daf-16/FOXO blocks adult cell fate in Caenorhabditis elegans dauer larvae via lin-41/TRIM71.

Many tissue-specific stem cells maintain the ability to produce multiple cell types during long periods of non-division, or quiescence. FOXO transcription factors promote quiescence and stem cell maintenance, but the mechanisms by which FOXO proteins promote multipotency during quiescence are still emerging. The single FOXO ortholog in C. elegans, daf-16, promotes entry into a quiescent and stress-resistant larval stage called dauer in response to adverse environmental cues. During dauer, stem and progenitor cells maintain or re-establish multipotency to allow normal development to resume after dauer. We find that during dauer, daf-16/FOXO prevents epidermal stem cells (seam cells) from prematurely adopting differentiated, adult characteristics. In particular, dauer larvae that lack daf-16 misexpress collagens that are normally adult-enriched. Using col-19p::gfp as an adult cell fate marker, we find that all major daf-16 isoforms contribute to opposing col-19p::gfp expression during dauer. By contrast, daf-16(0) larvae that undergo non-dauer development do not misexpress col-19p::gfp. Adult cell fate and the timing of col-19p::gfp expression are regulated by the heterochronic gene network, including lin-41 and lin-29. lin-41 encodes an RNA-binding protein orthologous to LIN41/TRIM71 in mammals, and lin-29 encodes a conserved zinc finger transcription factor. In non-dauer development, lin-41 opposes adult cell fate by inhibiting the translation of lin-29, which directly activates col-19 transcription and promotes adult cell fate. We find that during dauer, lin-41 blocks col-19p::gfp expression, but surprisingly, lin-29 is not required in this context. Additionally, daf-16 promotes the expression of lin-41 in dauer larvae. The col-19p::gfp misexpression phenotype observed in dauer larvae with reduced daf-16 requires the downregulation of lin-41, but does not require lin-29. Taken together, this work demonstrates a novel role for daf-16/FOXO as a heterochronic gene that promotes expression of lin-41/TRIM71 to contribute to multipotent cell fate in a quiescent stem cell model.

MRI of a middle ear cholesteatoma in a cat.

A 9-year-old Maine coon cat presented with right-sided Horner and facial nerve paralysis. Magnetic resonance imaging (MRI) showed a heterogeneously contrast-enhancing mass occupying the right dorsolateral compartment of the tympanic cavity and extending into the ventromedial compartment, which was expanded and fluid filled. A ventral bulla osteotomy was performed to debulk the soft tissue mass. Histopathology revealed a cholesteatoma. The cat showed slow improvement of clinical signs and was euthanized eleven months postoperatively for unrelated causes. The MRI characteristics of middle ear cholesteatoma in the present case varied from those described in humans and dogs.

Donor-specific phenotypic variation in hiPSC cardiomyocyte-derived exosomes impacts endothelial cell function.

Exosomes are an important mechanism of cell-cell interaction in the cardiovascular system, both in maintaining homeostasis and in stress response. Interindividual differences that alter content in exosomes may play a role in cardiovascular disease pathology. To study the effect of interindividual cardiomyocyte (CM) variation, we characterized exosomal content in phenotypically diverse human induced pluripotent stem cell-derived CMs (hiPSC-CMs). Cell lines were generated from six participants in the HyperGEN cohort: three with left ventricular hypertrophy (LVH) and three with normal left ventricular mass (LVM). Sequence analysis of the intracellular and exosomal RNA populations showed distinct expression pattern differences between hiPSC-CM lines derived from individuals with LVH and those with normal LVM. Functional analysis of hiPSC-endothelial cells (hiPSC-ECs) treated with exosomes from both hiPSC-CM groups showed significant variation in response, including differences in tube formation, migration, and proliferation. Overall, treatment of hiPSC-ECs with exosomes resulted in significant expression changes associated with angiogenesis and endothelial cell vasculogenesis. However, the hiPSC-ECs treated with exosomes from the LVH-affected donors exhibited significantly increased proliferation but decreased tube formation and migration, suggesting angiogenic dysregulation.NEW & NOTEWORTHY The intracellular RNA and the miRNA content in exosomes are significantly different in hiPSC-CMs derived from LVH-affected individuals compared with those from unaffected individuals. Treatment of endothelial cells with these exosomes functionally affects cellular phenotypes in a donor-specific manner. These findings provide novel insight into underlying mechanisms of hypertrophic cell signaling between different cell types. With a growing interest in stem cells and exosomes for cardiovascular therapeutic use, this also provides information important for regenerative medicine.

Functional analysis of microRNA pathway genes in the somatic gonad and germ cells during ovulation in C. elegans.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that play critical roles in animal development and physiology, though functions for most miRNAs remain unknown. Worms with reduced miRNA biogenesis due to loss of Drosha or Pasha/DGCR8 activity are sterile and fail to ovulate, indicating that miRNAs are required for the process of oocyte maturation and ovulation. Starting with this penetrant sterile phenotype and using new strains created to perform tissue specific RNAi, we characterized the roles of the C. elegans Pasha, pash-1, and two miRNA-specific Argonautes, alg-1 and alg-2, in somatic gonad cells and in germ cells in the regulation of ovulation. Conditional loss of pash-1 activity resulted in a reduced rate of ovulation and in basal and ovulatory sheath contractions. Similarly, knockdown of miRNA-specific Argonautes in the cells of the somatic gonad by tissue-specific RNAi results in a reduction of the ovulation rate and in basal and ovulatory sheath contractions. Reduced miRNA pathway gene activity resulted in a range of defects, including oocytes that were pinched upon entry of the oocyte into the distal end of the spermatheca in about 42% of the ovulation events observed following alg-1 RNAi. This phenotype was not observed on worms exposed to control RNAi. In contrast, knockdown of alg-1 and alg-2 in germ cells results in few defects in oocyte maturation and ovulation. These data identify specific steps in the process of ovulation that require miRNA pathway gene activity in the somatic gonad cells.

Uropathogenic Escherichia coli infection-induced epithelial trained immunity impacts urinary tract disease outcome.

Previous urinary tract infections (UTIs) can predispose one to future infections; however, the underlying mechanisms affecting recurrence are poorly understood. We previously found that UTIs in mice cause differential bladder epithelial (urothelial) remodelling, depending on disease outcome, that impacts susceptibility to recurrent UTI. Here we compared urothelial stem cell (USC) lines isolated from mice with a history of either resolved or chronic uropathogenic Escherichia coli (UPEC) infection, elucidating evidence of molecular imprinting that involved epigenetic changes, including differences in chromatin accessibility, DNA methylation and histone modification. Epigenetic marks in USCs from chronically infected mice enhanced caspase-1-mediated cell death upon UPEC infection, promoting bacterial clearance. Increased Ptgs2os2 expression also occurred, potentially contributing to sustained cyclooxygenase-2 expression, bladder inflammation and mucosal wounding-responses associated with severe recurrent cystitis. Thus, UPEC infection acts as an epi-mutagen reprogramming the urothelial epigenome, leading to urothelial-intrinsic remodelling and training of the innate response to subsequent infection.

Longitudinal multi-omics analyses link gut microbiome dysbiosis with recurrent urinary tract infections in women.

Recurrent urinary tract infections (rUTIs) are a major health burden worldwide, with history of infection being a significant risk factor. While the gut is a known reservoir for uropathogenic bacteria, the role of the microbiota in rUTI remains unclear. We conducted a year-long study of women with (n = 15) and without (n = 16) history of rUTI, from whom we collected urine, blood and monthly faecal samples for metagenomic and transcriptomic interrogation. During the study 24 UTIs were reported, with additional samples collected during and after infection. The gut microbiome of individuals with a history of rUTI was significantly depleted in microbial richness and butyrate-producing bacteria compared with controls, reminiscent of other inflammatory conditions. However, Escherichia coli gut and bladder populations were comparable between cohorts in both relative abundance and phylogroup. Transcriptional analysis of peripheral blood mononuclear cells revealed expression profiles indicative of differential systemic immunity between cohorts. Altogether, these results suggest that rUTI susceptibility is in part mediated through the gut-bladder axis, comprising gut dysbiosis and differential immune response to bacterial bladder colonization, manifesting in symptoms.

Sperm fate is promoted by the mir-44 microRNA family in the Caenorhabditis elegans hermaphrodite germline.

Posttranscriptional regulation of gene expression, typically effected by RNA-binding proteins, microRNAs (miRNAs), and translation initiation factors, is essential for normal germ cell function. Numerous miRNAs have been detected in the germline; however, the functions of specific miRNAs remain largely unknown. Functions of miRNAs have been difficult to determine as miRNAs often modestly repress target mRNAs and are suggested to sculpt or fine tune gene expression to allow for the robust expression of cell fates. In Caenorhabditis elegans hermaphrodites, cell fate decisions are made for germline sex determination during larval development when sperm are generated in a short window before the switch to oocyte production. Here, analysis of newly generated mir-44 family mutants has identified a family of miRNAs that modulate the germline sex determination pathway in C. elegans. Mutants with the loss of mir-44 and mir-45 produce fewer sperm, showing both a delay in the specification and formation of sperm as well as an early termination of sperm specification accompanied by a premature switch to oocyte production. mir-44 and mir-45 are necessary for the normal period of fog-1 expression in larval development. Through genetic analysis, we find that mir-44 and mir-45 may act upstream of fbf-1 and fem-3 to promote sperm specification. Our research indicates that the mir-44 family promotes sperm cell fate specification during larval development and identifies an additional posttranscriptional regulator of the germline sex determination pathway.

Whole-Exome Sequencing and hiPSC Cardiomyocyte Models Identify MYRIP, TRAPPC11, and SLC27A6 of Potential Importance to Left Ventricular Hypertrophy in an African Ancestry Population.

Background: Indices of left ventricular (LV) structure and geometry represent useful intermediate phenotypes related to LV hypertrophy (LVH), a predictor of cardiovascular (CV) disease (CVD) outcomes. Methods and Results: We conducted an exome-wide association study of LV mass (LVM) adjusted to height2.7, LV internal diastolic dimension (LVIDD), and relative wall thickness (RWT) among 1,364 participants of African ancestry (AAs) in the Hypertension Genetic Epidemiology Network (HyperGEN). Both single-variant and gene-based sequence kernel association tests were performed to examine whether common and rare coding variants contribute to variation in echocardiographic traits in AAs. We then used a data-driven procedure to prioritize and select genes for functional validation using a human induced pluripotent stem cell cardiomyocyte (hiPSC-CM) model. Three genes [myosin VIIA and Rab interacting protein (MYRIP), trafficking protein particle complex 11 (TRAPPC11), and solute carrier family 27 member 6 (SLC27A6)] were prioritized based on statistical significance, variant functional annotations, gene expression in the hiPSC-CM model, and prior biological evidence and were subsequently knocked down in the hiPSC-CM model. Expression profiling of hypertrophic gene markers in the knockdowns suggested a decrease in hypertrophic expression profiles. MYRIP knockdowns showed a significant decrease in atrial natriuretic factor (NPPA) and brain natriuretic peptide (NPPB) expression. Knockdowns of the heart long chain fatty acid (FA) transporter SLC27A6 resulted in downregulated caveolin 3 (CAV3) expression, which has been linked to hypertrophic phenotypes in animal models. Finally, TRAPPC11 knockdown was linked to deficient calcium handling. Conclusions: The three genes are biologically plausible candidates that provide new insight to hypertrophic pathways.