Biology subjects

Rajkovic, A.

Publications and source records attributed to Rajkovic, A..

7 recordsLinked to original sources

bioRxiv2020

Single Cell atlas of uterine myometrium and leiomyomas reveals diverse and novel cell types of non-monoclonal origin.

Uterine leiomyomas are the most common tumors of the female reproductive tract with significant morbidity that includes excessive bleeding, infertility and pregnancy complications. The origin and cellular composition of leiomyomas is controversial, yet very important in better understanding the pathogenesis of these tumors. We applied single-cell RNA sequencing to better understand cellular heterogeneity of uterine leiomyomas and normal myometrium at the molecular level. Our data reveal previously unknown heterogeneity in the smooth muscle cells, fibroblast cells, and endothelial cells of normal myometrium and leiomyomas. We discovered a novel lymphatic endothelial cell population in uterine leiomyomas and that the immune as well as transcriptional profile of leiomyomas is MED12 genotype-dependent. Moreover, we show that leiomyoma cell moiety is not monoclonal in nature. Our work describes unprecedented single cell resolution of normal uterine myometrium and leiomyoma tumors and provides insight into tumor specific hormone responsiveness and extracellular matrix accumulation.

genetics↗

bioRxiv2020

Loss of a subunit of vacuolar ATPase identifies unexpected biological signatures of reduced organelle acidification in vivo

The inability to maintain a strictly regulated endo(lyso)somal acidic pH through the proton-pumping action of the vacuolar-ATPases has been associated with various human diseases including heritable connective tissue disorders, neurodegenerative diseases and cancer. Multiple studies have investigated the pleiotropic effects of reduced acidification in vitro, but the mechanisms elicited by impaired endo(lyso)somal acidification in vivo remain poorly understood. Here, we show that loss of atp6v1e1b in zebrafish leads to early mortality, associated with craniofacial dysmorphisms, vascular anomalies, cardiac dysfunction, hypotonia and epidermal structural defects, reminiscent of the phenotypic manifestations in cutis laxa patients carrying a defect in the ATP6V1E1 gene. Mechanistically, we found that in vivo genetic depletion of atp6v1e1b leads to N-glycosylation defects and reduced maturation of endosomal and lysosomal vesicles, but retains the hypoxia-mediated response. In order to gain further insights into the processes affected by aberrant organelle acidification, we performed an untargeted analysis of the transcriptome and metabolome in early atp6v1e1b-deficient larvae. We report multiple affected pathways including but not limited to oxidative phosphorylation, sphingolipid and fatty acid metabolism with profound defects in mitochondrial respiration. Taken together, our results identify new complex biological effects of reduced organelle acidification in vivo, which likely contribute to the multisystemic manifestations observed in disorders caused by v-ATPase deficiency.

molecular biology↗

bioRxiv2020

Deletion of Gremlin-2 alters estrous cyclicity and disrupts female fertility in mice

Members of the differential screening-selected gene aberrative in neuroblastoma (DAN) protein family are developmentally conserved extracellular binding proteins that antagonize bone morphogenetic protein (BMP) signaling. This protein family includes the Gremlin proteins, GREM1 and GREM2, which are known to have key functions during embryogenesis and adult physiology. While BMPs play essential roles in adult female reproductive physiology, the role of the DAN family in ovarian function is less understood. We generated mice null for Grem2 to study its role in female fertility in addition to screening patients with primary ovarian insufficiency for variants in GREM2. Grem2-/- mice are viable and female Grem2-/- mice have diminished fecundity and irregular estrous cycles. This is accompanied by reduced serum levels of anti-Mullerian hormone, a marker of the ovarian reserve, in adult mice. Alterations in ovarian expression of inhibin and activin subunit genes, which are required for regulation of the hypothalamic-pituitary-ovarian (HPO) axis, were identified. While Grem2 mRNA transcript was not detected in the pituitary, Grem2 was expressed in the hypothalami of wild type female mice. Additionally, screening 106 women with primary ovarian insufficiency identified one individual with a heterozygous variant in GREM2 that lies within the predicted BMP-GREM2 interface. In total, these data suggest that Grem2 is necessary for female fecundity by playing a novel role in regulating the HPO axis and possibly contributing to female reproductive disease.

physiology↗

bioRxiv2020

Human Embryonic Expression Identifies Novel Essential Gene Candidates

Disruption of essential genes leads to pregnancy loss, early lethality, or severe disease. Current methods to predict genes that underlie severe phenotypes include knockout animal model systems, evolutionary conservation, and variation intolerance metrics. With existing methods, human lethal genes are missed due to interspecies differences or paucity of gene characterization. We analyzed global gene expression in stages of early human development (1-cell to the blastocyst). These data were integrated with all 4049 current murine knockout phenotypes, genome-wide evolutionary gene conservation, and human genic intolerance metrics. We found that currently established human essential genes and orthologs of murine essential genes demonstrate higher gene expression across developmental stages compared to non-essential genes (Wilcoxon rank sum test, p<8.5e-10), indicating that higher expression correlates with essentiality. Of 1438 unique genes candidates with the highest expression, an estimated 1115 (78%) have not yet been associated with human disease and are thus novel candidates. The essential gene candidates concur with four prediction metrics, further supporting essentiality. We also assessed gene-specific expression changes during early development for their ability to predict essentiality. Genes that increase in expression were more likely to be essential (Fishers exact test, p<2.4e-06), suggesting that dynamic temporal expression during development may be particularly important. We find that embryonic gene expression can be used to prioritize genes that currently lack a Mendelian phenotype. Human embryonic gene expression is readily available, and applied as a novel tool, it may identify highly conserved processes vital in development.

genetics↗

bioRxiv2020

Motifs of the C-terminal Domain of MCM9 Direct Localization to Sites of Mitomycin-C Damage for RAD51 Recruitment

The MCM8/9 complex is implicated in aiding fork progression and facilitating homologous recombination (HR) in response to several DNA damage agents. MCM9 itself is an outlier within the MCM family containing a long C-terminal extension (CTE) comprising 42% of the total length, but with no known functional components and high predicted disorder. In this report, we identify and characterize two unique motifs within the primarily unstructured CTE that are required for localization of MCM8/9 to sites of mitomycin C (MMC) induced DNA damage. First, an unconventional bipartite-like nuclear localization (NLS) motif consisting of two positively charged amino acid stretches separated by a long intervening sequence is required for the nuclear import of both MCM8 and MCM9. Second, a variant of the BRC motif (BRCv), similar to that found in other HR helicases, is necessary for localization to sites of MMC damage. The MCM9-BRCv directly interacts with and recruits RAD51 downstream to MMC-induced damage to aid in DNA repair. Patient lymphocytes devoid of functional MCM9 and discrete MCM9 knockout cells have a significantly impaired ability to form RAD51 foci after MMC treatment. Therefore, the disordered CTE in MCM9 is functionally important in promoting MCM8/9 activity and in recruiting downstream interactors; thus, requiring full length MCM9 for proper DNA repair.

biochemistry↗

bioRxiv2020

Dietary emulsifiers alter composition and activity of the human gut microbiota in vitro, irrespective of chemical or natural emulsifier origin.

The use of additives in food products has become an important public health concern. In recent reports, dietary emulsifiers have been shown to affect the gut microbiota, contributing to a pro-inflammatory phenotype and metabolic syndrome. So far, it is not yet known whether similar microbiome shifts are observable for a more diverse set of emulsifier types and to what extent these effects vary with the unique features of an individuals microbiome. To bridge this gap, we investigated the effect of five dietary emulsifiers on the fecal microbiota from 10 human individuals upon a 48 hour exposure. Community structure was assessed with quantative microbial profiling, functionality was evaluated by measuring fermentation metabolites and pro-inflammatory properties were assessed with the phylogenetic prediction algorythm PICRUSt, together with a TLR5 reporter cell assay for flagellin. A comparison was made between two mainstream chemical emulsifiers (carboxymethylcellulose and P80), a natural extract (soy lecithin) and biotechnological emulsifiers (sophorolipids and rhamnolipids). While fecal microbiota responded in a donor-dependent manner to the different emulsifiers, profound differences between emulsifier were observed. Rhamnolipids, sophorolipids and soy lecithin eliminated 91% {+/-} 0%, 89% {+/-} 1% and 87% {+/-} 1% of the viable bacterial population after 48 hours, yet they all selectively increased the proportional abundance of putative pathogens. Moreover, profound shifts in butyrate (-96% {+/-} 6 %, -73% {+/-} 24% and -34 {+/-} 25% respectively) and propionate (+13% {+/-} 24 %, +88% {+/-} 50% and +29% {+/-} 16% respectively) production were observed for these emulsifiers. Phylogenetic prediction indicated higher motility, which was, however, not confirmed by increased flagellin levels using the TLR5 reporter cell assay. We conclude that dietary emulsifiers can severely impact the gut microbiota and this seems to be proportional to their emulsifying strength, rather than emulsifier type or origin. As biotechnological emulsifiers were especially more impactful than chemical emulsifiers, caution is warranted when considering them as more natural alternatives for clean label strategies.

microbiology↗

bioRxiv2020

Female reproductive tract has low concentration of SARS-CoV2 receptors

There has been significant concern regarding fertility and reproductive outcomes during the SARS-CoV2 pandemic. Recent data suggests a high concentration of SARS-Cov2 receptors, ACE2 or TMPRSS2, in nasal epithelium and cornea, which explains person-to-person transmission. We investigated the prevalence of SARS-CoV2 receptors among reproductive tissues by exploring the single-cell sequencing datasets from uterus, myometrium, ovary, fallopian tube, and breast epithelium. We did not detect significant expression of either ACE2 or TMPRSS2 in the normal human myometrium, uterus, ovaries, fallopian tube, or breast. Furthermore, none of the cell types in the female reproductive organs we investigated, showed the co-expression of ACE2 with proteases, TMPRSS2, Cathepsin B (CTSB), and Cathepsin L (CTSL) known to facilitate the entry of SARS2-CoV2 into the host cell. These results suggest that myometrium, uterus, ovaries, fallopian tube, and breast are unlikely to be susceptible to infection by SARS-CoV2. Our findings suggest that COVID-19 is unlikely to contribute to pregnancy-related adverse outcomes such as preterm birth, transmission of COVID-19 through breast milk, oogenesis and female fertility.

genetics↗