Human placenta releases extracellular vesicles carrying corticotrophin releasing hormone mRNA into the maternal blood.

The human placenta releases diverse extracellular vesicles (EVs), including microvesicles (100-1000 nm) and exosomes (30-150 nm), into the maternal blood for feto-maternal communication. Exosomes and microvesicles contribute to normal pregnancy physiology and major pregnancy pathologies. Differences in miRNA expressions and protein content in placental exosomes have been reported in complicated pregnancies. During human pregnancy, Corticotropin-Releasing Hormone (CRH) is produced and released by the placenta into the maternal blood. CRH is involved in regulating gestational length and the initiation of labour. CRH mRNA levels in the maternal plasma rise with gestation. High levels of CRH mRNA are reported to be associated with preeclamptic and preterm pregnancies. However, the underlying mechanism of placental CRH mRNA secretion remains to be elucidated. We hypothesise that the placenta releases CRH mRNA packaged within extracellular vesicles (EVs) into the maternal blood. In this study, placental EVs (microvesicles and exosomes) were isolated from human term healthy placentas via villus washes and from explant culture media by differential centrifugation and purified by density gradient ultracentrifugation using a continuous sucrose gradient (0.25-2.5 M). Western blotting using placenta- and exosome-specific markers and electron microscopy confirmed exosomes and microvesicles in the placental wash and explant media samples. Real-time quantitative RT-PCR data detected CRH mRNA in placenta-derived EVs from placental washes and explants. We also sorted placenta-secreted EVs in maternal plasma samples (≥37 weeks) by high-resolution flow cytometry using a fluorescent-labelled PLAP antibody. CRH mRNA was demonstrated in placental EVs obtained from maternal blood plasma. We therefore show that human placental EVs carry CRH mRNA into the maternal blood. Our study implies that measuring CRH mRNA in placental EVs in the maternal plasma could beused for monitoring pregnancy.

Extracellular Polymeric Substances Drive Symbiotic Interactions in Bacterial‒Microalgal Consortia.

The importance of several factors that drive the symbiotic interactions between bacteria and microalgae in consortia has been well realised. However, the implication of extracellular polymeric substances (EPS) released by the partners remains unclear. Therefore, the present study focused on the influence of EPS in developing consortia of a bacterium, Variovorax paradoxus IS1, with a microalga, Tetradesmus obliquus IS2 or Coelastrella sp. IS3, all isolated from poultry slaughterhouse wastewater. The bacterium increased the specific growth rates of microalgal species significantly in the consortia by enhancing the uptake of nitrate (88‒99%) and phosphate (92‒95%) besides accumulating higher amounts of carbohydrates and proteins. The EPS obtained from exudates, collected from the bacterial or microalgal cultures, contained numerous phytohormones, vitamins, polysaccharides and amino acids that are likely involved in interspecies interactions. The addition of EPS obtained from V. paradoxus IS1 to the culture medium doubled the growth of both the microalgal strains. The EPS collected from T. obliquus IS2 significantly increased the growth of V. paradoxus IS1, but there was no apparent change in bacterial growth when it was cultured in the presence of EPS from Coelastrella sp. IS3. These observations indicate that the interaction between V. paradoxus IS1 and T. obliquus IS2 was mutualism, while commensalism was the interaction between the bacterial strain and Coelastrella sp. IS3. Our present findings thus, for the first time, unveil the EPS-induced symbiotic interactions among the partners involved in bacterial‒microalgal consortia.

Visualization of endogenous p27 and Ki67 reveals the importance of a c-Myc-driven metabolic switch in promoting survival of quiescent cancer cells.

Rationale: Recurrent and metastatic cancers often undergo a period of dormancy, which is closely associated with cellular quiescence, a state whereby cells exit the cell cycle and are reversibly arrested in G0 phase. Curative cancer treatment thus requires therapies that either sustain the dormant state of quiescent cancer cells, or preferentially, eliminate them. However, the mechanisms responsible for the survival of quiescent cancer cells remain obscure. Methods: Dual genome-editing was carried out using a CRISPR/Cas9-based system to label endogenous p27 and Ki67 with the green and red fluorescent proteins EGFP and mCherry, respectively, in melanoma cells. Analysis of transcriptomes of isolated EGFP-p27highmCherry-Ki67low quiescent cells was conducted at bulk and single cell levels using RNA-sequencing. The extracellular acidification rate and oxygen consumption rate were measured to define metabolic phenotypes. SiRNA and inducible shRNA knockdown, chromatin immunoprecipitation and luciferase reporter assays were employed to elucidate mechanisms of the metabolic switch in quiescent cells. Results: Dual labelling of endogenous p27 and Ki67 with differentiable fluorescent probes allowed for visualization, isolation, and analysis of viable p27highKi67low quiescent cells. Paradoxically, the proto-oncoprotein c-Myc, which commonly drives malignant cell cycle progression, was expressed at relatively high levels in p27highKi67low quiescent cells and supported their survival through promoting mitochondrial oxidative phosphorylation (OXPHOS). In this context, c-Myc selectively transactivated genes encoding OXPHOS enzymes, including subunits of isocitric dehydrogenase 3 (IDH3), whereas its binding to cell cycle progression gene promoters was decreased in quiescent cells. Silencing of c-Myc or the catalytic subunit of IDH3, IDH3α, preferentially killed quiescent cells, recapitulating the effect of treatment with OXPHOS inhibitors. Conclusion: These results establish a rigorous experimental system for investigating cellular quiescence, uncover the high selectivity of c-Myc in activating OXPHOS genes in quiescent cells, and propose OXPHOS targeting as a potential therapeutic avenue to counter cancer cells in quiescence.

Acid-adapted microalgae exhibit phenotypic changes for their survival in acid mine drainage samples.

Phenotypic plasticity or genetic adaptation in an organism provides phenotypic changes when exposed to the extreme environmental conditions. The resultant physiological and metabolic changes greatly enhance the organism's potential for its survival in such harsh environments. In the present novel approach, we tested the hypothesis whether acid-adapted microalgae, initially isolated from non-acidophilic environments, can survive and grow in acid-mine-drainage (AMD) samples. Two acid-adapted microalgal strains, Desmodesmus sp. MAS1 and Heterochlorella sp. MAS3, were tested individually or in combination (co-culture) for phenotypic changes during their growth in samples collected from AMD. The acid-adapted microalgae in AMD exhibited a two-fold increase in growth when compared with those grown at pH 3.5 in BBM up to 48 h and then declined. Furthermore, oxidative stress triggered several alterations such as increased cell size, granularity, and enhanced lipid accumulation in AMD-grown microalgae. Especially, the apparent limitation of phosphate in AMD inhibited the uptake of copper and iron in the cultures. Interestingly, growth of the acid-adapted microalgae in AMD downregulated amino acid metabolic pathways as a survival mechanism. This study demonstrates for the first time that acid-adapted microalgae can survive under extreme environmental conditions as exist in AMD by effecting significant phenotypic changes.

Sustainable production of biomass and biodiesel by acclimation of non-acidophilic microalgae to acidic conditions.

The overwhelming response towards algal biodiesel production has been well-recognized recently as a sustainable alternative to conventional fuels. Most microalgae cannot grow well at acidic pH. The present study, therefore, investigated whether non-acidophilic microalgae Desmodesmus sp. MAS1 and Heterochlorella sp. MAS3 can be acclimated to extreme-acidic pH for sustainable production of biomass and biodiesel. Growth analysis indicated that both the microalgal strains possessed a passive uptake of CO2 at pH 3.0 with biomass production of 0.25 g dry wt. L-1 in Desmodemus sp. and 0.45 g dry wt. L-1 in Heterochlorella sp.. Flow-cytometry analysis for reactive oxygen species, membrane permeability and neutral-lipids revealed the capabilities of both strains to adapt to the stress imposed by acidic pH. Lipid production was doubled in both the strains when grown at pH 3.0. In-situ transesterification of biomass resulted in 13-15% FAME yield in the selected microalgae, indicating their great potential in biofuel production.

A p53-Responsive miRNA Network Promotes Cancer Cell Quiescence.

: Cancer cells in quiescence (G0 phase) are resistant to death, and re-entry of quiescent cancer cells into the cell-cycle plays an important role in cancer recurrence. Here we show that two p53-responsive miRNAs utilize distinct but complementary mechanisms to promote cancer cell quiescence by facilitating stabilization of p27. Purified quiescent B16 mouse melanoma cells expressed higher levels of miRNA-27b-3p and miRNA-455-3p relative to their proliferating counterparts. Induction of quiescence resulted in increased levels of these miRNAs in diverse types of human cancer cell lines. Inhibition of miRNA-27b-3p or miRNA-455-3p reduced, whereas its overexpression increased, the proportion of quiescent cells in the population, indicating that these miRNAs promote cancer cell quiescence. Accordingly, cancer xenografts bearing miRNA-27b-3p or miRNA-455-3p mimics were retarded in growth. miRNA-27b-3p targeted cyclin-dependent kinase regulatory subunit 1 (CKS1B), leading to reduction in p27 polyubiquitination mediated by S-phase kinase-associated protein 2 (Skp2). miRNA-455-3p targeted CDK2-associated cullin domain 1 (CAC1), which enhanced CDK2-mediated phosphorylation of p27 necessary for its polyubiquitination. Of note, the gene encoding miRNA-27b-3p was embedded in the intron of the chromosome 9 open reading frame 3 gene that was transcriptionally activated by p53. Similarly, the host gene of miRNA-455-3p, collagen alpha-1 (XXVII) chain, was also a p53 transcriptional target. Collectively, our results identify miRNA-27b-3p and miRNA-455-3p as important regulators of cancer cell quiescence in response to p53 and suggest that manipulating miRNA-27b-3p and miRNA-455-3p may constitute novel therapeutic avenues for improving outcomes of cancer treatment. SIGNIFICANCE: Two novel p53-responsive microRNAs whose distinct mechanisms of action both stabilize p27 to promote cell quiescence and may serve as therapeutic avenues for improving outcomes of cancer treatment.