Static Assays Research Articles

A unifying model to estimate thermal tolerance limits in ectotherms across static, dynamic and fluctuating exposures to thermal stress

Temperature tolerance is critical for defining the fundamental niche of ectotherms and researchers classically use either static (exposure to a constant temperature) or dynamic (ramping temperature) assays to assess tolerance. The use of different methods complicates comparison between studies and here we present a mathematical model (and R-scripts) to reconcile thermal tolerance measures obtained from static and dynamic assays. Our model uses input data from several static or dynamic experiments and is based on the well-supported assumption that thermal injury accumulation rate increases exponentially with temperature (known as a thermal death time curve). The model also assumes thermal stress at different temperatures to be additive and using experiments with Drosophila melanogaster, we validate these central assumptions by demonstrating that heat injury attained at different heat stress intensities and durations is additive. In a separate experiment we demonstrate that our model can accurately describe injury accumulation during fluctuating temperature stress and further we validate the model by successfully converting literature data of ectotherm heat tolerance (both static and dynamic assays) to a single, comparable metric (the temperature tolerated for 1 h). The model presented here has many promising applications for the analysis of ectotherm thermal tolerance and we also discuss potential pitfalls that should be considered and avoided using this model.

Enhancement of pyocyanin production by subinhibitory concentration of royal jelly in Pseudomonas aeruginosa

Background: Pseudomonas aeruginosa, a multidrug-resistant Gram-negative bacterium, produces pyocyanin, a virulence factor associated with antibiotic tolerance. High concentrations of royal jelly have an antibacterial effect, which may potentially overcome antibacterial resistance. However, in some cases, antibiotic tolerance can occur due to prolonged stress of low-dose antibacterial agents. This study aimed to investigate the effect of subinhibitory concentrations of royal jelly on bacterial growth, pyocyanin production, and biofilm formation of P. aeruginosa. Methods: Pseudomonas aeruginosa ATCC 10145 and clinical isolates were cultured in a royal jelly-containing medium to test the antibacterial activity. Pyocyanin production was observed by measuring the absorbance at 690 nm after 36 h culture and determined using extinction coefficient 4310 M-1 cm-1. Static microtiter plate biofilm assay performed to detect the biofilm formation, followed by scanning electron microscopy. Results: Royal jelly effectively inhibited the viability of both strains from a concentration of 25%. The highest production of pyocyanin was observed in the subinhibitory concentration group 6.25%, which gradually decreased along with the decrease of royal jelly concentration. Results of one-way ANOVA tests differed significantly in pyocyanin production of the two strains between the royal jelly groups. Tukey HSD test showed concentrations of 12.5%, 6.25%, and 3.125% significantly increased pyocyanin production of ATCC 10145, and the concentrations of 12.5% and 6.25% significantly increased production of the clinical isolates. Concentrations of 12.5% and 6.125% significantly induced biofilm formation of P. aeruginosa ATCC 10145, in line with the results of the SEM analysis. Conclusions: The royal jelly concentration of 25% or higher inhibits bacterial growth; however, the subinhibitory concentration increases pyocyanin production and biofilm formation in P. aeruginosa . It is advisable to determine the appropriate concentration of royal jelly to obtain beneficial virulence inhibiting activity.

Analysis of Non-linear Pharmacokinetics of P-Glycoprotein Substrates in a Microfluidic Device Using a Mathematical Model that Includes an Unstirred Water Layer (UWL) Compartment

The purpose of this research is to analyze non-linear pharmacokinetics of P-glycoprotein (P-gp) substrates in a cell based assay of a microfluidic device, which might be affected by hydrodynamic barrier (unstirred water layer, UWL). Apparent permeability (Papp) were obtained using non-P-gp substrates (propranolol, metoprolol, and atenolol) and P-gp substrates (quinidine and talinolol) in a commercially available microfluidic device, organoplate ® of Caco-2 cell based assay. The previous UWL resistance model was well fitted to Papp of static and flow condition by assuming UWL including and negligible condition, while P-gp substrates of higher passive permeability (quinidine) was apart from the fitting curve. The concentration dependent non-linear kinetics of P-gp substrates, quinidine and talinolol, was more analyzed in detail, and apparent Vmax discrepancybetween static and flow assay condition in the quinidine assay was observed, while that was not observed in talinolol, the lower permeable substrate. Based on the experimental results, a mathematical model for P-gp substrates including UWL compartment on the previous 3-compartment model was developed, and it indicated that the apparent Vmax was variable along with the ratio between passive permeability and UWL permeability. The mathematical model adding UWL compartment well explained non-linear pharmacokinetics of apparent permeability of P-gp substrate in the microfluidic device. The model also has a potential to be applied to P-gp substrate permeability analysis in vivo.

Selective Somatostatin 5 (SST5) and Somatostatin 2 (SST2) Nonpeptide Agonists Potently Suppress Glucose- and Tolbutamide-Stimulated Dynamic Insulin Secretion From Isolated Human Islets

Congenital hyperinsulinism (HI) is the most common cause of persistent hypoglycemia in newborns and infants and arises from dysregulated insulin secretion. Rapid recognition and treatment are vital to prevent seizures, permanent developmental delays, coma, or even death. Very few medical options exist to treat congenital HI patients: the KATP channel activator diazoxide, the injectable somatostatin receptor peptide agonists octreotide and lanreotide, or chronic glucose infusions. However, side effects and/or limited efficacy render these therapies inadequate for many patients.Somatostatin is a 14-amino acid peptide hormone with a broad spectrum of biological actions, which are regulated through five somatostatin receptor subtypes (SST1-SST5). Somatostatin’s common physiological role is to down-regulate secretion of other hormones in various tissues. Its role in the maintenance of euglycemia is to regulate insulin and glucagon secretion from pancreatic β- and α-cells, respectively. Somatostatin regulates insulin secretion by decreasing the intracellular levels of cAMP, inhibition of voltage-gated calcium channels (VGCC), activation of the G protein-activated inward rectifier K+ channel (GIRK), and direct inhibition of insulin exocytosis.Several studies have evaluated the effect of somatostatin, somatostatin peptide analogs, and a limited number of nonpeptide somatostatin receptor agonists on insulin secretion in static assays using isolated human islets. However, the lack of highly selective agonists has made the interpretation of the contribution of SST receptor sub-types difficult to discern. Our programs for the treatment of hyperinsulinism, acromegaly, and other indications have led to the development of selective nonpeptide SST2, SST3, SST4, and SST5 agonists, possessing EC50s < 1 nM in cell-based assays of receptor activation and selectivity > 130 times over the other members of the family. The ability of these selective nonpeptide agonists to regulate glucose- and tolbutamide-stimulated dynamic insulin secretion from human islets was evaluated using a perifusion system (Biorep, FL).We found that selective SST2 and SST5 agonists potently suppressed dynamic insulin secretion in contrast to SST3 or SST4 selective agonists. Importantly, SST5 agonists were shown to have a greater effect than selective SST2 agonists or diazoxide, demonstrating their potential utility in human conditions such as congenital HI. In addition, SST5 activation is also known to have a smaller effect on glucagon secretion and is also less prone to agonist-driven desensitization than SST2 activation. Taken together, these studies support our program to identify, characterize, and develop potent, nonpeptide, orally-bioavailable, selective SST5 agonists with appropriate pharmaceutical and safety characteristics for the treatment of congenital HI.

Chemical Composition, and Evaluation of Antibacterial，Antibiofilm and Synergistic Effects with Conventional Antibiotics of Essential Oil from Mallotus repandus

The essential oil (EO) of aerial parts of Mallotus repandus (Willd.) Muell. Arg. was extracted by hydrodistillation and characterized by GC/FID and GC/MS. Fifty-one compounds comprising 97.1% of the EO were identified, of which α-humulene (18.7%), β-selinene (12.8%), aciphyllene (10.7%), (E)-caryophyllene (8.4%), α-copaene (5.5%), humulene epoxide II (4.9%) and caryophyllene oxide (4.3%) were the major compounds. The EO was evaluated for antibacterial properties using broth microdilution method and crystal-violet static biofilm formation assay. The M. repandus EO possessed a bactericidal effect against tested gram-positive bacteria strains (MIC = MBC: 0.05-0.10 mg/mL). Further, the EO showed the ability to inhibit the biofilm formation of Staphylococcus aureus. In addition, the potential synergistic effect was assessed by checkerboard method. Combination of the M. repandus EO with Streptomycin showed synergistic effects against the tested bacterial strains. This study demonstrates that M. repandus EO could be further explored as good alternative for potential pharmaceuticals.

Biocontrol potential of bacteriophage \u0278sp1 against bacterial wilt-causing Ralstonia solanacearum in Solanaceae crops

BackgroundBacteriophages are effective biocontrol strategy as well as ecofriendly remedy for the emerging antibiotic and chemical resistance in bacterial phytopathogens such as bacterial wilt-causing Ralstonia solanacearum. One of the major challenges in the use of bacteriophage therapy for agricultural phytopathogens is maintaining their viability even during variations in pH, temperature, ultraviolet irradiation, and desiccation during field application for sustainable agriculture.ResultsIn this study, the isolation and characterization of phage ɸsp1 for its efficacy against wilt-causing R. solanacearum performed on Solanum lycopersicum (tomato) seedlings and Solanum tuberosum (potato) tuber assay are reported. Bacteriophage was found to be viable and stable at a wide pH range (3.0–9.0) and at temperatures up to 55 °C. Phage ɸsp1 required ~15 min for adsorption and completed its life cycle in 25–30 min by host cell lysis with a burst size of ~250–300. Phage ɸsp1 eradicated 94.73% preformed R. solanacearum biofilm and inhibited biofilm formation by 73.68% as determined by the static crystal violet microtiter biofilm assay. Transmission electron microscope revealed the phage ɸsp1 to be approximately 208±15 nm in size, comprising of icosahedral head (100 ±15 nm) and tail, as belonging to Myoviridae family. Plant bioassays showed 81.39 and 87.75% reduction in pathogen count using phages ɸsp1 in potato tuber and tomato seedlings, respectively. Reversal in disease symptoms was 100% in phage-treated tuber and tomato plant (pot assay) compared to only pathogen-treated controls.ConclusionIsolated bacteriophage ɸsp1 was found to be highly host specific, effective in biofilm prevention, and capable of inhibiting bacterial wilt at low multiplicity of infection (1.0 MOI) in tomato as well as potato tuber bioassays. Phages ɸsp1 were environmentally stable as they survive at variable pH and temperature. Bacteriophage ɸsp1 shows a promise for development into a biocontrol formulation for the prevention of R. solanacearum bacterial wilt disease.

Acute exposure to an organochlorine pesticide alters locomotor behavior and cardiac function in the freshwater invertebrate Daphnia magna

ABSTRACT In this study, the acute impacts of aqueous methoxychlor exposure on the viability, locomotive behavior, and heartrate of Daphnia magna was assessed. Adults were exposed to 0 (ethanol control), 1, 2.5, 5, or 10 µg/L of methoxychlor for 48 hr using a static exposure assay. After 24 hr mobility was quantified using automated behavioral analysis software and differences in mortality and heartrate assessed after 24 and 48 hr. Exposure caused an increase in mortality after 48 hr, with effects seen in as little as 24 hr in the 10 µg/L treatment. Decreases in speed, total distance traveled, and frozen events were observed after 24 hr. Heartrate in the 10 µg/L treatment was depressed after 24 hr, with decreases in the 2.5 and 5 µg/L treatments observed after 48 hr. These results provide an expanded understanding of the impacts of a well-established legacy organochlorine pesticide on a model invertebrate.

Garlic (Allium sativum L.) Ethanolic Extract Capability to Inhibit Pseudomonas aeruginosa Biofilm Formation

Garlic extract (Allium sativum L.) is known to contain substances that can inhibitbacterial adhesion. This study aims to explore the ability of garlic (Allium sativum L.) extract toinhibit the adhesion of P. aeruginosa. Ethanolic garlic (Allium sativum L.) extract was made bythe maceration method. A bacterial adherence assay was performed used the static microtiter biofilm assay method on 96-well plates. Pseudomonas aeruginosa was inoculated in added garlic(Allium sativum L.) extract to brain heart infusion (BHI) broth in various concentrations. Biofilmbacteria on wall plates were stained with 0.1% Crystal violet and were extracted using 96%,subsequently were measured using a microplate reader with absorbance at 595 nm. Decreasedoptical density value equal with increased extract concentration. Statistical analysis using One-way ANOVA revealed a significant difference in the optical density (OD) value between thegroups (p&lt;0.05). The minimum concentration obtained in this study was 156.25 ? g/ml.Conclusion, ethanolic garlic (Allium sativum L.) extract can inhibit adherence to P. aeruginosa.Keywords: Garlic ethanolic extract; Pseudomonas aeruginosa; adherence.

Weak organic acid synergy towards the prevention of catheter blockages

Up to 50% of all long-term catheterized individuals experience recurrent episodes of urinary catheter infections and blockages, leading to urine retention, pyelonephritis and septicaemia if the catheter is left in situ. We have previously reported the synergistic activity of weak organic acid (WOA) combinations against nosocomial uropathogens. To investigate the efficacy of selected WOAs, citric acid and propionic acid, alone and in combination, on prevention of crystalline biofilm formation and catheter blockages. Static crystallization assays and dynamic invitro bladder model assays, with scanning electron microscopy, were performed for determination of bacterial viability, urinary pH and time to catheter blockage. The rate of encrustation around the catheter eyeholes was reduced in the presence of the citric acid/propionic acid combination, extending the time to blockage three-fold. Synergistic WOA combinations identified herein represent promising alternatives to antibiotics to combat the global healthcare burden of catheter-associated urinary tract infections and related blockages.

Bactericidal efficiency of micro- and nanostructured surfaces: a critical perspective.

Micro/nanostructured surfaces (MNSS) have shown the ability to inactivate bacterial cells by physical means. An enormous amount of research has been conducted in this area over the past decade. Here, we review the various surface factors that affect the bactericidal efficiency. For example, surface hydrophobicity of the substrate has been accepted to be influential on the bactericidal effect of the surface, but a review of the literature suggests that the influence of hydrophobicity differs with the bacterial species. Also, various bacterial viability quantification methods on MNSS are critically reviewed for their suitability for the purpose, and limitations of currently used protocols are discussed. Presently used static bacterial viability assays do not represent the conditions of which those surfaces could be applied. Such application conditions do have overlaying fluid flow, and bacterial behaviours are drastically different under flow conditions compared to under static conditions. Hence, it is proposed that the bactericidal effect should be assessed under relevant fluid flow conditions with factors such as shear stress and flowrate given due significance. This review will provide a range of opportunities for future research in design and engineering of micro/nanostructured surfaces with varying experimental conditions.

Resolving the Deceptive Isoform and Complex Selectivity of HDAC1/2 Inhibitors

SummaryThe histone deacetylase paralogs HDAC1/2/3 and their corepressor complexes serve as epigenetic master regulators of chromatin function. Over the past decades, HDACs have been widely pursued as pharmacological targets and considerable efforts have been invested in the development of small molecule drugs. Specifically, ortho-aminoanilide derived inhibitors, including CI-994 and Cpd-60, stand out with their attractive selectivity profiles and have been used extensively as tools to delineate the biological roles of specific HDAC isoforms and complexes. Here, we apply a suite of activity-independent strategies to investigate how dynamic processes that regulate HDAC complexes govern the isoform and complex selectivity of HDAC inhibitors. Importantly, we find that overreliance on static and simplified biochemical activity assays has confounded the determination of the biological selectivity of these inhibitors. Our data urge a comprehensive reinterpretation of numerous studies utilizing these tool compounds for the interrogation of epigenetic and other cellular processes.

A microfluidic platform for in situ investigation of biofilm formation and its treatment under controlled conditions

BackgroundStudying bacterial adhesion and early biofilm development is crucial for understanding the physiology of sessile bacteria and forms the basis for the development of novel antimicrobial biomaterials. Microfluidics technologies can be applied in such studies since they permit dynamic real-time analysis and a more precise control of relevant parameters compared to traditional static and flow chamber assays. In this work, we aimed to establish a microfluidic platform that permits real-time observation of bacterial adhesion and biofilm formation under precisely controlled homogeneous laminar flow conditions.ResultsUsing Escherichia coli as the model bacterial strain, a microfluidic platform was developed to overcome several limitations of conventional microfluidics such as the lack of spatial control over bacterial colonization and allow label-free observation of bacterial proliferation at single-cell resolution. This platform was applied to demonstrate the influence of culture media on bacterial colonization and the consequent eradication of sessile bacteria by antibiotic. As expected, the nutrient-poor medium (modified M9 minimal medium) was found to promote bacterial adhesion and to enable a higher adhesion rate compared to the nutrient-rich medium (tryptic soy broth rich medium ). However, in rich medium the adhered cells colonized the glass surface faster than those in poor medium under otherwise identical conditions. For the first time, this effect was demonstrated to be caused by a higher retention of newly generated bacteria in the rich medium, rather than faster growth especially during the initial adhesion phase. These results also indicate that higher adhesion rate does not necessarily lead to faster biofilm formation. Antibiotic treatment of sessile bacteria with colistin was further monitored by fluorescence microscopy at single-cell resolution, allowing in situ analysis of killing efficacy of antimicrobials.ConclusionThe platform established here represents a powerful and versatile tool for studying environmental effects such as medium composition on bacterial adhesion and biofilm formation. Our microfluidic setup shows great potential for the in vitro assessment of new antimicrobials and antifouling agents under flow conditions.

Virtual Screening for Novel SarA Inhibitors to Prevent Biofilm Formation of Staphylococcus aureus in Prosthetic Joint Infections.

Staphylococcus aureus is one of the predominant causes of periprosthetic joint infections (PJIs). Bacterial adhesion and biofilm formation are important factors in the pathogenesis of PJIs. S. aureus biofilm formation is regulated by several factors, including S. aureus regulator A (SarA). Previous studies have found that SarA mutants have limited ability to develop biofilms. In this study, we identified a SarA-targeting antibiofilm compound, ZINC00990144, and evaluated its efficacy and toxicity. According to static biofilm assay, the antibiofilm ability of the compound was concentration dependent. ZINC00990144 reduced biofilm in multiple strains by 40–86% at a concentration of 11.5 μM. Additionally, ZINC00990144 inhibited biofilm formation on different orthopedic implant materials including Titanium and UHMWPE disc. Furthermore, quantitative polymerase chain reaction results demonstrated that ZINC00990144 upregulated the expression of S. aureus exoproteases to inhibit the formation of biofilms. Moreover, ZINC00990144 prevented biofilm formation when exposed to sub-inhibitory doses of vancomycin, which is known to promote biofilm formation. CCK-8 results demonstrated ZINC00990144 has no significant effect on cell viability at concentration of 11.5 μM or below. Finally, we verified the antibiofilm function of the compound in vivo using implant infection mice model with/without exposure to sub-inhibitory vancomycin. In conclusion, ZINC00990144 acts by modulating between biofilm and planktonic state of S. aureus instead of being bactericidal. Therefore, it has the potential to be used in combination with other antibiotics to prevent PJIs.

Comparison of Static and Dynamic Assays When Quantifying Thermal Plasticity of Drosophilids.

Simple SummaryTemperature directly affects many biological processes, from enzymatic reactions to population growth, and thermal stress tolerance is central to our understanding of the global distribution and abundance of species and populations. Given the importance of thermal stress tolerance in ecophysiology and evolutionary biology it is important to be able to measure thermal stress resistance accurately and in ecologically relevant ways. Several methods for such quantification exist in the arthropod literature and the comparability of different methods is currently being debated. Here we reconcile the two most commonly used thermal assays (dynamic ramping and static knockdown assays) for quantifying insect heat tolerance limits and plastic responses using a newly suggested modeling technique. We find that results obtained on the basis of the two assays are highly correlated and that data from one assay can therefore reasonably well predict estimates from the other. These data are of general relevance to the study of thermal biology of ectotherms.Numerous assays are used to quantify thermal tolerance of arthropods including dynamic ramping and static knockdown assays. The dynamic assay measures a critical temperature while the animal is gradually heated, whereas the static assay measures the time to knockdown at a constant temperature. Previous studies indicate that heat tolerance measured by both assays can be reconciled using the time × temperature interaction from “thermal tolerance landscapes” (TTLs) in unhardened animals. To investigate if this relationship remains true within hardened animals, we use a static assay to assess the effect of heat hardening treatments on heat tolerance in 10 Drosophila species. Using this TTL approach and data from the static heat knockdown experiments, we model the expected change in dynamic heat knockdown temperature (CTmax: temperature at which flies enter coma) and compare these predictions to empirical measurements of CTmax. We find that heat tolerance and hardening capacity are highly species specific and that the two assays report similar and consistent responses to heat hardening. Tested assays are therefore likely to measure the same underlying physiological trait and provide directly comparable estimates of heat tolerance. Regardless of this compliance, we discuss why and when static or dynamic assays may be more appropriate to investigate ectotherm heat tolerance.

Corrosion resistance and biocompatibility of polydopamine/hyaluronic acid composite coating on AZ31 magnesium alloy

Magnesium (Mg) and its alloys have received widespread attention as implants for orthopedic surgery due to their degradability and appropriate mechanical properties. However, their poor corrosion resistance may lead to a decline of mechanical properties as well as changes in the surrounding microenvironment, which will eventually lead to their failure as implants. In this work, hyaluronic acid (HYA) was immobilized on AZ31 alloy via a mussel-inspired polydopamine (PDA) anchoring layer. The HYA layer closed the cracks caused by the PDA layer. In vitro corrosion behavior of the PDA/HYA composite coating was investigated via potentiodynamic polarization (PDP) and static immersion assay. The corrosion current density of PDA/HYA coating decreased from 8.48×10−5 A/cm2 to 5.71×10−6 A/cm2. During immersion, no obvious local alkalization was observed. Large amounts of calcium phosphate deposited on PDA/HYA coating, which enhanced the biological activity. MTT experiments show that the MC3T3-E1 cell survival rate of polydopamine coating exceeds 100% after cultivated for 5 days. It was found that the number of cells spreading on the surface of PDA/HYA composite coating was the most after the cell culture directly on the surface.

Effective Strategy Targeting Polymyxin-Resistant Gram-Negative Pathogens: Polymyxin B in Combination with the Selective Serotonin Reuptake Inhibitor Sertraline.

This study aimed to investigate synergistic antibacterial activity of polymyxin B in combination with the selective serotonin reuptake inhibitor, sertraline, against the Gram-negative pathogens Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The combination of polymyxin B and sertraline showed synergistic antibacterial activity in checkerboard and static time-kill assays at clinically relevant concentrations against both polymyxin-susceptible and polymyxin-resistant isolates. The potential antimicrobial mode of action of the combination was investigated against P.aeruginosa FADDI-PA024 using untargeted metabolomics alongside scanning and transmission electron microscopy (EM). Scanning and transmission EM revealed that the polymyxin B and sertraline combination resulted in greater damage to the bacterial cell compared to each drug alone. Metabolomics results showed that the combination significantly affected the bacterial ability to remodel its outer membrane. This was reflected by the major perturbation of glycerophospholipids and fatty acids and the pantothenate and coenzyme A (CoA) pathways, which feed fatty acid elongation (e.g., trans-hexadec-2-enoyl-CoA) as well as inhibit the biosynthesis of lipopolysaccharide and peptidoglycan. The combination also inhibited the polymyxin resistance phosphoethanolamine (pEtN) lipid A modification pathway, indicated by the declined levels of phosphoethanolamine. In summary, the present study highlights the potential possibilities of a polymyxin-sertraline combination for the treatment of infections caused by multidrug resistant Gram-negative bacteria such as central nervous system (CNS) infections via direct intraventricular/intrathecal delivery.

Upshaw-Schulman syndrome-associated ADAMTS13 variants possess proteolytic activity at the surface of endothelial cells and in simulated circulation

ADAMTS13 regulates the hemostatic activity of von Willebrand factor (VWF). Determined by static assays, proteolytic activity <10IU/dL in patient plasma, in absence of ADAMTS13 autoantibodies, indicates Upshaw-Schulman syndrome (USS); the congenital form of Thrombotic Thrombocytopenic Purpura (TTP). We have recently functionally characterized sixteen USS-associated ADAMTS13 missense variants under static conditions. Here, we used two assays under shear flow conditions to analyze the activity of those seven mutants with sufficiently high residual secretion plus two newly identified variants. One assay determines cleavage of VWF strings bound to the surface of endothelial cells. The other, light transmission aggregometry-based assay, mimics degradation of VWF-platelet complexes, which are likely to be present in the circulation during TTP bouts. We found that 100 ng/ml of all variants were able to cleave about 80-90% of VWF strings even though 5 out of 9 exhibited activity ≤1% in the state-of-the-art static assay at the same concentration. These data indicate underestimation of ADAMTS13 activity by the used static assay. In simulated circulation, two variants, with missense mutations in the vicinity of the catalytic domain, exhibited only minor residual activity while all other variants were able to effectively break down VWF-platelet complexes. In both assays, significant proteolytic activity could be observed down to 100 ng/ml ADAMTS13. It is thus intriguing to postulate that most variants would have ample activity if secretion of 10% of normal plasma levels could be achieved.

SIX2 Regulates Human β Cell Differentiation from Stem Cells and Functional Maturation In Vitro

SUMMARYGeneration of insulin-secreting β cells in vitro is a promising approach for diabetes cell therapy. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are differentiated to β cells (SC-β cells) and mature to undergo glucose-stimulated insulin secretion, but molecular regulation of this defining β cell phenotype is unknown. Here, we show that maturation of SC-β cells is regulated by the transcription factor SIX2. Knockdown (KD) or knockout (KO) of SIX2 in SC-β cells drastically limits glucose-stimulated insulin secretion in both static and dynamic assays, along with the upstream processes of cytoplasmic calcium flux and mitochondrial respiration. Furthermore, SIX2 regulates the expression of genes associated with these key β cell processes, and its expression is restricted to endocrine cells. Our results demonstrate that expression of SIX2 influences the generation of human SC-β cells in vitro.

Oxidative and biochemical responses as indicators of chemical stress in Clarias gariepinus (Burchell, 1822) juvenile exposed to glyphosate

A sub-lethal toxicity experiment was undertaken to study the response of some enzyme activities, tissue proteins and muscular stress proteins in Clarias gariepinus juveniles exposed to glyphosate herbicide. The juveniles were exposed to 0.19 mgL−1, 0.28 mgL−1and 0.57 mgL−1 of the test herbicide, which represented 1/9th, 1/6th and 1/3rd of the LC50, respectively in 24 h static renewal assay. The liver and muscle tissues of the exposed juveniles were sampled on day 1 and 14 during assay. The results showed that catalase, TBARS, ALP and ALT increased significantly (p < 0.05) with concentrations and time. Protein bands of different molecular weight ranging from 15 kDa to 250 kDa were expressed at different treatments. The results showed that glyphosate herbicides induce oxidative stress in fishes even at sub-lethal doses.

Iron deficiency-induced thrombocytosis increases thrombotic tendency in rats.

Iron deficiency (ID) is globally prevalent, and apart from anemia is associated with thrombocytosis. While considered benign, studies linking thrombotic events with prior ID anemia suggest otherwise. In this study we used animal models to assess the influence of ID on thrombotic tendency. Sprague-Dawley rats were fed control or iron-deficient diets and ferric carboxymaltose was used to reverse ID. Thrombosis was induced by stenosis of the inferior vena cava or damage to the right carotid artery using ferric chloride. Thrombi were evaluated histologically and by high frequency ultrasound in the venous model. ID consistently induced thrombocytosis alongside anemia. The growth of venous thrombi and the final dimensions of both arterial and venous thrombi were greater in animals with ID. In both models, platelet numbers correlated with the final thrombus size, with thrombi in iron-deficient animals having the largest platelet areas. Platelet function was also evaluated in surgically-naïve rats. Coagulability, determined by thromboelasto - graphy, and hemostasis, evaluated by tail transection, were enhanced in the animals with ID. Platelet P-selectin expression and plasma P-selectin levels were both higher in animals with ID. Platelet adhesion and aggregation in ID was impaired under shear flow but was intact in static assays. Iron replacement therapy reversed all ID-related changes in hematologic parameters, thrombus dimensions, and platelet assays. In summary, ID alone increases thrombotic tendency. Iron replacement therapy reverses these changes, making it a viable strategy for the prevention of ID-related thrombotic disease. This may be of importance in patients with chronic illnesses who may already be at increased risk of thrombosis, such as those with inflammatory bowel disease, chronic kidney disease, or cancer.