Zeitschriftenartikel zum Thema „Gels et hydrogels“

Guanosine is an important building block for supramolecular gels owing to the unique self-assembly property that results from the unique hydrogen bond acceptors and donor groups. Guanosine-derived supramolecular hydrogels have promise in the fields of drug delivery, targeted release, tissue engineering applications,etc.However, the property of poor longevity and the need for excess cations hinder the widespread applications of guanosine hydrogels. Although guanosine-derived supramolecular hydrogels have been reviewed previously by Dash et al., the structural framework of this review is different, as the modification of guanosine is described at the molecular level. In this review, we summarize the development and lifetime stability improvement of guanosine-based supramolecular hydrogels through optimized structure and elaborate on three aspects: sugar modification, base modification, and binary gels. Additionally, we introduce the concept and recent research progress of self-healing gels, providing inspiration for the development of guanosine-derived supramolecular hydrogels with longer lifespans, unique physicochemical properties, and biological activities.

Longer and stronger; stiff but not brittle Hydrogels are highly water-swollen, cross-linked polymers. Although they can be highly deformed, they tend to be weak, and methods to strengthen or toughen them tend to reduce stretchability. Two papers now report strategies to create tough but deformable hydrogels (see the Perspective by Bosnjak and Silberstein). Wang et al . introduced a toughening mechanism by storing releasable extra chain length in the stiff part of a double-network hydrogel. A high applied force triggered the opening of cycling strands that were only activated at high chain extension. Kim et al . synthesized acrylamide gels in which dense entanglements could be achieved by using unusually low amounts of water, cross-linker, and initiator during the synthesis. This approach improves the mechanical strength in solid form while also improving the wear resistance once swollen as a hydrogel. —MSL

Longer and stronger; stiff but not brittle Hydrogels are highly water-swollen, cross-linked polymers. Although they can be highly deformed, they tend to be weak, and methods to strengthen or toughen them tend to reduce stretchability. Two papers now report strategies to create tough but deformable hydrogels (see the Perspective by Bosnjak and Silberstein). Wang et al . introduced a toughening mechanism by storing releasable extra chain length in the stiff part of a double-network hydrogel. A high applied force triggered the opening of cycling strands that were only activated at high chain extension. Kim et al . synthesized acrylamide gels in which dense entanglements could be achieved by using unusually low amounts of water, cross-linker, and initiator during the synthesis. This approach improves the mechanical strength in solid form while also improving the wear resistance once swollen as a hydrogel. —MSL

Abstract Abstract 2172 Background: Previous studies on clot formation have shown that the mechanical properties of clots have direct effects on hemostasis and thrombosis, and alterations of those clot mechanics are associated with disease(Collet, et al. 2006) (Hvas, et al. 2007). As such, understanding the mechanical properties of clots is vital to understand hemostasis and thrombosis. As platelets drive this contraction phenomenon, single platelet measurements are required to obtain a mechanistic understanding of the retraction process and to identify specific therapeutic targets for disease states in which platelet/clot retraction is pathologically altered. In addition, as fibrin has recently been shown to have extremely complex material and mechanical properties (Brown, et al. 2009), single platelet studies would decouple the effects of fibrin from platelets when examining clot mechanics. However, few studies have focused on the biomechanical role of platelets in clot formation and clot mechanics, especially at the single cell level. Our group has recently published measurements of single platelet contraction (Lam, et al, Nature Mat, 2011), showing that platelets are capable of applying large forces and are quite varied in their response. However, the key barrier which has prevented the study of single platelets has been the lack of a technology with the sufficient precision and sensitivity to both manipulate and measure individual platelets in a high throughput manner. To that end, we have extended a technique (Polio, et al. 2012) that is capable of measuring the contraction of individual platelets in a high throughput manner. Results: Here we precisely pattern FITC conjugated fibrinogen dots in a geometrical array (Fig 1A) on polyacrylamide (PAA) gels. Thrombin activated platelets are incubated on the gel and contract upon contact with the micropatterned fibrinogen “dots”. When the platelet comes into contact with two dots and contracts, the distance in which the platelet moves the dots from their original position is used to determine the force. Conceptually, this is similar to the idea of a linear spring, in which a certain spring displacement corresponds to a known force. Using this technique, we measured 71 platelets which were attached to two fibrinogen dots each, and found that on low stiffness gels, that the average contractile force was approximately 4nN (Fig 2A). Platelets may attach to a maximum of four dots, but do so with a much lower frequency as compared to two dots (Fig 2B). Preliminary results indicate that as platelet area increases, as indicated by contact with additional protein “dots”, the total force exerted by the platelet increases, with a maximum contractile force achieved when touching three protein dots (Fig 2C). Based on this data, there may be an optimum platelet spread area that maximizes contractile force. Conclusions and Ongoing Efforts: We will determine how the biophysical parameters, such as micro-environmental stiffness and shear flow, quantitatively affect platelet contractility. As our current understanding of the underlying biological mechanisms of platelet contraction is solely qualitative, we will also quantitatively investigate the biological signaling pathways of platelet contraction using pharmacological agents and platelet agonists using our system. Pharmacologic agents including glycoprotein IIb/IIIa (integrin αIIbβ3) antagonists, Rho kinase inhibitors, calcium inhibitors, and myosin inhibitors, will be used to measure the quantitative effect each biological component has on platelet contraction. In addition, soluble agonists known to activate platelets including thrombin, ADP, thromboxane A2, and epinephrine will be investigated quantitatively and systematically to measure their interactive and synergistic effects on platelet contraction. Furthermore, this device represents a new platform which could be used in drug discovery and to test for changes in platelet contraction with differing pharmacological doses. Disclosures: No relevant conflicts of interest to declare.

Simulation studies of aqueous polymer solutions, and heuristic arguments by De Gennes for aqueous polyethylene oxide polymer solutions, have suggested that many-body interactions can give rise to the ‘anomalous’ situation in which the second osmotic virial coefficient is positive, while the third virial coefficient is negative. This phenomenon was later confirmed in analytic calculations of the phase behavior and the osmotic pressure of complex fluids exhibiting cooperative self-assembly into extended dynamic polymeric structures by Dudowicz et al. In the present study, we experimentally confirm the occurrence of this osmotic virial sign inversion phenomenon for several highly charged model polyelectrolyte gels (poly(acrylic acid), poly(styrene sulfonate), DNA, hyaluronic acid), where the virial coefficients are deduced from osmotic pressure measurements. Our observations qualitatively accord with experimental and simulation studies indicating that polyelectrolyte materials exhibit supramolecular assembly in solution, another symptomatic property of fluids exhibiting many-body interactions. We also find that the inversion in the variation of the second (A2) and third (A2) virial coefficients upon approach to phase separation does not occur in uncharged poly(vinyl acetate) gels. Finally, we briefly discuss the estimation of the osmotic compressibility of swollen polyelectrolyte gels from neutron scattering measurements as an alternative to direct, time-consuming and meticulous osmotic pressure measurements. We conclude by summarizing some general trends and suggesting future research directions of natural and synthetic polyelectrolyte hydrogels.

Contexte : Le chitosane, est un biopolymère qui présente beaucoup d’avantages en formulation à savoir sa biocompatibilité, sa biodégradabilité, sa non toxicité et surtout sa mucoadhésion Objectif : Etudier l’influence de la concentration en chitosane sur les caractéristiques rhéologiques du gel mixte obtenu. Méthodes : Diverses formulations de solutions de chitosane à 1%, 2%, 3% et 4% (g/g) et de mélanges Poloxamer 407/chitosane ont été préparés en solution aqueuse d’acide acétique 0,1M. Résultats : Lorsque la masse molaire en chitosane augmente, on n’observe pas de température de début de micellisation sur le plan rhéologique. Lorsque la concentration de chitosane augmente dans les systèmes, il n’y a pas de variations très importantes de la Tgel. L’ajout de chitosane semble désorganiser les micelles formées, effet non observé avec le chitosane thiolé (CT2) 145000g/mol. Les systèmes se comportent comme des gels élastiques et il n’y a pas d’effet de la teneur en chitosane Conclusion : la présence de chitosane fait varier la viscosité de la solution avant la gélification et très peu la température de gélification. Mots-clés: Chitosane, gel, libération contrôlée, thermosensible.

This article provides the review of the medical use of pH- and temperature-sensitive polymer hydrogels. Such polymers are characterised by their thermal and pH sensitivity in aqueous solutions at the functioning temperature of living organisms and can react to the slightest changes in environmental conditions. Due to these properties, they are called stimuli-sensitive polymers. This response to an external stimulus occurs due to the amphiphilicity (diphilicity) of these (co)polymers. The term hydrogels includes several concepts of macrogels and microgels. Microgels, unlike macrogels, are polymer particles dispersed in a liquid and are nano- or micro-objects. The review presents studies reflecting the main methods of obtainingsuch polymeric materials, including precipitation polymerisation, as the main, simplest, and most accessible method for mini-emulsion polymerisation, microfluidics, and layer-by-layer adsorption of polyelectrolytes. 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INTRODUCTION. Primary chronic lymphocytic leukemia (CLL) cells, despite originating from a proliferative disease, rapidly undergo apoptosis in vitro in absence of microenvironmental survival signals1. Although co-culture with stromal cells or the addition of soluble factors can increase and extend CLL survival, no system permits the long-term expansion of CLL cells in vitro2. The difficulties of mimicking a physiologic microenvironment supporting CLL cells hinder in vitro studies of proliferation, drug screens and prevent propagation of rare subclones. For other cancers, various types of 3D cultures have been introduced utilizing scaffolds, gels, spheroid cultures and fluidic systems, representing a more accurate representation of the in vivo microenvironment3. Unlike solid tumors, secondary lymphoid tissues where CLL cells proliferate in vivo, do not derive from a single stem cell progenitor. Developing an appropriate 3D in vitro culture system for CLL is of obvious importance and may contribute pathophysiological relevance to study long-term CLL proliferation and more accurate drug screening4,5. Within the field of CLL, attempts have focused on bone marrow stroma, but it may be biologically and clinically more relevant to investigate the lymph node niche as this is the critical site of CLL proliferation6. METHODS. Primary CLL cells were cultured in various 3D systems including hydrogels, hanging drop cultures and ultra-low attachment plates (ULA) plates in parallel to an optimal 2D system, consisting of the culture of primary CLL cells on a monolayer of CD40L-presenting fibroblasts (3T40) or 3T3 negative control fibroblasts. CLL cells were either cultured as PBMCs alone, with or without T cells, or co-cultured with 3T40 or primary lymph node fibroblasts. CLL cells were either stimulated directly with IL-2, IL-15, IL-21 and CpG and/or indirectly via a T cell stimulation of anti-CD3/CD28. RESULTS. After testing and comparing multiple systems for the in vitro culture of CLL cells, we optimized a novel CLL culture system utilizing ULA plates creating spheroids of PBMCs isolated from peripheral blood. Without the addition of soluble factors or stroma, primary CLL cells in the ULA 3D model could be maintained in culture for 6 weeks as opposed to 1 week in the 2D system. Aside from significantly promoting CLL survival, cultures could be expanded approximately 3-4-fold over a course of 6 weeks using the ULA 3D model. 3D cultures showed a more consistent and significantly increased CLL proliferation compared to 2D cultures, independent of IGHV mutation status, increasing the average proliferation index of 2.87 to 3.90 (n=10). Additionally, co-culture with LN-derived stromal cells further increased CLL proliferation, reaching a maximum of 8 generations (n=6) (Figure 1). Lastly, when PBMCs were stimulated with IL-2, IL-15, IL-21 and CpG, spheroids developed proliferation center-like structures after 4 weeks of culture. CONCLUSIONS. We established a lymph node-based 3D in vitro culture system for CLL leading to increased CLL proliferation and survival compared to 2D systems. The set-up allows long-term expansion of CLL cells in vitro, as well as formation of proliferation center-like structures. We are currently optimizing drug resistance studies, expansion of specific CLL subclones and performing competition experiments. References: 1. Hamilton et al., Mimicking the tumour microenvironment: three different co-culture systems induce a similar phenotype but distinct proliferative signals in primary chronic lymphocytic leukaemia cells, 2012. 2. Asslaber et al., Mimicking the microenvironment in chronic lymphocytic leukaemia - where does the journey go?, 2013. 3. Gurski et al., 3D Matrices for Anti-Cancer Drug Testing and Development, 2010. 4. Nunes et al., 3D tumor spheroids as in vitro models to mimic in vivo human solid tumors resistance to therapeutic drugs, 2019. 5. Aljitwai et al., A novel three-dimensional stromal-based model for in vitro chemotherapy sensitivity testing of leukemia cells, 2014. 6. Van Gent et al., In vivo dynamics of stable chronic lymphocytic leukemia inversely correlates with somatic hypermutation levels and suggest no major leukemic turnover in bone marrow, 2008. Disclosures Kater: Genentech: Research Funding; Abbvie: Research Funding; Roche: Research Funding; Janssen: Research Funding; Celgene: Research Funding. Eldering:Celgene: Research Funding; Janssen: Research Funding; Genentech: Research Funding.

Mucus is an ubiquitous polymer hydrogel that functions as a protective coat on the surface of integument and mucosa of species ranging from simple animals (such as coelenterates) to mammals. The polymer matrix of mucus is made out of long-chain glycoproteins called mucins that are tangled together, forming a randomly woven, highly polyionic network (Lee et al., 1977; Verdugo et al., 1983). Mucin-containing granules, produced by mammalian goblet cells in vitro, undergo massive post-exocytotic swelling. Their swelling kinetics is similar to the swelling of condensed artificial polymer gels (Verdugo, 1984; Tanaka and Fillmore, 1979). We had proposed that mucins must be condensed in the secretory granule and expand by hydration during or after exocytosis (Verdugo, 1984; Tam and Verdugo, 1981). However, the polyionic charges of mucins prevents condensation unless they (the mucins) are appropriately shielded. The present experiments were designed to assert the presence of an intragranular shielding cation and its role in secretion. Giant mucin granules of the slug (Ariolimax columbianus) are released intact from mucus-secreting cells of the slug's skin. They burst spontaneously outside the cell, forming, upon hydration, the typical slug mucus (Deyrup-Olsen et al., 1983). We report here that these granules contain from 2.5 to 3.6 moles calcium/kg dry material, and that calcium is released from the granules immediately before the burst that discharges their secretory product. Therefore, we propose that calcium functions as a shielding cation of poly ionic mucins, and that the bursting discharge of mucins from secretory granules must result from the release of calcium from the intragranular compartment. Calcium release would unshield the polyionic charges of mucins, driving the mutual repulsion of polymer chains and triggering a quick expansion of the mucin network (resembling a Jack-in-the-box mechanism). The existence of a poly ion associated with a shielding cation seems to be a common feature in a large variety of secretory granules. Thus, the proposed spring-loaded release system based on the unshielding of a condensed polyion may serve as a general model for explaining the molecular mechanism of product release in secretion.

e21062 Background: Acral melanoma is the main subtype in Asia, and tends to be insensitive to conventional and novel therapies compared with cutaneous melanomas. It has been proved that biomechanical alterations inside the tumor microenvironment significantly affect tumor progression in breast, pancreatic and other cancers(Hepatology. 2016 July ; 64(1): 261–275; Cold Spring Harbor Symposia on Quantitative Biology, Volume LXXXI) . Hence, we hypothesized that the biomechanical microenvironment plays critical roles in proliferation and invasion in acral melanoma. In this study, the main purpose is to observe whether melanoma cells have the same ability called “durotaxis” as other tumors, which to respond to a gradient of extracellular stiffness and migrate in a directed fashion. Methods: In this study, the stiffness was compared between the acral melanoma tissue and the normal tissue by using the atomic force microscope (AFM) with fresh samples and the freezing microtome sectioning specimen (J. Matthew Barnes et al., Nature Cell Biology 2018.) . A stiffness gradient polyacrylamide hydrogel system was constructed to observe how the acral melanoma cells response. Using automated tracking (Curr Protoc Cell Biol. ; 76: 12.12.1–12.12.16.) of positional data for large sample size of single migrating cells to systematically analyze polarized melanoma cell migration in response to stiffness gradient. Results: The results showed that 1) all the acral melanoma cell lines tested displayed strong anti-durotactic migratory response, which was in accordance with the AFM measurement that the melanoma microenvironment gradually softened; 2) Acral melanoma cells tend to move toward softer areas on gradient gels, which is contrary to the migration behavior of most other tumor cells. Conclusions: In this study, we presented the different migratory pattern of acral melanoma cells. It may illustrate the invasion mechanism for aral melanoma cells. It could also herald that extracellular matrix may be a potential therapeutic target in acral melanomas.

Green gram (Vigna radiata) is considered the chief legume in Pakistan. Thus, current study was conducted to examine the ameliorating effect of phytohormones pre-treatments under salt stress on proteome profile of green gram by sodium-dodecyl-sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The soluble green gram seedlings proteins were resolved on 4% stacking and 12% resolving gels. The SDS-PAGE resolved 24 polypeptide bands ranging from 200 to 17kDa. Among these, 12 out of 24 bands of proteins were essentials house-keeping or growth proteins of green grams. While, 120, 114.6, 51.8, 29.1, and 22.8 kDa bands were over-expressed under 50 to 350mM salt with phytohormones treatments. The others 104.5 kDa, 99.8 kDa, 95.3 kDa, 91.0 kDa, 55 kDa, 46 kDa, and 17kDa bands were related to the GAᴣ, IAA, and SA induced tolerance. Overall 120 kDa, 114.6 kDa, 104.5 kDa, 99.8, 95.3 kDa, 51.8 kDa, 29.1 kDa and 22.8kDa bands were first time identified in the current study. The information retrieved from NCBI protein database, the resolved peptides were principally belonging to 7S and 8S vicilin, 2S, 8S, 11S, and 16.5S globulins. It is determined that seed priming with SA enhanced tolerance in green gram by rapidly synthesizing stress alleviating peptides.Key word: Cluster analysis, dendrogram, mungbean, salt stress, SDS-PAGEINTRODUCTIONVarious world-wide health concerning organization recommended the use of high graded plant protein such as legumes to prevent the risk of metabolic disorder (Hou et al., 2019). Legumes are most important protein crop on the earth. Among the legumes, the green gram is the major pulses. Its seeds are rich in superior quality storage protein, which account 85% of the total protein while, another 15% have not been broadly studied (Yi-Shen et al., 2018). The soluble storage protein comprises of 60% globulins, 25% albumin and 15% prolamins. Globulins are further divided into 3.4% basic-type (7S), 7.6% legumin-type (11S), and 89% vicilin-type (8S) (Mendoza et al., 2001; Itoh et al., 2006). Other than proteins, the green gram seeds also contain starch, fiber, phenolic compound, saponins, vitamins, calcium zinc, potassium, folate, magnesium, manganese and very low in fat that made it meager man’s meat (Hou et al., 2019). It is also a good source of green manure and fodder (El-Kafafi et al., 2015). Its root has ability to fix 30 to 50 Kg/ha atmospheric nitrogen in the soil which is essential for maintaining soil fertility (Chadha, 2010). The green gram is the valuable and the major Rabi pulse crop of Pakistan. Its cultivation area in 2016-2017 was about 179,000 hectares with seed yield of 130,000 tones. In comparison during 2017-2018, it was cultivated on 161,800 hectares land with 118,800 tones seed yield (GOP, 2018). One of the reasons of this 9% decrease in both land and productivity is the shortage of irrigated land due to soil salinity. The salinity induce oxidative bust in the mungbean cells, caused by responsive oxygen species (ROS) such as hydrogen peroxide, singlet oxygen, hydroxyl radical and superoxide radical. The ROS create hindrance in various metabolic processes of plant via interacting with macromolecules like proteins (Alharby et al., 2016). However, phytohormones like gibberellic acid (GAᴣ), indole acetic acid (IAA), and salicylic acid (SA) take part in the biosynthesis of salt tolerance proteins under salinity. These salt tolerance proteins acclimate plants under salinity stress. Application of biotechnology plays a significant role in agriculture (Khan et al., 2017). Therefore, production of particular proteins under salt stress is a specific response of cell which can be analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE is the simple, valid, and cost-effective biochemical marker (Mushtaq et al., 2018). This marker has been widely used to determine the extent of evolutionary variations in crops (El-Kafafi et al., 2015).OBJECTIVES The present study was directed first time with the aim to investigate the toxic effect of sodium chloride (0-350 mM) and stress acclimation by pre-treatment of GAᴣ, IAA, and SA on the proteome profile of NM-92 cultivar of a Pakistani green gram.MATERIALS AND METHODSThe present study was replicated thrice in the plant laboratory of Department of Genetics, Faculty of Science, and University of Karachi. The seeds of mung bean cultivar NM-92 were acquired from National Agricultural Research Centre (NARC), Islamabad. These freshly collected 15 seedsˉ1 treatment / replication were divided into two sets. The first was named as sodium chloride (SC) stress treatments were imbibed in sterile distilled water (DW) whereas, second set soaked in gibberellic acid (GAᴣ) (BDH Chemicals, England), indole acetic acid (IAA) (Fluka, Switzerland), and salicylic acid (SA) (J.T. Baker, Holland) in the separate beaker for 24 hours under dark condition. After 24 hours, given ample time to both the sets at room temperature. After recovery, all 20 treatments were sown in the 150 X 30 mm sized petri-dishes containing 0, 50, 150, 250, and 350 millimolar (mM) sodium chloride solution (Fisher Scientific, UK) for 72 hours.Protein extraction: Protein extraction was done by taking 0.3g of seedlings in an ice chilled mortar and crushed by adding 600µL 0.2 M Tris-HCl buffer having pH 7.5 contained 5% SDS (w/v) and 5% 2-mercaptoethanol (v/v). The homogenate was incubated at 0oC for 30 min., boiled in the water bath for 3 min. at 100oC. Samples were centrifuged in Heraeus Biofuge D-37520, Germany for 30 min. at 8000 rpm. The protein supernatant was saved at below 0°C for quantitative and qualitative determination with minor modifications. The total soluble protein content of the samples was estimated via “Bovine Serum Albumin (BSA) standard curve” and explicit in µg protein milligramˉ1 fresh weight of mung seedlings.Bovine serum albumin standard curve (2000 μg/mL): Total protein standard curve was made by dissolving 0.05g of Bovine Serum Albumin (BSA) in 25mL of distilled water. Ten serial dilutions were made from 0.1 mL to 1mL by BSA solution then performed Lowry. A standard curve of total proteins was plotted by taking BSA absorbance at Y-axis and 2000 μg BSA / mL at X-axisSample preparation for SDS-PAGE: For qualitative assessment of total proteins; the 35μL of saved protein supernatant was combined with 15μL of sample diluting buffer (SDB). The SDB was made up of 0.0625 M Tris-HCl pH 6.8 with 2% of SDS, 10% of glycerol, 0.003% of bromophenol blue dye and 5% of 2-mercaptoethanol. Boil the 50μL protein SDB supernatant at 100oC in water bath for 3 min., centrifuged at 6000 rpm for 4 min. The supernatant was loaded on SDS-PAGE gel with the given formulae. The SDS- PAGE: Total proteins were fractionated via SDS-PAGE with 4% stacking and 12% resolving gel. The resolving gel of 12% was made by taking 6mL solution A, 1.8 mL 3 M Tris 1 M HCl buffer pH 8.8, 144μL 10% SDS, 5.74 mL sterile distilled water, 720μL 1.5% ammonium persulphate (APS) in deionized water and 10μL TEMED. While, stacking was composed of 1.25mL of solution A, 2.5mL of 0.5M Tris 1M HCl buffer pH 6.8, 100μL 10% SDS, 1.8 mL of distilled water, 500μL 1.5% APS and 12μL TEMED. Solution A was prepared by conjoining 30% acrylamide and 0.8% N, N’-methylene-bisacrylamide in deionized water. To avoid polymerization in the beaker; the prepared solution was quickly poured into the 3 mm thick gel plates after adding TEMED. The stacking was lined over resolving gel, then combs were inserted between the gel plates of SCIE-PLAS TV-100 separation system, UK, and allowed to polymerize for ½ an hour. After polymerization gel was placed in the tank which were filled with Tris-Glycine buffer (electrode buffer) pH 8.4 then combs were removed. The electrode buffer contained 0.3% Tris, 1.41% Glycine and 0.1% SDS in 2000mL d/w. The gel was pre-run for 15 min. at 60 volts and 120 mA currents. The prepared SDS-PAGE samples were loaded in wells with BlueStepTM Broad Range Protein Marker, AMRESCO, USA as standard and run at 60 volts & 120 mA for about 45 min. When samples entered in resolving gel, and then gave 100 volts and 200 mA currents for around 2.5 hours. Furthermore, electrophoresis was carried out at a constant watt.The Gel was washed with 30% ethanol on Uni Thermo Shaker NTS-1300 EYELA, Japan at the constant shaking for 30 min. Then gels were placed in 10% glacial acetic acid in 50% methanol solution (Fixative) for 24 hours. SDS Gel was stained until protein bands were visible thereat placed as 5% of Methanol in 7.5% acetic acid glacial solution to destain the bands background. SDS-PAGE stain composed of 0.125% coomassie brilliant blue R-250 dissolved in 40% of Methanol and 7% acetic acid glacial solution. The stain was stirred on Magnetic stirrer & hot plate M6/1, Germany for 6-10 hours before used. Photographs were taken by Sanyo digital camera VPC-T1284BL and bands were scored through numbering pattern. Gels preserved in 10% acetic acid solution at 4°C.Interpretation of bands and data analysis: The total soluble protein bands relative mobility calculated by below formulae and Dendrogram was constructed via SPSS v. 20Where,F=(Migrated distance of protein band)/(Migrated distance of dye front)Slop=(Log MW of protein marker lower limit band–log〖MW of protein marker upper limit band )/(RF protein marker lower limit band –RF of protein marker upper limit band)RESULTS:The total soluble proteins extracted from green gram were perceived by SDS-PAGE Blue StepTm broad range biochemical markers. The protein-based marker was used to evaluate the toxic effect of sodium chloride along with pre-treatments of GAᴣ, IAA, and SA on proteome assay. In the current work, seedlings total soluble proteome resolved 24 polypeptide bands ranging from 200 to 17.1 kDa were recognized by using SDS-PAGE. The figure 1 showed Dendrogram assay, which classified the 20 treatments of SC, GAᴣ, IAA and SA into two major clusters where, the cluster I was the largest one (figure 1). Cluster I consisted of 15 treatments that further divided into I-A, and I-B. The pre-treatments of SC50+SA, SC150+SA, SC250+SA, and SC350+IAA were grouped together into C-1 of sub-cluster I-A. The C-2 of sub-cluster I-A, pre-treatment SC350+SA was most diverse among 20 treatments. The C-1 treatments showed 99% homology when compared with each other while, it was 97% similar with C-2. The sub-cluster I-B comprised another 10 treatments, SC0+GAᴣ, SC50+GAᴣ, SC150+GAᴣ, SC250+GAᴣ, SC350+GAᴣ, SC0+IAA, SC50+IAA, SC150+IAA, SC250+IAA, and SC0+SA that were also 99% similar for total proteins. Sub-cluster I-B pre-treatments was exhibiting 94% homology with the sub-cluster I-A. The second cluster was the smallest one that was divided into two sub-clusters, II-A and II-B. The II-A was comprised of SC50, SC150, and SC250 while, sub-cluster II-B consisted of SC0 and SC350. Within each sub-cluster, pre-treatments expressed 99% homology whereas, II-A was 97 different from II-B. Furthermore, cluster I showed 75% similarities with cluster II (figure 1). The seedlings storage proteome profile of green gram was shown in table 1.The results showed that 120kDa, 114.6 kDa, 51.8 kDa, 29.1 kDa and 22.8 kDa proteins bands were not induced at 0 mM SC, GAᴣ, IAA, and SA. The table 1 depicted the presence of 120 kDa and 114.6 kDa bands only at 350 mM SC level with all phytohormones treatments. Similarly, 51.8 kDa protein bands were appearing at 150SC, 250SC and 350SC stress with phytohormones. Based on the information collected from the NCBI protein database, this peptide was related to the 8S globulin alpha subunits. The two other, 7S globulins sub-units having 29.1kDa and 22.8 kDa molecular weights bands were synthesized under 50mM, 150mM, 250mM, 350mM SC stress with phytohormones. Concerning protein polypeptide of molecular weight 104.5 kDa, 99.8 kDa, 91.0 kDa, 55.0 kDa, and 46.0 kDa, those were induced by GAᴣ, IAA and SA at 0 to 350 mM SC. While, 17kDa protein band was appearing in SA, and IAA treated samples and 95.3kDa band was only present in SA treatment. Other 12 protein bands were present in all treatments proved as house-keeping proteins of green gram (table 1).DISCUSSIONThe SDS-PAGE profiling for proteome is the reliable and applied biochemical approach that has been used as biochemical marker in various crop differentiation, and characterization. In the current study, first time SDS-PAGE was utilized to investigate the impact of GAᴣ, IAA, and SA pre-soaking on green gram under salt toxicity. The salt toxicity adversely affects all seed, seedling, and plant metabolic process (Parveen et al., 2016). At salt toxicity, the endogenous GAᴣ, IAA, and SA levels markedly decrease (El-Khallal et al., 2009). In such condition, exogenous application of GAᴣ, IAA, and SA enhance seedlings survival rate by increasing synthesis of seed storage proteins. Likewise, our Dendrogram characterization based on 20 treatments showed significant diversity under 0 to 350 mM SC stress. The salicylic acid treatments were grouped together except SC0+SA treatment, exhibiting a close relationship, which proved its acclimating role under salt stress. These findings will help plant breeder toward enhancing food quality and quantity of green gram in future breeding programme on saline sodic land.The SDS-PAGE assay revealed 200. kDa, 109.4 kDa, 77 kDa, 68 kDa, 49 kDa, 38 kDa, 33 kDa, 26 kDa, 24 kDa, 22 kDa, 21 kDa and 19 kDa fractions as essential green gram proteins. Among these, 68 kDa, 49 kDa, 33 kDa, 26 kDa, 24 kDa and 21 kDa peptides were seed biotinylated isoform protein (Riascos et al., 2009), putative NADH-ubiquinone oxidoreductase subunit H (Gostinčar et al., 2019), heat shock protein 33 (Hamidian et al., 2015), globulin protein, seed coat / maturation protein (Dhaubhadel et al., 2005), and protein for dimerization. While, 22 kDa proteins belonged to the class of prolamin alpha zein Z1C1_2, Z1C1_4, and Z1C1_8 precursors, and 19kDa peptide was related with Z1A1_2, Z1A2_2, and Z1B_6 precursors (Miclaus et al., 2011). Further, the 91 kDa peptide is sucrose synthase SS1 protein, and 77kDa protein is the NADPH-cytochrome P450 reductase (Wang et al., 2004). Also, the phosphatase-associated two other proteins having 46 and 55 kDa molecular weight were reported earlier in Mucuna pruriens. Hameed et al. (2012) and Malviya et al. (2008) found 55 and 46kDa peptides as 7S vicilin small sub-units and 17kDa as 11S globulins sub-unit in the studied Vigna radiata. Some other molecular weight proteome such as 68 kDa and 49kDa are 7S vicilin, 33kDa is 8S vicilin, 38 and 26kDa 8S globulins, 24kDa 11S globulins, and 22kDa 16.5S globulins. These proteins required for germination and seed establishment of green gram plant (Hameed et al., 2012).The vast accumulation of 23kDa and 22kDa peptides under salt stress by salicylic acid, were reported previously in the mangrove Bruguiera parviffora and Zea mays (El-Khallal et al., 2009). Correspondingly, El-Kafafi et al. (2015) reported the presence of 115kDa, 23kDa, and 22kDa bands in the salt tolerant lines of green gram. These proteomes induced under salt stress may play a pivotal part in the stress acclimation and osmotic adjustment. Similarly, the induction of 104 kDa and 100kDa MW polypeptide by SC stress in the salt tolerant genotypes of green gram indicated the functional role of phytohormones in various metabolic and defense response El-Kafafi et al. (2015); Alharby et al. (2016), El-Khallal et al. (2009), Qados (2010). Ali et al. (2007), Alharby et al. (2016), and El-Kafafi et al. (2015) observed 17kDa, 26kDa, 33kDa and 77kDa bands involving in salt tolerance and can be considered as a positive biochemical marker for salt stress. Further, 26 kDa MW peptide also functions as osmotin under the salt stress that involved in enhancing the accumulation of glycine betaine and proline in the cells. Hence, proteome assay of green gram showed that GAᴣ, IAA, and SA could regulate the expression of salt stress proteins that are anticipated to play a crucial part in the salt tolerance mechanism. Likewise, the involvement of phytohormones in the induction of changes in the proteome profile pattern was attributed to their part in managing cell division by regulating some genes of apical meristems.CONCLUSIONFinally, the results revealed the presence of the ten new bands with MW of 200kDa, 120 kDa, 114.6 kDa, 109.4kDa, 104.5kDa, 99.8kDa, 95.3kDa, 51.8kDa, 29.1kDa and 22.8kDa have not reported previously under salt stress with phytohormones treatments in green gram. Furthermore, it was observed that phytohormones alleviate the negative impact of salt stress on green gram by enhancing synthesis of salt defense polypeptides. Hence, higher accumulation of proteins was observed in salicylic acid treated seedlings. Thus, present work recommended the pre-soaking of phytohormones to overcome the toxic impact of sodium chloride on green gram. Further research is needed on a biomolecular level to reveal the mechanism of signalling pathways under sever salt stress.CONFLICT OF INTERESTBoth authors have declared that no disagreement of interest regarding this research.REFERENCES Alharby, H. F., E. M. Metwali, M. P. Fuller and A. Y. Aldhebiani, 2016. The alteration of mRNA expression of sod and gpx genes, and proteins in tomato (Lycopersicon esculentum Mill) under stress of Nacl and/or ZnO nanoparticles. Saudi journal of biological sciences, 23(6): 773-781.Ali, A., M. Mageed, I. Ahmed and S. Mariey, 2007. Genetic and molecular studies on barley salt tolerance. In: African crop science conference proceedings. pp: 669-682.Chadha, M., 2010. Short duration mungbean: A new success in South Asia. Asia-Pacific association of agricultural research institutions.Dhaubhadel, S., K. Kuflu, M. C. Romero and M. Gijzen, 2005. A soybean seed protein with carboxylate-binding activity. 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AbstractThe glucose responsive hydrogels developed by Horbett et al. [1, 2] are potentially useful as materials for constructing glucose sensors. The construction of microsensors presents challenges in developing processes for preparing these gels that are compatible with semiconductor fabrication technology.We have lithographically patterned a pH-sensitive copolymer (poly-(2-hydroxyethyl methacrylate-co-dimethyl aminoethyl methacrylate) 90:10 (mole ratio) (HEMA/DMA) from a prepolymer solution of monomers, crosslinker, solvent, and a viscosity agent (p(HEMA)). Varying the spin speed and p(HEMA) concentration resulted in reproducible thicknesses between 5 and 25 μm. pH sensors produced by this method showed sensitivity ranging from 20 to 50%/pH and a response time (10%–90%) of 6.8 minutes. Sensitivity dropped by a factor of two over a 24 day testing period. The limited lifetime is thought to be due to loss of adhesion of the polymer to the substrate.Derivatizing glucose oxidase with a photoactivatible crosslinker allowed it to be covalently immobilized onto the surface of the gel layer in a spatially specific manner. After immobilization, GOX specific staining showed that enzyme activity was preserved, however the amount of enzyme immobilized was insufficient to create a working glucose sensor.

The syntheses of poly[2]catenane gels reported to date all rely on the polymer as the backbone. We reported poly[2]catenane gels prepared by entirely sequential self‐assembly of small molecules via noncovalent and dynamic covalent bonds. Due to the presence of hydrogen bonds in the poly[2]catenane gels, the gels also possessed stimulus responsiveness and self‐healing properties. More details are discussed in the article by Ji et al. on pages 2699—2704. image

Summary The exploration of a multipressure well is often faced with the problem of blowout and loss of circulation coexisting. Using a high-density temporary plugging agent to plug the lower layer is an effective method to ensure the normal circulation of the upper kill fluid and realize the safe and efficient development of the reservoir. In a previous study, we reported a solid-free flexible colloidal completion fluid (SFCCF; Jia et al. 2022b). In this paper, an ultrahigh-temperature (180°C)-resistant, curable solid-free flexible microgel pill (SFMP) with variable density was prepared based on SFCCF. SFMP is mainly composed of flexible microgel absorbent copolymer material (abbreviated as KA01), and the water absorption law follows Flory’s elastic gel theory. SFMP breaks through the upper density limit of traditional solid-free brine-weighted polymer gels, and its curing density is up to 1.8 g/cm3. The strength of SFMP was increased by crosslinking the stabilizer with KA01 through an acylation reaction. In addition, the hydrogen bonding between stabilizer and phosphate and the phosphorylation between phosphate and KA01 jointly promoted the curing of SFMP. The curing of SFMP is the manifestation of a multistage reaction, and the internal reaction rate increased with the increase in heating rate. SFMP has good performance of dynamic temporary plugging and pressure bearing and can be completely degraded by the chemical breaker. The formation pressure coefficients of the multipressure well in the East China Sea range from 1.20 to 1.42. The SFMP with 1.5 g/cm3 was used to plug the lower perforation layer of the Well AX, which achieved a reservoir protection effect. After the flowback of SFMP, the Well AX met production allocation requirements. The developed SMFP provides a new way for downhole operation in high-temperature, high-pressure (HTHP) multipressure wells.

Abstract: Low density systems or adaptively coordinated systems that has enough buoyancy to float on over contents of the stomach for a significant length of time without noticeably decelerating the rate of gastric emptying are known as floating systems. To accomplish stomach retention, the principle process of flotation was specifically examined in this drug delivery system. It is advantageous to construct medications in an oral sustained release gastro-retentive dose form for those that are absorbed in the upper portions of the GIT. The development of dynamically controlled systems depends on the rate at which the stomach empties. The most recent FDDS advancements are strategies to reduce the variability that lengthens the drug delivery system's retention period to more than 12 hours. This paper also contains an overview of several contemporary in-vitro methods that demonstrate the correct. This review on floating drug delivery systems (FDDS) was written with the intention of gathering the most recent research with a particular focus on the main mechanism of flotation to induce stomach retention. The most recent changes in A detailed discussion of FDDS is provided, covering the physiological and formulation factors impacting stomach retention, design methods for single-unit and multiple-unit floating systems, and their classification and formulation characteristics. The techniques used in vitro, the in vivo tests used to gauge the effectiveness and use Sharma N, Agarwal D, Gupta MK, Khinchi M. A Comprehensive Review On Floating Drug Delivery System. Int J Res Pharm Biomed Sci. 2011;2:428-41. Gohel MC, Mehta PR, Dave RK, Bariya NH. A More Relevant Dissolution Method For Evaluation Of A Floating Drug Delivery System. Diss Technol. 2004;11(4):22-5. Doi: 14227/DT110404P22. Yeole PG, Khan S, Patel VF. Floating Drug Delivery Systems: Need And Development. 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Regional Gastrointestinal Transit And Ph Studied In 215 Healthy Volunteers Using The Wireless Motility Capsule: Influence Of Age, Gender, Study Country And Testing Protocol. Aliment Pharmacol Ther. 2015;42(6):761-72. Doi: 1111/Apt.13329, PMID 26223837. Feldman M, Barnett C. Fasting Gastric Ph And Its Relationship To True Hypochlorhydria In Humans. Dig Dis Sci. 1991;36(7):866-9. Doi: 1007/BF01297133, PMID 2070698. Deshpande AA, Shah NH, Rhodes CT, Malick W. Development Of A Novel Cont Rolled Release System For Gastric Retention. P Harm Res. 1997;14(6):815-9. of floating systems, and the applications of these systems are all summarised in this paper. These systems are helpful for a number of issues that arise during the creation of a pharmaceutical dosage form.