EYA-1 is required for genomic integrity independent of H2AX signalling in Caenorhabditis elegans.

BACKGROUND: Resolving genomic insults is essential for the survival of any species. In the case of eukaryotes, several pathways comprise the DNA damage repair network, and many components have high evolutionary conservation. These pathways ensure that DNA damage is resolved which prevents disease associated mutations from occurring in a de novo manner. In this study, we investigated the role of the Eyes Absent (EYA) homologue in Caenorhabditis elegans and its role in DNA damage repair. Current understanding of mammalian EYA1 suggests that EYA1 is recruited in response to H2AX signalling to dsDNA breaks. C. elegans do not possess a H2AX homologue, although they do possess homologues of the core DNA damage repair proteins. Due to this, we aimed to determine if eya-1 contributes to DNA damage repair independent of H2AX. METHODS AND RESULTS: We used a putative null mutant for eya-1 in C. elegans and observed that absence of eya-1 results in abnormal chromosome morphology in anaphase embryos, including chromosomal bridges, missegregated chromosomes, and embryos with abnormal nuclei. Additionally, inducing different types of genomic insults, we show that eya-1 mutants are highly sensitive to induction of DNA damage, yet show little change to induced DNA replication stress and display a mortal germline resulting in sterility over successive generations. CONCLUSIONS: Collectively, this study suggests that the EYA family of proteins may have a greater involvement in maintaining genomic integrity than previously thought and unveils novel roles of EYA associated DNA damage repair.

atz-1 Influences meiosis to maintain germline chromosomal stability in Caenorhabditis elegans.

Exchange of genetic information during meiosis occurs in all sexually reproducing species to produce haploid gametes from diploid cells. This process involves tight coordination of a meiotic specific cohesin complex, the synaptonemal complex, and DNA damage repair mechanisms. In this study, we describe a putative myosin heavy chain protein orthologous to human myosin 1, F28D1.2, which we named Abnormal Transition Zone (atz-1). Deletion of atz-1 results in embryonic lethality and a depleted transition zone, accompanied by reduced expression of the meiotic cohesin protein, REC-8. atz-1 mutants display disorganized and aggregated chromosomal bodies in diakinetic oocytes. In addition to this, atz-1 mutants are hypersensitive to mild inhibition of DNA damage repair, suggesting that DNA replication in atz-1 mutants is impaired. Moreover, the atz-1 mutant phenotype is germline specific and resupplying somatically expressed atz-1 does not rescue the reproductive defects associated with atz-1 mutants. Overall, our data suggest that atz-1 contributes to meiosis and maintains germline chromosomal stability.

Oogenesis in Caenorhabditis elegans.

BACKGROUND: The nematode, Caenorhabditis elegans has proven itself as a valuable model for investigating metazoan biology. C. elegans have a transparent body, an invariant cell lineage, and a high level of genetic conservation which makes it a desirable model organism. Although used to elucidate many aspects of somatic biology, a distinct advantage of C. elegans is its well annotated germline which allows all aspects of oogenesis to be observed in real time within a single animal. C. elegans hermaphrodites have two U-shaped gonad arms which produce their own sperm that is later stored to fertilise their own oocytes. These two germlines take up much of the internal space of each animal and germ cells are therefore the most abundant cell present within each animal. This feature and the genetic phenotypes observed for mutant worm gonads have allowed many novel findings that established our early understanding of germ cell dynamics. The mutant phenotypes also allowed key features of meiosis and germ cell maturation to be unveiled. SUMMARY: This review will focus on the key aspects that make C. elegans an outstanding model for exploring each feature of oogenesis. This will include the fundamental steps associated with germline function and germ cell maturation and will be of use for those interested in exploring reproductive metazoan biology. KEY MESSAGES: Since germ cell biology is highly conserved in animals, much can be gained from study of a simple metazoan like C. elegans. Past findings have enhanced understanding on topics that would be more laborious or challenging in more complex animal models.

Stubborn Exercise Responders-Where to Next?

There is a wide variance in the magnitude of physiological adaptations after resistance or endurance training. The incidence of "non" or "poor" responders to training has been reported to represent as high as 40% of the project's sample. However, the incidence of poor responders to training can be ameliorated with manipulation of either the training frequency, intensity, type and duration. Additionally, global non-response to cardio-respiratory fitness training is eliminated when evaluating several health measures beyond just the target variables as at least one or more measure improves. More research is required to determine if altering resistance training variables results in a more favourable response in individuals with an initial poor response to resistance training. Moreover, we recommend abandoning the term "poor" responders, as ultimately the magnitude of change in cardiorespiratory fitness in response to endurance training is similar in "poor" and "high" responders if the training frequency is subsequently increased. Therefore, we propose "stubborn" responders as a more appropriate term. Future research should focus on developing viable physiological and lifestyle screening tests that identify likely stubborn responders to conventional exercise training guidelines before the individual engages with training. Exerkines, DNA damage, metabolomic responses in blood, saliva and breath, gene sequence, gene expression and epigenetics are candidate biomarkers that warrant investigation into their relationship with trainability. Crucially, viable biomarker screening tests should show good construct validity to distinguish between different exercise loads, and possess excellent sensitivity and reliability. Furthermore "red flag" tests of likely poor responders to training should be practical to assess in clinical settings and be affordable and non-invasive. Early identification of stubborn responders would enable optimization of training programs from the onset of training to maintain exercise motivation and optimize the impact on training adaptations and health.

Gut Microbiome and Metabolome Variations in Self-Identified Muscle Builders Who Report Using Protein Supplements.

Muscle builders frequently consume protein supplements, but little is known about their effect on the gut microbiota. This study compared the gut microbiome and metabolome of self-identified muscle builders who did or did not report consuming a protein supplement. Twenty-two participants (14 males and 8 females) consumed a protein supplement (PS), and seventeen participants (12 males and 5 females) did not (No PS). Participants provided a fecal sample and completed a 24-h food recall (ASA24). The PS group consumed significantly more protein (118 ± 12 g No PS vs. 169 ± 18 g PS, p = 0.02). Fecal metabolome and microbiome were analyzed by using untargeted metabolomics and 16S rRNA gene sequencing, respectively. Metabolomic analysis identified distinct metabolic profiles driven by allantoin (VIP score = 2.85, PS 2.3-fold higher), a catabolic product of uric acid. High-protein diets contain large quantities of purines, which gut microbes degrade to uric acid and then allantoin. The bacteria order Lactobacillales was higher in the PS group (22.6 ± 49 No PS vs. 136.5 ± 38.1, PS (p = 0.007)), and this bacteria family facilitates purine absorption and uric acid decomposition. Bacterial genes associated with nucleotide metabolism pathways (p < 0.001) were more highly expressed in the No PS group. Both fecal metagenomic and metabolomic analyses revealed that the PS group's higher protein intake impacted nitrogen metabolism, specifically altering nucleotide degradation.

Zinc transporters maintain longevity by influencing insulin/IGF-1 activity in Caenorhabditis elegans.

Adequate dietary intake of essential metals such as zinc is important for maintaining homeostasis. Abnormal zinc intake in Caenorhabditis elegans has been shown to increase or decrease normal lifespan by influencing the insulin/IGF-1 pathway. Distribution of zinc is achieved by a family of highly conserved zinc transport proteins (ZIPT in C. elegans). This study investigated the role of the zipt family of genes and showed that depletion of individual zipt genes results in a decreased lifespan. Moreover, zipt-16 and zipt-17 mutants synthetically interact with the insulin/IGF cofactors daf-16 and skn-1, and cause abnormal localisation of DAF-16. This study suggests that the zipt family of genes are required for maintaining normal lifespan through influencing the insulin/IGF-1 pathway.

GCNA Interacts with Spartan and Topoisomerase II to Regulate Genome Stability.

GCNA proteins are expressed across eukarya in pluripotent cells and have conserved functions in fertility. GCNA homologs Spartan (DVC-1) and Wss1 resolve DNA-protein crosslinks (DPCs), including Topoisomerase-DNA adducts, during DNA replication. Here, we show that GCNA mutants in mouse and C. elegans display defects in genome maintenance including DNA damage, aberrant chromosome condensation, and crossover defects in mouse spermatocytes and spontaneous genomic rearrangements in C. elegans. We show that GCNA and topoisomerase II (TOP2) physically interact in both mice and worms and colocalize on condensed chromosomes during mitosis in C. elegans embryos. Moreover, C. elegans gcna-1 mutants are hypersensitive to TOP2 poison. Together, our findings support a model in which GCNA provides genome maintenance functions in the germline and may do so, in part, by promoting the resolution of TOP2 DPCs.

The TRIM-NHL protein NHL-2 is a co-factor in the nuclear and somatic RNAi pathways in C. elegans.

Proper regulation of germline gene expression is essential for fertility and maintaining species integrity. In the C. elegans germline, a diverse repertoire of regulatory pathways promote the expression of endogenous germline genes and limit the expression of deleterious transcripts to maintain genome homeostasis. Here we show that the conserved TRIM-NHL protein, NHL-2, plays an essential role in the C. elegans germline, modulating germline chromatin and meiotic chromosome organization. We uncover a role for NHL-2 as a co-factor in both positively (CSR-1) and negatively (HRDE-1) acting germline 22G-small RNA pathways and the somatic nuclear RNAi pathway. Furthermore, we demonstrate that NHL-2 is a bona fide RNA binding protein and, along with RNA-seq data point to a small RNA independent role for NHL-2 in regulating transcripts at the level of RNA stability. Collectively, our data implicate NHL-2 as an essential hub of gene regulatory activity in both the germline and soma.

Exploring Potential Germline-Associated Roles of the TRIM-NHL Protein NHL-2 Through RNAi Screening.

TRIM-NHL proteins are highly conserved regulators of developmental pathways in vertebrates and invertebrates. The TRIM-NHL family member NHL-2 in Caenorhabditis elegans functions as a miRNA cofactor to regulate developmental timing. Similar regulatory roles have been reported in other model systems, with the mammalian ortholog in mice, TRIM32, contributing to muscle and neuronal cell proliferation via miRNA activity. Given the interest associated with TRIM-NHL family proteins, we aimed to further investigate the role of NHL-2 in C. elegans development by using a synthetic RNAi screening approach. Using the ORFeome library, we knocked down 11,942 genes in wild-type animals and nhl-2 null mutants. In total, we identified 42 genes that produced strong reproductive synthetic phenotypes when knocked down in nhl-2 null mutants, with little or no change when knocked down in wild-type animals. These included genes associated with transcriptional processes, chromosomal integrity, and key cofactors of the germline small 22G RNA pathway.

MicroRNAs: Not "Fine-Tuners" but Key Regulators of Neuronal Development and Function.

MicroRNAs (miRNAs) are a class of short non-coding RNAs that operate as prominent post-transcriptional regulators of eukaryotic gene expression. miRNAs are abundantly expressed in the brain of most animals and exert diverse roles. The anatomical and functional complexity of the brain requires the precise coordination of multilayered gene regulatory networks. The flexibility, speed, and reversibility of miRNA function provide precise temporal and spatial gene regulatory capabilities that are crucial for the correct functioning of the brain. Studies have shown that the underlying molecular mechanisms controlled by miRNAs in the nervous systems of invertebrate and vertebrate models are remarkably conserved in humans. We endeavor to provide insight into the roles of miRNAs in the nervous systems of these model organisms and discuss how such information may be used to inform regarding diseases of the human brain.

Comparative high-throughput RNAi screening methodologies in C. elegans and mammalian cells.

The discovery of RNAi in Caenorhabditis elegans has generated a paradigm shift in how research is performed. Targeted gene knockdown using high throughput screening approaches is becoming a routine feature of the scientific landscape, and researchers can now evaluate the function of each gene in the genome in a relatively short period of time. This review compares and contrasts high throughput screening methodologies in C. elegans and mammalian cells and highlights the breadth of applications of this technology.