Covid-19 in cystic fibrosis patients compared to the general population: Severity and virus-host cell interactions.

BACKGROUND: People with cystic fibrosis (pwCF) are considered at risk of developing severe forms of respiratory viral infections. We studied the consequences of COVID-19 and virus-host cell interactions in CF vs. non-CF individuals. METHODS: We enrolled CF and non-CF individuals, with /without COVID-like symptoms, who underwent nasopharyngeal swab for detection of SARS-CoV-2. Gene expression was evaluated by RNA sequencing on the same nasopharyngeal swabs. Criteria for COVID-19 severity were hospitalization and requirement or increased need of oxygen therapy. RESULTS: The study included 171 patients (65 pwCF and 106 non-CF individuals). Among them, 10 pwCF (15.4 %) and 43 people without CF (40.6 %) tested positive at RT-PCR. Symptomatic infections were observed in 8 pwCF (with 2 requiring hospitalization) and in 11 individuals without CF (6 requiring hospitalization). Host transcriptomic analysis revealed that genes involved in protein translation, particularly ribosomal components, were downregulated in CF samples irrespective of SARS-CoV-2 status. In SARS-CoV-2 negative individuals, we found a significant difference in genes involved with motile cilia expression and function, which were upregulated in CF samples. Pathway enrichment analysis indicated that interferon signaling in response to SARS-CoV-2 infection was upregulated in both pwCF and non-CF subjects. CONCLUSIONS: COVID-19 does not seem to be more severe in CF, possibly due to factors intrinsic to this population: the lower expression of ribosomal genes may downregulate the protein translation machinery, thus creating an unfavorable environment for viral replication.

Single-cell guided prenatal derivation of primary fetal epithelial organoids from human amniotic and tracheal fluids.

Isolation of tissue-specific fetal stem cells and derivation of primary organoids is limited to samples obtained from termination of pregnancies, hampering prenatal investigation of fetal development and congenital diseases. Therefore, new patient-specific in vitro models are needed. To this aim, isolation and expansion of fetal stem cells during pregnancy, without the need for tissue samples or reprogramming, would be advantageous. Amniotic fluid (AF) is a source of cells from multiple developing organs. Using single-cell analysis, we characterized the cellular identities present in human AF. We identified and isolated viable epithelial stem/progenitor cells of fetal gastrointestinal, renal and pulmonary origin. Upon culture, these cells formed clonal epithelial organoids, manifesting small intestine, kidney tubule and lung identity. AF organoids exhibit transcriptomic, protein expression and functional features of their tissue of origin. With relevance for prenatal disease modeling, we derived lung organoids from AF and tracheal fluid cells of congenital diaphragmatic hernia fetuses, recapitulating some features of the disease. AF organoids are derived in a timeline compatible with prenatal intervention, potentially allowing investigation of therapeutic tools and regenerative medicine strategies personalized to the fetus at clinically relevant developmental stages.

Cellular population dynamics shape the route to human pluripotency.

Human cellular reprogramming to induced pluripotency is still an inefficient process, which has hindered studying the role of critical intermediate stages. Here we take advantage of high efficiency reprogramming in microfluidics and temporal multi-omics to identify and resolve distinct sub-populations and their interactions. We perform secretome analysis and single-cell transcriptomics to show functional extrinsic pathways of protein communication between reprogramming sub-populations and the re-shaping of a permissive extracellular environment. We pinpoint the HGF/MET/STAT3 axis as a potent enhancer of reprogramming, which acts via HGF accumulation within the confined system of microfluidics, and in conventional dishes needs to be supplied exogenously to enhance efficiency. Our data suggest that human cellular reprogramming is a transcription factor-driven process that it is deeply dependent on extracellular context and cell population determinants.

Esrrb guides naive pluripotent cells through the formative transcriptional programme.

During embryonic development, naive pluripotent epiblast cells transit to a formative state. The formative epiblast cells form a polarized epithelium, exhibit distinct transcriptional and epigenetic profiles and acquire competence to differentiate into all somatic and germline lineages. However, we have limited understanding of how the transition to a formative state is molecularly controlled. Here we used murine embryonic stem cell models to show that ESRRB is both required and sufficient to activate formative genes. Genetic inactivation of Esrrb leads to illegitimate expression of mesendoderm and extra-embryonic markers, impaired formative expression and failure to self-organize in 3D. Functionally, this results in impaired ability to generate formative stem cells and primordial germ cells in the absence of Esrrb. Computational modelling and genomic analyses revealed that ESRRB occupies key formative genes in naive cells and throughout the formative state. In so doing, ESRRB kickstarts the formative transition, leading to timely and unbiased capacity for multi-lineage differentiation.

EGR1 drives cell proliferation by directly stimulating TFEB transcription in response to starvation.

The stress-responsive transcription factor EB (TFEB) is a master controller of lysosomal biogenesis and autophagy and plays a major role in several cancer-associated diseases. TFEB is regulated at the posttranslational level by the nutrient-sensitive kinase complex mTORC1. However, little is known about the regulation of TFEB transcription. Here, through integrative genomic approaches, we identify the immediate-early gene EGR1 as a positive transcriptional regulator of TFEB expression in human cells and demonstrate that, in the absence of EGR1, TFEB-mediated transcriptional response to starvation is impaired. Remarkably, both genetic and pharmacological inhibition of EGR1, using the MEK1/2 inhibitor Trametinib, significantly reduced the proliferation of 2D and 3D cultures of cells displaying constitutive activation of TFEB, including those from a patient with Birt-Hogg-Dubé (BHD) syndrome, a TFEB-driven inherited cancer condition. Overall, we uncover an additional layer of TFEB regulation consisting in modulating its transcription via EGR1 and propose that interfering with the EGR1-TFEB axis may represent a therapeutic strategy to counteract constitutive TFEB activation in cancer-associated conditions.

Improved SARS-CoV-2 sequencing surveillance allows the identification of new variants and signatures in infected patients.

BACKGROUND: Genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the only approach to rapidly monitor and tackle emerging variants of concern (VOC) of the COVID-19 pandemic. Such scrutiny is crucial to limit the spread of VOC that might escape the immune protection conferred by vaccination strategies or previous virus exposure. It is also becoming clear now that efficient genomic surveillance would require monitoring of the host gene expression to identify prognostic biomarkers of treatment efficacy and disease progression. Here we propose an integrative workflow to both generate thousands of SARS-CoV-2 genome sequences per week and analyze host gene expression upon infection. METHODS: In this study we applied an integrated workflow for RNA extracted from nasal swabs to obtain in parallel the full genome of SARS-CoV-2 and transcriptome of host respiratory epithelium. The RNA extracted from each sample was reverse transcribed and the viral genome was specifically enriched through an amplicon-based approach. The very same RNA was then used for patient transcriptome analysis. Samples were collected in the Campania region, Italy, for viral genome sequencing. Patient transcriptome analysis was performed on about 700 samples divided into two cohorts of patients, depending on the viral variant detected (B.1 or delta). RESULTS: We sequenced over 20,000 viral genomes since the beginning of the pandemic, producing the highest number of sequences in Italy. We thus reconstructed the pandemic dynamics in the regional territory from March 2020 to December 2021. In addition, we have matured and applied novel proof-of-principle approaches to prioritize possible gain-of-function mutations by leveraging patients' metadata and isolated patient-specific signatures of SARS-CoV-2 infection. This allowed us to (i) identify three new viral variants that specifically originated in the Campania region, (ii) map SARS-CoV-2 intrahost variability during long-term infections and in one case identify an increase in the number of mutations in the viral genome, and (iii) identify host gene expression signatures correlated with viral load in upper respiratory ways. CONCLUSION: In conclusion, we have successfully generated an optimized and cost-effective strategy to monitor SARS-CoV-2 genetic variability, without the need of automation. Thus, our approach is suitable for any lab with a benchtop sequencer and a limited budget, allowing an integrated genomic surveillance on premises. Finally, we have also identified a gene expression signature defining SARS-CoV-2 infection in real-world patients' upper respiratory ways.

The role of NSP6 in the biogenesis of the SARS-CoV-2 replication organelle.

SARS-CoV-2, like other coronaviruses, builds a membrane-bound replication organelle to enable RNA replication1. The SARS-CoV-2 replication organelle is composed of double-membrane vesicles (DMVs) that are tethered to the endoplasmic reticulum (ER) by thin membrane connectors2, but the viral proteins and the host factors involved remain unknown. Here we identify the viral non-structural proteins (NSPs) that generate the SARS-CoV-2 replication organelle. NSP3 and NSP4 generate the DMVs, whereas NSP6, through oligomerization and an amphipathic helix, zippers ER membranes and establishes the connectors. The NSP6(ΔSGF) mutant, which arose independently in the Alpha, Beta, Gamma, Eta, Iota and Lambda variants of SARS-CoV-2, behaves as a gain-of-function mutant with a higher ER-zippering activity. We identified three main roles for NSP6: first, to act as a filter in communication between the replication organelle and the ER, by allowing lipid flow but restricting the access of ER luminal proteins to the DMVs; second, to position and organize DMV clusters; and third, to mediate contact with lipid droplets (LDs) through the LD-tethering complex DFCP1-RAB18. NSP6 thus acts as an organizer of DMV clusters and can provide a selective means of refurbishing them with LD-derived lipids. Notably, both properly formed NSP6 connectors and LDs are required for the replication of SARS-CoV-2. Our findings provide insight into the biological activity of NSP6 of SARS-CoV-2 and of other coronaviruses, and have the potential to fuel the search for broad antiviral agents.

Synchronization between peripheral circadian clock and feeding-fasting cycles in microfluidic device sustains oscillatory pattern of transcriptome.

The circadian system cyclically regulates many physiological and behavioral processes within the day. Desynchronization between physiological and behavioral rhythms increases the risk of developing some, including metabolic, disorders. Here we investigate how the oscillatory nature of metabolic signals, resembling feeding-fasting cycles, sustains the cell-autonomous clock in peripheral tissues. By controlling the timing, period and frequency of glucose and insulin signals via microfluidics, we find a strong effect on Per2::Luc fibroblasts entrainment. We show that the circadian Per2 expression is better sustained via a 24 h period and 12 h:12 h frequency-encoded metabolic stimulation applied for 3 daily cycles, aligned to the cell-autonomous clock, entraining the expression of hundreds of genes mostly belonging to circadian rhythms and cell cycle pathways. On the contrary misaligned feeding-fasting cycles synchronize and amplify the expression of extracellular matrix-associated genes, aligned during the light phase. This study underlines the role of the synchronicity between life-style-associated metabolic signals and peripheral clocks on the circadian entrainment.

Single-Cell RNA Sequencing Analysis: A Step-by-Step Overview.

Thanks to innovative sample-preparation and sequencing technologies, gene expression in individual cells can now be measured for thousands of cells in a single experiment. Since its introduction, single-cell RNA sequencing (scRNA-seq) approaches have revolutionized the genomics field as they created unprecedented opportunities for resolving cell heterogeneity by exploring gene expression profiles at a single-cell resolution. However, the rapidly evolving field of scRNA-seq invoked the emergence of various analytics approaches aimed to maximize the full potential of this novel strategy. Unlike population-based RNA sequencing approaches, scRNA seq necessitates comprehensive computational tools to address high data complexity and keep up with the emerging single-cell associated challenges. Despite the vast number of analytical methods, a universal standardization is lacking. While this reflects the fields' immaturity, it may also encumber a newcomer to blend in.In this review, we aim to bridge over the abovementioned hurdle and propose four ready-to-use pipelines for scRNA-seq analysis easily accessible by a newcomer, that could fit various biological data types. Here we provide an overview of the currently available single-cell technologies for cell isolation and library preparation and a step by step guide that covers the entire canonical analytic workflow to analyse scRNA-seq data including read mapping, quality controls, gene expression quantification, normalization, feature selection, dimensionality reduction, and cell clustering useful for trajectory inference and differential expression. Such workflow guidelines will escort novices as well as expert users in the analysis of complex scRNA-seq datasets, thus further expanding the research potential of single-cell approaches in basic science, and envisaging its future implementation as best practice in the field.

Epigenomic landscape of human colorectal cancer unveils an aberrant core of pan-cancer enhancers orchestrated by YAP/TAZ.

Cancer is characterized by pervasive epigenetic alterations with enhancer dysfunction orchestrating the aberrant cancer transcriptional programs and transcriptional dependencies. Here, we epigenetically characterize human colorectal cancer (CRC) using de novo chromatin state discovery on a library of different patient-derived organoids. By exploring this resource, we unveil a tumor-specific deregulated enhancerome that is cancer cell-intrinsic and independent of interpatient heterogeneity. We show that the transcriptional coactivators YAP/TAZ act as key regulators of the conserved CRC gained enhancers. The same YAP/TAZ-bound enhancers display active chromatin profiles across diverse human tumors, highlighting a pan-cancer epigenetic rewiring which at single-cell level distinguishes malignant from normal cell populations. YAP/TAZ inhibition in established tumor organoids causes extensive cell death unveiling their essential role in tumor maintenance. This work indicates a common layer of YAP/TAZ-fueled enhancer reprogramming that is key for the cancer cell state and can be exploited for the development of improved therapeutic avenues.