Characterization of sub-nuclear changes in Caenorhabditis elegans embryos exposed to brief, intermediate and long-term anoxia to analyze anoxia-induced cell cycle arrest

BACKGROUND: The soil nematode C. elegans survives oxygen-deprived conditions (anoxia; <.001 kPa O(2)) by entering into a state of suspended animation in which cell cycle progression reversibly arrests. The majority of blastomeres of embryos exposed to anoxia arrest at interphase, prophase and metaphase. The spindle checkpoint proteins SAN-1 and MDF-2 are required for embryos to survive 24 hours of anoxia. To further investigate the mechanism of cell-cycle arrest we examined and compared sub-nuclear changes such as chromatin localization pattern, post-translational modification of histone H3, spindle microtubules, and localization of the spindle checkpoint protein SAN-1 with respect to various anoxia exposure time points. To ensure analysis of embryos exposed to anoxia and not post-anoxic recovery we fixed all embryos in an anoxia glove box chamber. RESULTS: Embryos exposed to brief periods to anoxia (30 minutes) contain prophase blastomeres with chromosomes in close proximity to the nuclear membrane, condensation of interphase chromatin and metaphase blastomeres with reduced spindle microtubules density. Embryos exposed to longer periods of anoxia (1–3 days) display several characteristics including interphase chromatin that is further condensed and in close proximity to the nuclear membrane, reduction in spindle structure perimeter and reduced localization of SAN-1 at the kinetochore. Additionally, we show that the spindle checkpoint protein SAN-1 is required for brief periods of anoxia-induced cell cycle arrest, thus demonstrating that this gene product is vital for early anoxia responses. In this report we suggest that the events that occur as an immediate response to brief periods of anoxia directs cell cycle arrest. CONCLUSION: From our results we conclude that the sub-nuclear characteristics of embryos exposed to anoxia depends upon exposure time as assayed using brief (30 minutes), intermediate (6 or 12 hours) or long-term (24 or 72 hours) exposures. Analyzing these changes will lead to an understanding of the mechanisms required for initiation and maintenance of cell cycle arrest in respect to anoxia exposure time as well as order the events that occur to bring about anoxia-induced cell cycle arrest

Genetic analysis of the spindle checkpoint genes san-1, mdf-2, bub-3 and the CENP-F homologues hcp-1 and hcp-2 in Caenorhabditis elegans

<p>Abstract</p> <p>Background</p> <p>The spindle checkpoint delays the onset of anaphase until all sister chromatids are aligned properly at the metaphase plate. To investigate the role <it>san-1</it>, the MAD3 homologue, has in <it>Caenorhabditis elegans </it>embryos we used RNA interference (RNAi) to identify genes synthetic lethal with the viable <it>san-1(ok1580) </it>deletion mutant.</p> <p>Results</p> <p>The <it>san-1(ok1580) </it>animal has low penetrating phenotypes including an increased incidence of males, larvae arrest, slow growth, protruding vulva, and defects in vulva morphogenesis. We found that the viability of <it>san-1(ok1580) </it>embryos is significantly reduced when HCP-1 (CENP-F homologue), MDF-1 (MAD-1 homologue), MDF-2 (MAD-2 homologue) or BUB-3 (predicted BUB-3 homologue) are reduced by RNAi. Interestingly, the viability of <it>san-1(ok1580) </it>embryos is not significantly reduced when the paralog of HCP-1, HCP-2, is reduced. The phenotype of <it>san-1(ok1580);hcp-1(RNAi) </it>embryos includes embryonic and larval lethality, abnormal organ development, and an increase in abnormal chromosome segregation (aberrant mitotic nuclei, anaphase bridging). Several of the <it>san-1(ok1580);hcp-1(RNAi) </it>animals displayed abnormal kinetochore (detected by MPM-2) and microtubule structure. The survival of <it>mdf-2(RNAi);hcp-1(RNAi) </it>embryos but not <it>bub-3(RNAi);hcp-1(RNAi) </it>embryos was also compromised. Finally, we found that <it>san-1(ok1580) </it>and <it>bub-3(RNAi)</it>, but not <it>hcp-1(RNAi) </it>embryos, were sensitive to anoxia, suggesting that like SAN-1, BUB-3 has a functional role as a spindle checkpoint protein.</p> <p>Conclusion</p> <p>Together, these data suggest that in the <it>C. elegans </it>embryo, HCP-1 interacts with a subset of the spindle checkpoint pathway. Furthermore, the fact that <it>san-1(ok1580);hcp-1(RNAi) </it>animals had a severe viability defect whereas in the <it>san-1(ok1580);hcp-2(RNAi) </it>and <it>san-1(ok1580);hcp-2(ok1757) </it>animals the viability defect was not as severe suggesting that <it>hcp-1 </it>and <it>hcp-2 </it>are not completely redundant.</p

Consumption of a High-Fat Meal Alters Post-Prandial SIRT mRNA Expression in Blood Leukocytes

Introduction. Sirtuins (SIRT) are protein deacetylases, hypothesized to regulate the transcription of various genes involved in the prevention of atherogenesis and diet induced obesity. Previous research from our laboratory has demonstrated that consumption of a single, high-fat meal increases various CVD risk factors for up to 5-h post-prandial. Given the importance of SIRT to metabolic disorders, it is reasonable to speculate that a single, high-fat meal also disrupts SIRT. Methods. The purpose of this study was to determine the effect of a high-fat meal (75% of daily kcals & 80% of daily fat needs), on SIRT mRNA expression in blood leukocytes during a 5-h post-prandial period. Men and women (N=17) were recruited to report to the lab following an overnight fast. Venous blood samples were collected prior to the meal, 1, 3, and 5-h post-meal. White buffy coat aliquot was frozen in RNALater solution. At the end of the study samples were thawed and RNA was isolated using a phenol/chloroform method. RNA was reverse transcribed and mRNA expression for SIRT 1-7 was determined using a Taqman qPCR technique with 18S rRNA as a normalizer, under standard PCR cycling conditions. An additional aliquot of serum was used to measure triglyceride, total cholesterol, and glucose responses were measured using enzymatic assays on an automated chemistry analyzer (ChemWell T; P.C., FL). Data was analyzed using a RM ANOVA with P\u3c0.05. Results. Consistent with previous results, the meal caused an increase in triglycerides, total cholesterol and glucose that reached peaked values at 3-h post-prandial. We also observed significant expression changes in the mRNA of the SIRT 1 (P=0.02) and SIRT 6 (P=0.03) during the 5-h post-prandial period. Both SIRT 1 and SIRT 6 showed the greatest decreased expression at 3-h post-prandial compared to baseline, 51.8% and 46.2% respectfully. Conclusion. To our knowledge, this is the 1st study to report that consumption of a high-fat meal transiently alters SIRT mRNA expression consistent with changes in serum triglyceride and glucose concentration. More research is needed to understand how transient, post-prandial changes in SIRT mRNA expression contribute to increased disease risk

Phylogenetic analysis of eukaryotic NEET proteins uncovers a link between a key gene duplication event and the evolution of vertebrates

NEET proteins belong to a unique family of iron-sulfur proteins in which the 2Fe-2S cluster is coordinated by a CDGSH domain that is followed by the “NEET” motif. They are involved in the regulation of iron and reactive oxygen metabolism, and have been associated with the progression of diabetes, cancer, aging and neurodegenerative diseases. Despite their important biological functions, the evolution and diversification of eukaryotic NEET proteins are largely unknown. Here we used the three members of the human NEET protein family (CISD1, mitoNEET; CISD2, NAF-1 or Miner 1; and CISD3, Miner2) as our guides to conduct a phylogenetic analysis of eukaryotic NEET proteins and their evolution. Our findings identified the slime mold Dictyostelium discoideum’s CISD proteins as the closest to the ancient archetype of eukaryotic NEET proteins. We further identified CISD3 homologs in fungi that were previously reported not to contain any NEET proteins, and revealed that plants lack homolog(s) of CISD3. Furthermore, our study suggests that the mammalian NEET proteins, mitoNEET (CISD1) and NAF-1 (CISD2), emerged via gene duplication around the origin of vertebrates. Our findings provide new insights into the classification and expansion of the NEET protein family, as well as offer clues to the diverged functions of the human mitoNEET and NAF-1 proteins

C.elegans as a Diabetes & Ischemia Model: Identification of Genetic and Cellular Changes that Modulate the Survival of Hyperglycemia and Oxygen-Deprivation

Diet represents an exogenous influence that often yields colossal effects on an individual’s phenotype, physiology, long-term health and disease risk. The overconsumption of dietary sugars for example, has contributed to significant increases in obesity and type 2 diabetes, health issues that are costly both in terms of dollars and human life. Additionally, individuals with these conditions have compromised oxygen delivery and thus, an increased vulnerability to other oxygen-deprivation related disease states, including cardiovascular disease, ischemic strokes, vascular and coronary diseases and myocardial infarction. While human and other mammalian studies have shown that individuals with type 2 diabetes have a worse prognosis and recovery after being challenged with an oxygen-deprivation related injury, mechanistic understanding regarding why this is the case is lacking. We are using C. elegans to identify genetic and cellular changes that modulate responses to the combinatory stress of hyperglycemia and oxygen-deprivation. We have determined that C. elegans fed a high glucose diet have increased cellular glucose (hyperglycemia), increased lipid content and increased sensitivity to oxygen-deprivation (anoxia) and ROS induction. We have determined that the insulin-like signaling pathway, via fatty acid and ceramide synthesis, modulates the increased sensitivity to anoxia. In mammalian systems, specific ceramide species increase after an ischemic event and are also linked to detrimental effects observed in diabetic patients, underscoring the potential role these molecules have in modulating oxygen-deprivation and hyperglycemia responses in individuals. Specific fatty acids also have known roles as both signaling molecules and as integral membrane components, thus, we hypothesize that a high-glucose diet disrupts fatty acid and ceramide homeostasis resulting in aberrations in metabolic processes and stress response pathways that are essential for the survival of oxygen-deprivation. Additionally, gene expression analysis (via RNAseq) on C. elegans fed either a standard or glucose-supplemented diet revealed that glucose impacts the expression of genes involved with multiple cellular processes, including lipid and carbohydrate metabolism, stress responses, cell division and extracellular functions. Several of the genes we identified are also differentially regulated in obese and type-2 diabetic human individuals, indicating a high degree of conserved gene expression changes between C. elegans fed a glucose-supplemented diet and in diabetic and/or obese human individuals. Together this work underscores how both diet and genotype impact stress responses and supports the use of C. elegans as a model for further elucidating the molecular mechanisms regulating dietary-induced metabolic diseases

Western Diet-Fed, Aortic-Banded Ossabaw Swine: A Preclinical Model of Cardio-Metabolic Heart Failure.

The development of new treatments for heart failure lack animal models that encompass the increasingly heterogeneous disease profile of this patient population. This report provides evidence supporting the hypothesis that Western Diet-fed, aortic-banded Ossabaw swine display an integrated physiological, morphological, and genetic phenotype evocative of cardio-metabolic heart failure. This new preclinical animal model displays a distinctive constellation of findings that are conceivably useful to extending the understanding of how pre-existing cardio-metabolic syndrome can contribute to developing HF

Environmental and Genetic Preconditioning for Long-Term Anoxia Responses Requires AMPK in Caenorhabditis elegans

Article on environmental and genetic preconditioning for long-term anoxia responses requires AMPK in Caenorhabditis elegans

Characterization in vitro and in vivo of a pandemic H1N1 influenza virus from a fatal case

Pandemic 2009 H1N1 (pH1N1) influenza viruses caused mild symptoms in most infected patients. However, a greater rate of severe disease was observed in healthy young adults and children without co-morbid conditions. Here we tested whether influenza strains displaying differential virulence could be present among circulating pH1N1 viruses. The biological properties and the genotype of viruses isolated from a patient showing mild disease (M) or from a fatal case (F), both without known co-morbid conditions were compared in vitro and in vivo. The F virus presented faster growth kinetics and stronger induction of cytokines than M virus in human alveolar lung epithelial cells. In the murine model in vivo, the F virus showed a stronger morbidity and mortality than M virus. Remarkably, a higher proportion of mice presenting infectious virus in the hearts, was found in F virus-infected animals. Altogether, the data indicate that strains of pH1N1 virus with enhanced pathogenicity circulated during the 2009 pandemic. In addition, examination of chemokine receptor 5 (CCR5) genotype, recently reported as involved in severe influenza virus disease, revealed that the F virus-infected patient was homozygous for the deleted form of CCR5 receptor (CCR5Δ32).Funding Statement: This work was supported by Instituto de Salud Carlos III (Programa especial de investigación sobre la gripe pándemica GR09/0023, GR09/0040, GR09/0039) and Ciber de Enfermedades Respiratorias. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Mechanism of KMT5B haploinsufficiency in neurodevelopment in humans and mice.

Pathogenic variants in KMT5B, a lysine methyltransferase, are associated with global developmental delay, macrocephaly, autism, and congenital anomalies (OMIM# 617788). Given the relatively recent discovery of this disorder, it has not been fully characterized. Deep phenotyping of the largest (n = 43) patient cohort to date identified that hypotonia and congenital heart defects are prominent features that were previously not associated with this syndrome. Both missense variants and putative loss-of-function variants resulted in slow growth in patient-derived cell lines. KMT5B homozygous knockout mice were smaller in size than their wild-type littermates but did not have significantly smaller brains, suggesting relative macrocephaly, also noted as a prominent clinical feature. RNA sequencing of patient lymphoblasts and Kmt5b haploinsufficient mouse brains identified differentially expressed pathways associated with nervous system development and function including axon guidance signaling. Overall, we identified additional pathogenic variants and clinical features in KMT5B-related neurodevelopmental disorder and provide insights into the molecular mechanisms of the disorder using multiple model systems