Exploring the Impact of PQN-75 and GLH-1/Vasa on Germline Development, Maintenance, and GSC Reprogramming Using Caenorhabditis elegans as a Model

This thesis combines research on PQN-75 expression, functional motifs of GLH-1/Vasa, and germ granule components in Caenorhabditis elegans to provide a comprehensive understanding of germline development, maintenance, and reprogramming, while also examining the role of pharyngeal gland cells in stress resistance and thermotolerance. In C. elegans, pharyngeal gland cells secrete mucin-like proteins, such as PQN-75, with similarities to human PRB2. The expression of PQN-75 in gland cells confers stress resistance and thermotolerance but does not affect fertility, instead it plays a role in the organism\u27s ability to adapt to varying environmental conditions. While, GLH-1/Vasa, an ATP-dependent DEAD-box helicase, plays a critical role in safeguarding the germline by regulating translation and amplifying piwi-interacting RNAs. To elucidate the functions of GLH-1 and its role in germline development, CRISPR/Cas9 technology was employed to investigate its functional motifs in C. elegans by analyzing 28 endogenous mutant alleles. Results demonstrate that helicase activity is essential for GLH-1\u27s association with P granules, and removing glycine-rich repeats diminishes P-granule interactions at the nuclear periphery. Additional, mass spectrometry reveals an affinity between GLH-1 and three structurally conserved PCI complexes, along with a reciprocal aversion for assembled ribosomes and the 26S proteasome. Suggesting that P granules compartmentalize the cytoplasm to exclude large protein assemblies, effectively shielding associated transcripts from translation, contributing to germline maintenance. Germ granules are essential for maintaining germline integrity and stem cell totipotency. Depletion of core germ granule components in C. elegans leads to germ cell reprogramming and sterility. To better understand the initiation of somatic reprogramming and the role of GLH-1 in this process, total mRNA (transcriptome) and polysome-associated mRNA (translatome) changes in a precision full-length deletion of glh-1 where examined. Here two significant changes were observed: first, GLH-1 suppresses the expression of neuropeptide-encoding transcripts, suggesting a role in repressing somatic reprogramming and maintaining germline integrity; second, GLH-1 promotes Major Sperm Proteins levels, repressing spermatogenic expression during oogenesis and promoting MSP expression to drive spermiogenesis and sperm motility, highlighting its importance in fertility. Our findings contribute to understanding the roles of PQN-75 and GLH-1/Vasa in C. elegans germline development, maintenance, and germline stem cell reprogramming, while also shedding light on the organism\u27s stress resistance and thermotolerance mechanisms. With broader implications identifying early stem cell reprogramming processes and provides a platform for future research on germline biology in C. elegans

Germline Maintenance Through the Multifaceted Activities of GLH/Vasa in

Vasa homologs are ATP-dependent DEAD-box helicases, multipotency factors, and critical components that specify and protect the germline. They regulate translation, amplify piwi-interacting RNAs (piRNAs), and act as RNA solvents; however, the limited availability of mutagenesis-derived alleles and their wide range of phenotypes have complicated their analysis. Now, with clustered regularly interspaced short palindromic repeats (CRISPR/Cas9), these limitations can be mitigated to determine why protein domains have been lost or retained throughout evolution. Here, we define the functional motifs of GLH-1/Vasa i

PQN-75 is expressed in the pharyngeal gland cells of Caenorhabditis elegans and is dispensable for germline development

In Caenorhabditis elegans, five pharyngeal gland cells reside in the terminal bulb of the pharynx and extend anterior processes to five contact points in the pharyngeal lumen. Pharyngeal gland cells secrete mucin-like proteins thought to facilitate digestion, hatching, molting and assembly of the surface coat of the cuticle, but supporting evidence has been sparse. Here we show pharyngeal gland cell expression of PQN-75, a unique protein containing an N-terminal signal peptide, nucleoporin (Nup)-like phenylalanine/glycine (FG) repeats, and an extensive polyproline repeat domain with similarities to human basic salivary proline-rich pre-protein PRB2. Imaging of C-terminal tagged PQN-75 shows localization throughout pharyngeal gland cell processes but not the pharyngeal lumen; instead, aggregates of PQN-75 are occasionally found throughout the pharynx, suggesting secretion from pharyngeal gland cells into the surrounding pharyngeal muscle. PQN-75 does not affect fertility and brood size in C. elegans but confers some degree of stress resistance and thermotolerance through unknown mechanisms

Many Labs 2: Investigating Variation in Replicability Across Samples and Settings

We conducted preregistered replications of 28 classic and contemporary published findings, with protocols that were peer reviewed in advance, to examine variation in effect magnitudes across samples and settings. Each protocol was administered to approximately half of 125 samples that comprised 15,305 participants from 36 countries and territories. Using the conventional criterion of statistical significance (p < .05), we found that 15 (54%) of the replications provided evidence of a statistically significant effect in the same direction as the original finding. With a strict significance criterion (p < .0001), 14 (50%) of the replications still provided such evidence, a reflection of the extremely high-powered design. Seven (25%) of the replications yielded effect sizes larger than the original ones, and 21 (75%) yielded effect sizes smaller than the original ones. The median comparable Cohen’s ds were 0.60 for the original findings and 0.15 for the replications. The effect sizes were small (< 0.20) in 16 of the replications (57%), and 9 effects (32%) were in the direction opposite the direction of the original effect. Across settings, the Q statistic indicated significant heterogeneity in 11 (39%) of the replication effects, and most of those were among the findings with the largest overall effect sizes; only 1 effect that was near zero in the aggregate showed significant heterogeneity according to this measure. Only 1 effect had a tau value greater than .20, an indication of moderate heterogeneity. Eight others had tau values near or slightly above .10, an indication of slight heterogeneity. Moderation tests indicated that very little heterogeneity was attributable to the order in which the tasks were performed or whether the tasks were administered in lab versus online. Exploratory comparisons revealed little heterogeneity between Western, educated, industrialized, rich, and democratic (WEIRD) cultures and less WEIRD cultures (i.e., cultures with relatively high and low WEIRDness scores, respectively). Cumulatively, variability in the observed effect sizes was attributable more to the effect being studied than to the sample or setting in which it was studied