Transgenic Expression of Nrf2 Induces a Pro-Reductive Stress and Adaptive Cardiac Remodeling in the Mouse.

Nuclear factor, erythroid 2 like 2 (Nfe2l2 or Nrf2), is a transcription factor that protects cells by maintaining a homeostatic redox state during stress. The constitutive expression of Nrf2 (CaNrf2-TG) was previously shown to be pathological to the heart over time. We tested a hypothesis that the cardiac-specific expression of full length Nrf2 (mNrf2-TG) would moderately increase the basal antioxidant defense, triggering a pro-reductive environment leading to adaptive cardiac remodeling. Transgenic and non-transgenic (NTG) mice at 7−8 months of age were used to analyze the myocardial transcriptome, structure, and function. Next generation sequencing (NGS) for RNA profiling and qPCR-based validation of the NGS data, myocardial redox levels, and imaging (echocardiography) were performed. Transcriptomic analysis revealed that out of 14,665 identified mRNAs, 680 were differently expressed (DEG) in TG hearts. Of 680 DEGs, 429 were upregulated and 251 were downregulated significantly (FC > 2.0, p < 0.05). Gene set enrichment analysis revealed that the top altered pathways were (a) Nrf2 signaling, (b) glutathione metabolism and (c) ROS scavenging. A comparative analysis of the glutathione redox state in the hearts demonstrated significant differences between pro-reductive vs. hyper-reductive conditions (233 ± 36.7 and 380 ± 68.7 vs. 139 ± 8.6 µM/mg protein in mNrf2-TG and CaNrf2-TG vs. NTG). Genes involved in fetal development, hypertrophy, cytoskeletal rearrangement, histone deacetylases (HDACs), and GATA transcription factors were moderately increased in mNrf2-TG compared to CaNrf2-TG. Non-invasive echocardiography analysis revealed an increase in systolic function (ejection fraction) in mNrf2-TG, suggesting an adaptation, as opposed to pathological remodeling in CaNrf2-TG mice experiencing a hyper-reductive stress, leading to reduced survival (40% at 60 weeks). The effects of excess Nrf2-driven antioxidant transcriptome revealed a pro-reductive condition in the myocardium leading to an adaptive cardiac remodeling. While pre-conditioning the myocardial redox with excess antioxidants (i.e., pro-reductive state) could be beneficial against oxidative stress, a chronic pro-reductive environment in the myocardium might transition the adaptation to pathological remodeling.

CRISPR-Cas9-induced gene knockout in zebrafish.

The application of CRISPR has greatly facilitated genotype-phenotype studies of human disease models. In this protocol, we describe CRISPR-Cas9-induced gene knockout in zebrafish, utilizing purified Cas9 protein and in vitro-transcribed sgRNA. This protocol targets the PHLPP1 gene in an Indian wild-caught strain, but is broadly applicable. Major factors influencing protocol success include zebrafish health and fecundity, sgRNA efficiency and specificity, germline transmission, and mutant viability. For complete details on the use and execution of this protocol, please refer to Balamurugan et al. (2022).

Generation of a GLO-2 deficient mouse reveals its effects on liver carbonyl and glutathione levels.

OBJECTIVE: Hydroxyacylglutathione hydrolase (aka as GLO-2) is a component of the glyoxalase pathway involved in the detoxification of the reactive oxoaldehydes, glyoxal and methylglyoxal. These reactive metabolites have been linked to a variety of pathological conditions, including diabetes, cancer and heart disease and may be involved in the aging process. The objective of this study was to generate a mouse model deficient in GLO-2 to provide insight into the function of GLO-2 and to determine if it is potentially linked to endogenous oxalate synthesis which could influence urinary oxalate excretion. METHODS: A GLO-2 knock out mouse was generated using CRISPR/Cas 9 techniques. Tissue and 24-h urine samples were collected under baseline conditions from adult male and female animals for biochemical analyses, including chromatographic measurement of glycolate, oxalate, glyoxal, methylglyoxal, D-lactate, ascorbic acid and glutathione levels. RESULTS: The GLO-2 KO animals developed normally and there were no changes in 24-h urinary oxalate excretion, liver levels of methylglyoxal, glyoxal, ascorbic acid and glutathione, or plasma d-lactate levels. GLO-2 deficient males had lower plasma glycolate levels than wild type males while this relationship was not observed in females. CONCLUSIONS: The lack of a unique phenotype in a GLO-2 KO mouse model under baseline conditions is consistent with recent evidence, suggesting a functional glyoxalase pathway is not required for optimal health. A lower plasma glycolate in male GLO-2 KO animals suggests glyoxal production may be a significant contributor to circulating glycolate levels, but not to endogenous oxalate synthesis.

Transcriptional Regulation of Structural and Functional Adaptations in a Developing Adulthood Myocardium.

The development of the heart follows a synergic action of several signaling pathways during gestational, pre- & postnatal stages. The current study aimed to investigate whether the myocardium experiences transcriptional changes during the transition from post-natal to adult hood stages. Herein, we used C57/B16/J mice at 4 (28- days; post-natal/PN) and 20 weeks (adulthood/AH) of ages and employed the next generation RNAseq (NGS) to profile the transcriptome and echocardiography analysis to monitor the structural/functional changes in the heart. NGS-based RNA-seq revealed that 1215 genes were significantly upregulated and 2549 were down regulated in the AH versus PN hearts, indicating a significant transcriptional change during this transition. A synchronized cardiac transcriptional regulation through cell cycle, growth hormones, redox homeostasis and metabolic pathways was noticed in both PN and AH hearts. Echocardiography reveals significant structural and functional (i.e. systolic/diastolic) changes during the transition of PN to adult stage. Particularly, a progressive decline in ejection fraction and cardiac output was observed in AH hearts. These structural adaptations are in line with critical signaling pathways that drive the maturation of heart during AH. Overall, we have presented a comprehensive transcriptomic analysis along with structural-functional relationship during the myocardial development in adult mice.

Bringing Real-World Microbiology Experiences to Undergraduate Students in Resource-Limited Environments.

Undergraduate microbiology curriculum should be amenable to periodic changes to incorporate new developments and ideas. The curriculum should be used not merely as a way to disseminate facts but also as a way to allow students to experience the process of science. In the context of undergraduate microbiology education in Osmania University (Hyderabad, India), existing curriculum does not explicitly allow students to engage in deeper understanding of concepts and understanding of the process of science, both in lecture and laboratory courses. The assessment methods that are currently used are limited in scope as they only test factual recall and superficial understanding of the subject and very minimally assess critical thinking skills. Another factor hampering innovation in the broader context of undergraduate education is the unavailability and inaccessibility to adequate resources. To address the issue of resource-limitations in implementing activities that expose undergraduate students to real-world microbiology experiences, a collaboration between a research institute and two teaching colleges was formed. This collaboration involved teacher and student workshops on exploring microbial diversity using 16S rRNA analysis with a view of blending novel research questions with technical skills in the undergraduate microbiology lab. This effort is an example of educators providing students with authentic experiences and, helping them gain critical knowledge and research skills in microbiology even under resource constraints, and students demonstrating motivation to participate in similar activities in the future. The collaborative effort described here can be a broadly sustainable model to improve overall undergraduate education in relatively resource-limited environments.

Enhanced Keap1-Nrf2 signaling protects the myocardium from isoproterenol-induced pathological remodeling in mice.

Nuclear factor (erythroid-derived 2)-like 2 (NFE2L2/Nrf2) is an inducible transcription factor that is essential for maintenance of redox signaling in response to stress. This suggests that if Nrf2 expression response could be enhanced for a defined physiological pro-oxidant stress then it would be protective. This has important implications for the therapeutic manipulation of the Keap1/Nrf2 signaling pathway which is now gaining a lot of attention. We tested this hypothesis through the generation of Nrf2 transgene expression mouse model with and without isoproterenol-induced cardiac stress. Cardiac-specific mouse Nrf2 transgenic (mNrf2-TG) and non-transgenic (NTG) mice were subjected to isoproterenol (ISO) treatment and assessed for myocardial structure, function (echocardiography and electrocardiography), and glutathione redox state. Myocardial infarction and fibrosis along with increased inflammation leading to myocardial dysfunction was noted in NTG mice exposed to ISO, while mNrf2-TG hearts were resistant to the ISO insult. Preservation of myocardial structure and function in the mNrf2-TG mice was associated with the enhanced Nrf2 expression displayed in these hearts with an increased basal and post-treatment expression of redox modulatory genes and an overall enhanced antioxidant status. Of note, myocardium of ISO-treated TG mice displayed significantly increased stabilization of the KEAP1-NRF2 complex and enhanced release of NRF2 to the nucleus resulting in overall decreased pro-oxidant markers. Taken together, we suggest that a basal enhanced Nrf2 expression in mouse heart results in maintenance of redox homeostasis and counteracts ISO-induced oxidative stress, and suppresses pathological remodeling. These data suggest that an alternative therapeutic approach to enhance the efficacy of the Keap1-Nrf2 system is to stimulate basal expression of Nrf2.

First Year Course-Based Undergraduate Research Experience (CURE) Using the CRISPR/Cas9 Genome Engineering Technology in Zebrafish.

Genetic analysis in model systems can provide a rich context for conceptual understanding of gene structure, regulation, and function. With an intent to create a rich learning experience in molecular genetics, we developed a semester-long course-based undergraduate research experience (CURE) using the CRISPR-Cas9 gene editing system to disrupt specific genes in the zebrafish. The course was offered to freshman students; nine students worked in four groups (two to three members per group) to design, synthesize, and test the nuclease activity of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/sgRNAs for targeted disruption of specific genes in the zebrafish. Each group worked with a gene with an already known mutant phenotype that can be visually scored and a gene that had not been studied in zebrafish previously. Embedded in the course were a series of workshop-styled units or tutorials, including tours to core facilities. The focus was on introducing and developing skills that could be accommodated within the span of a semester. Each group successfully cloned at least one plasmid-encoding CRISPR/sgRNA template, visually analyzed injected embryos, and performed genotyping assays to detect CRISPR-Cas9 activity. In-class discussions, a final end-of-semester written test, and group oral presentations were assessed for an understanding of the CRISPR-Cas9 system, application of the CRISPR-Cas9 system as a gene manipulation tool, and experimental methods used to create plasmid vectors and synthesize sgRNA. In addition, poster presentations were evaluated by faculty, graduate students, and senior undergraduate students at a University research exposition. Self-reflections in the form of group conversations were video recorded. All students (9/9) distinctly showed learning gains after completing the activity, but the extent of the gains was variable, as seen from results of a written test and poster presentation assessment. Qualitative analysis of evaluations and self-reporting data indicated several gains, suggesting that all students found many aspects of the CURE valuable and gained project-specific (conceptual) and transferrable skills (science process and science identity).

Conservation and early expression of zebrafish tyrosine kinases support the utility of zebrafish as a model for tyrosine kinase biology.

Tyrosine kinases have significant roles in cell growth, apoptosis, development, and disease. To explore the use of zebrafish as a vertebrate model for tyrosine kinase signaling and to better understand their roles, we have identified all of the tyrosine kinases encoded in the zebrafish genome and quantified RNA expression of selected tyrosine kinases during early development. Using profile hidden Markov model analysis, we identified 122 zebrafish tyrosine kinase genes and proposed unambiguous gene names where needed. We found them to be organized into 39 nonreceptor and 83 receptor type, and 30 families consistent with human tyrosine kinase family assignments. We found five human tyrosine kinase genes (epha1, bmx, fgr, srm, and insrr) with no identifiable zebrafish ortholog, and one zebrafish gene (yrk) with no identifiable human ortholog. We also found that receptor tyrosine kinase genes were duplicated more often than nonreceptor tyrosine kinase genes in zebrafish. We profiled expression levels of 30 tyrosine kinases representing all families using direct digital detection at different stages during the first 24 hours of development. The profiling experiments clearly indicate regulated expression of tyrosine kinases in the zebrafish, suggesting their role during early embryonic development. In summary, our study has resulted in the first comprehensive description of the zebrafish tyrosine kinome.

Caffeine-induced effects on heart rate in zebrafish embryos and possible mechanisms of action: an effective system for experiments in chemical biology.

Zebrafish embryos are well suited as a model system to perform chemical biology experiments effectively in educational settings. We studied the effect of caffeine on heart rate (HR) and other phenotypes of zebrafish embryos using visual microscopy and simple imaging. Acute treatment with millimolar concentrations of caffeine in embryo medium caused a dose-dependent decrease in HR in 2-3-day-old zebrafish embryos, ultimately resulting in complete HR cessation. A characteristic pattern of decrease in HR was observed, with an initial acute drop in HR and a period of stabilization followed by complete cessation. The effects of caffeine were not reversed by cotreatment with ruthenium red and adenosine, agents known to be antagonistic to caffeine, or by changes in calcium concentration in embryo medium. Apparent cardiac arrhythmia and a typical kinking effect in the trunk/tail region were also observed because of caffeine treatment. Our results, taken together with previous reports, raise the possibility that caffeine exerts its effects on embryonic HR of zebrafish by inhibition of ether-a-go-go potassium channels. However, further experimentation is required to dissect the molecular basis of caffeine action. We demonstrate that such experiments can be used to explore the effect of small molecules, such as caffeine, on cardiovascular phenotypes and to encourage experimental design in chemical biology.