A 2-month field cohort study of SARS-CoV-2 in saliva of BNT162b2 vaccinated nursing home workers.

Background: Nursing home (NH) residents have been severely affected during the COVID-19 pandemic because of their age and underlying comorbidities. Infection and outbreaks in NHs are most likely triggered by infected workers. Screening for asymptomatic NH workers can prevent risky contact and viral transmission to the residents. This study examined the effect of the BNT162b2 mRNA COVID­19 (Comirnaty®; BioNTech and Pfizer) vaccination on the saliva excretion of SARS-CoV-2 among NH workers, through weekly saliva RT-qPCR testing. Methods: A 2-month cohort study was conducted among 99 NHs in the Walloon region (Belgium), at the start of February 2021. Three groups of workers, i.e., non-vaccinated (n = 1618), one-dosed vaccinated (n = 1454), and two-dosed vaccinated (n = 2379) of BNT162b2 mRNA COVID­19 vaccine, were followed-up weekly. Their saliva samples were used to monitor the shedding of SARS-CoV-2. All positive samples were sequenced and genotyped to identify the circulating wild-type virus or variants of concern. Results: The protection fraction against the excretion of the SARS-CoV-2 in the saliva samples of the workers after the second dose is estimated at 0.90 (95% CI: 0.18; 0.99) at 1 week and 0.83 (95% CI: 0.54; 0.95) at 8 weeks. We observe more circulating SARS-CoV-2 and a greater variability of viral loads in the unvaccinated group compared to those of the vaccinated group. Conclusions: This field cohort study advances our knowledge of the efficacy of the mRNA BNT162b2 COVID-19 vaccine on the viral shedding in the saliva specimens of vaccinated NH workers, contributing to better decision-making in public health interventions and management.

Inhibition of the mitochondrial protein Opa1 curtails breast cancer growth.

BACKGROUND: Mitochondrial fusion and fission proteins have been nominated as druggable targets in cancer. Whether their inhibition is efficacious in triple negative breast cancer (TNBC) that almost invariably develops chemoresistance is unknown. METHODS: We used a combination of bioinformatics analyses of cancer genomic databases, genetic and pharmacological Optic Atrophy 1 (OPA1) inhibition, mitochondrial function and morphology measurements, micro-RNA (miRNA) profiling and formal epistatic analyses to address the role of OPA1 in TNBC proliferation, migration, and invasion in vitro and in vivo. RESULTS: We identified a signature of OPA1 upregulation in breast cancer that correlates with worse prognosis. Accordingly, OPA1 inhibition could reduce breast cancer cells proliferation, migration, and invasion in vitro and in vivo. Mechanistically, while OPA1 silencing did not reduce mitochondrial respiration, it increased levels of miRNAs of the 148/152 family known to inhibit tumor growth and invasiveness. Indeed, these miRNAs were epistatic to OPA1 in the regulation of TNBC cells growth and invasiveness. CONCLUSIONS: Our data show that targeted inhibition of the mitochondrial fusion protein OPA1 curtails TNBC growth and nominate OPA1 as a druggable target in TNBC.

Repetitive saliva-based mass screening as a tool for controlling SARS-CoV-2 transmission in nursing homes.

Nursing home (NH) residents and staff have been severely affected by the COVID-19 pandemic. The aim of this study was to examine the use of weekly saliva RT-qPCR testing for SARS-CoV-2 detection among NH workers as a strategy to control disease transmission within NHs in Belgium. From 16 November to 27 December 2020, a voluntary and anonymous weekly screening was implemented in a cohort of 50,000 workers across 572 NHs in the Walloon region of Belgium to detect asymptomatic cases of SARS-CoV-2 via saliva RT-qPCR testing and using the Diagenode saliva sample collection device. Positive workers were isolated to avoid subsequent infections in residents and other staff. RT-qPCR testing was based on pooled saliva sampling techniques from three workers, followed by individual testing of each positive or inconclusive pool. The majority of NHs (85%) and 55% of their workers participated. Pooling did not affect sensitivity as it only induced a very decrease in sensitivity estimated as 0.33%. Significant decreases in the prevalence (34.4-13.4%) and incidence of NHs with either single (13.8-2%) or multiple positive workers (3.7-0%) were observed over time. In addition, deaths among NH residents and NH worker absences decreased significantly over time. Weekly saliva RT-qPCR testing for SARS-CoV-2 demonstrated large-scale feasibility and efficacy in disrupting the chain of transmission. Implementation of this testing strategy in NHs could also be extended to other settings with the aim to control viral transmission for maintaining essential activities.

Extracellular Vesicles Mediate Communication between Endothelial and Vascular Smooth Muscle Cells.

The recruitment of pericytes and vascular smooth muscle cells (SMCs) that enwrap endothelial cells (ECs) is a crucial process for vascular maturation and stabilization. Communication between these two cell types is crucial during vascular development and in maintaining vessel homeostasis. Extracellular vesicles (EVs) have emerged as a new communication tool involving the exchange of microRNAs between cells. In the present study, we searched for microRNAs that could be transferred via EVs from ECs to SMCs and vice versa. Thanks to a microRNA profiling experiment, we found that two microRNAs are more exported in each cell type in coculture experiments: while miR-539 is more secreted by ECs, miR-582 is more present in EVs from SMCs. Functional assays revealed that both microRNAs can modulate both cell-type phenotypes. We further identified miR-539 and miR-582 targets, in agreement with their respective cell functions. The results obtained in vivo in the neovascularization model suggest that miR-539 and miR-582 might cooperate to trigger the process of blood vessel coverage by smooth muscle cells in a mature plexus. Taken together, these results are the first to highlight the role of miR-539 and miR-582 in angiogenesis and communication between ECs and SMCs.

Developmental and Tumor Angiogenesis Requires the Mitochondria-Shaping Protein Opa1.

While endothelial cell (EC) function is influenced by mitochondrial metabolism, the role of mitochondrial dynamics in angiogenesis, the formation of new blood vessels from existing vasculature, is unknown. Here we show that the inner mitochondrial membrane mitochondrial fusion protein optic atrophy 1 (OPA1) is required for angiogenesis. In response to angiogenic stimuli, OPA1 levels rapidly increase to limit nuclear factor kappa-light-chain-enhancer of activated B cell (NFκB) signaling, ultimately allowing angiogenic genes expression and angiogenesis. Endothelial Opa1 is indeed required in an NFκB-dependent pathway essential for developmental and tumor angiogenesis, impacting tumor growth and metastatization. A first-in-class small molecule-specific OPA1 inhibitor confirms that EC Opa1 can be pharmacologically targeted to curtail tumor growth. Our data identify Opa1 as a crucial component of physiological and tumor angiogenesis.

Tcf7l2 plays pleiotropic roles in the control of glucose homeostasis, pancreas morphology, vascularization and regeneration.

Type 2 diabetes (T2D) is a disease characterized by impaired insulin secretion. The Wnt signaling transcription factor Tcf7l2 is to date the T2D-associated gene with the largest effect on disease susceptibility. However, the mechanisms by which TCF7L2 variants affect insulin release from ß-cells are not yet fully understood. By taking advantage of a tcf7l2 zebrafish mutant line, we first show that these animals are characterized by hyperglycemia and impaired islet development. Moreover, we demonstrate that the zebrafish tcf7l2 gene is highly expressed in the exocrine pancreas, suggesting potential bystander effects on ß-cell growth, differentiation and regeneration. Finally, we describe a peculiar vascular phenotype in tcf7l2 mutant larvae, characterized by significant reduction in the average number and diameter of pancreatic islet capillaries. Overall, the zebrafish Tcf7l2 mutant, characterized by hyperglycemia, pancreatic and vascular defects, and reduced regeneration proves to be a suitable model to study the mechanism of action and the pleiotropic effects of Tcf7l2, the most relevant T2D GWAS hit in human populations.

Generation and application of signaling pathway reporter lines in zebrafish.

In the last years, we have seen the emergence of different tools that have changed the face of biology from a simple modeling level to a more systematic science. The transparent zebrafish embryo is one of the living models in which, after germline transformation with reporter protein-coding genes, specific fluorescent cell populations can be followed at single-cell resolution. The genetically modified embryos, larvae and adults, resulting from the transformation, are individuals in which time lapse analysis, digital imaging quantification, FACS sorting and next-generation sequencing can be performed in specific times and tissues. These multifaceted genetic and cellular approaches have permitted to dissect molecular interactions at the subcellular, intercellular, tissue and whole-animal level, thus allowing integration of cellular and developmental genetics with molecular imaging in the resulting frame of modern biology. In this review, we describe a new step in the zebrafish road to system biology, based on the use of transgenic biosensor animals expressing fluorescent proteins under the control of signaling pathway-responsive cis-elements. In particular, we provide here the rationale and details of this powerful tool, trying to focus on its huge potentialities in basic and applied research, while also discussing limits and potential technological evolutions of this approach.

Characterization and regulation of the hb9/mnx1 beta-cell progenitor specific enhancer in zebrafish.

Differentiation of insulin producing beta-cells is a genetically well defined process that involves functions of various conserved transcription factors. Still, the transcriptional mechanisms underlying specification and determination of beta-cell fate are poorly defined. Here we provide the description of a beta-cell progenitor specific enhancer as a model to study initial steps of beta-cell differentiation. We show that evolutionary non-conserved upstream sequences of the zebrafish hb9 gene are required and sufficient for regulating expression in beta-cells prior to the onset of insulin expression. This enhancer contains binding sites for paired-box transcription factors and two E-boxes that in EMSA studies show interaction with Pax6b and NeuroD, respectively. We show that Pax6b is a potent activator of endodermal hb9 expression and that this activation depends on the beta-cell enhancer. Using genetic approaches we show that pax6b is crucial for maintenance but not induction of pancreatic hb9 transcription. As loss of Pax6b or Hb9 independently results in the loss of insulin expression, the data reveal a novel cross-talk between the two essential regulators of early beta-cell differentiation. While we find that the known pancreatic E-box binding proteins NeuroD and Ngn3 are not required for hb9 expression we also show that removal of both E-boxes selectively eliminates pancreatic specific reporter expression. The data provide evidence for an Ngn3 independent pathway of beta-cell specification that requires function of currently not specified E-box binding factors.

The Pax6b homeodomain is dispensable for pancreatic endocrine cell differentiation in zebrafish.

Pax6 is a well conserved transcription factor that contains two DNA-binding domains, a paired domain and a homeodomain, and plays a key role in the development of eye, brain, and pancreas in vertebrates. The recent identification of the zebrafish sunrise mutant, harboring a mutation in the pax6b homeobox and presenting eye abnormalities but no obvious pancreatic defects, raised a question about the role of pax6b in zebrafish pancreas. We show here that pax6b does play an essential role in pancreatic endocrine cell differentiation, as revealed by the phenotype of a novel zebrafish pax6b null mutant and of embryos injected with pax6b morpholinos. Pax6b-depleted embryos have almost no beta cells, a strongly reduced number of delta cells, and a significant increase of epsilon cells. Through the use of various morpholinos targeting intron-exon junctions, pax6b RNA splicing was perturbed at several sites, leading either to retention of intronic sequences or to deletion of exonic sequences in the pax6b transcript. By this strategy, we show that deletion of the Pax6b homeodomain in zebrafish embryos does not disturb pancreas development, whereas lens formation is strongly affected. These data thus provide the explanation for the lack of pancreatic defects in the sunrise pax6b mutants. In addition, partial reduction of Pax6b function in zebrafish embryos performed by injection of small amounts of pax6b morpholinos caused a clear rise in alpha cell number and in glucagon expression, emphasizing the importance of the fine tuning of the Pax6b level to its biological activity.