Current Understanding of Exosomal MicroRNAs in Glioma Immune Regulation and Therapeutic Responses

Exosomes, the small extracellular vesicles, are released by multiple cell types, including tumor cells, and represent a novel avenue for intercellular communication via transferring diverse biomolecules. Recently, microRNAs (miRNAs) were demonstrated to be enclosed in exosomes and therefore was protected from degradation. Such exosomal miRNAs can be transmitted to recipient cells where they could regulate multiple cancer-associated biological processes. Accumulative evidence suggests that exosomal miRNAs serve essential roles in modifying the glioma immune microenvironment and potentially affecting the malignant behaviors and therapeutic responses. As exosomal miRNAs are detectable in almost all kinds of biofluids and correlated with clinicopathological characteristics of glioma, they might be served as promising biomarkers for gliomas. We reviewed the novel findings regarding the biological functions of exosomal miRNAs during glioma pathogenesis and immune regulation. Furthermore, we elaborated on their potential clinical applications as biomarkers in glioma diagnosis, prognosis and treatment response prediction. Finally, we summarized the accessible databases that can be employed for exosome-associated miRNAs identification and functional exploration of cancers, including glioma.

A possible mechanism of farnesol tolerance in C. albicans biofilms implemented by activating the PKC signalling pathway and stabilizing ROS levels

Introduction. Biofilms are the natural growth state for most microorganisms. C. albicans biofilms are composed of multiple cell types (round budding yeast-form cells, oval pseudohyphal cells, and elongated hyphal cells) encased in an extracellular matrix. C. albicans biofilms are notorious for resistance to antimicrobial treatments, a property that might be determined by complex mechanisms. Exogenous farnesol exerts a certain antifungal activity against C. albicans with medical implications. Different from other microbes, C. albicans biofilms can tolerate exogenous farnesol at high concentration with some cells still surviving and even maintaining proliferation, but the mechanism is unclear. Hypothesis. The study hypothesizes that C. albicans resists farnesol by activating the PKC signalling pathway. Aim. The study aims to discuss the molecular mechanism of C. albicans resistance to farnesol. Methodology. The ROS levels, the genes and proteins of the PKC pathway were compared between the farnesol-tolerant and non-tolerant groups using ROS levels assay, q-RT PCR and Western blot, respectively. Further, the mutant strains (pkc1Δ/Δ and mkc1Δ/Δ) were constructed, then the survival rates and ROS levels of biofilms exposed to farnesol were compared between mutant and wild strains. The morphological changes were observed using TEM. Results. The survival rates of C. albicans biofilms decreased rapidly under the lower concentration of farnesol (P<0.05), and kept stable (P>0.05) as the concentration rose up to 200 µM. The gene expression of the PKC pathway increased, while ROS levels remained stable and even decreased in the farnesol-tolerant biofilms, compared with those in the farnesol-nontolerant biofilms after farnesol treatment (P<0.05); pkc1 and mkc1 were significantly upregulated by C. albicans during the development of biofilm tolerance to farnesol. The cell wall and cytoplasm of pkc1Δ/Δ and mkc1Δ/Δ were damaged, and the ROS level increased (P<0.05); meanwhile, the survival rate of biofilms decreased compared with that of wild-type strain under the same farnesol concentrations (P<0.05). ROS inhibitors reversed these changes in pkc1Δ/Δ and mkc1Δ/Δ when the mutant strains exposed to farnesol. Conclusion. C. albicans biofilms can tolerate high concentrations of farnesol by activating pkc1 and mkc1 of the PKC pathway and stabilizing ROS levels. The pkc1 and mkc1 are two key genes regulated by C. albicans in the process of biofilm tolerance to farnesol.

Bacillus subtilis Histidine Kinase KinC Activates Biofilm Formation by Controlling Heterogeneity of Single-Cell Responses

In many bacterial and eukaryotic systems, multiple cell fate decisions are activated by a single master regulator. Typically, the activities of the regulators are controlled posttranslationally in response to different environmental stimuli.

Epstein-Barr Virus BGLF2 commandeers RISC to interfere with cellular miRNA function

The Epstein-Barr virus (EBV) BGLF2 protein is a tegument protein with multiple effects on the cellular environment, including induction of SUMOylation of cellular proteins. Using affinity-purification coupled to mass-spectrometry, we identified the miRNA-Induced Silencing Complex (RISC), essential for miRNA function, as a top interactor of BGLF2. We confirmed BGLF2 interaction with the Ago2 and TNRC6 components of RISC in multiple cell lines and their co-localization in cytoplasmic bodies that also contain the stress granule marker G3BP1. In addition, BGLF2 expression led to the loss of processing bodies in multiple cell types, suggesting disruption of RISC function in mRNA regulation. Consistent with this observation, BGLF2 disrupted Ago2 association with multiple miRNAs. Using let-7 miRNAs as a model, we tested the hypothesis that BGLF2 interfered with the function of RISC in miRNA-mediated mRNA silencing. Using multiple reporter constructs with 3’UTRs containing let-7a regulated sites, we showed that BGLF2 inhibited let-7a miRNA activity dependent on these 3’UTRs, including those from SUMO transcripts which are known to be regulated by let-7 miRNAs. In keeping with these results, we showed that BGLF2 increased the cellular level of unconjugated SUMO proteins without affecting the level of SUMO transcripts. Such an increase in free SUMO is known to drive SUMOylation and would account for the effect of BGLF2 in inducing SUMOylation. We further showed that BGLF2 expression inhibited the loading of let-7 miRNAs into Ago2 proteins, and conversely, that lytic infection with EBV lacking BGLF2 resulted in increased interaction of let-7a and SUMO transcripts with Ago2, relative to WT EBV infection. Therefore, we have identified a novel role for BGLF2 as a miRNA regulator and shown that one outcome of this activity is the dysregulation of SUMO transcripts that leads to increased levels of free SUMO proteins and SUMOylation.

Long non-coding RNAs (lncRNAs) NEAT1 and MALAT1 are differentially expressed in severe COVID-19 patients: An integrated single-cell analysis

Hyperactive and damaging inflammation is a hallmark of severe rather than mild Coronavirus disease 2019 (COVID-19). To uncover key inflammatory differentiators between severe and mild COVID-19, we applied an unbiased single-cell transcriptomic analysis. We integrated two single-cell RNA-seq datasets with COVID-19 patient samples, one that sequenced bronchoalveolar lavage (BAL) cells and one that sequenced peripheral blood mononuclear cells (PBMCs). The combined cell population was then analyzed with a focus on genes associated with disease severity. The immunomodulatory long non-coding RNAs (lncRNAs) NEAT1 and MALAT1 were highly differentially expressed between mild and severe patients in multiple cell types. Within those same cell types, the concurrent detection of other severity-associated genes involved in cellular stress response and apoptosis regulation suggests that the pro-inflammatory functions of these lncRNAs may foster cell stress and damage. Thus, NEAT1 and MALAT1 are potential components of immune dysregulation in COVID-19 that may provide targets for severity related diagnostic measures or therapy.

Inhibition of glycogen synthase kinase-3-beta (GSK3β) blocks nucleocapsid phosphorylation and SARS-CoV-2 replication

GSK3β has been proposed to have an essential role in Coronaviridae infection. Screening of a targeted library of GSK3β inhibitors against SARS-CoV-2 and HCoV-229E resulted in identification of high proportion of active compounds with low toxicity to host cells. A select lead compound, T-1686568, showed dose-dependent activity against SARS-CoV-2 transcription, translation and viral particle release in multiple cell lines and primary organoids. A protein kinase substrate profiling assay combined with western blot analysis showed that SARS-CoV-2 nucleocapsid is phosphorylated by GSK3β on S180/S184, S190/S194 and T198 which have already been primed in the adjacent phospho-sites S188, T198 and S206 respectively. Inhibition by T-1686568 resulted in reduction of the S1 Spike protein levels, an accumulation of the Nucleocapsid (N) protein and maintenance of the non-structural (NSP2) level in infected Huh-7.5.1 cells, indicating that N phosphorylation might serve as a critical precursor for processing and release of mature viruses.

Generation of rat lungs by blastocyst complementation in Fgfr2b-deficient mouse model

Regenerative medicine is a tool to compensate for the shortage of lungs for transplantation, but it remains difficult to construct a lung in vitro due to the complex three-dimensional structures and multiple cell types required. A blastocyst complementation method using interspecies chimeric animals has been attracting attention as a way to create complex organs in animals, but successful lung formation has not yet been achieved. Here, we applied a reverse-blastocyst complementation method to clarify the conditions required to form lungs in an Fgfr2b-deficient mouse model. We then successfully formed a rat-derived lung in the mouse model without generating a mouse line by applying a tetraploid-based organ-complementation method. Importantly, rat lung epithelial cells retained their developmental timing even in the mouse body. This result provides useful insights regarding the need to overcome the barrier of species-specific developmental timing in order to generate functional lungs in interspecies chimeras.

Perfused Platforms to Mimic Bone Microenvironment at the Macro/Milli/Microscale: Pros and Cons

As life expectancy increases, the population experiences progressive ageing. Ageing, in turn, is connected to an increase in bone-related diseases (i.e., osteoporosis and increased risk of fractures). Hence, the search for new approaches to study the occurrence of bone-related diseases and to develop new drugs for their prevention and treatment becomes more pressing. However, to date, a reliable in vitro model that can fully recapitulate the characteristics of bone tissue, either in physiological or altered conditions, is not available. Indeed, current methods for modelling normal and pathological bone are poor predictors of treatment outcomes in humans, as they fail to mimic the in vivo cellular microenvironment and tissue complexity. Bone, in fact, is a dynamic network including differently specialized cells and the extracellular matrix, constantly subjected to external and internal stimuli. To this regard, perfused vascularized models are a novel field of investigation that can offer a new technological approach to overcome the limitations of traditional cell culture methods. It allows the combination of perfusion, mechanical and biochemical stimuli, biological cues, biomaterials (mimicking the extracellular matrix of bone), and multiple cell types. This review will discuss macro, milli, and microscale perfused devices designed to model bone structure and microenvironment, focusing on the role of perfusion and encompassing different degrees of complexity. These devices are a very first, though promising, step for the development of 3D in vitro platforms for preclinical screening of novel anabolic or anti-catabolic therapeutic approaches to improve bone health.

The Intricate Interplay between Cell Cycle Regulators and Autophagy in Cancer

In the past decade, cell cycle regulators have extended their canonical role in cell cycle progression to the regulation of various cellular processes, including cellular metabolism. The regulation of metabolism is intimately connected with the function of autophagy, a catabolic process that promotes the efficient recycling of endogenous components from both extrinsic stress, e.g., nutrient deprivation, and intrinsic sub-lethal damage. Mediating cellular homeostasis and cytoprotection, autophagy is found to be dysregulated in numerous pathophysiological contexts, such as cancer. As an adaptative advantage, the upregulation of autophagy allows tumor cells to integrate stress signals, escaping multiple cell death mechanisms. Nevertheless, the precise role of autophagy during tumor development and progression remains highly context-dependent. Recently, multiple articles has suggested the importance of various cell cycle regulators in the modulation of autophagic processes. Here, we review the current clues indicating that cell-cycle regulators, including cyclin-dependent kinase inhibitors (CKIs), cyclin-dependent kinases (CDKs), and E2F transcription factors, are intrinsically linked to the regulation of autophagy. As an increasing number of studies highlight the importance of autophagy in cancer progression, we finally evoke new perspectives in therapeutic avenues that may include both cell cycle inhibitors and autophagy modulators to synergize antitumor efficacy.