomas, currently has no standard of care in the relapsed setting. Exposure to molds diminishes the numbers of T-helpers cells in peripheral blood. Aspergillus and Penicillium are known to produce small molecules called mycotoxins. Mycotoxins have previously been associated with immunosuppressive effects (Sutton et al., 1994, 1995), and a reduction of cytokine secretion (Rossano et al., 1999.) This study investigates the effects of two of these mycotoxins, Gliotoxin and Patulin, on the cellular components of mononuclear cells in peripheral blood of normal donors and T-cell lymphoma cells, examining viability and apoptotic damage in CD3+ (T cells), CD4+ (cytotoxic t cells), CD8+ (t helper cells), and CD16+ (NK cells), BDCA1+ and BDCA2+ (dendritic cells), CD14+ (monocytes), and CD19+ (B cells). Methods: Two mycotoxins, Gliotoxin and Patulin were solubilized into methanol or ethanol and cultured with either normal ficolled blood samples, T-cell lymphoma or T-cell leukemic cells. We plated cells at a concentration of 1x 106 cells per well, treated them with concentrations of 0, 1, 10, 100, 1,000, or 10,000 ng/mL, and incubated for 24 hours. After incubation the cells were removed and washed, and stained with fluorescent antibodies. Cellular content was determined by flow cytometry (FACS). Prior to acquisition by FACS, counting beads were added to each sample to determine absolute counts of each population of cells. Populations of interest were CD3+, CD4+, CD8+, and CD16+, BDCA1+ and BDCA2+, CD14+, and CD19+. Annexin and Propidium Iodide (PI) were added to evaluate apoptotic and non-viable cells. FloJo software was used to analyze the FACS data. Absolute count of viable cells was then converted to percentages using solvent only control as 100% viable. Results: Cell Viability after 24 hr Incubation with Mycotoxins Concentration Normal Peripheral Blood (n=9) T Cell Lymphoma (n=2) Gliotoxin (ng/mL) CD3+/CD4+ CD3+/CD8+ DC1 DC2 NK B CD3+ Median Percent Viable when 0 ng/mL = 100% Viable 0 100 100 100 100 100 100 100 1 127 128 219 143 104 127 96 10 126 129 36 69 109 117 82 100 15 15 3 25 2 18 110 1,000 23 33 3 11 13 11 37 10,000 47 46 63 13 26 27 4 Patulin (ng/mL) 0 100 100 100 100 100 100 100 1 66 68 74 86 60 81 93 10 94 100 101 114 84 88 333 100 84 101 79 141 110 138 152 1,000 39 38 2 26 35 27 69 10,000 53 45 0 34 39 78 1 The dose of Gliotoxin, most effective for inducing apoptosis and cell death in normal donor T cells is 100 ng/mL, whereas the most effective dose of Patulin was 1,000 ng/mL. In the cell line, the lowest dose of both mycotoxins that produced significant apoptosis and cell death was 1,000 ng/mL. Conclusion: Our results demonstrate that the mycotoxins, Patulin and Gliotoxin kill PBMC, and confirm the results found by Stanzani et al that Gliotoxin is most cytotoxic against the antigen presenting cells (APC), which impairs T cell response. Our experiment showed that Patulin had the same effect against APC. T cell viability itself was greatly influenced by the addition of Gliotoxin or Patulin, which supports previous evidence that mycotoxins suppress some populations of T cells. Certain issues, such as a less toxic solvent must be addressed; the data, however, shows that mycotoxins do have the ability to kill both normal and T cell leukemic/lymphoma cells.

Abstract Background: Several studies suggest that smoking and obesity may increase the risk of developing NHL, but the impact of these factors on survival is relatively unexplored. One recent population-based Italian study found that smoking may negatively impact overall survival. Methods: We evaluated the association of body mass index (BMI) and cigarette smoking on overall survival in 1,286 patients who participated in a population-based case-control study conducted through the Surveillance, Epidemiology, and End Results (SEER) cancer registries in Detroit, Iowa, Seattle and Los Angeles. BMI (on 1189 participants) and a smoking history (on 471 participants) were obtained through in-person interviews. Histology, stage, presence of B symptoms, first course of therapy, date of last follow-up, and vital status were derived from linkage to registry databases at each study site in the spring of 2005. Hazard Ratios (HRs) and 95% confidence intervals were estimated using Cox proportional hazards models, while adjusting for demographic and clinical factors. Results: The median age at diagnosis was 58 years (range: 16 – 74), and the most common histologies were DLBCL (33%), follicular (25%), SLL/CLL (12%), marginal zone (9%), mantle cell (4%) and Peripheral T-cell (3%). The median BMI was 26.6 (range: 16.2 – 47.3), and 26% of patients were classified as obese (i.e. BMI &gt;30). Of patients with a smoking history, 47% had never smoked, 19% were active smokers at diagnosis, and 34% were former smokers. No significant differences were observed between those with and without tobacco use data in terms of primary lymphoma characteristics. Overall survival (OS) was defined as time from diagnosis to death due to any cause. Median follow-up on all living patients was 58 months (range: 4 – 78), and 333 patients had died. When BMI was assessed as a continuous measure, it was borderline significantly associated with OS (p=0.050). An indicator of whether or not BMI &gt; 30 was also significantly associated with OS (HR=1.30; 95% CI: 1.01 – 1.68; p=0.042). Ever vs. never smokers also exhibited a survival disadvantage (HR=1.66; 95% CI: 1.12 – 2.44; p=0.011). When broken down further, former smokers who had smoked for &gt;10 years (HR=1.61; 95% CI: 1.02 – 2.52; p=0.039) and current smokers (HR=1.79; 95% CI: 1.07 – 3.02; p=0.028) were at a survival disadvantage over those who had never smoked. When both smoking and BMI status were included in the model, only the indicator of being a current smoker remained statistically significant (p=0.031), with a trend toward decreased survival for former smokers who had smoked &gt;10 years (p=0.061) and those with BMI &gt;30 (p=0.058). Conclusions: NHL patients who smoke or are obese had a poorer overall survival. These observations warrant further investigation, including whether interventions for smoking cessation or weight loss can impact NHL survival.

Background. The problem of the origin of the Glagolitic alphabet, its origins and the first years of Slavic writing can rightly be called the “cursed question” (questio dia-bolica – B. Uspensky) of Slavic studies, as attempts to connect the Glagolitic alphabet with any of the existing alphabets did not lead to any convincing results. Until now, the only relevant ideas of P. Shafarik remain, expressed about 150 years ago. Purpose. The aim of the article is to systematize and analyze historical and modern ideas (from the 90s to the present day), which are expressed by researchers regarding the longer antiquity of the Glagolitic alphabet. Results. The authors consider the development of P. Shafarik’s ideas at the pre-sent stage of development of East Slavic paleolinguistics. Materials about the origins of Slavic writing, which have accumulated in science, do not facilitate the solution of ques-tions about: 1) which of the two Slavic alphabets was created and / or improved by Constantine the Philosopher, 2) how and when another Slavic alphabet appeared, and 3) how the Slavic script developed in the post -Constantine period. In this context, as the authors show, all the hypotheses and ideas of modern researchers in one way or another develop the arguments of the hypothesis of PY Shafarik and do not go beyond it. Keywords: Slavonic writing, Cyrillic, Glagolitic, P. Shafarik’s hypothesis. Cristiano, Diddi, 2015. In the footsteps of Glagolitic protographers of Pannonian leg-ends: Methodological notes on the critique of variants. ΠΟΛΥΙΣΤΩΡ. Scripta slavica Mar-io Capaldo dicata. Moskow, Indrik, рp. 80–98. (In Russian) Dodonov, I. Yu., 2008. The origins of Slavic writing. Moskow: Veche. Avialable at: http://www.libma.ru/istorija/istoki_slavjanskoi_pismennosti). 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Scientific approach to the origin of the Cyrillic alphabet. Ma-terialy XLVI naukovo-tekhnichnoyi konferentsiyi pidrozdiliv VNTU. Vinnytsya, 22–24 be-reznya 2017 r. Avialable at: https://conferences.vntu.edu.ua/index.php/all— hum/all— hum— 2017/paper/view/2328. [Accessed 22.08.2021 (In Ukrainian) Štec, M., 1996. Šafárik on Old Slavonic and Church Slavonic. In: Pavol Jozef Šafárik a slavistika. Zborník príspevkov z vedeckej konferencie a dokumentov z osláv 200. výročia narodenia P. J. Šafárika. Acta facultatis philosophicae universitatis šafarikianae Literárný zborník 12/ Jazykovedný zborník 13. Historický zborník 5 (AFPh UŠ 79). Filozofická fakulta. UPJŠ. Prešov. Matica slovenská. Martin, s.291–295.(In Slovak) Stepanov, Yu. S. and Proskurin, S.G., 1993. Constants of world culture. Alphabets and alphabetic texts during periods of dual faith. Moscow: The science. 158 p. (In Russian) Toporov, V., 1998. 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Introduction: Myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN) overlap syndromes consist of 5 distinct WHO-defined entities; namely chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia, BCR/ABL1- (aCML), juvenile myelomonocytic leukemia (JMML), MDS/MPN with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T), and MDS/MPN, unclassifiable (MDS/MPN-U) (Arber et al., Blood 2016). With the notable exception of JMML, a bona fide RASopathy, the other entities are characterized by clinical heterogeneity and molecular diversity. Loss of function TET2 mutations (TET2MT) are common in myeloid neoplasms, especially CMML (60%), and are known leukemogenic drivers. We carried out this study to assess the TET2 mutational landscape and phenotypic correlates in patients with MDS/MPN overlap syndromes. Methods: After approval by the institutional review board, adult patients with WHO defined MDS/MPN overlap syndromes were included; with the exception of JMML. The BM morphology, cytogenetics and 2016, WHO-diagnoses were retrospectively reviewed and all patients underwent targeted next generation sequencing for 29 myeloid-relevant genes, obtained on BM mononuclear cells, at diagnosis, or at first referral, by previously described methods (Patnaik et al., BCJ 2016). Results: Five hundred and four patients were included in the study; including 387 (77%) with CMML, 48 (10%) with MDS/MPN-RS-T, 17 (3%) with aCML and 52 (10%) with MDS/MPN-U. The median age at diagnosis was 71 (range, 18-99) years, and 333 (66%) were male. TET2MT were seen in 212 (42%) patients, with the frequency of other mutations being: ASXL1 45%, SRSF2 40%, NRAS 15%, SF3B1 13%, CBL, RUNX1 and SETBP1 12% each, and JAK2 V617F 11% (Figure B). Among the MDS/MPN overlap syndromes, TET2 was more frequently mutated in CMML (49%) and aCML (47%) compared to MDS/MPN-RS-T (10%) and MDS/MPN-U (15%). The prevalence of patients with TET2MT increased with age, a finding consistent across all MDS/MPN subtypes (Figure C). Overall, 341 TET2MT were identified in 212 patients (mean 1.6 variants/patient, range 0-5): 120 (24%) had >1 TET2MT, while 113 (22%), 5 (1%) and 2 (0.4%) had 2, 3 and 5 mutations, respectively. CMML and aCML patients were more likely to have an age-independent increase in multiple TET2MT (28% and 24%), in comparison to MDS/MPN-RS-T (4%) and MDS/MPN-U (8%). TET2 MT spanned the entire coding sequence and were mostly truncating (78%, Figure A): 59 (17%) were missense, 14 (4%) involved the splice-donor/acceptor sites, 2 (0.5%) were in-frame deletions, 129 (38%) were nonsense, and 137 (40%) were frameshift mutations. Overall, the distribution of TET2MT was superimposable across CMML, aCML, and MDS/MPN-U; the only exception being the absence of splice site mutations in the latter two. One hundred and eighty-seven (55%) TET2MT were secondary to pathogenic single nucleotide variants (SNV), while the remainders were secondary to deletions (25%) and insertions (15%). Transitions comprised the most frequent type of SNV (65%), with the C:G>T:A being the most common (56%). Patients with MDS/MPN overlap syndrome and TET2MT were more likely to have additional gene mutations compared to wild type patients (mean mutation number 3.1 vs 2.1, p<0.0001), with common co-mutations being SRSF2 (51%), ASXL1 (42%), and CBL (17%). The median overall survival (OS) of the entire cohort was 29 (range, 0-170) months; 29 months for CMML, 63 months for MDS/MPN-RS-T, 14 months for aCML, and 25 months for MDS/MPN-U. On univariate analysis, OS was superior in CMML patients with TET2MT (35 months) compared to wild type cases (21 months, p<0.0001, Figure D), and in CMML patients with >1 TET2MT (41 months) in comparison to wild type (21 months, p<0.0001) and single TET2MT (29 months, p=0.0476) cases (Figure E). These observations were not seen in patients with aCML, MDS/MPN-RS-T, and MDS/MPN-U. Conclusion: Our study demonstrates that TET2MT are among the most frequent mutations in patients with MDS/MPN overlap syndromes (42%), especially CMML (49%), with an age-dependent increase in the frequency and number of TET2MT. Mutations in TET2 were found to span the entire coding sequence, with truncating mutations being more common (78%). Importantly, in CMML, TET2MT, including number of TET2MT, were associated with favorable survival outcomes. Figure Disclosures Al-Kali: Astex Pharmaceuticals, Inc.: Research Funding. Patnaik:Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

Abstract Abstract 7 Background: Hispanic children with ALL have a significantly inferior outcome compared with non-Hispanic whites (Blood, 2002). Low systemic exposure to 6MP adversely affects prognosis (N Engl J Med 1990), and red cell (RBC) levels of its metabolite (thioguanine nucleotide [TGN]) correlate with survival (Blood, 1999). Significant inter-patient variability in RBC TGN levels exists due either to inherited difference in thiopurine methyltransferase (TPMT) activity, or failure to adhere to prescribed 6MP. Non-adherence to 6MP has been reported, and could be related to differences in cultural beliefs, possibly accounting for ethnic differences in ALL survival. Methods: We tested this hypothesis in Hispanic and non-Hispanic white children with ALL diagnosed at age ≤21 yrs, and receiving maintenance therapy at 78 participating COG institutions. 6MP adherence was measured over 6 months using Medication Event Management System (MEMS) and RBC TGN. Electronic MEMS cap collected real-time data on dates/ times when the 6MP bottle was opened. Whether the bottle opening was followed by ingestion of 6MP was assessed by monthly (6 assessments/ patient) measurement of TGN. TPMT activity was also measured. Self-reported sociodemographics and reasons for non-adherence were also collected. MEMS-based adherence (outcome of interest) was defined as the ratio of days that 6MP bottle was opened to days 6MP doses were prescribed, reported as a percentage: “% adherence”. All prescribed doses were reviewed for each patient, and the period of time when 6MP was withheld by the prescriber due to toxicity/ illness taken into account. Longitudinal analysis was performed using Generalized Estimating Equations. Results: 333 patients (173 Hispanics; 160 non-Hispanic whites) contributed 54,193 person-days of observation for 6MP adherence. Median age at study participation was 6 yrs (2-20); 67% were males; 38% presented with high-risk disease per NCI criteria. While age, sex and disease characteristics did not differ by ethnicity, Hispanics were significantly more likely to report indices of lower SES (Table). Mean % adherence over the 6 month study period was significantly lower among Hispanics (86±20%) compared with non-Hispanic whites (93±9%, p<0.0001). Multivariate longitudinal analysis identified the following at risk for lower adherence: Hispanic ethnicity (p<0.0001, Fig A); age ≥12 yrs at study entry (p=0.004, Fig B); single mothers as caregivers (p=0.003, Fig C); and time on study (p<0.0001). Reasons for missing 6MP doses included forgetfulness (55%), disruption of usual routine (22%), logistical barriers (15%), and side effects (5%). With a median follow-up of 4.5 yrs, disease-free survival (DFS) was significantly inferior for Hispanics (81±5% at 6 yrs) compared with non-Hispanic whites (94±2%, p=0.01, Fig D). After adjusting for sex, NCI risk, TPMT activity, and 6MP dose-intensity, each 1% decrease in adherence was associated with a 3% increase in risk of relapse (p=0.01). Most importantly, in the multivariate model without adherence, Hispanic ethnicity was associated with a significantly increased risk of relapse (HR=3.2, p=0.01); however upon inclusion of adherence in the model (p=0.01), the association between ethnicity and recurrence diminished in magnitude and significance (HR=2.1, p=0.18). A cutpoint in adherence at 85% was accompanied by a statistical separation in DFS (DFS: 92±2.2% vs. 77.0±5.1%, p<0.0001, Fig E), as well as a large separation in TGN levels (198 vs. 174, p=0.07), making 85% a clinically relevant level of adherence. Conclusion: Non-adherence to 6MP is prevalent in children with ALL. Hispanic children are more likely to be non-adherent, even after adjusting for sociodemographic variables. Non-adherence is associated with significantly inferior DFS. Non-adherence explains the ethnic differences in DFS. This study has informed a targeted intervention to reduce non-adherence to oral chemotherapy, with the goal to reduce disparities in ALL outcome. Disclosures: Relling: St. Jude Children's Research Hospital: Employment, Patents & Royalties; Enzon Pharmaceuticals: Research Funding.

Introduction Patients with myelodysplastic syndromes (MDS) are at risk of early death from cardiovascular complications due to the link between clonal hematopoiesis and endothelial dysfunction. EASIX (Endothelial Activation and Stress Index) has been established to predict endothelial complications after allogeneic stem cell transplantation (alloSCT). EASIX is an endothelial dysfunction related biomarker that can be easily obtained with routine laboratory markers (creatinine× LDH/thrombocytes). We analyzed whether EASIX can predict mortality in higher-risk and in lower-risk MDS patients. Since alloSCT can alter the natural history of MDS and may be an independent contributor to endothelial complications, this analysis was restricted to patients who did not undergo alloSCT during the course of their disease. Patients and Methods We investigated the impact of EASIX measured at first diagnosis on survival of patients with lower- and higher-risk MDS (no allogeneic transplantation) in two independent institutions: n=192 (training cohort) and n=333 (validation cohort). For the purposes of this study, patients with low and intermediate-1 according to IPSS and very low and low-risk according to IPSS-R, respectively, were considered to have lower-risk MDS, while patients with intermediate-2 and high-risk according to IPSS and intermediate, high and very-high according to IPSS-R, respectively, were considered to have higher-risk MDS. Serum markers of endothelial cell distress were measured and correlated to EASIX. Results While no effects of EASIX on survival were observed in higher-risk patients, EASIX was associated with shorter survival in patients with lower-risk MDS in both cohorts (univariate: training cohort: hazard ratio (HR): 1.46; 95% confidence interval (CI) 1.24-1.71; p-value < 0.001 / validation cohort: HR 1.31 [1.17-1.48]; p-value < 0.001). We visualized this continuous effect of EASIX in lower-risk disease by grouping patients into quartiles according to EASIX (Figure 1 A and B). In both cohorts, patients in the highest quartile had a shorter survival compared to patients in lower quartiles. Confining our analysis to lower-risk patients who did not develop AML within the observation period, EASIX similarly predicted OS in both cohorts (training: n=78, no. of events n=17, HR per log2 increase 1.43 [1.05-1.94]; p=0.02; validation: n=267, no. of events n=93, HR per log2 increase 1.33 [1.21-1.47]; p<0.00). Multivariate Cox regression analysis and prediction error analyses confirmed that EASIX remained a significant predictor of survival after adjustment for age, sex, cytogenetic abnormalities and bone marrow blasts in lower-risk patients. The model of the training cohort could be validated. No effect of EASIX on survival was found in higher-risk patients from both cohorts in univariate and multivariate analysis. Serum levels of Angiopioetin-2 correlated significantly with EASIX whereas S100A9 or IL-1b did not. However, we observed a strong intra-pathway correlation of S100A9 with IL1b, IL18, IL37, and HMGB1. Conclusion We introduce EASIX as an easily accessible and independent predictor for survival in patients with lower-risk MDS. The strong correlation of EASIX and ANG2 underlines the endothelial nature of this biomarker. Disclosures Germing: Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Jazz Pharmaceuticals: Honoraria; Amgen: Honoraria. Kobbe:Neovii: Honoraria, Other: Travel support; Pfizer: Honoraria, Other: Travel support; Medac: Honoraria, Other: Travel support; Abbvie: Honoraria, Other: Travel support; Biotest: Honoraria, Other: Travel support; Jazz: Honoraria, Other: Travel support; Celgene: Honoraria, Other: Travel support, Research Funding; Roche: Honoraria, Other: Travel support; Amgen: Honoraria, Other: Travel support, Research Funding; MSD: Honoraria, Other: Travel support; Novartis: Honoraria, Other: Travel support; Takeda: Honoraria, Other: Travel support. Kaivers:Jazz Pharmaceuticals: Other: Travel Support. Merz:Janssen: Other: Travel grants; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Travel grants; Abbvie: Other: Travel grants; Celgene: Other: Travel grants; Takeda Vertrieb GmbH: Other: Travel grants, Research Funding. Dreger:MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia; Neovii, Riemser: Research Funding; AbbVie, Gilead, Novartis, Riemser, Roche: Speakers Bureau; AbbVie, AstraZeneca, Gilead, Janssen, Novartis, Riemser, Roche: Consultancy.

Introduction: In the POLARIX study (NCT03274492), polatuzumab vedotin in combination with rituximab plus cyclophosphamide, doxorubicin, and prednisone (Pola-R-CHP) demonstrated prolonged progression-free survival (PFS) versus rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) in patients with previously untreated diffuse large B-cell lymphoma (DLBCL; Tilly et al. N Eng J Med 2022); the PFS benefit was maintained with longer follow-up (Herrera et al. Blood 2022). We previously validated the prognostic value of ctDNA at baseline, on treatment, and at the end of treatment in POLARIX (Herrera et al. Blood 2022). Here, we assess and compare the mutation landscape and molecular subtyping of DLBCL by plasma ctDNA versus tissue DNA using the LymphGen classifier. Methods: Baseline plasma ctDNA single nucleotide variants (SNVs) were identified using the AVENIO ctDNA NHL assay (Research Use Only) with an allelic frequency (AF) of 0.5% (Stokowski et al. Blood 2022). Tissue SNVs at baseline were determined by whole exome sequencing (WES) with an AF cutoff of 5%. Plasma-depleted whole blood at baseline was used as a source of germline DNA to filter non-tumor-specific variants for both ctDNA and WES assays. Mutation landscapes in tissue DNA and plasma ctDNA were compared, focusing on genomic regions covered by both assays and variants within the coding regions. LymphGen uses genetic mutations, copy number data, and BCL2/BCL6 rearrangements to classify samples into at least one of seven genetic subtypes (Wright et al. Cancer Cell 2020). Genetic subtypes were defined by the LymphGen classifier using tissue and ctDNA SNVs, per the following: A53 ( TP53 and TP53BP1 mutations), BN2 ( BCL6 translocations and NOTCH2 mutations), EZB ( EZH2 mutations and BCL2 translocations), MCD ( MYD88L265P and CD79B mutations), N1 ( NOTCH1 mutations), and ST2 ( SGK1 and TET2 mutations). Hazard ratios (HR) were adjusted for International Prognostic Index score (2 vs 3-5) and age (≤60 vs &gt;60 years). Results: At baseline, 443 patients had both WES and ctDNA data. When comparing mutation landscapes between tissue DNA and ctDNA, a median 82% (lower quartile: 0.5, upper quartile: 1.0) of tissue SNVs per patient were also found in their matched ctDNA samples. In total, tissue and ctDNA SNVs were found in 163 and 202 genes, respectively. Among these genes, 154 had SNVs in both tissue and ctDNA samples, representing 96% and 78% of mutated genes identified in tissue and ctDNA, respectively. Eleven genes showed statistically significant differences in distribution between tissue and ctDNA (Fisher's exact test, false discovery rate&lt;0.01; Benjamini and Hochberg. J R Stat Soc 1995), including MEF2BNB-MEF2B, MEF2B, BCL10, BNC2, TNFAIP3, RIMS2, CDH19, KIF2B, BCL6, KMT2D and MYC; all but one ( MEF2B) showed higher mutation frequencies in ctDNA than in tissue. The distribution of molecular subtypes is listed in Table 1; due to a lack of information on copy number alterations, the A53 subtype was not included. Based on the tissue SNV data, LymphGen was able to determine the molecular subtypes of 442 patients. Using LymphGen results from tissue SNVs as a reference, 333 (75.3%) patients had the same subtype designation by tissue and ctDNA SNVs, and 109 (24.7%) had different designations ( Table 2). Among the 109 patients, 9 (8.2%) were assigned to a different molecular subtype by ctDNA, 50 (45.9%) patients with tissue SNV-defined molecular subtype were assigned to the undetermined subtype by ctDNA SNVs, and 50 (45.9%) patients with undetermined status by tissue SNVs were classified into distinctive genetic subtypes by ctDNA variants. In a pooled analysis, the 2-year PFS estimates of individual subtypes by tissue versus ctDNA SNVs were consistent ( Table 1). Conclusion: Our analyses demonstrated that the mutation landscape of ctDNA in DLBCL characterized by the AVENIO ctDNA NHL assay resembles that of tumor tissue determined by WES. Patients with molecular subtypes defined by WES or ctDNA had similar PFS outcomes. Overall, these findings support the use of plasma ctDNA as an alternative to tumor tissue for the genotyping of DLBCL.

La infección del tracto urinario de origen bacteriano es una patología frecuente en la mujer embarazada. Constituye un importante factor de riesgo asociado con el desarrollo de sepsis neonatal. La sepsis neonatal es una infección invasiva generalmente acompañada de bacteriemia que se presenta en el neonato en el primer mes de vida. Es una de las causas principales de morbilidad y mortalidad neonatal. El objetivo de esta investigación fue mostrar la relación entre la infección en el tracto urinario en la madre embrazada y la morbilidad y mortalidad del neonato. Se realizó un estudio de casos y controles que incluyó una muestra intencional y no probabilística de 224 pacientes, dividida en dos grupos de interés: 70 casos de neonatos nacidos de mujeres diagnosticadas conla infección y 154 controles de neonatos nacidos de mujeres sanas. Se realizó un análisis bivariado aplicando la prueba Chi cuadrado y estimando el Odds Ratio con apoyo del software OpenEpi, v3. El estudio mostró que los neonatos nacidos de madres con infección del tracto urinario tienen mayor riesgo de desarrollar sepsis neonatal, y sugiere que esta patología, una vez diagnosticada, puede ser tratada con eficacia evitando consecuencias graves para la salud del recién nacido. Palabras Clave: Infección, tracto urinario, sepsis neonatal, riesgo en el embarazo. Referencias [1]W. Coronell, 2Sepsis neonatal", Revista de Enfermedades Infecciosas en Pediatría, vol. XXIII, nº 90, pp. 68-72, 2014. [2]J. Bogante, "Infecciones uribnarias en el embarazo", Revista Medica de Costa Rica, vol. 4, nº 593, pp. 233-236, 2010. [3]L. Gilstrap, "Medical Complications of pregnancy", Obstretic and ginecology clinics, vol. 28, nº 25, pp. 340-348, 2015. [4]O. Shefali, "Causas mundiales, regionales y nacionales de mortalidad infantil en 2000–13, con proyecciones para informar las prioridades post-2015: un análisis sistemático actualizado," The Lancet, vol. 385, nº 9966, pp. 440-445, 2015. [5]B. Gretzeklle y C. Muñoz, "Factores de riesgo asociados a sepsis neonatal", Revista de la Facultad de Medicina URP., vol. 1, nº 10, pp. 8-16, 2017. [6]UNESCO, Organizcion Mundial de la Salud, ONU, febrero 2018. [En línea]. Available: https://www.who.int/es/news-room/fact-sheets/detail/millennium-development. [7]Miniesterio de Salud, "Guía de Práctica Clínica", Ministerio de Salud de Ecuador, Quito, 2015. [8]P. Baique, "Sepsis en pediatría: nuevos conceptos", An. Fac. Med, vol. 78, nº 3, pp. 333- 342, 2017. [9]A. Verdecia, N. Antuch , S. Rousseaux y I. Reyes,"Riesgos maternos asociados a sepsis neonatal precoz", Rev Inf Cient, vol. 9, nº 1, p. 74‐83, 2017. [10]M. Singer, C. Deutschman, C. Seymour, M. ShankarHari, D. Annane y M. Bauer, "The Third International Consensus Definitions for Sepsis and Septic Shock",JAMA, vol. 315, nº 8, pp. 801-810, 2016. [11]G. Fajardo, R. Flores y A. Cárcamo , "Caracterización general de sepsis neonatal temprana", Rev. Fac. Cienc. Méd., vol. 5, nº 2, pp. 28-35, 2017. [12]R. Pérez , J. Lona , M. Quiles , M. Verdugo y E.Ascencio , "Sepsis neonatal temprana, incidencia y factores de riesgo asociados en un hospital público del occidente de México", Rev Chilena Infectol, vol. 32, nº 4, pp. 387-392, 2016. [13]O. Aríz, A. Clemades, J. Faure, Y. Pérez y N. García, "Sepsis neonatal de inicio precoz en una unidad de cuidados neonatales: gérmenes asociados", Acta Médica del Centro, vol. 13, nº 2, pp. 151-159, 2019. [14]G. Samudio, L. Monzón y G. Ortiz, "Sepsis neonatal tardía nosocomial en una unidad de terapia intensiva: agentes etiológicos y localización más frecuente", Rev. chil. infectol , vol. 35, nº 5, pp. 547- 552 , 2018. [15]W. Bank, "Child maoratlity",»Banco Mundial, 2013. [16]A. Garaboa, Y. Sarmiento, C. Marquéz y M. Portal, "El recién nacido pretérmino con infección de inicio precoz", Rev Ciencias Médicas , vol. 19, nº 6, pp. 1014-1027 , 2015 . [17]T. Zea-Vera, T. Ochoa y Turin C, "Unificando los criterios de sepsis neonatal tardía: propuesta de un algoritmo de vigilancia diagnóstica", Rev Peru Med ExpSalud Publica , vol. 31, nº 2, pp. 358-563 , 2014 . [18]I. Belleste, R. Alonso, M. González, A. Campo y R. Amador, "Repercusión de la sepsis neonatal tardía en la morbilidad y mortalidad", Revista Cubana de Obstetricia y Ginecología, vol. 44, nº 1, pp. 1-9, 2018. [19]C. Fernandez., "Sepsis de origen precoz", Asturias: Hospital Central de Asturias, 2017. [20]J. Martinez, "Consideraciones sobre el impacto de la morbilidad y mortalidad neonatal y pediátrica en la salud pública Ecuatoriana", Universidad de Ambato, Ambato, 2018. [21]G. Montoya, C. Luna y L. Correa, "Factores de riesgo asociados a sepsis neonatal temprana en prematuros de un Hospital Nacional Docente Madre Niño,2017",Rev. Fac. Med. Hum, vol. 19, nº 3, pp. 35-42, 2019. [22]K. Sullivan , A. Dean y M. Soe, "O pen E pi : una calculadora epidemiológica y estadística basada en la web para la salud pública", Rep Salud Pública , vol. 124, nº 3, pp. 471-474 doi: 10.1177 / 003335490912400320, 2009. [23]B. Fernández, "Sepsis del recién nacido", Servicio de Neonatología, Universidad de Asturias, Asturias, 2017.

Abstract Immune based strategies have shown early promise in multiple myeloma (MM), and are likely to prove a valuable addition to the therapeutic platforms for these patients. B cell maturation antigen (BCMA) is a promising therapeutic target with selective expression on plasma cells, including MM cells. In the first report of BCMA targeting therapies, an anti-BCMA scFv-based chimeric antigen receptor (CAR) had clinical activity, albeit with short lived responses (Ali et al, 2016). Observed challenges included low expression level of BCMA, shedding of BCMA and potential loss of expression. In addition to BCMA, MM cells also express transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) which functions in plasma cell survival and differentiation. TACI is selectively expressed within lymphoid tissue and at reduced levels compared to BCMA, as measured by RT-PCR on normal tissues. TACI and BCMA share a common ligand called a proliferating ligand (APRIL), a compact self-protein forming a trimer and binding with nanomolar affinity to TACI and BCMA. The natural trimerization of APRIL would serve to enhance cell binding, especially important in the case of low levels of target antigen. Concurrent targeting of two tumour antigens, by increasing antigen density, would improve efficacy as well as reducing the risk of antigen negative disease escape. We quantified APRIL targets on primary MM cells by flow cytometry and observed BCMA expression in all of 41 patients tested (median 1051, range 105-8323 antibodies bound per cell, ABC) with most patients (78%) also expressing TACI (333, 0-21301 ABC). We designed an APRIL based 3rd generation chimeric antigen receptor (APRIL-CAR) comprising an engineered APRIL protein as antigen-binding domain. Peripheral blood mononuclear cells (PBMCs) were activated, transduced with APRIL-CAR and CD56-depleted before testing against cell lines expressing a wide range of BCMA and TACI (1005-1.5x10^5 and 2200-1.6x10^5 ABC respectively). We confirmed the cytolytic capability of APRIL-CAR against engineered targets, even at the low antigen levels observed in primary cells. Using standard chromium release, there was significant cytolysis of the lowest antigen expressers, with 40.9±3.8% (mean±SEM) and 42.6±5.7% cell death in SUPT1BCMAlo and SUPT1TACIlo respectively cf 15.0±5.8% for control SUPT1NT targets, at an E:T ratio of 4:1 (n=4, p<0.01 and p<0.05). When we lowered the E:T ratio, APRIL-CAR continued to show potent activity, with cytolysis of SUPT1BCMAlo/ SUPT1TACIlo targets at 1:10 (71.7±3.8% and 70.4±6.3% cell death respectively cf 21.6±5.8% for SUPT1NT, n=4, p<0.001 for both) and human myeloma cell lines at 1:32 (MM.1s and U266, 56.2±3.9% and 25.1±3.9% target cytolysis by APRIL-CAR and 4.7±11.6% and 6.6±3.9% by PBMCs NT respectively, n=5, p<0.01 for both). We confirmed cytolytic activity of APRIL-CAR against primary MM cells. Allogeneic PBMCs transduced with APRIL-CAR and CD56-depleted were co-cultured 1:1 with CD138+ bone marrow MM cells from 5 patients. Despite variable BCMA and TACI expression between patient samples (BCMA 1224-7728 and TACI 563-1213 ABC), tumour cytolysis was evident in all cases (APRIL-CAR 72.9±12.2% specific cell death cf media control 2.8±15.3%; p<0.05). Finally we tested the in vivo activity of APRIL-CAR in an intramedullary model established with tail vein injection of MM.1s.Fluc cells into NOD scid gamma (NSG) mice. Animals were analysed by bioluminescent imaging and FACS analysis of bone marrow for tumour cells. Complete clearance of established intramedullary disease was seen 5 days after infusion of 5x10^6 APRIL-CAR positive PBMCs in 12/12 animals. Elimination of tumour cells was confirmed in the bone marrow (p<0.01) with concurrent detection of APRIL-CAR T cells by FACS analysis. In summary we report for the first time a 3rd generation CAR utilising a natural ligand to simultaneously target two antigens found on MM cells. Our APRIL-based CAR demonstrated potent activity at the low levels of BCMA and TACI found in primary MM cells, at low (physiological) E:T ratios, and in an intramedullary MM model. This APRIL based CAR holds particular promise and deserves testing in clinical studies. Disclosures Lee: Bloodwise: Research Funding; Autolus Ltd: Patents & Royalties: APRIL based chimeric antigen receptor. Draper:Autolus Ltd: Equity Ownership, Patents & Royalties: APRIL based CAR. Chaplin:Autolus Ltd: Equity Ownership. Philip:Autolus: Equity Ownership. Thomas:Autolus Ltd: Employment. Kokalaki:Autolus Ltd: Employment. Francis:Autolus Ltd: Employment. Yong:Autolus Ltd: Equity Ownership, Patents & Royalties: APRIL based chimeric antigen receptor; Janssen: Research Funding. Pule:Autolus Ltd: Employment, Equity Ownership, Research Funding; UCL Business: Patents & Royalties; Amgen: Honoraria; Roche: Honoraria.

ABSTRACT The purpose of this study was to analyze the short-term and long-term relationship between the Islamic capital markets of 5 selected countries in the Covid-19 era. This study used a quantitative approach using weekly secondary data from January 2020 to December 2022. The data analysis model used Vector Auto Regression (VAR) analysis. The results of this study show that in the short term, variants in the capital market will change due to shocks in other ASEAN Islamic capital markets, but short-term deviations in the Islamic capital market will adjust in the long term. meanwhile, in the long run, Islamic capital markets in ASEAN countries that were tested, have integrated despite the occurrence of a health crisis that also has an impact on the financial crisis. This provides an opportunity for investors to mitigate risks and increase profit margins, especially in Islamic stock exchanges in ASEAN countries. On the other hand, this research allows investors to reformulate a more diversified portfolio after the Covid-19 pandemic. Keywords: Integration, Islamic Capital Market, ASEAN, Covid 19 ABSTRAK Tujuan dari penelitian ini adalah untuk menganalisis hubungan jangka pendek dan jangka panjang antara pasar modal syariah 5 negara terpilih di era Covid-19. Penelitian ini menggunakan pendekatan kuantitatif dengan menggunakan data sekunder mingguan januari 2020 sampai dengan desember 2022. Model analisis data menggunakan analisis Vector Auto Regression (VAR). Hasil penelitian ini menunjukkan bahwa dalam jangka pendek, varian di pasar modal akan berubah karena guncangan di pasar modal syariah ASEAN lainnya, namun penyimpangan jangka pendek di pasar modal syariah akan menyesuaikan dalam jangka panjang. sementara dalam jangka panjang, pasar modal syariah di negara-negara ASEAN yang teruji, memiliki integrasi meskipun terjadi krisis kesehatan yang juga berdampak pada krisis keuangan. Hal ini memberikan suatu kesempatan bagi para investor untuk memitigasi risiko dan memperbesar margin keuntungan terutama di bursa saham syariah negara ASEAN. Disisi lain, dengan adanya penelitian ini memungkinkan membantu para investor untuk merumuskan kembali portofolio yang lebih terdiversifikasi setelah terjadinya pandemi covid-19. Kata Kunci: Integrasi, Pasar Modal Syariah, ASEAN, Covid 19 REFERENCES Abd. Majid, M. S. (2018). Who Co-Moves The Islamic Stock Market of Indonesia -The US, The UK, or Japan? Al-Iqtishad: Jurnal Ilmu Ekonomi Syariah, 10(2), 267–284. doi:10.15408/aiq.v10i2.7288 Abdul Karim, B., & Abdul-Rahman, A. (2020). Market integration in asean-5: Evidence of Islamic and conventional stock markets. Polish Journal of Management Studies, 21(1), 186–198. doi:10.17512/pjms.2020.21.1.14 Abdulkarim, F. M., Akinlaso, M. I., Hamid, B. A., & Ali, H. S. (2020). The nexus between oil price and Islamic stock markets in Africa: A wavelet and Multivariate-GARCH approach. 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Se analiza el sistema defensivo de La Fonteta, una de las fortificaciones fenicias mejor conocida de la península ibérica, en el marco de las nuevas tácticas de la poliorcética implantadas en el Mediterráneo Occidental a partir de la colonización fenicia, que supusieron la rápida adopción en las costas del mediodía y el sureste peninsular de estos novedosos sistemas de fortificación que pronto se generalizaron en todo el territorio peninsular de influencia oriental.La muralla de La Fonteta, erigida en torno al 600 a. C., presenta elementos característicos de una obra de carácter oriental, como su construcción mediante cajones macizos ajustada a parámetros métricos establecidos, la presencia de torres cuadrangulares o el uso de mampostería de piedra y alzados de adobe y tierra, rematados con almenas. Además, se han identificado forros ataludados, una rampa o glacis y un foso en “V”, algunos de indudable origen oriental, pero otros de claro influjo indígena. Palabras Clave: muralla, foso, glacis, antemural, FeniciosTopónimos: La Fonteta (Guardamar del Segura, Alicante),Periodo: Período orientalizante ABSTRACTIn this paper, the La Fonteta defensive system, one of the best-known Phoenician fortifications on the Iberian Peninsula, is analyzed within the framework of the new polyorcetic tactics introduced in the Western Mediterranean following Phoenician colonization. This involved the rapid deployment on the shores of Southern and Southeastern Iberia of these new fortification systems, which soon spread across throughout the entire Near Eastern influenced territory of the peninsula.The wall of La Fonteta, built around 600 BC., presents characteristic elements of a Near Eastern construction, such as the use of solid caissons adjusted to established metric parameters, the presence of quadrangular towers, or the employment of stone masonry and elevations of mudbricks and earth, topped with battlements. In addition, attached sloped walls, a ramp or glacis and a 'V'-shaped ditch have been identified, some of these features undoubtedly of Near Eastern origin, but others of clear indigenous influence. Keywords: wall, ditch, glacis, avant-mur, PhoeniciansPlane names: La Fonteta (Guardamar del Segura, Alicante),Period: Orientalizing Period REFERENCIASAlmagro-Gorbea, M.; Lorrio, A. J. y Torres, M. (2021), “Los focenses y la crisis de c. 500 a. C. en el Sudeste: de La Fonteta y Peña Negra a La Alcudia de Elche”, Lucentum, 40, pp. 63-110.Almagro-Gorbea, M. y Torres, M. (2007), “Las fortificaciones tartésicas en el Suroeste peninsular”, en L. Berrocal y P. Moret (eds.), Paisajes fortificados de la Edad del Hierro. Las murallas protohistóricas de la Meseta y la vertiente atlántica en su contexto europeo, Bibliotheca Archaeologica Hispana, 28, pp. 35-55.Arancibia, A. y Escalante M. M. 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Ключові слова: пам’ять, ідентичність, національна ідентичність, комеморація, кмеморативні практики. Анотація Національна ідентичність, з одного боку, є однією з фундаментальних ідентичностей, тобто, вона може служити орієнтиром для людини навіть тоді, коли інші (професійні, родинні, класові) швидко змінюються внаслідок трансформації суспільства. З іншого боку, зміни суспільства, його соціальних інститутів (як агентів впливу) змінюють зміст і форми національної ідентичності членів суспільства. Відтак усталення тієї чи іншої моделі ідентичності здійснюється в певному соціальному контексті, який задає не тільки спектр альтернатив, але й набір різних комеморативних практик. У статті обґрунтовується взаємообумовленість смислоконституювання пам’яттєвого та ідентичнісного дискурсів, із урахуванням того, що пам’яттєвий дискурс, з одного боку, віддзеркалює характерні риси національної ідентичності, а з іншого – формує своєрідну смислову основу останньої, моделює її, впливаючи на зміни з різним ступенем інтенсивності. Доведено, що національна ідентичність формується за допомогою колективної пам’яті та комеморативних практик. Посилання Anderson, 2001 – Anderson B. Voobrazhaemыe soobshchestva. Razmыshlenyia ob ystokakh y rasprostranenyy natsyonalyzma [Imaginary communities. Reflections on the origins and spread of nationalism]. M. : Kanon-press, 2001. 288 s. [in Russian] Assman, 2012 – Assman A. Prostory spohadu. Formy ta transformatsii kulturnoi pam’iati [Spaces of memory . Forms and transformations of cultural memory] / per. z nim. K. Dmytrenko, L. Doronicheva, O. Yudin. K.: Nika-Tsentr, 2012. 440 s. (Seriia «Zmina paradyhmy». Vyp. 15). 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Parents play an important role in the development of their children. This research reflects the role of parents in developing children. Through four stages of identification, screening, eligibility, and acceptable results, this method uses a systematic literature review using the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analyses) method. The findings from the fourteen articles examined show that parenting skills play an important role in a child's growth and development from birth to death. The determining factor in the development of physical, motoric, moral, language, social-emotional, and life skills aspects is the role of both parents as important teachers for children from birth to adulthood. Parents can also use a variety of parenting strategies and skills, many of which they have learned throughout their lives and passed on to their children, to help their children grow. 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Introdução: Disfunção Temporomandibular (DTM) é o termo para designar um quadro de desorganização neuromuscular identificada pela presença de cefaleias crônicas, sons na articulação temporomandibular, restrições dos movimentos mandibulares, hiperestesia e dor nos músculos da mastigação, da cabeça e do pescoço. Objetivo: realizar uma revisão de literatura sobre a DTM. Material e Método: foi feita uma seleção de artigos científicos a partir das bases de dados LILACS e SCIELO utilizando os descritores “Articulação Temporomandibular”, “Transtornos da Articulação Temporomandibular” e “Dor Facial”, usando como critérios de inclusão trabalhos brasileiros e inglês publicados em português e inglês no período de 2000 a 2018. Dos 798 artigos encontrados e delimitados pelos critérios inclusivos, foram selecionados 56 artigos como amostra, que apresentaram a temática elencada para a pesquisa e que foram discutidos nas seguintes sessões: a) Conceitos e epidemiologia; b) Etiologia; c) Sintomatologia; d) Diagnóstico; e) Tratamento. Conclusão: as causas da DTM são multifatoriais e seu diagnóstico deve ser minucioso. Observa-se a importância da anamnese para coleta de dados sintomatológicos da doença e o estudo de cada caso para melhor adequar a técnica de tratamento a ser utilizada. Há a necessidade de avaliações clínicas multidisciplinares nos indivíduos identificados com DTM para que o tratamento seja otimizado, minimizando a morbidade e diminuindo os custos do tratamento. Descritores: Articulação Temporomandibular; Transtornos da Articulação Temporomandibular; Dor Facial. Referências Capellini VK, Souza GS, Faria CRS. Massage therapy in the management of myogenic TMD: a pilot study. J Appl Oral Sci. 2006;14(1):21-6. Bastos LVW, Tesch RS, Denardin OV. Alterações cefalométricas presentes em crianças e adolescentes com desordens da ATM nas diferentes classificações sagitais de má oclusão. R Dental Press Ortodon Ortop Facial. 2008;13(2):40-8. Menezes MS, Bussadori SK, Fernandes KPS, Gonzalez DAB. Correlação entre cefaleia e disfunção temporomandibular. Fisioterapia e Pesquisa. 2008,15(2):183-7. Branco RS, Branco CS, Tesch RS, Rapoport A. Frequência de relatos de parafunções nos subgrupos diagnósticos de DTM de acordo com os critérios diagnósticos para pesquisa em disfunções temporomandibulares (RDC/TMD). R Dental Press Ortodon Ortop Facial. 2008;13(2):61-9. Ritzel CH, Diefenthaeler F, Rodrigues AM, Guimarães ACS, Vaz MA. Temporo-mandibular joint dysfunction and trapezius muscle fatigability. Rev Bras Fisioter. 2007;11(5):333-9. Kato MT, Kogawa EM, Santos CN, Conti PCR. Tens and low-level laser therapy in the management of temporo-mandibular disorders. J Appl Oral Sci. 2006;14(2):130-5. Tomacheski DF, Barboza VL, Fernandes MR, Fernandes F. Disfunção têmporo-mandibular: estudo introdutório visando estruturação de prontuário odontológico. Publ UEPG Ci Biol Saúde. 2004;10(2):17-25. Machado IM, Pialarissi PR, Minici TD, Rotondi J, Ferreira LP. Relação dos sintomas otológicos nas disfunções temporomandibulares. Arq Int Otorrinolaringol. 2010;14(3):274-9. Venancio RA, Camparis CM, Lizarelli RFZ. Laser no Tratamento de Desordens Temporomandibulares. J. Bras. Oclusão, ATM, Dor Orofac. 2002;7:229-34. Quinto CA. Classificação e tratamento das disfunções temporomandibulares: qual o papel do fonoaudió- logo no tratamento dessas disfunções? Rev CEFAC. 2000;2(2):15-22. Piozzi R, Lopes FC. Desordens temporomandibulares: aspectos clínicos e guia para a odontologia e fisioterapia. J. Bras. Oclusão, ATM Dor Orofacial. 2002;2(5):43-7. De Leeuw R. Dor orofacial: guia de avaliação, diagnóstico e tratamento 4ª ed. São Paulo: Quintessence;2010. 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J Oral Rehabil. 2009;36(6):403-7. Giannakopoulos NN, Keller L, Rammelsberg P, Kronmüller KT, Schmitter M. Anxiety and depression in patients with chronic temporomandibular pain and in controls. J Dent. 2010;38(5):369-376. Fernandes G, Gonçalves DA, De Siqueira JT, Camparis CM. Painful temporomandibular disorders, self reported tinnitus, and depression are highly associated. Arq Neuropsiquiatr. 2013;71(12):943-7. Mottaghi A, Razavi SM, Elham Zamani Pozveh E, Jahangirmoghaddam M. Assessment of the relationship between stress and temporomandibular joint disorder in female students before university entrance exam (Konkour exam). Dent Res J (Isfahan). 2011;8(Supl.1):76-9. Pizolato RA, Freitas-Fernandes FS, Gavião MB. Anxiety/depression and orofacial myofacial disorders as factors associated with TMD in children. Braz Oral Res 2013;27(2):156-162. Calixtre LB, Grüninger BLS, Chaves TC, Oliveira AB. Is there an association between anxiety/depression and Temporomandibular Disorders in college students? J Appl Oral Sci. 2014;22(1):15-21. Winocur E, Gavish A, Finkelshtein T, Halachmi M, Gazit E. Oral habits among adolescent girls and their association with symptoms of temporomandibulardisorders. J Oral Rehabil. 2001;28(7):624-629. Carvalho LPM, Piva MR, Santos TS, Ribeiro CF, Araújo CRF, Souza LB. Estadiamento clínico da disfunção temporomandibular: estudo de 30 casos. Odontol Clín-Cient. 2008;7(1):47-52. Medeiros SP, Batista AUD, Forte FDS. Prevalência de sintomas de disfunção temporomandibular e hábitos parafuncionais em estudantes universitários. RGO 2011;59(2):201-208. Valetic'-Peruzovic'm, Alajbeg I, Prpic'-Mehicic'g, Juros V, Illes D, Pelivan I. Acta Medica Croatica. 2008;62(2):179-187. Gavish A, Halachmi M, Winocur E, Gazit E. Oral habits and their association with signs and symptoms of temporomandibular disorders in adolescent girls. J Oral Rehabil. 2000;27(1):22-32. Thilander B, Rubio G, Pena L, Mayorga C. Prevalence of Temporomandibular Dysfunction and Its Association With Malocclusion in Children and Adolescents: An Epidemiologic Study Related to Specified Stages of Dental Development. Angle Orthod. 2002;72(2):146-154. Paulino MR, Moreira VG, Lemos GA, Silva PLP, Bonan PRF, Batista AUD. Prevalência de sinais e sintomas de disfunção temporomandibular em estudantes pré-vestibulandos: associação de fatores emocionais, hábitos parafuncionais e impacto na qualidade de vida. Ciência & Saúde Coletiva. 2018;23(1):173-186. Okeson, Jeffrey P. Etiologia e identifi cação dos distúrbios funcionais no sistema mastigatório. In:. Tratamento das desordens temporomandibulares e oclusão. 4. ed. São Paulo: Artes Médicas, 2000. p. 117-272. Greene, Charles S. The etiology of temporomandibular disorders: implications for treatment. Journal of Orofacial Pain. 2001;15(2)93-105. Bove SRV, Guimarães AS, Smith RL. Caracterização dos pacientes de um ambulatório de disfunção temporomandibular e dor orofacial. Rev Latino Enferm. 2005;13(5):686-91. Detamore MS, Athanasiou KA. Structure and function of the temporomandibular joint disc: implications for tissue engineering. J Oral Maxillofac Surg. 2003;61(4):494-506. Ramínez LM, Ballesterol LE, Sandoval GP. Otological symptoms among patients with temporimandibular joint disorders. Revista Médica de Chile. 2007;135(12):1582-90. Felício CM, Melchior MDEO, Ferreira CL, Silva MA. Otologic symptoms of temporomandibular disorder and effect of orofacial myofunctional disorder and effect of orofacial myofunctional therapy. Cranio. 2008;26(2):118-25. Bertoli, Elizangela de et al. Prevalence and impact of post-traumatic stress disorder symptoms in patients with masticatory muscle or temporomandibular joint pain: differences and similarities. Journal of Orofacial Pain, Carol Stream, v. 21, n. 2, p. 107-119, Spring 2007. Reissmann, Daniel R. et al. Functional and psychosocial impact related to specifi c temporomandibular disorder diagnoses. Journal of Dentistry, Guildford, v. 35, n. 8, p. 643-650, Aug. 2007. Aggarwal, Vishal R. et al. Psychosocial interventions for the management of chronic orofacial pain Psychosocial interventions for the management of chronic orofacial pain Psychosocial interventions for the management of chronic orofacial pain. Cochrane Database of Systematic Reviews, Oxford, v. 9, n. 11, CD008456, Nov. 2011. Costa, Max Dória; Froes Junior, Gontran da Rocha Torres; SANTOS, Carlos Neanes. Avaliação de fatores oclusais em pacientes com disfunção temporomandibular. Dental Press Journal of Orthodontics, Maringá, v. 17, n. 6, p. 61-68, nov./dez. 2012. Liao, Chun-Hui et al. The risk of temporomandibular disorder in patients with depression: a population-based cohort study. Community Dentistry and Oral Epidemiology, Copenhagen, v. 39, n. 6, p. 525-531, Dec. 2011. Conti PCR. 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Influence of temporomandibular disorder presence and severity on oral health-related quality of life. Rev Dor. 2015;16(1):10-14. Ballegaard V, Thede-Schmidt-Hansen P, Svensson P, Jensen R. Are headache and temporomandibular disorders related? A blinded study. Cephalalgia. 2008;28(8):832-41. Plesh O, Noonan C, Buchwald DS, Goldberg J, Afari N. Temporomandibular disorder-type pain and migraine headache in women: A preliminary twin study. J Orofac Pain. 2012;26(2):91-8. Melo GM, Barbosa JFS. Parafunção x DTM: a influ­ência dos hábitos parafuncionais na etiologia das de­sordens temporomandibulares. POS. 2009; 1(1):43-8. Guhur MLP, Alberto RN, Carniatto N. Influências bio­lógicas, psicológicas e sociais do vestibular na adoles­cência. Roteiro. 2010;35(1):115-38. Cuccia AM, Caradonna C, Caradonna D. Manual Therapy of the mandibular accessory ligaments for the management of temporomandibular joint disorders. JAOA. 2011;111(2):102-12. Pasinato F, Souza JA, Corrêa ECR, Silva AMT. 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RESUMENEl estudio histórico de la religiosidad islámica se ha encontrado tradicionalmente con el problema de la escasez de fuentes. Además, siempre se ha estudiado a partir de las fuentes islámicas, obviando las cristianas. Si es cierto que para la Edad Media las fuentes cristianas no ofrecen demasiada información y están además viciadas por su carácter polémico, también lo es que para la Edad Moderna, con el surgimiento de un tipo de literatura sobre el islam que está alejado de la tradición polémica, disponemos de un buen número de importantes fuentes cristianas que merecen ser tenidas en cuenta en cualquier análisis histórico de la religiosidad islámica. Es el caso especialmente de la Topographía e Historia general de Argel (1612), cuyas descripciones y observaciones resultan de una riqueza sin precedentes.PALABRAS CLAVE: Edad Moderna, cinco pilares del islam, religiosidad, España, Argel.ABSTRACTTraditionally, the historical study of Islamic religiosity has been faced with the problem of the scant amount of sources. Moreover, it has always been approached on the basis of Islamic sources, disregarding the Christian ones. If for the Middle Ages Christian sources do not present much information about the subject and this is tainted by its polemical character, for the Early Modern Age, with the emergence of a new kind of literature about Islam that does not belong to the polemical tradition, we have at our disposal a good number of sources of information. This is particularly thecase of the Topographía e Historia general de Argel (1612), with descriptions and observations of an unprecedented quality. It is an evident indication that, from the sixteenth century onwards, there are Christian sources that must be taken into account in any historical analysis of Islamic religiosity.KEY WORDS: Image of Islam, early modern Spain, five pillars of Islam, religiosity. BIBLIOGRAFÍAAfricanus, L., Descripción general del África y de las cosas peregrinas que allí hay, traducción y edición de S. Fanjul, Barcelona, Lunwerg, 1995.Alfonso, P., Diálogo contra los judíos, traducción de E. Ducay, Zaragoza, Instituto de Estudios Altoaragoneses, 1996.Berkey, J. P., The formation of islam: religion and society in the Near East, 600-1800, Cambridge, Cambridge University Press, 2003.Berque, J., Al-Yousi: problémes de la culture marocaine au XVIIème siècle, Paris, Moutin & Co., 1958.Bunes Ibarra, M. Á. de, La imagen de los musulmanes y del norte de África en la España de los siglos XVI y XVII: los caracteres de una hostilidad, Madrid, CSIC, 1989.Camamis, G., Estudios sobre el cautiverio en el Siglo de Oro, Madrid, Gredos, 1977.Cardaillac, L., Moriscos y cristianos: un enfrentamiento polémico (1442-1560), 2ª ed., Madrid, Fondo de Cultura Económica, 2016.Constable, O. R., “Regulating religious noise: the Council of Vienne, the mosque call and Muslim pilgrimage in the late medieval Mediterranean world”, Medieval Encounters, vol. 16, núm. 1 (2010), pp. 64–95.Cruz Hernández, M., El islam de al-Andalus: historia y estructura de su realidad social, 2ª ed., Madrid, AECI, 1996.Cruz Palma, Ó. de la, “Las cinco oraciones islámicas diarias (Salawat) en las fuentes latinas medievales”, en Martínez Gázquez, J. y Tolan, J. V. (eds.), Ritus infidelium. Miradas interconfesionales sobre las prácticas religiosas en la Edad Media, Madrid, Casa de Velázquez, 2013, pp. 133-149.— Machometus: la invención del profeta Mahoma en las fuentes medievales, Bellaterra, Institut d’Estudis Medievals, 2017.Daniel, N., Islam and the West: the making of an image, Oxford, Oneworld Pub., 1993.Eckhart, A., “Le cercueil flottant de Mahomet”, en Mélanges de philologie romane et de littérature offerts à Ernest Hoepffener, París, Les Belles Lettres, 1949, pp. 77-88.Eisenberg, D., “Cervantes, autor de la Topografía e historia general de Argel publicada por Diego de Haedo”, Cervantes: Bulletin of the Cervantes Society of America, vol. 16, núm. 1 (1996), pp. 32-53.Evans, R. J. W. y Marr, A. (eds.), Curiosity and wonder from the Renaissance to the Enlightenment, Abingdon y Nueva York, Routledge, 2016.Fierro, M., “Prácticas y creencias religiosas en al-Andalus”, Al-Qantara, vol. 13, núm. 2 (1992), pp. 463-474.Garcés, M. A., Cervantes en Argel: historia de un cautivo, Madrid, Gredos, 2005.— “Introduction”, en Sosa, A. de, An early modern dialogue with Islam: Antonio de Sosa’s Topography of Algiers (1612), Notre Dame, Ind., University of Notre Dame Press, 2011, pp. 1-78.García-Arenal, M., Inquisición y moriscos: los procesos del Tribunal de Cuenca, 3ª ed., Madrid, Siglo XXI, 1987.García Sanjuán, A., “La celebración de la navidad en al-Andalus y la convivencia entre cristianos y musulmanes”, en Miura, J. M. (ed.), Te cuento la navidad: visiones y miradas sobre las fiestas de invierno, Sevilla, Aconcagua, 2011, pp. 43-49.Gonzalbes, C., “Un ensayo para la catalogación de los amuletos de plomo andalusíes”, Boletín Arqueológico Medieval, 12 (2005), pp. 7-17.González de Clavijo, R., Embajada a Tamorlán, edición de F. 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Environments containing significant concentration of NaCl such as salt lakes harbor extremophiles microorganisms which have a great biotechnology interest. To explore the diversity of Bacteria in Chott Tinsilt (Algeria), an isolation program was performed. Water samples were collected from the saltern during the pre-salt harvesting phase. This Chott is high in salt (22.47% (w/v). Seven halophiles Bacteria were selected for further characterization. The isolated strains were able to grow optimally in media with 10–25% (w/v) total salts. Molecular identification of the isolates was performed by sequencing the 16S rRNA gene. It showed that these cultured isolates included members belonging to the Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus genera with less than 98% of similarity with their closest phylogenetic relative. The halophilic bacterial isolates were also characterized for the production of biosurfactant and industrially important enzymes. Most isolates produced hydrolases and biosurfactants at high salt concentration. In fact, this is the first report on bacterial strains (A4 and B4) which were a good biosurfactant and coagulase producer at 20% and 25% ((w/v)) NaCl. In addition, the biosurfactant produced by the strain B4 at high salinity (25%) was also stable at high temperature (30-100°C) and high alkalinity (pH 11).Key word: Salt Lake, Bacteria, biosurfactant, Chott, halophiles, hydrolases, 16S rRNAINTRODUCTIONSaline lakes cover approximately 10% of the Earth’s surface area. The microbial populations of many hypersaline environments have already been studied in different geographical regions such as Great Salt Lake (USA), Dead Sea (Israel), Wadi Natrun Lake (Egypt), Lake Magadi (Kenya), Soda Lake (Antarctica) and Big Soda Lake and Mono Lake (California). Hypersaline regions differ from each other in terms of geographical location, salt concentration and chemical composition, which determine the nature of inhabitant microorganisms (Gupta et al., 2015). Then low taxonomic diversity is common to all these saline environments (Oren et al., 1993). Halophiles are found in nearly all major microbial clades, including prokaryotic (Bacteria and Archaea) and eukaryotic forms (DasSarma and Arora, 2001). They are classified as slight halophiles when they grow optimally at 0.2–0.85 M (2–5%) NaCl, as moderate halophiles when they grow at 0.85–3.4 M (5–20%) NaCl, and as extreme halophiles when they grow at 3.4–5.1 M (20–30%) NaCl. Hyper saline environments are inhabited by extremely halophilic and halotolerant microorganisms such as Halobacillus sp, Halobacterium sp., Haloarcula sp., Salinibacter ruber , Haloferax sp and Bacillus spp. (Solomon and Viswalingam, 2013). There is a tremendous demand for halophilic bacteria due to their biotechnological importance as sources of halophilic enzymes. Enzymes derived from halophiles are endowed with unique structural features and catalytic power to sustain the metabolic and physiological processes under high salt conditions. Some of these enzymes have been reported to be active and stable under more than one extreme condition (Karan and Khare, 2010). Applications are being considered in a range of industries such as food processing, washing, biosynthetic processes and environmental bioremediation. Halophilic proteases are widely used in the detergent and food industries (DasSarma and Arora, 2001). However, esterases and lipases have also been useful in laundry detergents for the removal of oil stains and are widely used as biocatalysts because of their ability to produce pure compounds. Likewise, amylases are used industrially in the first step of the production of high fructose corn syrup (hydrolysis of corn starch). They are also used in the textile industry in the de-sizing process and added to laundry detergents. Furthermore, for the environmental applications, the use of halophiles for bioremediation and biodegradation of various materials from industrial effluents to soil contaminants and accidental spills are being widely explored. In addition to enzymes, halophilic / halotolerants microorganisms living in saline environments, offer another potential applications in various fields of biotechnology like the production of biosurfactant. Biosurfactants are amphiphilic compounds synthesized from plants and microorganisms. They reduce surface tension and interfacial tension between individual molecules at the surface and interface respectively (Akbari et al., 2018). Comparing to the chemical surfactant, biosurfactant are promising alternative molecules due to their low toxicity, high biodegradability, environmental capability, mild production conditions, lower critical micelle concentration, higher selectivity, availability of resources and ability to function in wide ranges of pH, temperature and salinity (Rocha et al., 1992). They are used in various industries which include pharmaceuticals, petroleum, food, detergents, cosmetics, paints, paper products and water treatment (Akbari et al., 2018). The search for biosurfactants in extremophiles is particularly promising since these biomolecules can adapt and be stable in the harsh environments in which they are to be applied in biotechnology.OBJECTIVESEastern Algeria features numerous ecosystems including hypersaline environments, which are an important source of salt for food. The microbial diversity in Chott Tinsilt, a shallow Salt Lake with more than 200g/L salt concentration and a superficies of 2.154 Ha, has never yet been studied. The purpose of this research was to chemically analyse water samples collected from the Chott, isolate novel extremely or moderate halophilic Bacteria, and examine their phenotypic and phylogenetic characteristics with a view to screening for biosurfactants and enzymes of industrial interest.MATERIALS AND METHODSStudy area: The area is at 5 km of the Commune of Souk-Naâmane and 17 km in the South of the town of Aïn-Melila. This area skirts the trunk road 3 serving Constantine and Batna and the railway Constantine-Biskra. It is part the administrative jurisdiction of the Wilaya of Oum El Bouaghi. The Chott belongs to the wetlands of the High Plains of Constantine with a depth varying rather regularly without never exceeding 0.5 meter. Its length extends on 4 km with a width of 2.5 km (figure 1).Water samples and physico-chemical analysis: In February 2013, water samples were collected from various places at the Chott Tinsilt using Global Positioning System (GPS) coordinates of 35°53’14” N lat. and 06°28’44”E long. Samples were collected randomly in sterile polythene bags and transported immediately to the laboratory for isolation of halophilic microorganisms. All samples were treated within 24 h after collection. Temperature, pH and salinity were measured in situ using a multi-parameter probe (Hanna Instruments, Smithfield, RI, USA). The analytical methods used in this study to measure ions concentration (Ca2+, Mg2+, Fe2+, Na+, K+, Cl−, HCO3−, SO42−) were based on 4500-S-2 F standard methods described elsewhere (Association et al., 1920).Isolation of halophilic bacteria from water sample: The media (M1) used in the present study contain (g/L): 2.0 g of KCl, 100.0/200.0 g of NaCl, 1.0 g of MgSO4.7HO2, 3.0 g of Sodium Citrate, 0.36 g of MnCl2, 10.0 g of yeast extract and 15.0 g agar. The pH was adjusted to 8.0. Different dilutions of water samples were added to the above medium and incubated at 30°C during 2–7 days or more depending on growth. Appearance and growth of halophilic bacteria were monitored regularly. The growth was diluted 10 times and plated on complete medium agar (g/L): glucose 10.0; peptone 5.0; yeast extract 5.0; KH2PO4 5.0; agar 30.0; and NaCl 100.0/200.0. Resultant colonies were purified by repeated streaking on complete media agar. The pure cultures were preserved in 20% glycerol vials and stored at −80°C for long-term preservation.Biochemical characterisation of halophilic bacterial isolates: Bacterial isolates were studied for Gram’s reaction, cell morphology and pigmentation. Enzymatic assays (catalase, oxidase, nitrate reductase and urease), and assays for fermentation of lactose and mannitol were done as described by Smibert (1994).Optimization of growth conditions: Temperature, pH, and salt concentration were optimized for the growth of halophilic bacterial isolates. These growth parameters were studied quantitatively by growing the bacterial isolates in M1 medium with shaking at 200 rpm and measuring the cell density at 600 nm after 8 days of incubation. To study the effect of NaCl on the growth, bacterial isolates were inoculated on M1 medium supplemented with different concentration of NaCl: 1%-35% (w/v). The effect of pH on the growth of halophilic bacterial strains was studied by inoculating isolates on above described growth media containing NaCl and adjusted to acidic pH of 5 and 6 by using 1N HCl and alkaline pH of 8, 9, 10, 11 and 12 using 5N NaOH. The effect of temperature was studied by culturing the bacterial isolates in M1 medium at different temperatures of incubation (4°C–55°C).Screening of halophilic bacteria for hydrolytic enzymes: Hydrolase producing bacteria among the isolates were screened by plate assay on starch, tributyrin, gelatin and DNA agar plates respectively for amylase, lipase, protease and DNAse activities. Amylolytic activity of the cultures was screened on starch nutrient agar plates containing g/L: starch 10.0; peptone 5.0; yeast extract 3.0; agar 30.0; NaCl 100.0/250.0. The pH was 7.0. After incubation at 30 ºC for 7 days, the zone of clearance was determined by flooding the plates with iodine solution. The potential amylase producers were selected based on ratio of zone of clearance diameter to colony diameter. Lipase activity of the cultures was screened on tributyrin nutrient agar plates containing 1% (v/v) of tributyrin. Isolates that showed clear zones of tributyrin hydrolysis were identified as lipase producing bacteria. Proteolytic activity of the isolates was similarly screened on gelatin nutrient agar plates containing 10.0 g/L of gelatin. The isolates showing zones of gelatin clearance upon treatment with acidic mercuric chloride were selected and designated as protease producing bacteria. The presence of DNAse activity on plates was determined on DNAse test agar (BBL) containing 10%-25% (w/v) total salt. After incubation for 7days, the plates were flooded with 1N HCl solution. Clear halos around the colonies indicated DNAse activity (Jeffries et al., 1957).Milk clotting activity (coagulase activity) of the isolates was also determined following the procedure described (Berridge, 1952). Skim milk powder was reconstituted in 10 mM aqueous CaCl2 (pH 6.5) to a final concentration of 0.12 kg/L. Enzyme extracts were added at a rate of 0.1 mL per mL of milk. The coagulation point was determined by manual rotating of the test tube periodically, at short time intervals, and checking for visible clot formation.Screening of halophilic bacteria for biosurfactant production. Oil spread Assay: The Petridis base was filled with 50 mL of distilled water. On the water surface, 20μL of diesel and 10μl of culture were added respectively. The culture was introduced at different spots on the diesel, which is coated on the water surface. The occurrence of a clear zone was an indicator of positive result (Morikawa et al., 2000). The diameter of the oil expelling circles was measured by slide caliber (with a degree of accuracy of 0.02 mm).Surface tension and emulsification index (E24): Isolates were cultivated at 30 °C for 7 days on the enrichment medium containing 10-25% NaCl and diesel oil as the sole carbon source. The medium was centrifuged (7000 rpm for 20 min) and the surface tension of the cell-free culture broth was measured with a TS90000 surface tensiometer (Nima, Coventry, England) as a qualitative indicator of biosurfactant production. The culture broth was collected with a Pasteur pipette to remove the non-emulsified hydrocarbons. The emulsifying capacity was evaluated by an emulsification index (E24). The E24 of culture samples was determined by adding 2 mL of diesel oil to the same amount of culture, mixed for 2 min with a vortex, and allowed to stand for 24 h. E24 index is defined as the percentage of height of emulsified layer (mm) divided by the total height of the liquid column (mm).Biosurfactant stability studies : After growth on diesel oil as sole source of carbone, cultures supernatant obtained after centrifugation at 6,000 rpm for 15 min were considered as the source of crude biosurfactant. Its stability was determined by subjecting the culture supernatant to various temperature ranges (30, 40, 50, 60, 70, 80 and 100 °C) for 30 min then cooled to room temperature. Similarly, the effect of different pH (2–11) on the activity of the biosurfactant was tested. The activity of the biosurfactant was investigated by measuring the emulsification index (El-Sersy, 2012).Molecular identification of potential strains. DNA extraction and PCR amplification of 16S rDNA: Total cellular DNA was extracted from strains and purified as described by Sambrook et al. (1989). DNA was purified using Geneclean® Turbo (Q-BIO gene, Carlsbad, CA, USA) before use as a template in polymerase chain reaction (PCR) amplification. For the 16S rDNA gene sequence, the purified DNA was amplified using a universal primer set, forward primer (27f; 5′-AGA GTT TGA TCM TGG CTC AG) and a reverse primer (1492r; 5′-TAC GGY TAC CTT GTT ACG ACT T) (Lane, 1991). Agarose gel electrophoresis confirmed the amplification product as a 1400-bp DNA fragment.16S rDNA sequencing and Phylogenic analysis: Amplicons generated using primer pair 27f-1492r was sequenced using an automatic sequencer system at Macrogene Company (Seoul, Korea). The sequences were compared with those of the NCBI BLAST GenBank nucleotide sequence databases. Phylogenetic trees were constructed by the neighbor-joining method using MEGA version 5.05 software (Tamura et al., 2011). Bootstrap resembling analysis for 1,000 replicates was performed to estimate the confidence of tree topologies.Nucleotide sequence accession numbers: The nucleotide sequences reported in this work have been deposited in the EMBL Nucleotide Sequence Database. The accession numbers are represented in table 5.Statistics: All experiments were conducted in triplicates. Results were evaluated for statistical significance using ANOVA.RESULTSPhysico-chemical parameters of the collected water samples: The physicochemical properties of the collected water samples are reported in table 1. At the time of sampling, the temperature was 10.6°C and pH 7.89. The salinity of the sample, as determined in situ, was 224.70 g/L (22,47% (w/v)). Chemical analysis of water sample indicated that Na +and Cl- were the most abundant ions (table 1). SO4-2 and Mg+2 was present in much smaller amounts compared to Na +and Cl- concentration. Low levels of calcium, potassium and bicarbonate were also detected, often at less than 1 g/L.Characterization of isolates. Morphological and biochemical characteristic feature of halophilic bacterial isolates: Among 52 strains isolated from water of Chott Tinsilt, seven distinct bacteria (A1, A2, A3, A4, B1, B4 and B5) were chosen for further characterization (table 2). The colour of the isolates varied from beige, pale yellow, yellowish and orange. The bacterial isolates A1, A2, A4, B1 and B5 were rod shaped and gram negative (except B5), whereas A3 and B4 were cocci and gram positive. All strains were oxidase and catalase positive except for B1. Nitrate reductase and urease activities were observed in all the bacterial isolates, except B4. All the bacterial isolates were negative for H2S formation. B5 was the only strain positive for mannitol fermentation (table 2).We isolated halophilic bacteria on growth medium with NaCl supplementation at pH 7 and temperature of 30°C. We studied the effect of NaCl, temperature and pH on the growth of bacterial isolates. All the isolates exhibited growth only in the presence of NaCl indicating that these strains are halophilic. The optimum growth of isolates A3 and B1 was observed in the presence of 10% NaCl, whereas it was 15% NaCl for A1, A2 and B5. A4 and B4 showed optimum growth in the presence of 20% and 25% NaCl respectively. A4, B4 and B5 strains can tolerate up to 35% NaCl.The isolate B1 showed growth in medium supplemented with 10% NaCl and pH range of 7–10. The optimum pH for the growth B1 was 9 and they did not show any detectable growth at or below pH 6 (table 2), which indicates the alkaliphilic nature of B1 isolate. The bacterial isolates A1, A2 and A4 exhibited growth in the range of pH 6–10, while A3 and B4 did not show any growth at pH greater than 8. The optimum pH for growth of all strains (except B1) was pH 7.0 (table 2). These results indicate that A1, A2, A3, A4, B4 and B5 are neutrophilic in nature. All the bacterial isolates exhibited optimal growth at 30°C and no detectable growth at 55°C. Also, detectable growth of isolates A1, A2 and A4 was observed at 4°C. However, none of the bacterial strains could grow below 4°C and above 50°C (table 2).Screening of the halophilic enzymes: To characterize the diversity of halophiles able to produce hydrolytic enzymes among the population of microorganisms inhabiting the hypersaline habitats of East Algeria (Chott Tinsilt), a screening was performed. As described in Materials and Methods, samples were plated on solid media containing 10%-25% (w/v) of total salts and different substrates for the detection of amylase, protease, lipase and DNAse activities. However, coagulase activity was determined in liquid medium using milk as substrate (figure 3). Distributions of hydrolytic activity among the isolates are summarized in table 4.From the seven bacterial isolates, four strains A1, A2, A4 and B5 showed combined hydrolytic activities. They were positive for gelatinase, lipase and coagulase. A3 strain showed gelatinase and lipase activities. DNAse activities were detected with A1, A4, B1 and B5 isolates. B4 presented lipase and coagulase activity. Surprisingly, no amylase activity was detected among all the isolates.Screening for biosurfactant producing isolates: Oil spread assay: The results showed that all the strains could produce notable (>4 cm diameter) oil expelling circles (ranging from 4.11 cm to 4.67 cm). The average diameter for strain B5 was 4.67 cm, significantly (P < 0.05) higher than for the other strains.Surface tension and emulsification index (E24): The assimilation of hydrocarbons as the sole sources of carbon by the isolate strains led to the production of biosurfactants indicated by the emulsification index and the lowering of the surface tension of cell-free supernatant. Based on rapid growth on media containing diesel oil as sole carbon source, the seven isolates were tested for biosurfactant production and emulsification activity. The obtained values of the surface tension measurements as well as the emulsification index (E24) are shown in table 3. The highest reduction of surface tension was achieved with B5 and A3 isolates with values of 25.3 mN m−1 and 28.1 mN m−1 respectively. The emulsifying capacity evaluated by the E24 emulsification index was highest in the culture of isolate B4 (78%), B5 (77%) and A3 (76%) as shown in table 3 and figure 2. These emulsions were stable even after 4 months. The bacteria with emulsification indices higher than 50 % and/or reduction in the surface tension (under 30 mN/m) have been defined as potential biosurfactant producers. Based on surface tension and the E24 index results, isolates B5, B4, A3 and A4 are the best candidates for biosurfactant production. It is important to note that, strains B4 and A4 produce biosurfactant in medium containing respectively 25% and 20% (w/v) NaCl.Stability of biosurfactant activities: The applicability of biosurfactants in several biotechnological fields depends on their stability at different environmental conditions (temperatures, pH and NaCl). For this study, the strain B4 appear very interesting (It can produce biosurfactant at 25 % NaCl) and was choosen for futher analysis for biosurfactant stability. The effects of temperature and pH on the biosurfactant production by the strain B4 are shown in figure 4.biosurfactant in medium containing respectively 25% and 20% (w/v) NaCl.Stability of biosurfactant activities: The applicability of biosurfactants in several biotechnological fields depends on their stability at different environmental conditions (temperatures, pH and NaCl). For this study, the strain B4 appear very interesting (It can produce biosurfactant at 25 % NaCl) and was chosen for further analysis for biosurfactant stability. The effects of temperature and pH on the biosurfactant production by the strain B4 are shown in figure 4. The biosurfactant produced by this strain was shown to be thermostable giving an E-24 Index value greater than 78% (figure 4A). Heating of the biosurfactant to 100 °C caused no significant effect on the biosurfactant performance. Therefore, the surface activity of the crude biosurfactant supernatant remained relatively stable to pH changes between pH 6 and 11. At pH 11, the value of E24 showed almost 76% activity, whereas below pH 6 the activity was decreased up to 40% (figure 4A). The decreases of the emulsification activity by decreasing the pH value from basic to an acidic region; may be due to partial precipitation of the biosurfactant. This result indicated that biosurfactant produced by strain B4 show higher stability at alkaline than in acidic conditions.Molecular identification and phylogenies of potential isolates: To identify halophilic bacterial isolates, the 16S rDNA gene was amplified using gene-specific primers. A PCR product of ≈ 1.3 kb was detected in all the seven isolates. The 16S rDNA amplicons of each bacterial isolate was sequenced on both strands using 27F and 1492R primers. The complete nucleotide sequence of 1336,1374, 1377,1313, 1305,1308 and 1273 bp sequences were obtained from A1, A2, A3, A4, B1, B4 and B5 isolates respectively, and subjected to BLAST analysis. The 16S rDNA sequence analysis showed that the isolated strains belong to the genera Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus as shown in table 5. The halophilic isolates A2 and A4 showed 97% similarity with the Halomonas variabilis strain GSP3 (accession no. AY505527) and the Halomonas sp. M59 (accession no. AM229319), respectively. As for A1, it showed 96% similarity with the Halomonas venusta strain GSP24 (accession no. AY553074). B1 and B4 showed for their part 96% similarity with the Salinivibrio costicola subsp. alcaliphilus strain 18AG DSM4743 (accession no. NR_042255) and the Planococcus citreus (accession no. JX122551), respectively. The bacterial isolate B5 showed 98% sequence similarity with the Halobacillus trueperi (accession no. HG931926), As for A3, it showed only 95% similarity with the Staphylococcus arlettae (accession no. KR047785). The 16S rDNA nucleotide sequences of all the seven halophilic bacterial strains have been submitted to the NCBI GenBank database under the accession number presented in table 5. The phylogenetic association of the isolates is shown in figure 5.DICUSSIONThe physicochemical properties of the collected water samples indicated that this water was relatively neutral (pH 7.89) similar to the Dead Sea and the Great Salt Lake (USA) and in contrast to the more basic lakes such as Lake Wadi Natrun (Egypt) (pH 11) and El Golea Salt Lake (Algeria) (pH 9). The salinity of the sample was 224.70 g/L (22,47% (w/v). This range of salinity (20-30%) for Chott Tinsilt is comparable to a number of well characterized hypersaline ecosystems including both natural and man-made habitats, such as the Great Salt Lake (USA) and solar salterns of Puerto Rico. Thus, Chott Tinsilt is a hypersaline environment, i.e. environments with salt concentrations well above that of seawater. Chemical analysis of water sample indicated that Na +and Cl- were the most abundant ions, as in most hypersaline ecosystems (with some exceptions such as the Dead Sea). These chemical water characteristics were consistent with the previously reported data in other hypersaline ecosystems (DasSarma and Arora, 2001; Oren, 2002; Hacěne et al., 2004). Among 52 strains isolated from this Chott, seven distinct bacteria (A1, A2, A3, A4, B1, B4 and B5) were chosen for phenotypique, genotypique and phylogenetique characterization.The 16S rDNA sequence analysis showed that the isolated strains belong to the genera Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus. Genera obtained in the present study are commonly occurring in various saline habitats across the globe. Staphylococci have the ability to grow in a wide range of salt concentrations (Graham and Wilkinson, 1992; Morikawa et al., 2009; Roohi et al., 2014). For example, in Pakistan, Staphylococcus strains were isolated from various salt samples during the study conducted by Roohi et al. (2014) and these results agreed with previous reports. Halomonas, halophilic and/or halotolerant Gram-negative bacteria are typically found in saline environments (Kim et al., 2013). The presence of Planococcus and Halobacillus has been reported in studies about hypersaline lakes; like La Sal del Rey (USA) (Phillips et al., 2012) and Great Salt Lake (Spring et al., 1996), respectively. The Salinivibrio costicola was a representative model for studies on osmoregulatory and other physiological mechanisms of moderately halophilic bacteria (Oren, 2006).However, it is interesting to note that all strains shared less than 98.7% identity (the usual species cut-off proposed by Yarza et al. (2014) with their closest phylogenetic relative, suggesting that they could be considered as new species. Phenotypic, genetic and phylogenetic analyses have been suggested for the complete identification of these strains. Theses bacterial strains were tested for the production of industrially important enzymes (Amylase, protease, lipase, DNAse and coagulase). These isolates are good candidates as sources of novel enzymes with biotechnological potential as they can be used in different industrial processes at high salt concentration (up to 25% NaCl for B4). Prominent amylase, lipase, protease and DNAase activities have been reported from different hypersaline environments across the globe; e.g., Spain (Sánchez‐Porro et al., 2003), Iran (Rohban et al., 2009), Tunisia (Baati et al., 2010) and India (Gupta et al., 2016). However, to the best of our knowledge, the coagulase activity has never been detected in extreme halophilic bacteria. Isolation and characterization of crude enzymes (especially coagulase) to investigate their properties and stability are in progress.The finding of novel enzymes with optimal activities at various ranges of salt concentrations is of great importance. Besides being intrinsically stable and active at high salt concentrations, halophilic and halotolerant enzymes offer great opportunities in biotechnological applications, such as environmental bioremediation (marine, oilfiel) and food processing. The bacterial isolates were also characterized for production of biosurfactants by oil-spread assay, measurement of surface tension and emulsification index (E24). There are few reports on biosurfactant producers in hypersaline environments and in recent years, there has been a greater increase in interest and importance in halophilic bacteria for biomolecules (Donio et al., 2013; Sarafin et al., 2014). Halophiles, which have a unique lipid composition, may have an important role to play as surface-active agents. The archae bacterial ether-linked phytanyl membrane lipid of the extremely halophilic bacteria has been shown to have surfactant properties (Post and Collins, 1982). Yakimov et al. (1995) reported the production of biosurfactant by a halotolerant Bacillus licheniformis strain BAS 50 which was able to produce a lipopeptide surfactant when cultured at salinities up to 13% NaCl. From solar salt, Halomonas sp. BS4 and Kocuria marina BS-15 were found to be able to produce biosurfactant when cultured at salinities of 8% and 10% NaCl respectively (Donio et al., 2013; Sarafin et al., 2014). In the present work, strains B4 and A4 produce biosurfactant in medium containing respectively 25% and 20% NaCl. To our knowledge, this is the first report on biosurfactant production by bacteria under such salt concentration. Biosurfactants have a wide variety of industrial and environmental applications (Akbari et al., 2018) but their applicability depends on their stability at different environmental conditions. The strain B4 which can produce biosurfactant at 25% NaCl showed good stability in alkaline pH and at a temperature range of 30°C-100°C. Due to the enormous utilization of biosurfactant in detergent manufacture the choice of alkaline biosurfactant is researched (Elazzazy et al., 2015). On the other hand, the interesting finding was the thermostability of the produced biosurfactant even after heat treatment (100°C for 30 min) which suggests the use of this biosurfactant in industries where heating is of a paramount importance (Khopade et al., 2012). To date, more attention has been focused on biosurfactant producing bacteria under extreme conditions for industrial and commercial usefulness. In fact, the biosurfactant produce by strain B4 have promising usefulness in pharmaceutical, cosmetics and food industries and for bioremediation in marine environment and Microbial enhanced oil recovery (MEOR) where the salinity, temperature and pH are high.CONCLUSIONThis is the first study on the culturable halophilic bacteria community inhabiting Chott Tinsilt in Eastern Algeria. Different genera of halotolerant bacteria with different phylogeneticaly characteristics have been isolated from this Chott. Culturing of bacteria and their molecular analysis provides an opportunity to have a wide range of cultured microorganisms from extreme habitats like hypersaline environments. Enzymes produced by halophilic bacteria show interesting properties like their ability to remain functional in extreme conditions, such as high temperatures, wide range of pH, and high salt concentrations. These enzymes have great economical potential in industrial, agricultural, chemical, pharmaceutical, and biotechnological applications. Thus, the halophiles isolated from Chott Tinsilt offer an important potential for application in microbial and enzyme biotechnology. In addition, these halo bacterial biosurfactants producers isolated from this Chott will help to develop more valuable eco-friendly products to the pharmacological and food industries and will be usefulness for bioremediation in marine environment and petroleum industry.ACKNOWLEDGMENTSOur thanks to Professor Abdelhamid Zoubir for proofreading the English composition of the present paper.CONFLICT OF INTERESTThe authors declare that they have no conflict of interest.Akbari, S., N. H. Abdurahman, R. M. Yunus, F. Fayaz and O. R. Alara, 2018. Biosurfactants—a new frontier for social and environmental safety: A mini review. Biotechnology research innovation, 2(1): 81-90.Association, A. P. H., A. W. W. Association, W. P. C. 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Abstract Background Glycated albumin (GA) is an alternate marker of glucose control that is recommended in situations when hemoglobin A1c is unreliable. One of us (JPC) has examined its use in pregnant women with diabetes since 2019 and regularly observed GA values of 8.0–19.4%, with occasional outliers. However, in a recently recruited cohort of 149 women with and without gestational diabetes, 58% of the samples showed GA results &lt;8.0%, despite no apparent physiological differences. Here we further investigate a subset of these samples using a liquid chromatography-mass spectrometry method (LC-MS/MS). Methods Glycated serum proteins (GSP) were measured using the GlycoGap kit (Diazyme Labs, Poway, CA), an enzymatic method, and subsequently converted to percent glycated albumin (GA%) using the equation provided by the manufacturer.The LC-MS/MS assay was performed according to a previously published protocol. The samples were first reduced and alkylated, then spiked with a blend of two isotope-labeled standards and digested using Glu-C enzyme. The resulting digests were analyzed using the QTRAP 6500+ (AB Sciex, Foster City, CA) coupled with a LC system (Shimadzu, Japan) in MRM mode to provide values representing the percentage of glycated to non-glycated Lys-525 in albumin (MSGA%).Samples from a previously collected cohort (A, n = 78) of pregnant women with and without diabetes, with GA% range of 8.5–24.2%, were analyzed using both methods, and a standard curve (MSGA% vs GA%) was established. Thirty-eight samples with GA &lt;8.0% (cohort B) were then analyzed using the LC-MS/MS method, and the standard curve was used to generate the expected enzymatic GA% results. Results The comparison of the LC-MS/MS and Diazyme methods resulted in a straight-line equation (r2 = 0.84, slope = 1.81, y-intercept = 0.79). For the cohort B samples analyzed by LC-MS/MS, the median MSGA% result was 5.89% (range: 4.32–8.95%). Based on the equation derived from the method comparison (MSGA% vs GA%), the expected median value with the Diazyme enzymatic method would have been 11.48% (range: 8.63–17.02%). These GA% values are well within the typically expected range. Conclusion LC-MS/MS results and GA% calculated from a method comparison curve were within the expected range while original enzymatic results were not, suggesting the presence of an interfering substance. Our hypothesis is that prenatal supplements may lead to elevated ascorbic acid, which is known to cause interference in certain enzymatic assays. We are continuing to investigate this possibility.

Oilseed rape (Brassica napus L.) is one of the most important oilseed crops in China. It is widely cultivated in China, with winter oilseed rape in Yangtze River basin and in southern China, and spring oilseed rape in northern China. In August 2017, a survey for Leptosphaeria spp. on spring oilseed rape was conducted in Minle county, Zhangye city, Gansu Province, China. The symptoms typical of blackleg on basal stems of oilseed rape were observed in the field. A large number of black fruiting bodies (pycnidia) were present on the lesions (Fig. 1A). The disease incidence of basal stem infection in the surveyed field was 19%. A total of 19 diseased stems were collected to isolate the pathogen. After surface sterilizing (75% ethanol for 30 s, 5% NaOCl for 60 s, followed by rinsing in sterilized water three times), diseased tissues were cultured on acidified potato dextrose agar (PDA) plates at 20°C for 7 days. Twelve fungal isolates were obtained. All fungal isolates produced typical tan pigment on PDA medium, and produced pycnidia after two weeks (Fig. 1B). Colony morphological characteristics indicated that these isolates might belong to Leptosphaeria biglobosa. To confirm identification, multiple PCR was conducted using the species-specific primers LmacF, LbigF, LmacR (Liu et al. 2006). Genomic DNA of each isolate was extracted using the cetyltrimethylammonium bromide (CTAB) method. DNA samples of L. maculans isolate UK-1 and L. biglobosa isolate W10 (Cai et al. 2015) were used as references. Only a 444-bp DNA band was detected in all 12 isolates and W10, whereas a 333-bp DNA band was detected only in the UK-1 isolate (Fig. 1C). PCR results suggested that these 12 isolates all belong to L. biglobosa. In addition, the internal transcribed spacer (ITS) region of these 12 isolates was analyzed for subspecies identification (Vincenot et al. 2008). Phylogenetic analysis based on ITS sequence showed that five isolates (Lb1134, Lb1136, Lb1138, Lb1139 and Lb1143) belonged to L. biglobosa ‘brassicae’ (Lbb) with 78% bootstrap support, and the other seven isolates (Lb1135, Lb1137, Lb1140, Lb1141, Lb1142, Lb1144 and Lb1145) belonged to L. biglobosa ‘canadensis’ (Lbc) with 95% bootstrap support (Fig. 1D). Two Lbb isolates (Lb1134 and Lb1136) and two Lbc isolates (Lb1142 and Lb1144) were randomly selected for pathogenicity testing on B. napus cultivar Zhongshuang No. 9 (Wang et al. 2002). Conidial suspensions (10 μL, 1 × 107 conidia mL-1) of these four isolates were inoculated on needle-wounded cotyledons (14-day-old seedling), with 10 cotyledons (20 wounded sites) per isolate. A further 10 wounded cotyledons were inoculated with water and served as controls. Seedlings were maintained in a growth chamber at 20°C with 100% relative humidity and a 12-h photoperiod. After 7 days, cotyledons inoculated with the four isolates showed necrotic lesions in the inoculated wounds. Control cotyledons had no symptoms (Fig. 2). Fungi re-isolated from the infected cotyledons showed similar colony morphology as the original isolates. Therefore, L. biglobosa ‘brassicae’ and L. biglobosa ‘canadensis’ appear to be the pathogens causing the observed blackleg symptoms on spring oilseed rape in Gansu, China. In previous studies, L. biglobosa ‘brassicae’ has been found in many crops in China, including oilseed rape (Liu et al. 2014; Cai et al. 2015), Chinese radish (Raphanus sativus) (Cai et al. 2014a), B. campestris ssp. chinensis var. purpurea (Cai et al. 2014b), broccoli (B. oleracea var. italica) (Luo et al. 2018), ornamental kale (B. oleracea var. acephala) (Zhou et al. 2019a), B. juncea var. multiceps (Zhou et al. 2019b), B. juncea var. tumida (Deng et al. 2020) and Chinese cabbage (B. rapa subsp. pekinensis) (Yu et al. 2021 accepted). To the best of our knowledge, this is the first report of L. biglobosa ‘canadensis’ causing blackleg on B. napus in China.

The Trp64Arg (rs4994) polymorphism in codon 64 of ADRB3 (beta3-adrenergic receptor) gene is involved in the regulation of energy metabolism. This study optimizes the genotyping method of ADRB3 rs4994 polymorphism and determines the allele and genotype frequencies of this polymorphism in 3-5 years old children in Hanoi. A cross-sectional study was conducted on 100 three-to-five-year-old Hanoi children, using DNA extraction method from cheek mucosa cells. The genotyping of this polymorphism was performed by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method. The study optimized the method of genotyping of ADRB3 rs4994 polymorphism with Mval enzyme. In 3-5 years old children in Hanoi, T/T genotype accounted for the highest proportion (78%), C/C genotype accounted for the lowest proportion (3%). T and C allele frequencies were 0.785 and 0.125, ​​respectively. The genotypes observed were in agreement with those expected under Hardy-Weinberg equilibrium. It is necessary to apply the genotyping method and genetic distribution for genotyping the ADRB3 rs4994 polymorphism in large-scale studies in Vietnam. Keywords ADRB3, rs4994, genotyping, RFLP. References [1] T. Rankinen, A. Zuberi, Y.C. Chagnon, S.J. Weisnagel, G. Argyropoulos, B. Walts and C. Bouchard, The human obesity gene map: the 2005 update, Obesity. 14 (2006) 529-644. https://doi.org/10.1038/oby.2006.71. [2] S. Krief, F. Lönnqvist, S. Raimbault, B. Baude, A. Van Spronsen and P. Arner, Tissue distribution of β3-adrenergic receptor mRNA in man, J Clin Invest. 91 (1993) 344-349. Doi: 10.2337/db18- https://doi.org/10.2337/db18- 0462.[3] F.Lönnqvist, S. Krief, A.D. Strsberg, S. Nyberg, L.J. Emorine and P. Amer, A pathogenic role of visceral fat β3-adrenoceptors in obesity, J Clin Invest. 95 (1995) 1109-1116. Doi: https://doi.org/10.1172/JCI117758.[4] J. Walston, K. Silver, C. 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Daghistani, A. Eldali, Z.K. Hassan, M.H. Elamin and A. Warsy, ADRB3 polymorphism rs4994 (Trp64Arg) associates significantly with bodyweight elevation and dyslipidaemias in Saudis but not rs1801253 (Arg389Gly) polymorphism in ARDB1, Lipids in health and disease. 17 (2018) 58-66. https://doi.org/10.1186/s12944-018-0679-7[9] J. Walston, K. Silver, C. Bogardus, W.C. Knowler, F.S. Celi, S. Austin, Time of onset of non-insulin-dependent diabetes mellitus and genetic variation in the beta 3-adrenergic-receptor gene, N Engl J Med. 333 (1995) 343-347. https://doi.org/10.1056/NEJM199508103330603[10] K. Clement, C. Vaisse, B.S. Manning, A. Basdevant, B. Guy-Granda and J. Ruiz, Genetic variation in the beta 3-adrenergic receptor and an increased capacity to gain weight in patients with morbid obesity, N Engl J Med. 333 (1995) 352-354. https://doi.org/10.1056/NEJM199508103330605[11] H. Kim-Motoyama, K. Yasuda, T. Yamaguchi, N. Yamada, T. Katakura and A.R. Shuldiner, A mutation of the β3-adrenergic receptor is associated with visceral obesity but decreased serum triglyceride, Diabetologia. 40 (1997) 469-472. [12] S.G. Malik, M.R. Saraswati, K. Suastika, H. Trimarsanto, S. Oktavianthi and H. Sudoyo, Association of beta3-adrenergic receptor (ADRB3) Trp64Arg gene polymorphism with obesity and metabolic syndrome in the Balinese: a pilot study, BMC research notes 4 (2011) 167-173. https://doi.org/10.1186/1756-0500-4-167[13] E. Widen, M. Lehto, T. Kanninen, J. Walston, A.R. Shuldiner, L.C. Groop, Association of a polymorphism in the beta 3-adrenergic-receptor gene with features of the insulin resistance syndrome in Finns, N Engl J Med 333 (1995) 348-351. https://doi.org/10.1056/NEJM199508103330604[14] A.J. Biery, S.O.E. Ebbesson, A.R. Shuldiner, B.B. Boyer, The β 3-adrenergic receptor TRP64ARG polymorphism and obesity in Alaskan Eskimos, International journal of obesity. 21 (1997) 1176-1179.[15] X. Yuan, K. Yamada, K.I. Koyama, F. Ichikawa, S. Ishiyama, A. Koyanagi and K. Nonaka, β3-adrenergic receptor gene polymorphism is not a major genetic determinant of obesity and diabetes in Japanese general population, Diabetes research and clinical practice, 37 (1997) 1-7. https://doi.org/10.1016/S0168-8227(97)00064-8[16] N. Sakane, T. Yoshida, T. Umekawa, A. Kogure, Y. Takakura and M. Kondo, Effects of Trp64Arg mutation in the β3-adrenergic receptor gene on weight loss, body fat distribution, glycemic control, and insulin resistance in obese type 2 diabetic patients, Diabetes care 20 (1997) 1887-1890. https://doi.org/10.2337/diacare.20.12.1887[17] R. Bracale, F. Pasanisi, G. Labruna, C. Finelli, C. Nardelli, P. Buono and G. Oriani, Metabolic syndrome and ADRB3 gene polymorphism in severely obese patients from South Italy, European journal of clinical nutrition. 61 (2007) 1213-1219. https://doi.org/10.1038/sj.ejcn.1602640[18] G.T. Marth, E. Czabarka, J. Murvai, S.T. Sherry, The Allele Frequency Spectrum in Genome-Wide Human Variation Data Reveals Signals of Differential Demographic History in Three Large World Populations, Genetics. 166 (2004) 351-372. [19] National Center for Biotechnology Information. http://hapmap. ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?do_not_redirect&rs=rs4994 (accessed 6 March 2019).[20] L.T. Tuyet, B.T.N. Anh, T.Q. Binh, Application of restriction fragment length polymorphirm method for genotyping BDNF rs6265 polymorphism. Journal of science of HNUE, Chemical and Biological Science, 59 (2014) 123-130. https://doi.org/10.15625/0866-7160/v37n1se.6095[21] L.T. Tuyết, T.Q. Bình, Bước đầu nghiên cứu đa hình nucleotide đơn MC4R-rs17782313 ở trẻ 5-6 tuổi Hà Nội bằng phương pháp PCR-RFLP. Tạp chí Khoa học Đại học Quốc gia Hà Nội, chuyên san KHTN và Công nghệ. 31 (2015) 57-63. https://js.vnu.edu.vn/NST/article/view/84[22] L.T. Tuyết, T.Q. Bình, Associations of Single Nucleotide Polymorphism rs17782313 in Melanocortin 4 Receptor Gene with Anthropometric Indices in Normal and Obesity Primary School Children in Hanoi. NU Journal of Science: Medical and Pharmaceutical Sciences. 34 (2018) 1-7. https://doi.org/10.25073/2588 - https://doi.org/10.25073/2588-1132/vnumps.4107

Abstract Background and Aims Chronic kidney disease (CKD) and heart failure share a complex, bidirectional interaction, and are therefore an area of crucial interest in nephrology due to their prevalence and effect on mortality. It is basic to identify the risk of heart failure in patients with CKD as soon as possible to improve treatment outcomes and complications. It is important to note that while there is wide experience in prediction of heart failure, the use of models based on artificial intelligence is practically non-existent with this objective in mind. Based on the experience of our group in the prediction of mortality in patients with CKD and in CKD progression with machine learning algorithms, this study aims to predict the onset of heart failure in CKD patients. This study addresses this issue using machine learning techniques to offer new opportunities for accurate prediction of cardiac disease. This approach facilitates the individualization of risk assessment and allows the implementation of early and personalized interventions that can significantly influence the management of both diseases. Method Design: Retrospective observational study of a historical cohort from the Registry of Renal Patients of Catalonia (RMRC) and the Catalan Agency for Health Quality and Evaluation. Sample: 5000 patients with CKD, selected for having the minimum number of missing data. Follow-up: 10 years, from January 2010 to December 2020. Inclusion criteria: patients older than 18 years with CKD. Variables: 333 variables: a) Age, gender, weight, height (4); b) Status on the transplant waiting list (2); c) Renal replacement therapy (3); d) Diagnoses (ICD-10) excluding group I50 diagnoses, used as the outcome (146); e) Laboratory variables (78); f) Pharmacological treatments (100). Label and models: The algorithm used was the Light Gradient Boosting Machine (LGBM), with which four models were trained to perform a binary classification to predict whether a patient would develop heart failure in a period of 3, 4, 5 or 6 years. Label 0 was used to represent the absence of heart failure, while label 1 was used to represent the presence of heart failure. The methodology used to train the models was as follows: 1. Pre-processing of the data to manage missing data and data errors. 2. Creating different datasets, one for each prediction horizon. 3. Training and evaluating the LGBM models (with 5-fold CV). Results Age: 63 ± 13 years, Gender: 66% male and 34% were female. Different prediction horizons were tested, and the best results were obtained for 6 years where was achieved an area under the curve of 0.86 and an accuracy of 0.76. The 5 variables with major relevance according to SHAP values (SHapley Additive exPlanations) and in this order are: age, CKD diagnosis, hypertension, haemoglobin, and lymphocytes. The results presented in Fig. 1 and Table 1 correspond to the mean obtained for the 5-folds of the Group Cross-Validation. Conclusion –Advanced artificial intelligence algorithms present promising results for the prediction of heart failure in CKD patients. –This enables early, personalized, and more effective treatments for CKD patients.

This essay aims to elucidate issues related to the origin, definition and contributions of number sense to mathematical learning. We discuss the controversies involved in the origin and conceptualization of the construct, as well as the understanding of number sense that characterizes this work. We present studies that highlight the relevant role that number sense plays in the construction of initial mathematics and the relation that this construct has evidenced in children’s later mathematical performance. These findings are then related to educational actions developed by researchers in the field, showing the effectiveness of the investment in developing mathematical interventions. In conclusion, it is evidenced the controversy that characterizes the concept of number sense, which reflects the need for more research in this field. A strong predictive relationship between the construct and later mathematical knowledge is also highlighted, which has led to the realization of intervention studies in this field. Such studies have shown encouraging results for kindergarten and elementary school students who have difficulties in learning mathematics.ResumoEste ensaio tem como objetivo elucidar questões relativas à origem, definição e contribuições do senso numérico para a aprendizagem matemática. Abordam-se as controvérsias envolvidas na origem e conceituação do constructo, assim como a compreensão de senso numérico que caracteriza este trabalho. São apresentados estudos que destacam o papel relevante que o senso numérico desempenha para a construção da matemática inicial e a relação que este constructo tem evidenciado com o desempenho matemático posterior das crianças. Após, relaciona-se o que foi exposto às ações educacionais desenvolvidas por pesquisadores da área, mostrando a efetividade do investimento no desenvolvimento de intervenções matemáticas. Em conclusão, fica evidenciada a controvérsia que caracteriza a conceituação de senso numérico, o que reflete a necessidade e relevância de mais pesquisa nesta área. É destacada também a forte relação preditiva entre o constructo e o conhecimento matemático posterior, o que tem impulsionado a realização de estudos de intervenção neste campo, com resultados animadores, para alunos da Educação Infantil e dos anos iniciais do Ensino Fundamental que apresentam dificuldades de aprendizagem na matemática.ResumenEste ensayo tiene como objetivo dilucidar cuestiones relacionadas con el origen, la definición y las contribuciones del sentido numérico al aprendizaje matemático. Discutimos las controversias involucradas en el origen y la conceptualización del constructo, así como la comprensión del sentido numérico que caracteriza este trabajo. Presentamos estudios que destacan el papel relevante que juega el sentido numérico en la construcción de las matemáticas iniciales y la relación que este constructo ha evidenciado en el rendimiento matemático posterior de los niños. Estos hallazgos se relacionan luego con acciones educativas desarrolladas por investigadores en este campo, evidenciando la efectividad de los esfuerzos en el desarrollo de intervenciones matemáticas. En conclusión, queda evidenciada la controversia que caracteriza el concepto de sentido numérico, lo que refleja la necesidad de más investigación en este campo. También se destaca una fuerte relación predictiva entre este constructo y el conocimiento matemático posterior, lo que ha llevado a la realización de estudios de intervención en esta área. 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Previous studies contend that Philippines is still a ‘collectivist’ society (Cf. Hofstede Center; Cukur et al. 2004:613-634). In this collectivist or community-oriented society, individualism is not something that is highly valued. Being ‘individualistic’ is often associated to being narcissistic, loner, asocial, selfish, etc. However, one may ask whether the youth in the Philippines are not spared from this insidious culture of individualism, notwithstanding the seemingly dominant collective and communitarian character of the society. Although the overwhelming poverty is still the main problem in the Philippines, where according to Wostyn (2010:26) “only the wonderland of movies gives some respite to their consciousness of suffering and oppression”, the Filipino youth of today are also exposed to the consumeristic values of the ‘city’ and are not spared from the contradictions and insecurities posed by the pluralistic society. They are citizens of an increasing social and cultural pluralism characteristic of many liberal societies. Is it possible that individualism may also exist within this culture, especially among the younger generation? Is individualism slowly creeping in as caused by their exposure and easy access to modern technology, to higher education, mobility, interactions with other cultures, etc. Would this individualistic tendency have any influence on their religious beliefs, especially their belief on salvation? What would be the implications and challenges of these findings to the families in the Philippines? These are the questions we wish to answer in this study. 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Interactivity (or reciprocity) is a key dimension to assess the deliberative quality of online discussions. In quantitative content analyses, this dimension measures if participants engage in dialog with each other and refer to each other. Field of application/Theoretical foundation Most studies on online discussions draw on deliberative norms to measure the quality of their discourse (e.g., Esau et al., 2017; Friess et al., 2021; Rowe, 2015; Ziegele et al., 2020; Zimmermann, 2017). Deliberation is an important concept for the study of (political) online discussions (Ziegele et al., 2020). It focuses on a free and equal exchange of arguments to bridge social differences and legitimize political decisions (Dryzek et al., 2019; Fishkin, 1991, Habermas, 2015). Interactivity is a key dimension of deliberative quality, since deliberation is always a reciprocal and dialogical process (Goodin, 2000; Zimmermann, 2017). Participants engage in a dialogic exchange with each other, reflecting on other views and perspectives, and referring to each other (Friess et al., 2021; Ziegele et al., 2020). This reciprocal process includes both responding and listening (Barber, 1984; Graham, 2009). Interactivity is considered essential for desirable effects of deliberation such as learning, tolerance building and opinion change (Estlund & Landmore, 2018; Friess et al., 2021). References/Combination with other methods Besides quantitative content analyses, the (deliberative) quality of online discussions is examined with qualitative content analyses and discourse analyses (e.g., Graham & Witschge, 2003; Price & Capella, 2002). Furthermore, participants’ perceptions of the quality of online discussions are investigated with qualitative interviews (e.g., Engelke, 2019; Ziegele, 2016) or a combination of qualitative interviews and content analy­sis (Díaz Noci et al., 2012). Cross-references Interactivity is one of five dimensions of deliberative quality in this database written by the same author. Accordingly, there are overlaps with the entries on inclusivity, rationality, explicit civility, and storytelling regarding theoretical background, references/combinations with other methods, and some example studies. Information on Esau et al. (2017) Authors: Katharina Esau, Dennis Friess, & Christiane Eilders Research question: “How does platform design affect the level of deliberative quality?” (p. 323) Object of analysis: “We conducted a quantitative content analysis of user comments left in a news forum, on news websites, and on Facebook news pages concerning the same journalistic content on two topics […] A sample of news articles […] with related user comments, was drawn from the online platforms of four German news media […] The first step of the sampling process consisted of 18 news articles from which 3,341 comments were collected […] In the second step for each article, up to 100 sequential comments were randomly selected for content analysis, leading to a total sample of 1,801 comments (979 on Facebook, 591 on news websites, and 231 in the news forum)” (p. 331). Time frame of analysis: December 2015 Info about variables Level of analysis: individual comment Variables and reliability: see Table 1 Table 1: Variables and Reliability (Esau et al., 2017, pp. 332-333): Dimension Measure Definition RCA Cohen’s Kappa Reciprocity General engagement This measure captures whether a comment addresses another comment. .92 - Argumentative engagement This measure captures whether a comment addresses a specific argument made in another comment. .77 .542 Critical engagement This measure captures whether a comment is critical of another comment. .89 - n = 40; 12 coders Values: Dichotomous measures (yes, no) Information on Heinbach & Wilms (2022) Authors: Dominique Heinbach & Lena K. Wilms (Codebook by Dominique Heinbach, Marc Ziegele, & Lena K. Wilms) Research question: Which attributes differentiate moderated from unmoderated comments? Object of analysis: The quantitative content analysis was based on a stratified random sample of moderated and not moderated comments (N = 1.682) from the German online participation platform “#meinfernsehen202” [#myTV2021], a citizen participation platform to discuss the future of public broadcasting in Germany. Time frame of analysis: November 24, 2020 to March 3, 2021 Info about variables Level of analysis: User comment Variables and reliability: see Table 2 Table 2: Variables and reliability (Heinbach & Wilms, 2022) Dimension Measure Definition Krippendorff’s α Reciprocity Reference to other users or to the community Does the comment refer to at least one other user, a group of users, or all users in the community? .78 Reference to the content of other comments Does the comment refer to content, arguments or positions in other comments? .78 Critical reference Does the comment refer to other comments in a critical manner? .86 n = 159, 3 coders Values: All variables were coded on a four-point scale (1 = clearly not present; 2 = rather not present; 3 = rather present; 4 = clearly present). Detailed explanations and examples for each value are provided in the Codebook (in German). Codebook: in the appendix of this entry (in German) Information on Stromer-Galley (2007) Author: Jennifer Stromer-Galley Research question: The aim of the paper was developing a coding scheme for academics and practitioners of deliberation to systematically measure what happens during group deliberations (p. 1; p. 7). Object of analysis: The author conducted a secondary analysis of online group discussions (23 groups with 5-12 participants) in an experiment called “The Virtual Agora Project” at Carnegie Mellon Unversitiy in Pittsburgh, Pennsylvania. Participants attended the discussions from dormitory rooms that were equipped with a computer, headphones, and microphone. The group discussions were recorded and transcribed for analysis (pp. 7-8). Although strictly speaking the study does not analyze media content, the coding scheme has provided the basis for numerous other studies on the deliberative quality of online discussions (e.g., Rowe, 2015; Stroud et al., 2015; Ziegele et al., 2020). Time frame of analysis: Three weeks in July 2004 (p. 7). Info about variables Level of analysis: Level of the turn: Speaking contribution of a participant. Participants had to get “in line” to speak. When a speaker had finished their turn, the software activated the next speaker (max. 3 minutes per turn) (p. 8). Level of the thought: Coders segmented each turn into thought units before coding the categories. “A thought is defined as an utterance (from a single sentence to multiple sentences) that expresses an idea on a topic. A change in topic signaled a change in thought. A second indicator of a change in thought was a change in the type of talk. The distinct types of talk that this coding captured were the following: talk about the problem of public schools, talk about the process of the talk, talk about the process of the deliberation, and social talk” (p. 9). Variables an values: see Table 3 Reliability: “Two coders spent nearly two months developing and training with the coding scheme. The intercoder agreement measures […] were established from coding 3 of the 23 groups, which were randomly selected. […] Cohen’s Kappas of the coding elements described above are as follows: thought statements on the problem of public schools, .95; […] turn type (new topic, continuing self, responding to others) .97; meta-talk, 1.0 […]” (p. 13-14). Codebook: in the appendix (pp. 22-33) Table 3: Variables and values of the dimension “engagement” (Stromer-Galley, 2007, p.12; pp. 24-26). Category Level Description Value Definition Turn-type Turn Identify whether and to whom this turn is referring. Starting a new topic A new topic (not prompted by the moderator). Respond on topic A turn that is in response to a prior speaker or is on a topic that has been discussed. Includes responding to multiple speakers. Respond to moderator A turn that is a response to a prompt or question from the moderator. Continue self A turn that seems not to respond to anything a prior speaker said but to continue the current speaker’s ideas from one of his or her prior turns. Problem Thought Talk about the problem is talk that focuses on the issue under consideration. Question A genuine question directed to another speaker that is trying to seek information or an opinion from others. Metatalk Thought Metatalk is talk about the talk. It attempts to step back and assess what has transpired or is transpiring in the interaction. Consensus Consensus metatalk is talk about the speaker’s sense of consensus of the group (“I think we all agree that . . . .”), including an explanation for the collective’s opinions or the collective’s behavior (We’re asking you these questions because . .). Conflict Highlighting some disagreement or conflict in the group (“I sense some disagreement around . . . .”). Clarify own Clarify the speaker’s own opinion or fact statement (“what I’m trying to say is”). It’s an attempt to clarify what the speaker means. This will arise ONLY after they’ve provided an opinion, NOT a question, and are now trying to clarify their original opinion on the problem, likely because they believe someone has misunderstood them. Clarify other Clarify someone else’s argument/opinion or fact statement (“Sally, so, what you’re saying is . . . “). It is an attempt to clarify what someone else means. Pay attention to the use of another participants’ name. That can be a sign of metatalk of another’s position. Example studies Esau, K., Fleuß, D. & Nienhaus, S.‑M. (2021). Different Arenas, Different Deliberative Quality? Using a Systemic Framework to Evaluate Online Deliberation on Immigration Policy in Germany. Policy & Internet, 13(1), 86–112. https://doi.org/10.1002/poi3.232 Esau, K., Friess, D. & Eilders, C. (2017). Design Matters! An Empirical Analysis of Online Deliberation on Different News Platforms. Policy & Internet, 9(3), 321–342. https://doi.org/10.1002/poi3.154 Esau, K., Frieß, D. & Eilders, C. (2019). Online-Partizipation jenseits klassischer Deliberation: Eine Analyse zum Verhältnis unterschiedlicher Deliberationskonzepte in Nutzerkommentaren auf Facebook-Nachrichtenseiten und Beteiligungsplattformen. In I. Engelmann, M. Legrand & H. Marzinkowski (Hrsg.), Digital Communication Research: Bd. 6. Politische Partizipation im Medienwandel (S. 221–245). Friess, D., Ziegele, M. & Heinbach, D. (2021). Collective Civic Moderation for Deliberation? Exploring the Links between Citizens’ Organized Engagement in Comment Sections and the Deliberative Quality of Online Discussions. Political Communication, 38(5), 624–646. https://doi.org/10.1080/10584609.2020.1830322 Heinbach, D. & Wilms, L. K. (2022): Der Einsatz von Moderation bei #meinfernsehen2021 [The deployment of moderation at #meinfernsehen2021]. In: F. Gerlach, C. Eilders & K. Schmitz (Eds.): #meinfernsehen2021. Partizipationsverfahren zur Zukunft des öffentlich-rechtlichen Fernsehens. Baden-Baden: Nomos. Rowe, I. (2015). Deliberation 2.0: Comparing the Deliberative Quality of Online News User Comments Across Platforms. Journal of Broadcasting & Electronic Media, 59(4), 539–555. https://doi.org/10.1080/08838151.2015.1093482 Stromer Galley, J. (2007). Measuring Deliberation's Content: A Coding Scheme. Journal of Public Deliberation, 3(1), Article 12. Ziegele, M., Quiring, O., Esau, K. & Friess, D. (2020). Linking News Value Theory With Online Deliberation: How News Factors and Illustration Factors in News Articles Affect the Deliberative Quality of User Discussions in SNS’ Comment Sections. Communication Research, 47(6), 860-890. https://doi.org/10.1177/0093650218797884 Zimmermann, T. (2017). Digitale Diskussionen: Über politische Partizipation mittels Online-Leserkommentaren. Edition Politik: Bd. 44. transcript Verlag. http://www.content-select.com/index.php?id=bib_view&ean=9783839438886 Further references Barber, B. R. (1984). Strong democracy: Participatory politics for a new age. University of California Press. Díaz Noci, J., Domingo, D., Masip, P., Micó, J. L. & Ruiz, C. (2012). Comments in news, democracy booster or journalistic night­mare: Assessing the quality and dynamics of citizen debates in Catalan online new­spapers. #ISOJ, 2(1), 46–64. https://isoj.org/ wp-content/uploads/2016/10/ISOJ_Jour­nal_V2_N1_2012_Spring.pdf#page=46 Dryzek, J. S., Bächtiger, A., Chambers, S., Cohen, J., Druckman, J. N., Felicetti, A., Fishkin, J. S., Farrell, D. M., Fung, A., Gutmann, A., Landemore, H., Mansbridge, J., Marien, S., Neblo, M. A., Niemeyer, S., Setälä, M., Slothuus, R., Suiter, J., Thompson, D. & Warren, M. E. (2019). The crisis of democracy and the science of deliberation. Science (New York, N.Y.), 363(6432), 1144–1146. https://doi.org/10.1126/science.aaw2694 Engelke, K. M. (2019). Enriching the Conversation: Audience Perspectives on the Deliberative Nature and Potential of User Comments for News Media. Digital Journalism, 8(4), 1–20. https://doi.org/10.1080/21670811.2019.1680567 Estlund, D. & Landemore, H. (2018). The epistemic value of democratic deliberation. In A. Bächtiger, J. S. Dryzek, J. J. Mansbridge & M. E. Warren (Hrsg.), Oxford handbooks online. The Oxford handbook of deliberative democracy: An introduction (S. 113–131). Oxford University Press. Fishkin, J. S. (1991). Democracy and deliberation: New directions for democratic reform. Yale University Press. http://www.jstor.org/stable/10.2307/j.ctt1dt006v https://doi.org/10.2307/j.ctt1dt006v Goodin, R. E. (2000). Democratic Deliberation Within. Philosophy & Public Affairs, 29(1), 81–109. https://doi.org/10.1111/j.1088-4963.2000.00081.x Graham, T. (2009). What's Wife Swap got to do with it? Talking politics in the net-based public sphere Amsterdam: University of Amsterdam DOI: 10.13140/RG.2.1.3413.0088 Graham, T. & Witschge, T. (2003). In Search of Online Deliberation: Towards a New Method for Examining the Quality of Online Discussions. Communications, 28(2). https://doi.org/10.1515/comm.2003.012 Habermas, J. (2015). Between facts and norms: Contributions to a discourse theory of law and democracy (Reprinted.). Polity Press. Price, V. & Cappella, J. N. (2002). Online deliberation and its influence: The Electronic Dialogue Project in Campaign 2000. IT&Society, 1(1), 303–329. https://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.9.5945&rep=rep1&type=pdf Stroud, N. J., Scacco, J. M., Muddiman, A., & Curry, A. L. (2015). Changing Deliberative Norms on News Organizations' Facebook Sites. Journal of Computer-Mediated Communication, 20(2), 188–203. https://doi.org/10.1111/jcc4.12104 Ziegele, M. (2016). Nutzerkommentare als Anschlusskommunikation: Theorie und qualitative Analyse des Diskussionswerts von Online-Nachrichten [The Discussion Value of Online News. An Analysis of User Comments on News Platforms]. Springer VS.

IntroductionThe common origin of the adjective “corporeal” and the noun “corpse” in the Latin root corpus points to the value of mortuary practices for investigating how the human body is objectified. In post-mortem rituals, the body—formerly the manipulator of objects—becomes itself the object that is manipulated. Thus, these funerary rituals provide a type of double reflexivity, where the object and subject of manipulation can be used to reciprocally illuminate one another. To this extent, any consideration of corporeality can only benefit from a discussion of how the body is objectified through mortuary practices. This paper offers just such a discussion with respect to a selection of two contrasting mortuary practices, in the context of the prehistoric past and the Classical Era respectively. At the most general level, we are motivated by the same intellectual impulse that has stimulated expositions on corporeality, materiality, and incarnation in areas like phenomenology (Merleau-Ponty 77–234), Marxism (Adorno 112–119), gender studies (Grosz vii–xvi), history (Laqueur 193–244), and theology (Henry 33–53). That is to say, our goal is to show that the body, far from being a transparent frame through which we encounter the world, is in fact a locus where historical, social, cultural, and psychological forces intersect. On this view, “the body vanishes as a biological entity and becomes an infinitely malleable and highly unstable culturally constructed product” (Shilling 78). However, for all that the cited paradigms offer culturally situated appreciations of corporeality; our particular intellectual framework will be provided by cognitive science. Two reasons impel us towards this methodological choice.In the first instance, the study of ritual has, after several decades of stagnation, been rewarded—even revolutionised—by the application of insights from the new sciences of the mind (Whitehouse 1–12; McCauley and Lawson 1–37). Thus, there are good reasons to think that ritual treatments of the body will refract historical and social forces through empirically attested tendencies in human cognition. In the present connection, this means that knowledge of these tendencies will reward any attempt to theorise the objectification of the body in mortuary rituals.In the second instance, because beliefs concerning the afterlife can never be definitively judged to be true or false, they give free expression to tendencies in cognition that are otherwise constrained by the need to reflect external realities accurately. To this extent, they grant direct access to the intuitive ideas and biases that shape how we think about the world. Already, this idea has been exploited to good effect in areas like the cognitive anthropology of religion, which explores how counterfactual beings like ghosts, spirits, and gods conform to (and deviate from) pre-reflective cognitive patterns (Atran 83–112; Barrett and Keil 219–224; Barrett and Reed 252–255; Boyer 876–886). Necessarily, this implies that targeting post-mortem treatments of the body will offer unmediated access to some of the conceptual schemes that inform thinking about human corporeality.At a more detailed level, the specific methodology we propose to use will be provided by conceptual blending theory—a framework developed by Gilles Fauconnier, Mark Turner, and others to describe how structures from different areas of experience are creatively blended to form a new conceptual frame. In this system, a generic space provides the ground for coordinating two or more input spaces into a blended space that synthesises them into a single output. Here this would entail using natural or technological processes to structure mortuary practices in a way that satisfies various psychological needs.Take, for instance, W.B. Yeats’s famous claim that “Too long a sacrifice / Can make a stone of the heart” (“Easter 1916” in Yeats 57-8). Here, the poet exploits a generic space—that of everyday objects and the effort involved in manipulating them—to coordinate an organic input from that taxonomy (the heart) with an inorganic input (a stone) to create the blended idea that too energetic a pursuit of an abstract ideal turns a person into an unfeeling object (the heart-as-stone). Although this particular example corresponds to a familiar rhetorical figure (the metaphor), the value of conceptual blending theory is that it cuts across distinctions of genre, media, language, and discourse level to provide a versatile framework for expressing how one area of human experience is related to another.As indicated, we will exploit this versatility to investigate two ways of objectifying the body through the examination of two contrasting mortuary practices—cremation and inhumation—against different cultural horizons. The first of these is the conceptualisation of the body as an object of a technical process, where the post-mortem cremation of the corpse is analogically correlated with the metallurgical refining of ore into base metal. Our area of focus here will be Bronze Age cremation practices. The second conceptual scheme we will investigate focuses on treatments of the body as a vegetable object; here, the relevant analogy likens the inhumation of the corpse to the planting of a seed in the soil from which future growth will come. This discussion will centre on the Classical Era. Burning: The Body as Manufactured ObjectThe Early and Middle Bronze Age in Western Europe (2500-1200 BCE) represented a period of change in funerary practices relative to the preceding Neolithic, exemplified by a move away from the use of Megalithic monuments, a proliferation of grave goods, and an increase in the use of cremation (Barrett 38-9; Cooney and Grogan 105-121; Brück, Material Metaphors 308; Waddell, Bronze Age 141-149). Moreover, the Western European Bronze Age is characterised by a shift away from communal burial towards single interment (Barrett 32; Bradley 158-168). Equally, the Bronze Age in Western Europe provides us with evidence of an increased use of cist and pit cremation burials concentrated in low-lying areas (Woodman 254; Waddell, Prehistoric 16; Cooney and Grogan 105-121; Bettencourt 103). This greater preference for lower-lying location appears to reflect a distinctive change in comparison to the distribution patterns of the Neolithic burials; these are often located on prominent, visible aspects of a landscape (Cooney and Grogan 53-61). These new Bronze Age burial practices appear to reflect a distancing in relation to the territories of the “old ancestors” typified by Megalithic monuments (Bettencourt 101-103). Crucially, the Bronze Age archaeological record provides us with evidence that indicates that cremation was becoming the dominant form of deposition of human remains throughout Central and Western Europe (Sørensen and Rebay 59-60).The activities associated with Bronze Age cremations such as the burning of the body and the fragmentation of the remains have often been considered as corporeal equivalents (or expressions) of the activities involved in metal (bronze) production (Brück, Death 84-86; Sørensen and Rebay 60–1; Rebay-Salisbury, Cremations 66-67). There are unequivocal similarities between the practices of cremation and contemporary bronze production technologies—particularly as both processes involve the transformation of material through the application of fire at temperatures between 700 ºC to 1000 ºC (Musgrove 272-276; Walker et al. 132; de Becdelievre et al. 222-223).We assert that the technologies that define the European Bronze Age—those involved in alloying copper and tin to produce bronze—offered a new conceptual frame that enabled the body to be objectified in new ways. The fundamental idea explored here is that the displacement of inhumation by cremation in the European Bronze Age was motivated by a cognitive shift, where new smelting technologies provided novel conceptual metaphors for thinking about age-old problems concerning human mortality and post-mortem survival. The increased use of cremation in the European Bronze Age contrasts with the archaeological record of the Near Eastern—where, despite the earlier emergence of metallurgy (3300–3000 BCE), we do not see a notable proliferation in the use of cremation in this region. Thus, mortuary practices (i.e. cremation) provide us with an insight into how Western European Bronze Age cultures mediated the body through changes in technological objects and processes.In the terminology of conceptual blending, the generic space in question centres on the technical manipulation of the material world. The first input space is associated with the anxiety attending mortality—specifically, the cessation of personal identity and the extinction of interpersonal relationships. The second input space represents the technical knowledge associated with bronze production; in particular, the extraction of ore from source material and its mixing with other metals to form an alloy. The blended space coordinates these inputs to objectify the human body as an object that is ritually transformed into a new but more durable substance via the cremation process. In this contention we use the archaeological record to draw a conceptual parallel between the emergence of bronze production technology—centring on transition of naturally occurring material to a new subsistence (bronze)—and the transitional nature of the cremation process.In this theoretical framework, treating the body as a mixture of substances that can be reduced to its constituents and transformed through technologies of cremation enabled Western European Bronze Age society to intervene in the natural process of putrefaction and transform the organic matter into something more permanent. This transformative aspect of the cremation is seen in the evidence we have for secondary burial practices involving the curation and circulation of cremated bones of deceased members of a group (Brück, Death 87-93). This evidence allows us to assert that cremated human remains and objects were considered products of the same transformation into a more permanent state via burning, fragmentation, dispersal, and curation. Sofaer (62-69) states that the living body is regarded as a person, but as soon as the transition to death is made, the body becomes an object; this is an “ontological shift in the perception of the body that assumes a sudden change in its qualities” (62).Moreover, some authors have proposed that the exchange of fragmented human remains was central to mortuary practices and was central in establishing and maintaining social relations (Brück, Death 76-88). It is suggested that in the Early Bronze Age the perceptions of the human body mirrored the perceptions of objects associated with the arrival of the new bronze technology (Brück, Death 88-92). This idea is more pronounced if we consider the emergence of bronze technology as the beginning of a period of capital intensification of natural resources. Through this connection, the Bronze Age can be regarded as the point at which a particular natural resource—in this case, copper—went through myriad intensive manufacturing stages, which are still present today (intensive extraction, production/manufacturing, and distribution). Unlike stone tool production, bronze production had the addition of fire as the explicit method of transformation (Brück, Death 88-92). Thus, such views maintain that the transition achieved by cremation—i.e. reducing the human remains to objects or tokens that could be exchanged and curated relatively soon after the death of the individual—is equivalent to the framework of commodification connected with bronze production.A sample of cremated remains from Castlehyde in County Cork, Ireland, provides us with an example of a Bronze Age cremation burial in a Western European context (McCarthy). This is chosen because it is a typical example of a Bronze Age cremation burial in the context of Western Europe; also, one of the authors (MG) has first-hand experience in the analysis of its associated remains. The Castlehyde cremation burial consisted of a rectangular, stone-lined cist (McCarthy). The cist contained cremated, calcined human remains, with the fragments generally ranging from a greyish white to white in colour; this indicates that the bones were subject to a temperature range of 700-900ºC. The organic content of bone was destroyed during the cremation process, leaving only the inorganic matrix (brittle bone which is, often, described as metallic in consistency—e.g. Gejvall 470-475). There is evidence that remains may have been circulated in a manner akin to valuable metal objects. First of all, the absence of long bones indicates that there may have been a practice of removing salient remains as curatable records of ancestral ties. Secondly, remains show traces of metal staining from objects that are no longer extant, which suggests that graves were subject to secondary burial practices involving the removal of metal objects and/or human bone. To this extent, we can discern that human remains were being processed, curated, and circulated in a similar manner to metal objects.Thus, there are remarkable similarities between the treatment of the human body in cremation and bronze metal production technologies in the European Bronze Age. On the one hand, the parallel between smelting and cremation allowed death to be understood as a process of transformation in which the individual was removed from processes of organic decay. On the other hand, the circulation of the transformed remains conferred a type of post-mortem survival on the deceased. In this way, cremation practices may have enabled Bronze Age society to symbolically overcome the existential anxiety concerning the loss of personhood and the breaking of human relationships through death. In relation to the former point, the resurgence of cremation in nineteenth century Europe provides us with an example of how the disposal of a human body can be contextualised in relation to socio-technological advancements. The (re)emergence of cremation in this period reflects the post-Enlightenment shift from an understanding of the world through religious beliefs to the use of rational, scientific approaches to examine the natural world, including the human body (and death). The controlled use of fire in the cremation process, as well as the architecture of crematories, reflected the industrial context of the period (Rebay-Salisbury, Inhumation 16).With respect to the circulation of cremated remains, Smith suggests that Early Medieval Christian relics of individual bones or bone fragments reflect a reconceptualised continuation of pre-Christian practices (beginning in Christian areas of the Roman Empire). In this context, it is claimed, firstly, that the curation of bone relics and the use of mobile bone relics of important, saintly individuals provided an embodied connection between the sacred sphere and the earthly world; and secondly, that the use of individual bones or fragments of bone made the Christian message something portable, which could be used to reinforce individual or collective adherence to Christianity (Smith 143-167). Using the example of the Christian bone relics, we can thus propose that the curation and circulation of Bronze Age cremated material may have served a role similar to tools for focusing religiously oriented cognition. Burying: The Body as a Vegetable ObjectGiven that the designation “the Classical Era” nominates the entirety of the Graeco-Roman world (including the Near East and North Africa) from about 800 BCE to 600 CE, there were obviously no mortuary practices common to all cultures. Nevertheless, in both classical Greece and Rome, we have examples of periods when either cremation or inhumation was the principal funerary custom (Rebay-Salisbury, Inhumation 19-21).For instance, the ancient Homeric texts inform us that the ancient Greeks believed that “the spirit of the departed was sentient and still in the world of the living as long as the flesh was in existence […] and would rather have the body devoured by purifying fire than by dogs or worms” (Mylonas 484). However, the primary sources and archaeological record indicate that cremation practices declined in Athens circa 400 BCE (Rebay-Salisbury, Inhumation 20). With respect to the Roman Empire, scholarly opinion argues that inhumation was the dominant funerary rite in the eastern part of the Empire (Rebay-Salisbury, Inhumation 17-21; Morris 52). Complementing this, the archaeological and historical record indicates that inhumation became the primary rite throughout the Roman Empire in the first century CE. Inhumation was considered to be an essential rite in the context of an emerging belief that a peaceful afterlife was reflected by a peaceful burial in which bodily integrity was maintained (Rebay-Salisbury, Inhumation 19-21; Morris 52; Toynbee 41). The question that this poses is how these beliefs were framed in the broader discourses of Classical culture.In this regard, our claim is that the growth in inhumation was driven (at least in part) by the spread of a conceptual scheme, implicit in Greek fertility myths that objectify the body as a seed. The conceptual logic here is that the post-mortem continuation of personal identity is (symbolically) achieved by objectifying the body as a vegetable object that will re-grow from its own physical remains. Although the dominant metaphor here is vegetable, there is no doubt that the motivating concern of this mythological fabulation is human mortality. As Jon Davies notes, “the myths of Hades, Persephone and Demeter, of Orpheus and Eurydice, of Adonis and Aphrodite, of Selene and Endymion, of Herakles and Dionysus, are myths of death and rebirth, of journeys into and out of the underworld, of transactions and transformations between gods and humans” (128). Thus, such myths reveal important patterns in how the post-mortem fate of the body was conceptualised.In the terminology of mental mapping, the generic space relevant to inhumation contains knowledge pertaining to folk biology—specifically, pre-theoretical ideas concerning regeneration, survival, and mortality. The first input space attaches to human mortality; it departs from the anxiety associated with the seeming cessation of personal identity and dissolution of kin relationships subsequent to death. The second input space is the subset of knowledge concerning vegetable life, and how the immersion of seeds in the soil produces a new generation of plants with the passage of time. The blended space combines the two input spaces by way of the funerary script, which involves depositing the body in the soil with a view to securing its eventual rebirth by analogy with the sprouting of a planted seed.As indicated, the most important illustration of this conceptual pattern can be found in the fertility myths of ancient Greece. The Homeric Hymns, in particular, provide a number of narratives that trace out correspondences between vegetation cycles, human mortality, and inhumation, which inform ritual practice (Frazer 223–404; Carney 355–65; Sowa 121–44). The Homeric Hymn to Demeter, for instance, charts how Persephone is abducted by Hades, god of the dead, and taken to his underground kingdom. While searching for her missing daughter, Demeter, goddess of fertility, neglects the earth, causing widespread devastation. Matters are resolved when Zeus intervenes to restore Persephone to Demeter. However, having ingested part of Hades’s kingdom (a pomegranate seed), Persephone is obliged to spend half the year below ground with her captor and the other half above ground with her mother.The objectification of Persephone as both a seed and a corpse in this narrative is clearly signalled by her seasonal inhumation in Hades’ chthonic realm, which is at once both the soil and the grave. And, just as the planting of seeds in autumn ensures rebirth in spring, Persephone’s seasonal passage from the Kingdom of the Dead nominates the individual human life as just one season in an endless cycle of death and rebirth. A further signifying element is added by the ingestion of the pomegranate seed. This is evocative of her being inseminated by Hades; thus, the coordination of vegetation cycles with life and death is correlated with secondary transition—that from childhood to adulthood (Kerényi 119–183).In the examples given, we can see how the Homeric Hymn objectifies both the mortal and sexual destiny of the body in terms of thresholds derived from the vegetable world. Moreover, this mapping is not merely an intellectual exercise. Its emotional and social appeal is visible in the fact that the Eleusinian mysteries—which offered the ritual homologue to the Homeric Hymn to Demeter—persisted from the Mycenaean period to 396 CE, one of the longest recorded durations for any ritual (Ferguson 254–9; Cosmopoulos 1–24). In sum, then, classical myth provided a precedent for treating the body as a vegetable object—most often, a seed—that would, in turn, have driven the move towards inhumation as an important mortuary practice. The result is to create a ritual form that makes key aspects of human experience intelligible by connecting them with cyclical processes like the seasons of the year, the harvesting of crops, and the intergenerational oscillation between the roles of parent and child. Indeed, this pattern remains visible in the germination metaphors and burial practices of contemporary religions such as Christianity, which draw heavily on the symbolism associated with mystery cults like that at Eleusis (Nock 177–213).ConclusionWe acknowledge that our examples offer a limited reflection of the ethnographic and archaeological data, and that they need to be expanded to a much greater degree if they are to be more than merely suggestive. Nevertheless, suggestiveness has its value, too, and we submit that the speculations explored here may well offer a useful starting point for a larger survey. In particular, they showcase how a recurring existential anxiety concerning death—involving the fear of loss of personal identity and kinship relations—is addressed by different ways of objectifying the body. Given that the body is not reducible to the objects with which it is identified, these objectifications can never be entirely successful in negotiating the boundary between life and death. In the words of Jon Davies, “there is simply no let-up in the efforts by human beings to transcend this boundary, no matter how poignantly each failure seemed to reinforce it” (128). For this reason, we can expect that the record will be replete with conceptual and cognitive schemes that mediate the experience of death.At a more general level, it should also be clear that our understanding of human corporeality is rewarded by the study of mortuary practices. 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