Mutations in the DMD gene lead to Duchenne muscular dystrophy, a severe X-linked neuromuscular disorder which manifests itself as young boys acquire motor functions. DMD is diagnosed after 2 to 4 years, but the absence of dystrophin has an impact before symptoms appear in patients, which poses a serious challenge in the optimization of standards of care. In this report, we investigated the early consequences of dystrophin deficiency during skeletal muscle development. We used single-cell transcriptome profiling to characterize the myogenic trajectory of human pluripotent stem cells and showed that DMD cells bifurcate to an alternative branch when they reach the somite stage. Here, dystrophin deficiency was linked to marked dysregulations of cell junction families involved in the cell state transitions characteristic of somitogenesis. Altogether, this work demonstrates that in vitro, dystrophin deficiency has early consequences during myogenic development, which should be considered in future therapeutic strategies for DMD. less

Lewy body (LB) disorders, characterized by the aggregation of misfolded alpha-synuclein proteins, exhibit notable clinical heterogeneity. This may be due to variations in accumulation patterns of LB neuropathology. By applying data-driven disease progression modelling to regional neuropathological LB density scores from 814 brain donors, we describe three inferred trajectories of LB pathology that were characterized by differing clinicopathological presentation and longitudinal antemortem clinical progression. Most donors (81.9%) showed earliest pathology in the olfactory bulb, followed by accumulation in either limbic (60.8%) or brainstem (21.1%) regions. The remaining donors (18.1%) exhibited the first abnormalities in brainstem regions. Early limbic pathology was associated with Alzheimer\'s disease-associated characteristics. Meanwhile, brainstem-first pathology was associated with progressive motor impairment and substantial LB pathology outside of the brain. Our data provides evidence for heterogeneity in the temporal spread of LB pathology, possibly explaining some of the clinical disparities observed in LBDs. less

The RGB trichrome staining method has been used to highlight two major components of the extracellular matrix, collagen and glycosaminoglycans. While the RGB trichrome efficiently stains extracellular matrix components, it lacks a nuclear stain, limiting its application in histopathology. To address this issue, a modification of the original stain, named HemRGB trichrome, has been developed. This modification incorporates iron hematoxylin for improving nuclear staining while retaining specificity for the staining of extracellular matrix. The application of HemRGB trichrome staining to samples from both normal colonic tissues and colorectal adenocarcinomas (CRC) provides a robust nuclear staining, together with a high-contrasted staining of tumor microenvironmental components, such as infiltrating immune cells, collagen and ground substance, extracellular mucins, as well as contrasted interfaces between CRC metastases and liver parenchyma. This study underscores the potential of HemRGB trichrome as a valuable tool for histopathological studies, especially for cancer evaluation, where nuclear characteristics are particularly relevant. less

Xanthomonas euvesicatoria (X. euvesicatoria) is the causal agent of bacterial spot disease that threatens pepper and tomato production around the globe. X. euvesicatoria gene Xe4428 encodes a type III effector (T3E) that shares 89.67% amino acid identity with Xanthomonas oryzae pv. oryzicola (Xoc) T3E AvrRxo1. Deletion of Xe4428 in the genome of X. euvesicatoria (strain Xcv85-10) compromised its virulence to infect pepper and Nicotiana benthamiana plants. Transient co-expression of Xe4428 and Rxo1 on pepper and N. benthamiana plant leaves results in a robust hypersensitive reaction. Thus, Xe4428, renamed as XeAvrRxo1, is a bona fide orthologue of XocAvrRxo1 that possesses both virulence and avirulence functions. Expression of XeAvrRxo1 in E. coli and X. euvesicatoria is toxic to both bacterial cells. Another X. euvesicatoria gene Xe4429, encodes a putative chaperone of XeAvrRxo1, which can interact with XeAvrRxo1 to suppress its toxicity in X. euvesicatoria and E. coli bacterial cells. Xe4429 also binds to the promoter region of XeavrRxo1 and represses its transcription/translation in X. euvesicatoria bacterial cells. In addition, expression of Xe4429 can enhance the secretion and translocation of XeAvrRxo1 into plant cells. Therefore, Xe4429 functions as an antitoxin, a transcription repressor, and a type III chaperone that is capable of enhancing the secretion and translocation of XeAvrRxo1 during pathogenesis. less

Atherosclerosis is a pathology affecting the arteries, characterized by the buildup of plaques in the blood vessel walls. Atherosclerosis is the main cause of cardiovascular diseases, which constitute the leading cause of death in the world. Cholesterol crystals are the main components of the plaques, which actively participate in plaque growth and rupture and do not dissolve in aqueous environments. Employing novel cryo-scanning electron microscopy techniques, we examined human atherosclerotic plaques at high resolution, in 3D, and in close to native conditions. We show that cholesterol crystal clearance occurs in advanced human plaques through the activity of cells. We suggest that this occurs by enzymatic esterification of cholesterol to cholesteryl ester, which aggregates into intra- and extra- cellular pools. This discovery provides further understanding of the disease process in atherosclerosis, and may inspire new therapeutic approaches. less

Background: Hepatocyte nuclear factor 4 alpha (HNF4) is a nuclear factor essential for liver function and regeneration. HNF4 negatively regulates the expression of cMyc, which plays an important role in proliferation and differentiation during liver regeneration. This study investigated the role of HNF4-cMyc interaction in regulating liver injury and regeneration using the choline-deficient and ethionine-supplemented (0.15%) (CDE) diet feeding model, which exhibits characteristics of chronic liver diseases including liver injury, inflammation, early fibrotic changes along with hepatocyte and biliary epithelial cell regeneration, and activation of hepatic progenitor cells (HPC). Methods: Wild-type (WT), hepatocyte-specific knockout of HNF4 (HNF4-KO), cMyc (cMyc-KO), and HNF4-cMyc double knockout (DKO) mice were fed a CDE diet for one week to induce subacute liver injury. To study regeneration and recovery, mice were fed a one-week CDE diet followed by a one-week recovery period on a normal chow diet. Results: WT mice showed significant liver injury and decreased HNF4 mRNA and protein expression after one week of a CDE diet. WT mice also showed an increase in markers of proliferation and HPC activation, but no major change in markers of inflammation or fibrosis. The HNF4-KO mice exhibited baseline hepatomegaly, which significantly declined during the recovery period. HNF4 deletion resulted in significantly higher injury compared to WT mice after one week of CDE diet feeding but similar recovery. Markers of inflammation, fibrosis, proliferation, and HPC activation were significantly higher in HNF4-KO mice during the injury period but declined during the recovery period. The cMyc-KO mice showed increased injury after one week of the CDE diet, but it was substantially lower than the WT and HNF4-KO mice. Deletion of cMyc resulted in a significant activation of inflammatory genes higher than in the WT and HNF4-KO mice. Whereas fibrosis and proliferation markers increased in cMyc-KO mice, they were substantially lower than in HNF4-KO mice and similar to WT mice. cMyc-KO also showed an increase in HPC markers following one week of CDE-induced injury. Deletion of both HNF4 and cMyc in DKO mice resulted in significant liver injury comparable to the HNF4-KO mice after one week of CDE diet feeding, but led to complete recovery. Markers of inflammation, fibrosis, and proliferation increased after CDE diet feeding, were higher than WT mice, and comparable to HNF4-KO mice. Interestingly, DKO mice showed a significant increase in HPC markers both following one week of CDE-induced injury and after one week of recovery. Conclusions: These data indicate that deletion of HNF4 increases and deletion of cMyc decreases subacute liver injury induced by a one week CDE diet feeding. Deletion of HNF4 results in increased inflammation, fibrosis, proliferation, and HPC activation, all of which except inflammation are reduced following cMyc deletion. Simultaneous deletion of HNF4 and cMyc results in a phenotype similar to HNF4 deletion but with higher HPC activation. Taken together, these data show that HNF4 protects against inflammatory and fibrotic change following CDE diet-induced injury, which is driven by cMyc. less

Background: Recently we have shown alterations in the anticoagulant response to recombinant activated factor VII (rFVIIa)-induced coagulation activation in patients with thrombophilia. Objectives: Here we extended this in vivo model to study fibrinolysis biomarkers. Methods: The study population included 56 patients with thrombophilia and a history of venous thromboembolism (VTE+), 38 asymptomatic patients with thrombophilia (VTE-) and 35 healthy controls. Plasma levels of D-dimer, plasmin-2-antiplasmin complex (PAP), and plasminogen activator inhibitor-1 (PAI-1) were monitored over 8 hours after rFVIIa infusion (0.015 mg/kg) along with thrombin activation markers and activated protein C (APC). Results: In all cohorts, PAP increased (P<3.9*10-10) and PAI-1 decreased (P<3.5*10-8). In contrast to thrombin-antithrombin complex (TAT), which also increased temporarily in all cohorts (P<3.6*10-6), changes of PAP and PAI-1 did not reverse during the observation period. The area under the curve (AUC) of PAP (respectively TAT), as measure of plasmin (respectively thrombin) formation, was greater in the VTE+ cohort than in healthy controls (PAP AUC P=0.003, TAT AUC P=2.5*10-4) and showed correlation (r=0.554). As evidenced by the respective AUCs, asymptomatic factor V Leiden (FVL) carriers in the VTE- cohort showed less PAP formation (P=9*10-4), more pronounced PAI-1 decline (P=0.010), and increased APC formation (P=0.020) than those within the VTE+ group (n=19 each). This was not observed in prothrombin 20210G>A carriers or patients with unexplained familial thrombophilia. Conclusion: rFVIIa-induced thrombin formation is associated with fibrinolysis parameter changes outlasting the concomitant anticoagulant response. Both correlate with thrombosis history in FVL and might help to explain its variable clinical expressivity. less

Objective: Angiotensin-II (Ang-II) drives pathological vascular wall remodeling in hypertension and abdominal aortic aneurysm (AAA). Previous studies showed that the phosphatase activity of calcineurin (Cn) mediates Ang-II-induced AAA, but the cell type involved in the action of Cn in AAA formation remained unknown. Methods: Smooth muscle cell (SMC)-specific and endothelial cell (EC)-specific Cn-deficient mice (SM-Cn-/- and EC-Cn-/- mice, respectively) were created and assessed for Ang-II-induced AAA formation and hypertension vs controls. Osmotic minipumps were used to administer Ang-II and cyclosporine A (CsA), a pharmaceutical Cn inhibitor. AAA formation and hypertension were monitored by ultrasonography, arterial blood pressure monitoring, and histological analysis. Deep RNA sequencing was used to identify the Ang-II-regulated transcriptome sensitive to Cn deletion or pharmacological inhibition. Arterial and SMC contractility were also assessed. Results: Cn expressed in SMCs, but not ECs, was required for Ang-II-induced AAA. Unexpectedly, SMC Cn played a structural role in the early onset and maintenance of Ang-II-induced hypertension independently of Cn phosphatase activity. Nearly 90% of the genes regulated by Ang-II in the aorta required Cn expression in SMCs. Cn orchestrated, independently of its enzymatic activity, the induction by Ang-II of a gene expression program closely related to SMC contractility and hypertension. Cn deletion in SMCs, but not its pharmacological inhibition, impaired the regulation of arterial contractility. Among the genes whose regulation by Ang-II required Cn expression but not its phosphatase activity, we discovered that Serpine1 was critical for Ang-II-induced hypertension. Indeed, pharmacological inhibition of PAI-1, the protein encoded by Serpine1, impaired SMCs contractility and readily regressed hypertension. Conclusions: Whereas the phosphatase activity of Cn mediates Ang-II-induced AAA, a phosphatase-independent action of SMC Cn mediates hypertension by orchestrating a gene expression program closely related to contractility and blood pressure regulation. Our results urge the evaluation of PAI-1 as a candidate therapeutic target for hypertension. less

Multiplex staining enables simultaneous detection of multiple protein markers within a tissue sample. However, the increased marker count increased the likelihood of staining and imaging failure, leading to higher resource usage in multiplex staining and imaging. We address this by proposing a deep learning-based MArker imputation model for multipleX IMages (MAXIM) that accurately impute protein markers by leveraging latent biological relationships between markers. The model\'s imputation ability is extensively evaluated at pixel and cell levels across various cancer types. Additionally, we present a comparison between imputed and actual marker images within the context of a downstream cell classification task. The MAXIM model\'s interpretability is enhanced by gaining insights into the contribution of individual markers in the imputation process. In practice, MAXIM can reduce the cost and time of multiplex staining and image acquisition by accurately imputing protein markers affected by staining issues. less

{beta}-coronaviruses cause acute infection in the upper respiratory tract, resulting in various symptoms and clinical manifestations. OC43 is a human {beta}-coronavirus that induces mild clinical symptoms and can be safely studied in the BSL2 laboratory. Due to its low risk, OC43 can be a valuable and accessible model for understanding {beta}-coronavirus pathogenesis. One potential target for limiting virus infectivity could be gap junction-mediated communication. This study aims to unveil the status of cell-to-cell communications through gap junctions in human {beta}-coronavirus infection. Infection with OC43 leads to reduced expression of Cx43 in A549, a lung epithelial carcinoma cell line. Infection with this virus also showed a significant ER and oxidative stress increase. Internal localization of Cx43 is observed post OC43 infection in the ERGIC region, which impairs the gap junction communication between two adjacent cells, confirmed by Lucifer yellow dye transfer assay. It also affects hemichannel formation, as depicted by the EtBr uptake assay. Altogether, these results suggest that several physiological changes accompany OC43 infection in A549 cells and can be considered an appropriate model system for understanding the differences in gap junction communication post-viral infections. This model system can provide valuable insights for developing therapies against human {beta}-coronavirus infections. less