Biology subjects

Mark Welch, D. B.

Publications and source records attributed to Mark Welch, D. B..

6 recordsLinked to original sources

bioRxiv2025

Hyperosmotic Stress of HL-60/S4 Cells: Gene Set Enrichment Analysis and Microscopy Changes

Cellular homeostasis requires that internal cell conditions (e.g., pH, ionic and non-ionic solute concentrations and hydration levels) maintain a dynamic stability. To better understand the mechanisms enforcing this homeostasis, we examined the transcript abundance and cellular morphology of undifferentiated myeloid HL-60/S4 cells exposed to 30 or 60 minutes of hyperosmotic stress, resulting from 300 mM sucrose added to the tissue culture medium. We used Gene Set Expression Analyses (GSEA) to examine various cell functions (Phenotypes) of interest. Microscopy was also employed, where possible, to visualize the nuclear and chromatin physical states. We observed that hyperosmotic stress resulted in congelation of interphase and mitotic chromatin. In parallel, GSEA indicated a reduction of heterochromatin, histone lysine methyltransferase and mRNA transcription. Mitosis ceases during hyperosmotic stress, with the intact tubulin spindle remaining attached to clustered centromeres on the congealed chromosomes. GSEA indicated that ribosome biosynthesis in nucleoli and oxidative activity in mitochondria are significantly increased. In addition, the proteasome phenotype is increased, suggesting that protein synthesis and destruction are both occurring at an increased pace during hyperosmotic stress. Nuclear envelope-associated chromatin also appears to be affected by hyperosmotic stress: LBR and Heterochromatin Protein 1 alpha (CBX5) disperse into the cytoplasm. Evidence is also presented that 300 mM sucrose leads to a reduction of DNA methylation and aberrant cellular localization of MeCP2. The effects of acute hyperosmotic stress on HL-60/S4 cells are very diverse and very profound. In many respects, the stress response resembles a frantic attempt for cell survival in the face of inevitable cell death.

cell biology↗

bioRxiv2024

The Effects of Lamin B Receptor knockdown on a Myeloid Leukemia Cell

In an effort to extend our understanding of the genetic functions of the nuclear envelope protein Lamin B Receptor (LBR), we examined the effect of a stable short hairpin (sh1) RNAi knockdown of LBR on the transcriptome and immunostained morphology of the human myeloid leukemia cell line (HL-60/S4). This examination was on sh1 cells induced to granulocytic form with Retinoic Acid (RA) versus sh1 cells maintained undifferentiated (0). By comparison to control cells (i.e., not sh1), we obtained gene lists that were differentially expressed only in the LBR knockdown cell line (i.e., "only-sh1-down" and "only-sh1-up"), in RA versus 0 cells. These curated gene lists were examined by Gene Ontology (GO) analysis. Aside from chromatin related GO terms, the most surprising finding was a significant downregulation of Golgi related genes only in the sh1 cells. Possible relationships between the "Cis-Golgi-Network" and LBR are discussed. Another surprise was a significant upregulation of "Ribosome" protein transcripts only in the sh1 cells. In parallel to these findings, an immunostaining comparison of nucleoli in S4 and sh1 cells demonstrated that the number and location of nucleoli in a single nucleus differs, depending upon the presence of LBR. Speculations on the influence of LBR levels upon the liquid-liquid phase separation model of nucleolar condensation are presented.

cell biology↗

bioRxiv2024

The Multifaceted Phenotype of Senescent HL-60/S4 Macrophages

Every cell has a multifaceted phenotype. Transcriptional analysis of functionally defined groups of genes can provide insight into this phenotypic complexity. In the present study, the mRNA transcriptome of phorbol ester (TPA) differentiated HL-60/S4 macrophage cells was scrutinized using Gene Set Enrichment Analysis (GSEA), which evaluates the strengths of various cellular phenotypes by examining the enrichment of functionally different gene sets. Employing GSEA, we obtained supporting evidence that HL-60/S4 macrophages are senescent, probably a consequence of enriched TGF{beta} and NOTCH signaling transcripts. There appears to be a reduction of transcripts for heterochromatin, nucleosome formation, and chromatin remodeling phenotypes. In addition, despite upregulated oxidative stress gene transcription, we observed a reduction of DNA damage and repair transcripts. GSEA indicated that transcripts for autophagy, extracellular matrix, and inflammation/inflammasomes are enriched. We also observed that the HL-60/S4 macrophage is enriched for apoptosis gene transcripts, which may promote necrotic death by pyroptosis. The long-term goal of this research direction is to see whether this complex multifaceted phenotypic pattern is shared with other types of macrophages and to determine what mechanisms might exist to coordinate these phenotypic facets within a single cell.

cell biology↗

bioRxiv2021

Genome structure of Brachionus asplanchnoidis, a Eukaryote with intrapopulation variation in genome size

Eukaryotic genomes vary greatly in size due to variation in the proportion of non-coding DNA, a pattern that emerges both in comparisons at a larger taxonomic scale and at the level of individuals within a species. The rotifer Brachionus asplanchnoidis represents one of the most extreme cases of intraspecific genome size variation among Eukaryotes, displaying almost 2-fold variation within a geographic population. Here we used a whole-genome sequencing approach to identify the underlying DNA sequence differences by assembling a high-quality reference genome draft for one individual of the population and aligning short-reads of 15 individuals from the same geographic population. We identified large, contiguous copy number variable regions (CNVs), which exhibited significant coverage differences among individuals, and whose coverage overall scaled with genome size. CNVs were mainly composed of tandemly repeated satellite DNA, with only few interspersed genes or other sequences, and were characterized by an elevated GC-content. Judging from their distributions across contigs, some CNVs are fragments of accessory (B-)chromosomes while others resemble large insertions to normal chromosomes. CNV patterns in offspring of two parents with divergent genome size, and CNV patterns in several individuals from an inbred line differing in genome size demonstrated inheritance and accumulation of CNVs across generations. Our study provides unprecedented insights into genome size evolution at microevolutionary time scales and thus paves the way for studying genome size evolution in contemporary populations rather than inferring patterns and processes a posteriori from species comparisons.

evolutionary biology↗

bioRxiv2020

Intergenerational microbial transmission in the little skate (Leucoraja erinacea)

BackgroundMicrobial transmission from parent to offspring is hypothesized to be widespread in vertebrates. However, evidence for this is limited as many evolutionarily important clades remain unexamined. There is currently no data on the microbiota associated with any Chondrichthyan species during embryonic development, despite the global distribution, ecological importance, and phylogenetic position of this clade. In this study, we take the first steps towards filling this gap by investigating the microbiota associated with embryonic development in the little skate, Leucoraja erinacea, a common North Atlantic species and popular system for chondrichthyan biology. MethodsTo assess the potential for bacterial transmission in an oviparous chondrichthyan, we used 16S rRNA amplicon sequencing to characterize the microbial communities associated with the skin, gill, and egg capsule of the little skate, at six points during ontogeny. Community composition was analyzed using the QIIME2 pipeline and microbial continuity between stages was tracked using FEAST. ResultsWe identify site-specific and stage-specific microbiota dominated by the bacterial phyla Proteobacteria and Bacteroidetes. This composition is similar to, but distinct from, that of previously published data on the adult microbiota of other chondrichthyan species. Our data reveal that the skate egg capsule harbors a highly diverse bacterial community-particularly on the internal surface of the capsule-and facilitates intergenerational microbial transfer to the offspring. Embryonic skin and external gill tissues host similar bacterial communities; the skin and gill communities later diverge as the internal gills and skin denticles develop. ConclusionsOur study is the first exploration of the chondrichthyan microbiota throughout ontogeny and provides the first evidence of vertical transmission in this group.

microbiology↗

bioRxiv2020

The complex evolution of the HSP70 gene family

The metazoan 70-kDa heat shock protein (HSP70) family contains several members localized in different subcellular compartments. The cytosolic members have been classified into inducible HSP70s and constitutive heat shock cognates (HSC70s), but their distinction and evolutionary relationship remain unclear because of occasional reports of "constitutive HSP70s" and the lack of cross-phylum comparisons. Here we provide novel insights into the evolution of these important molecular chaperones. Phylogenetic analyses of [~]100 full-length HSP70s revealed an ancient duplication that gave rise to two lineages from which all metazoan cytosolic HSP70s descend. One lineage (A) contains a relatively small number of Lophotrochozoan and Ecdysozoan genes, none of which have been shown to be constitutively expressed (i.e., either inducible or unknown). The other lineage (B) included both inducible and constitutive genes from diverse phyla. Species-specific duplications are present in both lineages, and Lineage B contains well-supported phylum-specific clades for Rotifera, Nematoda, and Chordata. Some genes in Lineage B have likely independently acquired inducibility, which may explain the sporadic distribution of "HSP70" or "HSC70" in previous analyses. Consistent with the diversification history within each group, inducible members show lower purifying selection pressure compared to constitutive members. These results illustrate the evolutionary history of the HSP70 family, encouraging us to propose a new nomenclature: "HSP70 + subcellular localization + linage + copy number in the organism + inducible or constitutive, if known." e.g., HSP70cA1i for cytosolic Lineage A, copy 1, inducible.

evolutionary biology↗