p53 Regulates the Extent of Fibroblast Proliferation and Fibrosis in Left Ventricle Pressure Overload.

BACKGROUND: Cardiomyopathy is characterized by the pathological accumulation of resident cardiac fibroblasts that deposit ECM (extracellular matrix) and generate a fibrotic scar. However, the mechanisms that control the timing and extent of cardiac fibroblast proliferation and ECM production are not known, hampering the development of antifibrotic strategies to prevent heart failure. METHODS: We used the Tcf21 (transcription factor 21)MerCreMer mouse line for fibroblast-specific lineage tracing and p53 (tumor protein p53) gene deletion. We characterized cardiac physiology and used single-cell RNA-sequencing and in vitro studies to investigate the p53-dependent mechanisms regulating cardiac fibroblast cell cycle and fibrosis in left ventricular pressure overload induced by transaortic constriction. RESULTS: Cardiac fibroblast proliferation occurs primarily between days 7 and 14 following transaortic constriction in mice, correlating with alterations in p53-dependent gene expression. p53 deletion in fibroblasts led to a striking accumulation of Tcf21-lineage cardiac fibroblasts within the normal proliferative window and precipitated a robust fibrotic response to left ventricular pressure overload. However, excessive interstitial and perivascular fibrosis does not develop until after cardiac fibroblasts exit the cell cycle. Single-cell RNA sequencing revealed p53 null fibroblasts unexpectedly express lower levels of genes encoding important ECM proteins while they exhibit an inappropriately proliferative phenotype. in vitro studies establish a role for p53 in suppressing the proliferative fibroblast phenotype, which facilitates the expression and secretion of ECM proteins. Importantly, Cdkn2a (cyclin-dependent kinase inhibitor 2a) expression and the p16Ink4a-retinoblastoma cell cycle control pathway is induced in p53 null cardiac fibroblasts, which may eventually contribute to cell cycle exit and fulminant scar formation. CONCLUSIONS: This study reveals a mechanism regulating cardiac fibroblast accumulation and ECM secretion, orchestrated in part by p53-dependent cell cycle control that governs the timing and extent of fibrosis in left ventricular pressure overload.

A riboswitch separated from its ribosome-binding site still regulates translation.

Riboswitches regulate downstream gene expression by binding cellular metabolites. Regulation of translation initiation by riboswitches is posited to occur by metabolite-mediated sequestration of the Shine-Dalgarno sequence (SDS), causing bypass by the ribosome. Recently, we solved a co-crystal structure of a prequeuosine1-sensing riboswitch from Carnobacterium antarcticum that binds two metabolites in a single pocket. The structure revealed that the second nucleotide within the gene-regulatory SDS, G34, engages in a crystal contact, obscuring the molecular basis of gene regulation. Here, we report a co-crystal structure wherein C10 pairs with G34. However, molecular dynamics simulations reveal quick dissolution of the pair, which fails to reform. Functional and chemical probing assays inside live bacterial cells corroborate the dispensability of the C10-G34 pair in gene regulation, leading to the hypothesis that the compact pseudoknot fold is sufficient for translation attenuation. Remarkably, the C. antarcticum aptamer retained significant gene-regulatory activity when uncoupled from the SDS using unstructured spacers up to 10 nucleotides away from the riboswitch-akin to steric-blocking employed by sRNAs. Accordingly, our work reveals that the RNA fold regulates translation without SDS sequestration, expanding known riboswitch-mediated gene-regulatory mechanisms. The results infer that riboswitches exist wherein the SDS is not embedded inside a stable fold.

Bulk RNA sequencing of human pediatric lung cell populations reveals unique transcriptomic signature associated with postnatal pulmonary development.

Postnatal lung development results in an increasingly functional organ prepared for gas exchange and pathogenic challenges. It is achieved through cellular differentiation and migration. Changes in the tissue architecture during this development process are well-documented and increasing cellular diversity associated with it are reported in recent years. Despite recent progress, transcriptomic and molecular pathways associated with human postnatal lung development are yet to be fully understood. In this study, we investigated gene expression patterns associated with healthy pediatric lung development in four major enriched cell populations (epithelial, endothelial, and nonendothelial mesenchymal cells, along with lung leukocytes) from 1-day-old to 8-yr-old organ donors with no known lung disease. For analysis, we considered the donors in four age groups [less than 30 days old neonates, 30 days to < 1 yr old infants, toddlers (1 to < 2 yr), and children 2 yr and older] and assessed differentially expressed genes (DEG). We found increasing age-associated transcriptional changes in all four major cell types in pediatric lung. Transition from neonate to infant stage showed highest number of DEG compared with the number of DEG found during infant to toddler- or toddler to older children-transitions. Profiles of differential gene expression and further pathway enrichment analyses indicate functional epithelial cell maturation and increased capability of antigen presentation and chemokine-mediated communication. Our study provides a comprehensive reference of gene expression patterns during healthy pediatric lung development that will be useful in identifying and understanding aberrant gene expression patterns associated with early life respiratory diseases.NEW & NOTEWORTHY This study presents postnatal transcriptomic changes in major cell populations in human lung, namely endothelial, epithelial, mesenchymal cells, and leukocytes. Although human postnatal lung development continues through early adulthood, our results demonstrate that greatest transcriptional changes occur in first few months of life during neonate to infant transition. These early transcriptional changes in lung parenchyma are particularly notable for functional maturation and activation of alveolar type II cell genes.

B Cell Activation and Plasma Cell Differentiation Are Promoted by IFN-λ in Systemic Lupus Erythematosus.

Type I IFN is essential for viral clearance but also contributes to the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus (SLE), via aberrant nucleic acid-sensing pathways, leading to autoantibody production. Type III IFN (IFN-λ) is now appreciated to have a nonredundant role in viral infection, but few studies have addressed the effects of IFN-λ on immune cells given the more restricted expression of its receptor primarily to the epithelium. In this study, we demonstrate that B cells display a prominent IFN gene expression profile in patients with lupus. Serum levels of IFN-λ are elevated in SLE and positively correlate with B cell subsets associated with autoimmune plasma cell development, including CD11c+T-bet+CD21- B cells. Although B cell subsets express all IFN receptors, IFNLR1 strongly correlates with the CD11c+CD21- B cell expansion, suggesting that IFN-λ may be an unappreciated driver of the SLE IFN signature and B cell abnormalities. We show that IFN-λ potentiates gene transcription in human B cells typically attributed to type I IFN as well as expansion of T-bet-expressing B cells after BCR and TLR7/8 stimulation. Further, IFN-λ promotes TLR7/8-mediated plasmablast differentiation and increased IgM production. CD11c+ B cells demonstrate IFN-λ hyperresponsive signaling compared with other B cell subsets, suggesting that IFN-λ accelerates plasma cell differentiation through this putative extrafollicular pathway. In summary, our data support type III IFN-λ as a cytokine promoting the Ab-secreting cell pool in human viral and autoimmune disease.

Analysis of a preQ1-I riboswitch in effector-free and bound states reveals a metabolite-programmed nucleobase-stacking spine that controls gene regulation.

Riboswitches are structured RNA motifs that recognize metabolites to alter the conformations of downstream sequences, leading to gene regulation. To investigate this molecular framework, we determined crystal structures of a preQ1-I riboswitch in effector-free and bound states at 2.00 Å and 2.65 Å-resolution. Both pseudoknots exhibited the elusive L2 loop, which displayed distinct conformations. Conversely, the Shine-Dalgarno sequence (SDS) in the S2 helix of each structure remained unbroken. The expectation that the effector-free state should expose the SDS prompted us to conduct solution experiments to delineate environmental changes to specific nucleobases in response to preQ1. We then used nudged elastic band computational methods to derive conformational-change pathways linking the crystallographically-determined effector-free and bound-state structures. Pathways featured: (i) unstacking and unpairing of L2 and S2 nucleobases without preQ1-exposing the SDS for translation and (ii) stacking and pairing L2 and S2 nucleobases with preQ1-sequestering the SDS. Our results reveal how preQ1 binding reorganizes L2 into a nucleobase-stacking spine that sequesters the SDS, linking effector recognition to biological function. The generality of stacking spines as conduits for effector-dependent, interdomain communication is discussed in light of their existence in adenine riboswitches, as well as the turnip yellow mosaic virus ribosome sensor.

Ontogenetic shifts in the nesting behaviour of female crocodiles.

Body size and age are crucial factors influencing reproductive capacity and success. As females grow, their reproductive investment and success often increase due to improved overall physiological condition and experience gained through successive reproductive events. While much of this work has been conducted on birds and mammals, surprisingly little is known on how body size affects nesting decisions in other long-lived vertebrates. We monitored the movements and nesting behaviour of 57 wild female estuarine crocodiles Crocodylus porosus over a 10-year period (and across consecutive nesting seasons) using externally mounted satellite tags, implanted acoustic transmitters and a network of submerged acoustic receivers. Applying Hidden Markov models to the telemetry-derived location data revealed that female nesting behaviours could be split into three distinct states: (i) ranging movements within home ranges and at nesting sites; (ii) migrations to and from nesting sites; (iii) and nesting/nest guarding. We found that during migration events, larger females migrated further and remained away from dry season territories for longer periods than smaller individuals. Furthermore, not only were migratory movements stimulated by increases in rainfall, larger females migrated to nest sites at lower rainfall thresholds than smaller females. We provide some of the first evidence of body size influencing nesting decisions in an ectothermic vertebrate, with shifts likely resulting from an increased willingness to invest in nest protection among larger and more experienced females.

Automated protein motif generation in the structure-based protein function prediction tool ProMOL.

ProMOL, a plugin for the PyMOL molecular graphics system, is a structure-based protein function prediction tool. ProMOL includes a set of routines for building motif templates that are used for screening query structures for enzyme active sites. Previously, each motif template was generated manually and required supervision in the optimization of parameters for sensitivity and selectivity. We developed an algorithm and workflow for the automation of motif building and testing routines in ProMOL. The algorithm uses a set of empirically derived parameters for optimization and requires little user intervention. The automated motif generation algorithm was first tested in a performance comparison with a set of manually generated motifs based on identical active sites from the same 112 PDB entries. The two sets of motifs were equally effective in identifying alignments with homologs and in rejecting alignments with unrelated structures. A second set of 296 active site motifs were generated automatically, based on Catalytic Site Atlas entries with literature citations, as an expansion of the library of existing manually generated motif templates. The new motif templates exhibited comparable performance to the existing ones in terms of hit rates against native structures, homologs with the same EC and Pfam designations, and randomly selected unrelated structures with a different EC designation at the first EC digit, as well as in terms of RMSD values obtained from local structural alignments of motifs and query structures. This research is supported by NIH grant GM078077.

RETRACTED: Reduced self-harm on acute mental health wards following the implementation of a vision-based patient monitoring system: Evidence from five NHS trusts.

WHAT IS KNOWN ON THE SUBJECT?: Inpatient mental health settings pose unique challenges for patient safety, in part because psychiatric disorders are well-known risk factors for self-harm and suicide. Ward staff follow protocols to try and keep patients safe including carrying out in-person safety checks, usually every 15 min, but patients unfortunately still find opportunities to harm themselves. A vision-based patient monitoring system (VBPMS) is a contact-free technology that can help mental health nurses to monitor patients in hospital more effectively. For example, the tool alerts staff to situations where a patient might need assistance, like when they spend a long period of time in their ensuite bathroom. Research has shown that the VBPMS can support staff to prevent safety incidents from occurring and a recent study found a decrease in self-harm after the tool was implemented at one NHS mental health trust. WHAT DOES THE PAPER ADD TO EXISTING KNOWLEDGE?: This paper presents data from five NHS mental health trusts and suggests that the VBPMS can consistently help staff to prevent self-harm on acute mental health wards. WHAT ARE THE IMPLICATIONS FOR PRACTICE?: Managing self-harm behaviours is a challenge for mental health nursing staff. Standard safety protocols are not effective enough - staff need new ways to monitor patients. In general hospitals, technology is used every day to support care. This research shows that the VBPMS can be used to improve patient safety in mental health hospitals. ABSTRACT: Introduction Self-harm is sadly relatively common in mental health inpatient settings, and the effectiveness and appropriateness of strategies used to prevent it have long been disputed. The use of vision-based patient monitoring systems (VBPMS) has been shown to improve various safety outcomes. Their potential to support staff in preventing inpatient self-harm warrants further exploration. Aim This research investigated whether implementing a VBPMS-as an adjunct to existing clinical practice-within acute inpatient services at five NHS England mental health trusts led to reductions in self-harm. Method Five quasi-experimental studies assessed the number of bedroom self-harm incidents, across 12 adult wards, before and 5-12 months after a VBPMS was deployed. Percentage changes were calculated. Four studies used control groups and were included in a fixed-effect meta-analysis. Results In the meta-analysis, a significant inverse-weighted average relative risk percentage change of -38.9% was recorded. For the study with no control groups, reductions in self-harm were seen across all wards (range: -9.1% to -29.8%). Discussion This research demonstrates that VBPMS can support clinicians to create safer ward environments by preventing incidents of self-harm from occurring. Implications for Practice The results provide preliminary support for wider rollout of the technology on acute mental health wards.

Corrigendum: Single cell RNA sequencing analysis of mouse cochlear supporting cell transcriptomes with activated ERBB2 receptor indicates a cell-specific response that promotes CD44 activation.

[This corrects the article DOI: 10.3389/fncel.2022.1096872.].

Temporal Single Cell Analysis of Leukemia Microenvironment Identifies Taurine-Taurine Transporter Axis as a Key Regulator of Myeloid Leukemia.

Signals from the microenvironment are known to be critical for development, sustaining adult stem cells, and for oncogenic progression. While candidate niche-driven signals that can promote cancer progression have been identified1-6, concerted efforts to comprehensively map microenvironmental ligands for cancer stem cell specific surface receptors have been lacking. Here, we use temporal single cell RNA-sequencing to identify molecular cues from the bone marrow stromal niche that engage leukemia stem cells (LSC) during oncogenic progression. We integrate these data with our RNA-seq analysis of human LSCs from distinct aggressive myeloid cancer subtypes and our CRISPR based in vivo LSC dependency map7 to develop a temporal receptor-ligand interactome essential for disease progression. These analyses identify the taurine transporter (TauT)-taurine axis as a critical dependency of myeloid malignancies. We show that taurine production is restricted to the osteolineage population during cancer initiation and expansion. Inhibiting taurine synthesis in osteolineage cells impairs LSC growth and survival. Our experiments with the TauT genetic loss of function murine model indicate that its loss significantly impairs the progression of aggressive myeloid leukemias in vivo by downregulating glycolysis. Further, TauT inhibition using a small molecule strongly impairs the growth and survival of patient derived myeloid leukemia cells. Finally, we show that TauT inhibition can synergize with the clinically approved oxidative phosphorylation inhibitor venetoclax8, 9 to block the growth of primary human leukemia cells. Given that aggressive myeloid leukemias continue to be refractory to current therapies and have poor prognosis, our work indicates targeting the taurine transporter may be of therapeutic significance. Collectively, our data establishes a temporal landscape of stromal signals during cancer progression and identifies taurine-taurine transporter signaling as an important new regulator of myeloid malignancies.

Single-Cell Transcriptomic Profiling Identifies Molecular Phenotypes of Newborn Human Lung Cells.

While animal model studies have extensively defined the mechanisms controlling cell diversity in the developing mammalian lung, there exists a significant knowledge gap with regards to late-stage human lung development. The NHLBI Molecular Atlas of Lung Development Program (LungMAP) seeks to fill this gap by creating a structural, cellular and molecular atlas of the human and mouse lung. Transcriptomic profiling at the single-cell level created a cellular atlas of newborn human lungs. Frozen single-cell isolates obtained from two newborn human lungs from the LungMAP Human Tissue Core Biorepository, were captured, and library preparation was completed on the Chromium 10X system. Data was analyzed in Seurat, and cellular annotation was performed using the ToppGene functional analysis tool. Transcriptional interrogation of 5500 newborn human lung cells identified distinct clusters representing multiple populations of epithelial, endothelial, fibroblasts, pericytes, smooth muscle, immune cells and their gene signatures. Computational integration of data from newborn human cells and with 32,000 cells from postnatal days 1 through 10 mouse lungs generated by the LungMAP Cincinnati Research Center facilitated the identification of distinct cellular lineages among all the major cell types. Integration of the newborn human and mouse cellular transcriptomes also demonstrated cell type-specific differences in maturation states of newborn human lung cells. Specifically, newborn human lung matrix fibroblasts could be separated into those representative of younger cells (n = 393), or older cells (n = 158). Cells with each molecular profile were spatially resolved within newborn human lung tissue. This is the first comprehensive molecular map of the cellular landscape of neonatal human lung, including biomarkers for cells at distinct states of maturity.

13Ccarbene nuclear magnetic resonance chemical shift analysis confirms CeIV[double bond, length as m-dash]C double bonding in cerium(iv)-diphosphonioalkylidene complexes.

Diphosphonioalkylidene dianions have emerged as highly effective ligands for lanthanide and actinide ions, and the resulting formal metal-carbon double bonds have challenged and developed conventional thinking about f-element bond multiplicity and covalency. However, f-element-diphosphonioalkylidene complexes can be represented by several resonance forms that render their metal-carbon double bond status unclear. Here, we report an experimentally-validated 13C Nuclear Magnetic Resonance computational assessment of two cerium(iv)-diphosphonioalkylidene complexes, [Ce(BIPMTMS)(ODipp)2] (1, BIPMTMS = {C(PPh2NSiMe3)2}2-; Dipp = 2,6-diisopropylphenyl) and [Ce(BIPMTMS)2] (2). Decomposing the experimental alkylidene chemical shifts into their corresponding calculated shielding (σ) tensor components verifies that these complexes exhibit Ce[double bond, length as m-dash]C double bonds. Strong magnetic coupling of Ce[double bond, length as m-dash]C σ/π* and π/σ* orbitals produces strongly deshielded σ11 values, a characteristic hallmark of alkylidenes, and the largest 13C chemical shift tensor spans of any alkylidene complex to date (1, 801 ppm; 2, 810 ppm). In contrast, the phosphonium-substituent shielding contributions are much smaller than the Ce[double bond, length as m-dash]C σ- and π-bond components. This study confirms significant Ce 4f-orbital contributions to the Ce[double bond, length as m-dash]C bonding, provides further support for a previously proposed inverse-trans-influence in 2, and reveals variance in the 4f spin-orbit contributions that relate to the alkylidene hybridisation. This work thus confirms the metal-carbon double bond credentials of f-element-diphosphonioalkylidenes, providing quantified benchmarks for understanding diphosphonioalkylidene bonding generally.

Single cell RNA sequencing analysis of mouse cochlear supporting cell transcriptomes with activated ERBB2 receptor indicates a cell-specific response that promotes CD44 activation.

Hearing loss caused by the death of cochlear hair cells (HCs) might be restored through regeneration from supporting cells (SCs) via dedifferentiation and proliferation, as observed in birds. In a previous report, ERBB2 activation in a subset of cochlear SCs promoted widespread down-regulation of SOX2 in neighboring cells, proliferation, and the differentiation of HC-like cells. Here we analyze single cell transcriptomes from neonatal mouse cochlear SCs with activated ERBB2, with the goal of identifying potential secreted effectors. ERBB2 induction in vivo generated a new population of cells with de novo expression of a gene network. Called small integrin-binding ligand n-linked glycoproteins (SIBLINGs), these ligands and their regulators can alter NOTCH signaling and promote cell survival, proliferation, and differentiation in other systems. We validated mRNA expression of network members, and then extended our analysis to older stages. ERBB2 signaling in young adult SCs also promoted protein expression of gene network members. Furthermore, we found proliferating cochlear cell aggregates in the organ of Corti. Our results suggest that ectopic activation of ERBB2 signaling in cochlear SCs can alter the microenvironment, promoting proliferation and cell rearrangements. Together these results suggest a novel mechanism for inducing stem cell-like activity in the adult mammalian cochlea.

Characterizing Neonatal Heart Maturation, Regeneration, and Scar Resolution Using Spatial Transcriptomics.

The neonatal mammalian heart exhibits a remarkable regenerative potential, which includes fibrotic scar resolution and the generation of new cardiomyocytes. To investigate the mechanisms facilitating heart repair after apical resection in neonatal mice, we conducted bulk and spatial transcriptomic analyses at regenerative and non-regenerative timepoints. Importantly, spatial transcriptomics provided near single-cell resolution, revealing distinct domains of atrial and ventricular myocardium that exhibit dynamic phenotypic alterations during postnatal heart maturation. Spatial transcriptomics also defined the cardiac scar, which transitions from a proliferative to secretory phenotype as the heart loses regenerative potential. The resolving scar is characterized by spatially and temporally restricted programs of inflammation, epicardium expansion and extracellular matrix production, metabolic reprogramming, lipogenic scar extrusion, and cardiomyocyte restoration. Finally, this study revealed the emergence of a regenerative border zone defined by immature cardiomyocyte markers and the robust expression of Sprr1a. Taken together, our study defines the spatially and temporally restricted gene programs that underlie neonatal heart regeneration and provides insight into cardio-restorative mechanisms supporting scar resolution.

Coordination of endothelial cell positioning and fate specification by the epicardium.

The organization of an integrated coronary vasculature requires the specification of immature endothelial cells (ECs) into arterial and venous fates based on their localization within the heart. It remains unclear how spatial information controls EC identity and behavior. Here we use single-cell RNA sequencing at key developmental timepoints to interrogate cellular contributions to coronary vessel patterning and maturation. We perform transcriptional profiling to define a heterogenous population of epicardium-derived cells (EPDCs) that express unique chemokine signatures. We identify a population of Slit2+ EPDCs that emerge following epithelial-to-mesenchymal transition (EMT), which we term vascular guidepost cells. We show that the expression of guidepost-derived chemokines such as Slit2 are induced in epicardial cells undergoing EMT, while mesothelium-derived chemokines are silenced. We demonstrate that epicardium-specific deletion of myocardin-related transcription factors in mouse embryos disrupts the expression of key guidance cues and alters EPDC-EC signaling, leading to the persistence of an immature angiogenic EC identity and inappropriate accumulation of ECs on the epicardial surface. Our study suggests that EC pathfinding and fate specification is controlled by a common mechanism and guided by paracrine signaling from EPDCs linking epicardial EMT to EC localization and fate specification in the developing heart.

Innovative Optogenetic Strategies for Vision Restoration.

The advent of optogenetics has ushered in a new era in neuroscience where spatiotemporal control of neurons is possible through light application. These tools used to study neural circuits can also be used therapeutically to restore vision. In order to recapitulate the broad spectral and light sensitivities along with high temporal sensitivity found in human vision, researchers have identified and developed new optogenetic tools. There are two major kinds of optogenetic effectors employed in vision restoration: ion channels and G-protein coupled receptors (GPCRs). Ion channel based optogenetic therapies require high intensity light that can be unsafe at lower wavelengths, so work has been done to expand and red-shift the excitation spectra of these channels. Light activatable GPCRs are much more sensitive to light than their ion channel counterparts but are slower kinetically in terms of both activation and inactivation. This review article examines the latest optogenetic ion channel and GPCR candidates for vision restoration based on light and temporal sensitivity.

Expression of Fyn kinase modulates EMT in oral cancer cells.

Oral squamous cell carcinoma (SCC) is an aggressive tumor with a poor 5-year survival rate. Oral SCC can undergo epithelial to mesenchymal transition (EMT). We previously showed that the epithelial integrin alphavbeta6 complexes with Fyn kinase in oral SCC to promote EMT. Using immunofluorescence microscopy and Western blotting, we evaluated whether the expression of specific markers of EMT were influenced by modulating serum concentration (ie. growth factors). The SCC cultures were grown under contrasting levels of serum. In low serum (1%), Fyn promoted EMT; whereas suppression of Fyn kinase promoted the epithelial phenotype. However, when the SCC cells were grown in 10% serum, activation of Fyn had the reverse effect. Lastly, cell migration was evaluated under low serum conditions (1% FBS). Activation of Fyn promoted SCC cell migration and its suppression thwarted SCC migration toward FN. These results indicate that the activation of Fyn kinase as well as local growth factor concentration modulate EMT in oral SCC.