A New Drug for Obesity: Tirzepatide.

Obesity is now recognised as an emerging public health problem across the globe. Its incidence has been growing in the last two decades. Furthermore, as per the obesity treatment guidelines, a comprehensive approach that incorporates behavioural treatment, medications, lifestyle modifications, and/or bariatric surgery is the best way to manage weight. A novel dual agonist of Glucose-dependent insulinotropic peptide (GIP) and Glucagon-like peptide -1 (GLP- 1) receptors, Tirzepatide, was recently approved for the management of obesity. Tirzepatide manages blood sugar levels and enhances weight loss more than GLP-1 receptor agonists.

Continuous addition kinetic elucidation: catalyst and reactant order, rate constant, and poisoning from a single experiment.

Kinetic analysis of catalytic reactions is a powerful tool for mechanistic elucidation but is often challenging to perform, limiting understanding and therefore development of these reactions. Establishing order in a catalyst is usually achieved by running several reactions at different loadings, which is both time-consuming and complicated by the challenge of maintaining consistent run-to-run experimental conditions. Continuous addition kinetic elucidation (CAKE) was developed to circumvent these issues by continuously injecting a catalyst into a reaction, while monitoring reaction progress over time. For reactions that are mth order in a single yield-limiting reactant and nth order in catalyst, a plot of reactant concentration against time has a shape dependent only on the orders m and n. Therefore, fitting experimental CAKE data (using open access code or a convenient web tool) allows the reactant and catalyst orders, rate constant, and the amount of complete catalyst inhibition to be determined from a single experiment. Kinetic information obtained from CAKE experiments showed good agreement with the literature.

Jigsaw Classroom: Perceptions of Students and Teachers.

Background: Various innovative teaching methods have been designed in an attempt to provide millennial students an interactive and stimulating learning environment. A jigsaw method is one such form of cooperative learning that enables students to develop critical-thinking and ability for effective communication and promotes positive student attitudes toward their own learning. The present study was thus undertaken to introduce a jigsaw-based cooperative learning method and to understand the perception of the students and faculty. Methodology: This was a cross-sectional study conducted among second year MBBS students. Students were randomly divided into 10 parent groups, with 10 students each. The topic was divided into 10 subtopics. Each student in the parent group was allotted a sub-topic. The students who were given the same sub-topic assembled to form an expert group. They interacted and discussed the subtopic. Students finally returned to their parent groups for peer teaching and presentation. A validated questionnaire was used to gather students and faculty feedback. Descriptive statistics were used to analyze responses to the Likert scale questions. Qualitative data was analyzed using thematic analysis. Results: The present study included 95 s year MBBS students. Majority (81%) students agreed that this teaching learning method enhanced their communication skills. Seventy-five percent of the students agreed that the activity helped in overcoming shyness and hesitation in the class. Overall the faculty also agreed that the jigsaw method was helpful for the students. Conclusions: Cooperative learning like jigsaw facilitates learning allowing student-student discussion, improving communication and teaching skills.

Adverse Drug Reactions in Corona Virus Disease Patients Admitted to Intensive Care Unit: An Observational Study.

BACKGROUND: Occurrence of adverse drug reactions (ADRs) in COVID-19 patients has not been extensively studied. AIM: The present study was conducted to analyze the pattern of suspected ADRs in the COVID-19 Intensive Care Unit (ICU). METHODS: In this cross-sectional study, all the individual case study reports of patients admitted to the COVID ICU (August-October 2020) were analyzed for type of ADRs, system involved, suspected drug, onset time, time to revert and management. RESULTS: Thirty six patients (out of 395 patients admitted) experienced 44 ADRs. Dermatological manifestations were the most frequent ADRs. Remdesivir was the most common drug associated with ADRs. The female gender, polypharmacy (>5 drugs) and presence of comorbidities were the independent risk factors for the occurrence of ADRs. CONCLUSION: Use of many of these drugs in COVID-19 is experimental and the literature does not guarantee their safety and efficacy. During these times of uncertainty, the results from the present study reinforce the importance of monitoring patients.

Head-to-Head Comparison of Tazarotene and Calcitriol with or without Sequential Therapy in Mild-to-Moderate Psoriasis: A Randomized Open-label Study.

Objective: Psoriasis is an autoimmune disease that causes rapid turnover of skin cells. It is a chronic disease that affects a patient's quality of life significantly and frequently requires long-term treatment. The study on sequential therapy with tazarotene 0.1% and calcitriol 0.0003% has not been tried so far; hence, we designed this study to compare the safety and efficacy of sequential therapy with tazarotene 0.1% cream and calcitriol 0.0003% ointment versus monotherapy in mild-to-moderate stable plaque psoriasis (SPP). The objective of this study was to compare the safety and efficacy of topical sequential treatment with tazarotene followed by calcitriol, topical calcitriol followed by tazarotene, tazarotene monotherapy, calcitriol monotherapy, and compare the safety and efficacy of the sequential therapies with monotherapies. Methods: The study was a single center, prospective parallel-group, active control, randomized study of 16 weeks duration (treatment for 8 weeks and follow-up for 16 weeks), randomized to either of the four groups, i.e., tazarotene 0.1% for 4 weeks followed by calcitriol 0.0003% for 4 weeks or calcitriol 0.0003% for 4 weeks followed by tazarotene 0.1% for 4 weeks or tazarotene 0.1% for 8 weeks or calcitriol 0.0003% for 8 weeks. Both tazarotene and calcitriol were applied once daily in all the groups. Findings: There was no significant difference with regard to age and duration of illness among the four treatment groups. Statistically significant improvement was observed in erythema, scaling, and induration scores, and Physician`s global assessment scale at 8 weeks and 16 weeks as compared to baseline in tazarotene - calcitriol, calcitriol - tazarotene, and calcitriol versus tazarotene groups. Conclusion: This study concluded that topical treatment with tazarotene 0.1% and calcitriol 0.003% was efficacious in treating mild-to-moderate SPP as both sequential and monotherapy. However, topical treatment with tazarotene as monotherapy was the least efficacious.

Chimeric antigen receptor T cell: A cancer immunotherapy.

During present decade, targeted drug therapy has been the epitome for treatment of cancer. Drugs like Imatinib, a tyrosine kinase receptor inhibitor and Trastuzumab, an human epidermal growth factor receptor-2/neu inhibitor, has been developed and accepted widely for management of chronic myeloid leukaemia and breast cancer respectively. Recent development among the various immunotherapies is adoptive cell transfer (ACT). Research on development of various types of ACT immunotherapy is going on, but so far, Chimeric antigen receptors T cell therapy (CAR-T) has achieved the maximum advancement in terms of clinical development. CARs are the modified receptors that integrates specificity and responsiveness onto immune cells to enhance the recognition of cancer cells. For the CAR-T, the T cells are sequestered from a blood of a participant via apheresis. DNA of particular antigen is injected into harvested T cells to generate CARs on cell surface. Following surface manifestation of receptors, multiplication is carried out in enriched media followed by infusion into patient. After infusion, CAR-T cells targeted and exterminate the cancer cells. Initially, only two drugs targeting CD19 as genetically modified autologous immunotherapy has been approved in CAR-T therapy i.e., Tisagenlecleucel and Axicabtagene Ciloleucel, which are discussed in detail in current review. Recently two more drugs got approval i.e., brexucabtagene ciloleucel and lisocabtagene maraleucel, both are directed against CD19, similar to tisagenlecleucel. CAR-T cell therapy is approved for management of Acute Lymphoblastic Leukaemia, Chronic Lymphocytic Leukaemia and lymphoma. CAR-T cell persistence responsible for effectiveness and safety concerns are barriers for their wide application among patients. Growth factor receptors and cluster of differentiation are new drugs targets that are being explored as effective immunotherapy against cancers.

Hydroxychloroquine: Adverse Drug Reaction Profile of an Old Drug in a New Situation.

BACKGROUND: Hydroxychloroquine (HCQ) has been extensively used during the COVID-19 pandemic both as a therapeutic and prophylactic drug. HCQ is generally well tolerated; however, adverse drug reactions (ADRs) in COVID-19 need further exploration. In this study, we have determined the type and pattern of ADRs of HCQ as a prophylactic and therapeutic drug in COVID-19. METHODS: All spontaneous suspected ADR reports due to HCQ in COVID-19 patients submitted to the ADR monitoring of a tertiary care hospital were included. Additionally, a survey was designed for active surveillance of ADRs among healthcare professionals (HCPs) who were on prophylaxis with HCQ. The ADRs were analyzed to determine severity, causality, and preventability using the Hartwig Scale, World Health Organisation-Uppasala Monitoring Centre (WHO-UMC) scale, and modified Schumock and Thornton criterion respectively. RESULTS: Sixty-four ADR reports were received from COVID-19 patients. A total of 78 ADRs were reported by 49 HCPs who were on HCQ prophylaxis. The majority of the patients had ADRs related to skin and soft tissues (37.5%), whereas the HCPs on prophylaxis mostly had gastrointestinal complaints (42.3%). ADRs observed in HCPs on prophylaxis were mild, not requiring any intervention. However, 50% of ADRs in patients were of "moderate" category. CONCLUSION: Undiscerning and unsupervised use of HCQ can expose the general population as well as patients to serious adverse drug effects. Utmost care is necessary before using HCQ prophylactically or for treatment in COVID-19.

Adverse Drug Reactions in Psychiatry Outpatient Department of a Tertiary Care Hospital in Western Uttar Pradesh: An Observational Study.

Objective: Psychiatric disorders are chronic in nature which require medications for a long duration. These medications have been associated with many adverse events. Failure to recognize an adverse drug reaction (ADR) exposes the patient to continuing risk of ADR, leading to a significant impact on patient's quality of life. Thus, the present study carried out to identify the pattern of ADRs reported due to psychotropic medication. Methods: This was a cross-sectional study conducted to analyze ADRs reported from the psychiatry department of a tertiary care teaching hospital from October 2021 to March 2022. Findings: A total of 137 ADRs were identified from 102 patients. Majority of the ADRs were reported from antidepressants, with paroxetine being the leading offending drug. The central nervous system was most commonly affected, and dizziness (13.13%) was the most common ADR noted. On causality assessment, 97 ADRs (70.8%) were of "possible" type. Almost half of the patients with ADRs (47.5%) recovered spontaneously. No ADR encountered turned out to be fatal. Conclusion: The present study revealed that the majority of ADRs reported from psychiatry OPD were mild in nature. We reinforce the identification of ADR is crucial in the hospital setting process as it gives an insight into the risk-benefit ratio for rational use of the drug.

CRISPR-Cas9 gene editing and rapid detection of gene-edited mutants using high-resolution melting in the apple scab fungus, Venturia inaequalis.

Apple scab, caused by the fungal pathogen Venturia inaequalis, is the most economically important disease of apple (Malus x domestica) worldwide. To develop durable control strategies against this disease, a better understanding of the genetic mechanisms underlying the growth, reproduction, virulence and pathogenicity of V. inaequalis is required. A major bottleneck for the genetic characterization of V. inaequalis is the inability to easily delete or disrupt genes of interest using homologous recombination. Indeed, no gene deletions or disruptions in V. inaequalis have yet been published. Using the melanin biosynthesis pathway gene trihydroxynaphthalene reductase (THN) as a target for inactivation, which has previously been shown to result in a light-brown colony phenotype when transcriptionally silenced using RNA interference, we show, for the first time, that the CRISPR-Cas9 gene editing system can be successfully applied to the apple scab fungus. More specifically, using a CRISPR-Cas9 single guide RNA (sgRNA) targeted to the THN gene, delivered by a single autonomously replicating Golden Gate-compatible plasmid, we were able to identify six of 36 stable transformants with a light-brown phenotype, indicating an ∼16.7% gene inactivation efficiency. Notably, of the six THN mutants, five had an independent mutation. As part of our pipeline, we also report a high-resolution melting (HRM) curve protocol for the rapid detection of CRISPR-Cas9 gene-edited mutants of V. inaequalis. This protocol identified a single base pair deletion mutation in a sample containing only 5% mutant genomic DNA, indicating high sensitivity for mutant screening. In establishing CRISPR-Cas9 as a tool for gene editing in V. inaequalis, we have provided a strong starting point for studies aiming to decipher gene function in this fungus. The associated HRM curve protocol will enable CRISPR-Cas9 transformants to be screened for gene inactivation in a high-throughput and low-cost manner, which will be particularly powerful in cases where the CRISPR-Cas9-mediated gene inactivation efficiency is low.

Prevalence of self-reported anxiety and self-medication among upper and middle socioeconomic strata amidst COVID-19 pandemic.

BACKGROUND: Amidst corona virus disease-2019 (COVID-19) pandemic, disruption to the usual ways of life can lead to anxiety and feeling of being unsafe, which may be associated with self-medication. The study was planned to investigate the prevalence of anxiety and self-medication during COVID-19 pandemic. MATERIALS AND METHODS: In this cross-sectional study, data were collected through an anonymous online survey with questionnaire consisting of four sections: demographic data of the participants, validated COVID-19 Anxiety Inventory Items, Self-rating Anxiety Scale, and self-medication practices during COVID-19 pandemic. The data were organized and analyzed using Windows Microsoft excel and SPSS software (Version 21). Descriptive statistics were calculated for all patient characteristics and survey responses. Analytical analysis included relationship between various parameters using Chi-square test. RESULTS: A total of 1100 responses were received. Approximately half of the participants were worried about getting severely ill. Majority reported reduced social contact (71.3%, n = 784) and also communicated that the pandemic has influenced their use of safety measures (86.5%, n = 951). The prevalence of mild-to-moderate, moderate-to severe, and extremely severe anxiety cases were 23.1%, 4.7%, and 0.54%, respectively. One-fourth of the responders reported self-medication, out of which 60% were found to be anxious. Nonsteroidal anti-inflammatory drugs were most commonly used. CONCLUSIONS: Rising to the present challenge will require integration across different sectors with the immediate aim of getting people safely through the pandemic. As the world focuses on containing and delaying the spread of the COVID-19, with burdened health-care systems, we should not miss out on people with anxiety disorders and self-medication problem, which may worsen with the pandemic.

The perioperative administration of dexamethasone and infection (PADDI) trial protocol: rationale and design of a pragmatic multicentre non-inferiority study.

INTRODUCTION: The intraoperative administration of dexamethasone for prophylaxis against postoperative nausea and vomiting is a common and recommended practice. The safety of the administration of this immunosuppressive agent at a time of significant immunological disruption has not been rigorously evaluated in terms of infective complications. METHODS/ANALYSIS: This is a pragmatic, multicentre, randomised, controlled, non-inferiority trial. A total of 8880 patients undergoing elective major surgery will be enrolled. Participants will be randomly allocated to receive either dexamethasone 8 mg or placebo intravenously following the induction of anaesthesia in a 1:1 ratio, stratified by centre and diabetes status. Patient enrolment into the trial is ongoing. The primary outcome is surgical site infection at 30 days following surgery, defined according to the Centre for Disease Control criteria. ETHICS/DISSEMINATION: The PADDI trial has been approved by the ethics committees of over 45 participating sites in Australia, New Zealand, Hong Kong, South Africa and the Netherlands. The trial has been endorsed by the Australia and New Zealand College of Anaesthetists Clinical Trials Network and the Australian Society for Infectious Diseases Clinical Research Network. Participant recruitment began in March 2016 and is expected to be complete in mid-2019. Publication of the results of the PADDI trial is anticipated to occur in early 2020. TRIAL REGISTRATION NUMBER: ACTRN12614001226695.

Oviposition behavior of Diatraea saccharalis (Lepidoptera: Crambidae) on different rice cultivars in Louisiana.

The sugarcane borer, Diatraea saccharalis (F.), is a pest of a variety of graminaceous crops in the southern United States, including sugarcane, maize, and rice in Louisiana. This study examined several aspects of D. saccharalis oviposition behavior on rice (Oryza sativa). The vertical distribution of egg masses on four phenological stages of rice in the field showed that D. saccharalis prefers to oviposit on the uppermost portions of rice plants, regardless of plant stage. In greenhouse choice experiments, females strongly preferred plants at the boot and panicle differentiation stages over plants at the tillering stage for oviposition. Greenhouse studies were also conducted to quantify the oviposition preference of D. saccharalis for different cultivars of. When plants were at the tillering stage, cultivars Cocodrie, Priscilla, Bengal, Cheniere, and CL161 were more preferred than cultivars Jupiter, XL723, and XP744. When plants reached the panicle initiation stage, cultivars Cocodrie, CL161, and Priscilla were more preferred than Bengal, Cheniere, Jupiter, XL723, and XP744. Females also oviposited significantly more egg masses on the adaxial surfaces of rice leaves than on the abaxial surfaces. These results will facilitate scouting and management of sugarcane borer and can be used as a foundation for the development of sugarcane borer resistant cultivars.

Cadmium induced p53-dependent activation of stress signaling, accumulation of ubiquitinated proteins, and apoptosis in mouse embryonic fibroblast cells.

The tumor suppressor oncoprotein, p53, is a critical regulator of stress-induced growth arrest and apoptosis. p53 activity is regulated through the ubiquitin proteasome system (UPS) with stress-induced disruption leading to increased accumulation of p53, resulting in growth arrest. In the present study, we investigate the role of p53 to determine sensitivity to cadmium (Cd) and whether induction of stress signaling responses and perturbation of the UPS are involved in Cd-induced cytotoxicity and apoptosis. We treated synchronously cultured p53 transgenic mouse embryonic fibroblasts, both wild-type p53+/+ and knockout p53-/- cells, with cadmium chloride (Cd, 0.5-20µM) for 24 h. Cd-induced cytotoxicity was assessed by cellular morphology disruption and neutral red dye uptake assay. Proteins in the stress signaling pathway, including p38 mitogen-activated protein kinase (MAPK) and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK); ubiquitination, such as high-molecular weight of polyubiquitinated proteins (HMW-polyUb); and apoptotic pathways, were all measured. We found that Cd induced p53-dependent cytotoxicity in the p53+/+ cells, which exhibited a twofold greater sensitivity. We observed a dose-dependent stimulation of p38 MAPK and SAPK/JNK phosphorylation that corresponded to accumulation of HMW-polyUb conjugates and lead to the induction of apoptosis, as evidenced by the elevation of cleaved caspase-3. Our study suggests that Cd-mediated cytotoxicity and induction of stress signaling responses, elevated accumulation of HMW-polyUb conjugates, and resulting apoptosis are all dependent on p53 status.

A system-based comparison of gene expression reveals alterations in oxidative stress, disruption of ubiquitin-proteasome system and altered cell cycle regulation after exposure to cadmium and methylmercury in mouse embryonic fibroblast.

Environmental and occupational exposures to heavy metals such as methylmercury (MeHg) and cadmium (Cd) pose significant health risks to humans, including neurotoxicity. The underlying mechanisms of their toxicity, however, remain to be fully characterized. Our previous studies with Cd and MeHg have demonstrated that the perturbation of the ubiquitin-proteasome system (UPS) was associated with metal-induced cytotoxicity and apoptosis. We conducted a microarray-based gene expression analysis to compare metal-altered gene expression patterns with a classical proteasome inhibitor, MG132 (0.5 microM), to determine whether the disruption of the UPS is a critical mechanism of metal-induced toxicity. We treated mouse embryonic fibroblast cells at doses of MeHg (2.5 microM) and Cd (5.0 microM) for 24 h. The doses selected were based on the neutral red-based cell viability assay where initial statistically significant decreases in variability were detected. Following normalization of the array data, we employed multilevel analysis tools to explore the data, including group comparisons, cluster analysis, gene annotations analysis (gene ontology analysis), and pathway analysis using GenMAPP and Ingenuity Pathway Analysis (IPA). Using these integrated approaches, we identified significant gene expression changes across treatments within the UPS (Uchl1 and Ube2c), antioxidant and phase II enzymes (Gsta2, Gsta4, and Noq1), and genes involved in cell cycle regulation pathways (ccnb1, cdc2a, and cdc25c). Furthermore, pathway analysis revealed significant alterations in genes implicated in Parkinson's disease pathogenesis following metal exposure. This study suggests that these pathways play a critical role in the development of adverse effects associated with metal exposures.

Gene expression profiling analysis reveals arsenic-induced cell cycle arrest and apoptosis in p53-proficient and p53-deficient cells through differential gene pathways.

Arsenic (As) is a well-known environmental toxicant and carcinogen as well as an effective chemotherapeutic agent. The underlying mechanism of this dual capability, however, is not fully understood. Tumor suppressor gene p53, a pivotal cell cycle checkpoint signaling protein, has been hypothesized to play a possible role in mediating As-induced toxicity and therapeutic efficiency. In this study, we found that arsenite (As(3+)) induced apoptosis and cell cycle arrest in a dose-dependent manner in both p53(+/+) and p53(-/-) mouse embryonic fibroblasts (MEFs). There was, however, a distinction between genotypes in the apoptotic response, with a more prominent induction of caspase-3 in the p53(-/-) cells than in the p53(+/+) cells. To examine this difference further, a systems-based genomic analysis was conducted comparing the critical molecular mechanisms between the p53 genotypes in response to As(3+). A significant alteration in the Nrf2-mediated oxidative stress response pathway was found in both genotypes. In p53(+/+) MEFs, As(3+) induced p53-dependent gene expression alterations in DNA damage and cell cycle regulation genes. However, in the p53(-/-) MEFs, As(3+) induced a significant up-regulation of pro-apoptotic genes (Noxa) and down-regulation of genes in immune modulation. Our findings demonstrate that As-induced cell death occurs through a p53-independent pathway in p53 deficient cells while apoptosis induction occurs through p53-dependent pathway in normal tissue. This difference in the mechanism of apoptotic responses between the genotypes provides important information regarding the apparent dichotomy of arsenic's dual mechanisms, and potentially leads to further advancement of its utility as a chemotherapeutic agent.

Sensitivity of mouse lung fibroblasts heterozygous for GPx4 to oxidative stress.

Phospholipid hydroperoxide glutathione peroxidase (GPx4) is a member of the family of selenium-dependent enzymes that catalyze the reduction of cell membrane-bound phospholipid hydroperoxides in situ and thus protects against membrane damage. Overexpression of GPx4 protects cultured cells from phosphatidylcholine hydroperoxide (PCOOH)-induced loss of mitochondrial membrane potential and blocks cell death induced by treatment with various apoptotic agents. We have generated mice that are heterozygous for a GPx4 null allele (GPx4 +/-); the homozygous null genotype is embryonic lethal. We report that cultured lung fibroblasts (LFs) isolated from adult GPx4 +/- mice had approximately 50% of the GPx4 activity of LFs from GPx4 +/+ mice and were significantly more susceptible to H2O2, cadmium, and cumene hydroperoxide-induced cytotoxicity, as measured by neutral red assay. Both GPx4 +/+ and GPx4 +/- LFs were susceptible to PCOOH-induced cytotoxicity at a high PCOOH concentration. We also found that GPx4 +/- LFs have lower mitochondrial membrane potential, greater cardiolipin oxidation, and lower amounts of reduced thiols relative to GPx4 +/+ LFs, but are more resistant than GPx4 +/+ LFs to further decrements in these endpoints following PCOOH treatment. These results suggest that adult lung fibroblasts deficient in GPx4 may have upregulated compensatory mechanisms to deal with the highly oxidized environment in which they developed.

Cell cycle inhibition by sodium arsenite in primary embryonic rat midbrain neuroepithelial cells.

Arsenite (As3+) exposure during development has been associated with neural tube defects and other structural malformations, and with behavioral alterations including altered locomotor activity and operant learning. The molecular mechanisms underlying these effects are uncertain. Because arsenic can cross the placenta and accumulate in the developing neuroepithelium, we examined cell cycling effects of sodium arsenite (As3+ 0, 0.5, 1, 2, and 4 microM) on embryonic primary rat midbrain (gestational day [GD] 12) neuroepithelial cells over 48 h. There was a concentration- and time-dependent As3+-induced reduction in cell viability assessed by neutral red dye uptake assay but minimal apoptosis at concentrations below 4 microM. Morphologically, apoptosis was not apparent until 4 microM at 24 h, which was demonstrated by a marginal but statistically significant increase in cleaved caspase-3/7 activity. Cell cycling effects over several rounds of replication were determined by continuous 5-bromo-2'-deoxyuridine (BrdU) labeling and bivariate flow cytometric Hoechst-Propidium Iodide analysis. We observed a time- and concentration-dependent inhibition of cell cycle progression as early as 12 h after exposure (> or =0.5 microM). In addition, data demonstrated a concentration-dependent increase in cytostasis within all cell cycle phases, a decreased proportion of cells able to reach the second cell cycle, and a reduced cell cycle entry from gap 1 phase (G1). The proportion of affected cells and the severity of the cell cycle perturbation, which ranged from a decreased transition probability to complete cytostasis in all cell cycle phases, were also found to be concentration-dependent. Together, these data support a role for perturbed cell cycle progression in As3+ mediated neurodevelopmental toxicity.

Essential role of extracellular matrix (ECM) overlay in establishing the functional integrity of primary neonatal rat Sertoli cell/gonocyte co-cultures: an improved in vitro model for assessment of male reproductive toxicity.

The development of in vitro models for testicular toxicity may provide important tools for investigating specific mechanisms of toxicity in the testis. Although various systems have been reported, their application in toxicological studies has been limited by the poor ability to replicate the complex biochemical, molecular, and functional interactions observed in the testis. In the present study, we evaluated a significantly improved Sertoli cell/gonocyte co-culture (SGC) system that employs a 3-dimensional extracellular matrix Matrigel (ECM) applied as an overlay instead of a substratum. We explored the dose- and time-dependent effects of the addition of such an ECM overlay on cytoskeletal and morphological changes in the SGC system, and the resulting effects on cellular integrity. Furthermore, we correlated the latter effects with the ECM-dependent modulation of stress and survival signaling pathways and, most critically, the expression levels of the spermatogonia-specific protein, c-Kit. Finally, we applied this co-culture system to investigate the dose- and time-dependent effects on the morphology and induction of apoptosis of cadmium. We observed that the dose-dependent addition of an ECM overlay led to an enhanced attachment of Sertoli cells and facilitated the establishment of SGC communication and cytoskeletal structure, with a dramatic improvement in cell viability. The latter was consistent with the observed dose- and time-dependent modulation of both stress signaling pathways (SAPK/JNK) and survival signaling pathways (ERK and AKT) in the presence of the ECM overlay. Furthermore, the dose-dependent stabilization of c-Kit protein expression confirmed the functional integrity of this co-culture system. We conclude that this modified SGC system will provide investigators with a simple, efficient, and highly reproducible alternative in the screen for testicular cell-specific cytotoxicity and the assessment of molecular mechanisms associated with both normal development and reproductive toxicity induced by environmental toxicants.

Phenobarbital responsiveness as a uniquely sensitive indicator of hepatocyte differentiation status: requirement of dexamethasone and extracellular matrix in establishing the functional integrity of cultured primary rat hepatocytes.

We used a serum-free, highly defined primary hepatocyte culture model to investigate the mechanisms whereby dexamethasone (Dex) and extracellular matrix (ECM) coordinate cell differentiation and transcriptional responsiveness to the inducer, phenobarbital (PB). Low nanomolar levels of Dex and dilute concentrations of ECM overlay were essential in the maintenance of normal hepatocyte physiology, as assessed by cell morphology, LDH release, expression of the hepatic nuclear factors C/EBPalpha, -beta, -gamma, HNF-1alpha, -1beta, -4alpha, and RXRalpha, expression of prototypical hepatic marker genes, including albumin and transferrin, and ultimately, cellular capacity to respond to PB. The loss of hepatocyte integrity produced by deficiency of these components correlated with the activation of several stress signaling pathways including the MAPK, SAPK/JNK, and c-Jun signaling pathways, with resulting nuclear recruitment of the activated protein-1 (AP-1) complex. In Dex-deficient cultures, normal cellular function, including the PB induction response, was largely restored in a dose-dependent manner by reintroduction of nanomolar additions of the hormone, in the presence of ECM. Our results demonstrate critical and cooperative roles for Dex and ECM in establishing hepatocyte integrity and in the coordination of an array of liver-specific functions. These studies further establish the PB gene induction response as an exceptionally sensitive indicator of hepatocyte differentiation status.