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Alzheimer's disease (AD) affects more than 10% of the population ≥65 y of age, but the underlying biological risks of most AD cases are unclear. We show anti-poly-glycine-arginine (a-polyGR) positive aggregates frequently accumulate in sporadic AD autopsy brains (45/80 cases). We hypothesize that these aggregates are caused by one or more polyGR-encoding repeat expansion mutations. We developed a CRISPR/deactivated-Cas9 enrichment strategy to identify candidate GR-encoding repeat expansion mutations directly from genomic DNA isolated from a-polyGR(+) AD cases. Using this approach, we isolated an interrupted (GGGAGA) n intronic expansion within a SINE-VNTR-Alu element in CASP8 ( CASP8 -GGGAGA EXP ). Immunostaining using a-polyGR and locus-specific C-terminal antibodies demonstrate that the CASP8 -GGGAGA EXP expresses hybrid poly(GR)n(GE)n(RE)n proteins that accumulate in CASP8 -GGGAGA EXP (+) AD brains. In cells, expression of CASP8 -GGGAGA EXP minigenes leads to increased p-Tau (Ser202/Thr205) levels. Consistent with other types of repeat-associated non-AUG (RAN) proteins, poly(GR)n(GE)n(RE)n protein levels are increased by stress. Additionally, levels of these stress-induced proteins are reduced by metformin. Association studies show specific aggregate promoting interrupted CASP8 -GGGAGA EXP sequence variants found in ~3.6% of controls and 7.5% AD cases increase AD risk [ CASP8 -GGGAGA-AD-R1; OR 2.2, 95% CI (1.5185 to 3.1896), P = 3.1 × 10 -5 ]. Cells transfected with a high-risk CASP8 -GGGAGA-AD-R1 variant show increased toxicity and increased levels of poly(GR)n(GE)n(RE)n aggregates. Taken together, these data identify polyGR(+) aggregates as a frequent and unexpected type of brain pathology in AD and CASP8 -GGGAGA-AD-R1 alleles as a relatively common AD risk factor. Taken together, these data support a model in which CASP8 -GGGAGA EXP alleles combined with stress increase AD risk., Competing Interests: Competing interests statement:T.E.G. is a cofounder and head of scientific advisory board for Andante Biologics. H.B.C. served as a consultant for Janssen Research and Development in 2021 and 2022. A.G. serves on the SAB of Genetech and Muna Therapeutics. B.T.H. serves on the SAB of Dewpoint and has an option for stock. He serves on a scientific advisory board or is a consultant for AbbVie, Alexion, Ambagon, Aprinoia Therapeutics, Arvinas, Avrobio, AstraZenica, Biogen, Bioinsights, BMS, Cure Alz Fund, Cell Signaling, Dewpoint, Latus, Novartis, Pfizer, Sanofi, Sofinnova, Vigil, Violet, Voyager, WaveBreak. T.E.G. owns stock and stock options in Andante Biologics. B.T.H. has an option for stock in Dewpoint. B.T.H. owns stock in Novartis. T.E.G. is an inventor on multiple patents and patent applications that relate to AD therapeutics. L.P.W.R. is an inventor on patents that are related to RAN translation. L.N. is an inventor on patents that are related to RAN translation.

Coconut water is used as an alternative to conventional sports drinks for hydration during endurance cycling; however, evidence supporting its use is limited. This study determined if drinking coconut water compared to a sports drink altered cycling performance and physiology. In a randomized crossover trial, 19 experienced male ( n = 15) and female ( n = 4) cyclists (age 30 ± 9 years, body mass 79 ± 11 kg, V̇O 2 peak 55 ± 8 mL·kg -1 ·min -1 ) completed two experimental trials, consuming either a commercially available sports drink or iso-calorific coconut water during 90 min of sub-maximal cycling at 70% of their peak power output, followed by a simulated, variable gradient, 20 km time trial. Blood glucose, lactate, sweat loss, and heart rate were monitored throughout the 90 min of sub-maximal cycling, as well as the time trial performance (seconds) and average power (watts). A repeated measures analysis of variance and effect sizes (Cohen's d) analysis were applied. There were no significant differences ( p ≥ 0.05) between the treatments for any of the measured physiological or performance variables. Additionally, the effect size analysis showed only trivial (d ≤ 0.2) differences between the treatments for all the measured variables, except blood glucose, which was lower in the coconut water trial compared to the sports drink trial (d = 0.31). Consuming coconut water had a similar effect on the cycling time trial performance and the physiological responses to consuming a commercially available sports drink.

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.

The Caenorhabditis elegans genes dyf-6, daf-10, and osm-1 are among the set of genes that affect chemotaxis and the ability of certain sensory neurons to take up fluorescent dyes from the environment. Some genes in this category are known to be required for intraflagellar transport (IFT), which is the bidirectional movement of raft-like particles along the axonemes of cilia and flagella. The cloning of dyf-6, daf-10, and osm-1 are described here. The daf-10 and osm-1 gene products resemble each other and contain WD and WAA repeats. DYF-6, the product of a complex locus, lacks known motifs, but orthologs are present in flies and mammals. Phenotypic analysis of dyf-6 mutants expressing an OSM-6::GFP reporter indicates that the cilia of the amphid and phasmid dendritic endings are foreshortened. Consistent with genetic mosaic analysis, which indicates that dyf-6 functions in neurons of the amphid sensilla, DYF-6::GFP is expressed in amphid and phasmid neurons. Movement of DYF-6::GFP within the ciliated endings of the neurons indicates that DYF-6 is involved in IFT. In addition, IFT can be observed in dauer larvae.

On the basis of synthetic lethality, five genes in Caenorhabditis elegans are known to be redundant with the mec-8 gene, which encodes a protein that contains two copies of an RNA recognition motif (RRM) and affects alternative RNA splicing. The molecular identities of two of the redundant genes, sym-1 and sym-5, were previously reported. The remaining three genes have now been cloned, and their synthetically lethal phenotypes with mec-8 are described in more detail. Animals homozygous for mec-8 and sym-2 loss-of-function mutations die during late embryogenesis. The SYM-2 predicted protein contains three RRMs; we propose that SYM-2 and MEC-8 can substitute for each other in promoting the maturation of the transcripts of a vital gene. Animals homozygous for mutations in mec-8 and in either sym-3 or sym-4 have the same striking defect: they arrest development just prior to or just after hatching with a pharynx that appears fully formed but is not properly attached to the body cuticle. sym-3 encodes a protein of unknown function with orthologs in Drosophila and mammals. sym-4 encodes a WD-repeat protein and may also have orthologs in Drosophila and mammals. We propose that SYM-3 and SYM-4 contribute to a common developmental pathway that is redundant with a MEC-8-dependent pathway.

Sex-lethal (SXL) is an RNA binding protein that acts as a regulator of both alternative pre-mRNA splicing and translation. Because SXL must sometimes function at some distance from its binding sites, it is believed that it must interact with other proteins. We used a yeast two-hybrid screen to isolate a novel Drosophila protein, SIN (SXL interactor), that interacts specifically with SXL. A direct physical association was demonstrated in vitro, and a single SXL RNA binding domain was sufficient for the interaction. SIN shows a high degree of similarity to a mammalian protein of unknown function. The cytogenetic location of Sin is 78A2-4. The transcript, which is abundant in early embryos, appears to be of maternal origin.

Sex-lethal (Sxl) is an RNA-binding protein, containing two conserved RNA binding domains (RBDs) and a glycine-rich region, which functions as a regulator of alternative splicing in Drosophila sex determination. Previous work demonstrated that Sxl monomers interact cooperatively upon binding to target RNAs and that the cooperativity depends on the glycine-rich N terminus. Here we use band shift experiments to show that RNA binding patterns are altered when Sxl is combined with other proteins having similar glycine-rich domains, including mammalian heterogeneous nuclear (hn) RNP L and Drosophila Hrb87F (an hnRNP A/B homolog). Direct involvement of the Sxl glycine-rich region in protein interactions was verified by Far-Western analysis. Two interaction domains, the Sxl N terminus and the Sxl first RNA binding domain, were suggested by the yeast two-hybrid assay. In a systematic examination of the RNA binding properties of Sxl domains, it was found that the Sxl termini as well as the RBDs influence RNA binding specificity. Finally, selection of the Sxl optimal binding site (SELEX) confirms the importance of U-runs in the Sxl binding site and suggests a second type of non-U-run target that may be associated with RNA secondary structure.

The Sex-lethal (Sxl) early transcripts have a unique 5' exon and a splicing pattern that differs from that of the late transcripts. While the late transcripts are regulated sex specifically by control of exon 3 inclusion, the early transcripts are not. While the late transcripts include exon 3 by default, the early transcripts skip exon 3. Splicing patterns of a reporter gene that mimics the early transcript, and its variants, were analyzed in Drosophila transformants and tissue culture cells. The results demonstrate that the early, in contrast to the late, splicing pattern is not regulated by stage-specific or sex-specific trans-acting factors, and so the pattern appears to arise from some type of intrinsic splice site preference or compatibility. Inclusion or exclusion of exon 3 is determined by the identity of the upstream 5' splice site region as late or early. The important region of the early exon lies within 233 nucleotides of the immediately adjacent intron.

Sex-lethal (Sxl) acts as a binary switch that regulates Drosophila sexual differentiation and dosage compensation and also maintains a stable female state through autoregulation. As part of a cascade of genes that are regulated by sex-specific splicing, Sxl controls the sex-specific splicing of transformer (tra) RNA and also its own RNA. Sxl contains two RNP-CS (RNA-binding) domains and is known to bind tra pre-mRNA near the alternative 3' splice site, thus blocking use of that site to give the female-specific splicing pattern. Here, we test how Sxl protein interacts with Sxl RNA during autoregulation. We show that Sxl not only binds Sxl pre-mRNA near the alternative 3' splice site but also at distant, multiple sites surrounding the Sxl alternative exon. Moreover, Sxl binds cooperatively at these multiple sites. The Sxl amino terminus is essential for the cooperative interaction and is also required for regulatory activity in vivo. It appears that this region of Sxl protein, which resembles regions in some other RNA-binding proteins, is a domain that mediates protein-protein interactions during RNA binding and plays an important role in splicing regulation.

The binary switch gene Sex-lethal (Sxl) must be on in females and off in males to allow the proper elaboration of the appropriate sexual developmental pathway in Drosophila melanogaster. Previous studies suggested a mechanism in which the on/off regulation of Sxl occurs post-transcriptionally at the level of RNA splicing. A critical prediction of this model is that functional Sxl proteins are absent in males but present in females. In this report we show that the expected full-length proteins are only present in female animals. Multiple forms of Sxl protein are found in females, some of which are expressed in a stage- and tissue-specific pattern. Consistent with a role of Sxl proteins in regulating alternate splicing, the proteins are localized in the nucleus where they exhibit a punctate staining pattern. Surprisingly, several minor Sxl proteins appear to be present in specific tissues of both sexes of adults. The possible origin of these species is discussed. We also show that Sxl expression in the early embryo is sex specific and depends on maternal daughterless and zygotic sisterless-b activity in accordance with the established roles of these genes as positive regulators of Sxl. The onset of Sxl expression in the germ line occurs later than that in the soma.