MicroRNAs (miRNAs) contribute to the progressive changes in gene expression that occur during development. The combined loss of all miRNAs results in embryonic lethality in all animals analyzed, illustrating the crucial role that miRNAs play collectively. However, although the loss of some individual miRNAs also results in severe developmental defects, the roles of many other miRNAs have been challenging to uncover. This has been mostly attributed to their proposed function as tuners of gene expression or providers of robustness. Here, we present a view of miRNAs in the context of development as a hierarchical and canalized series of gene regulatory networks. In this scheme, only a fraction of embryonic miRNAs act at the top of this hierarchy, with their loss resulting in broad developmental defects, whereas most other miRNAs are expressed with high cellular specificity and play roles at the periphery of development, affecting the terminal features of specialized cells. This view could help to shed new light on our understanding of miRNA function in development, disease and evolution.

MicroRNAs (miRNAs) play an essential role in the post-transcriptional regulation of animal development and physiology. However, in vivo studies aimed at linking miRNA-function to the biology of distinct cell types within complex tissues remain challenging, partly because in vivo miRNA-profiling methods lack cellular resolution. We report microRNome by methylation-dependent sequencing (mime-seq), an in vivo enzymatic small RNA-tagging approach that enables high-throughput sequencing of tissue- and cell-type-specific miRNAs in animals. The method combines cell-type-specific 3´-terminal 2´-O-methylation of animal miRNAs by a genetically encoded, plant-specific methyltransferase (HEN1), with chemoselective small RNA cloning and high-throughput sequencing. We show that mime-seq uncovers the miRNomes of specific cells within C. elegans and Drosophila at unprecedented specificity and sensitivity, enabling miRNA profiling with single-cell resolution in whole animals. Mime-seq overcomes current challenges in cell-type-specific small RNA profiling and provides novel entry points for understanding the function of miRNAs in spatially restricted physiological settings.

The epidermal growth factor receptor (EGFR), a member of the ErbB family of receptor tyrosine kinases, is expressed in up to 70% of epithelial ovarian cancers (EOCs), where it correlates with poor prognosis. The majority of EOCs are diagnosed at an advanced stage, and at least 50% present malignant ascites. High levels of IL-6 have been found in the ascites of EOC patients and correlate with shorter survival. Herein, we investigated the signaling cascade led by EGFR activation in EOC and assessed whether EGFR activation could induce an EOC microenvironment characterized by pro-inflammatory molecules. In vitro analysis of EOC cell lines revealed that ligand-stimulated EGFR activated NFkB-dependent transcription and induced secretion of IL-6 and plasminogen activator inhibitor (PAI-1). IL-6/PAI-1 expression and secretion were strongly inhibited by the tyrosine kinase inhibitor AG1478 and EGFR silencing. A significant reduction of EGF-stimulated IL-6/PAI-1 secretion was also obtained with the NFkB inhibitor dehydroxymethylepoxyquinomicin. Of 23 primary EOC tumors from advanced-stage patients with malignant ascites at surgery, 12 co-expressed membrane EGFR, IL-6 and PAI-1 by immunohistochemistry; both IL-6 and PAI-1 were present in 83% of the corresponding ascites. Analysis of a publicly available gene-expression data set from 204 EOCs confirmed a significant correlation between IL-6 and PAI-1 expression, and patients with the highest IL-6 and PAI-1 co-expression showed a significantly shorter progression-free survival time (P ¼ 0.028). This suggests that EGFR/NFkB/IL-6-PAI-1 may have a significant impact on the therapy of a particular subset of EOC, and that IL-6/PAI-1 co-expression may be a novel prognostic marker.

Immune profiling of COVID-19 patients has identified numerous alterations in both innate and adaptive immunity. However, whether those changes are specific to SARS-CoV-2 or driven by a general inflammatory response shared across severely ill pneumonia patients remains unknown. Here, we compared the immune profile of severe COVID-19 with non-SARS-CoV-2 pneumonia ICU patients using longitudinal, high-dimensional single-cell spectral cytometry and algorithm-guided analysis. COVID-19 and non-SARS-CoV-2 pneumonia both showed increased emergency myelopoiesis and displayed features of adaptive immune paralysis. However, pathological immune signatures suggestive of T cell exhaustion were exclusive to COVID-19. The integration of single-cell profiling with a predicted binding capacity of SARS-CoV-2-petides to the patients’ HLA profile further linked the COVID-19 immunopathology to impaired virus recognition. Towards clinical translation, circulating NKT cell frequency was identified as a predictive biomarker for patient outcome. Our comparative immune map serves to delineate treatment strategies to interfere with the immunopathologic cascade exclusive to severe COVID-19.

We previously found that neurons are able to affect the ability of brain capillary endothelial cells to form in vitro a monolayer with properties resembling the blood-brain barrier. We then looked, by immunofluorescence and western analysis, for factors, produced by neurons, with the potential to influence growth and differentiation of endothelial cells. In the present paper, we report that neurons produce both vascular endothelial growth factor and fibroblast growth factor 2, two well-known angiogenic factors. More interestingly, we gained evidence that both factors are released by neurons, at least in part, by shedding of extracellular vesicles, that contain β1 integrin, a membrane protein already known to be part of extracellular vesicles released by tumour cells. Shedding of extracellular vesicles by neurons was also confirmed by scanner electron microscopy.

Anti-CCR5 Abs shed light on the immunological mechanisms involved in the control of HIV-1 infection in a model that can be considered an experimentum naturae for resistance to HIV. They could be useful in the design of new strategies against HIV infection at mucosal sites.

No abstract

The transcription factor Pax5 controls B cell development, but its role in mature B cells is largely enigmatic. Here, we demonstrated that the loss of Pax5 by conditional mutagenesis in peripheral B lymphocytes led to the strong reduction of B-1a, marginal zone (MZ), and germinal center (GC) B cells as well as plasma cells. Follicular (FO) B cells tolerated the loss of Pax5 but had a shortened half-life. The Pax5-deficient FO B cells failed to proliferate upon B cell receptor or Toll-like receptor stimulation due to impaired PI3K-AKT signaling, which was caused by increased expression of PTEN, a negative regulator of the PI3K pathway. Pax5 restrained PTEN protein expression at the posttranscriptional level, likely involving Pten-targeting microRNAs. Additional PTEN loss in Pten,Pax5 double-mutant mice rescued FO B cell numbers and the development of MZ B cells but did not restore GC B cell formation. Hence, the posttranscriptional down-regulation of PTEN expression is an important function of Pax5 that facilitates the differentiation and survival of mature B cells, thereby promoting humoral immunity.