Probes for INS

Cardiac myosin-binding protein C (cMyBP-C) is an important regulator of cardiac muscle contraction and is commonly implicated in hypertrophic cardiomyopathy (HCM). However, the mechanism of regulation by cMyBP-C remains unclear due to experimental challenges in dissecting the proposed weak, transient interactions with its binding partners. Here, we utilized a nanosurf assay, containing a synthetic b-cardiac myosin thick filament, to systematically probe cMyBP-C interactions with actin and myosin. Recombinant human b-cardiac myosin subfragments (HMM or S1) were attached to DNA nanotubes, with 14 or 28 nm spacing, similar to the myosin head spacing on native thick filaments. No significant difference in thin filament velocity was observed with 14 nm vs. 28 nm motor spacing. Various N-terminal fragments of cMyBP-C were interdigitated with b-cardiac myosin on DNA nanotubes via encoded SNAP tags labeled with sequence-specific oligo attachment strands. We recapitulated inhibition of thin filament motility on b-cardiac myosin HMM and S1 nanotubes by C0-C2 (4-6 fold) and C1-C2 (4-8 fold) N-terminal cMyBP-C fragments. Equivalent inhibition of b-cardiac myosin HMM and S1 subfragments suggests the actin-cMyBP-C interaction dominates this inhibitory mechanism. We found that a C0-C1f fragment lacking the majority of the M-domain did not inhibit b-cardiac myosin nanotube motility, confirming the importance of the M-domain in regulatory interactions. Diminished inhibition by C0-C2 and C1-C2 phosphomimetic fragments (2-3 fold higher velocity compared to their phosphonull counterparts) further highlights the importance of the phosphorylatable serines in the regulatory Mdomain. These results shed light on the mechanism of cMyBP-C and highlight the utility of the nanosurf as

Hemochorial placentation is characterized by the development of trophoblast cells specialized to interact with the uterine vascular bed. We utilized trophoblast stem (TS) cell and mutant rat models to investigate regulatory mechanisms controlling trophoblast cell development. TS cell differentiation was characterized by acquisition of transcript signatures indicative of an endothelial cell-like phenotype, which was highlighted by the expression of anticoagulation factors including tissue factor pathway inhibitor (TFPI). TFPI localized to invasive endovascular trophoblast cells of the rat placentation site. Disruption of TFPI in rat TS cells interfered with development of the endothelial cell-like endovascular trophoblast cell phenotype. Similarly, TFPI was expressed in human invasive/extravillous trophoblast (EVT) cells situated within first-trimester human placental tissues and following differentiation of human TS cells. TFPI was required for human TS cell differentiation to EVT cells. We next investigated the physiological relevance of TFPI at the placentation site. Genome-edited global TFPI loss-of-function rat models revealed critical roles for TFPI in embryonic development, resulting in homogeneous midgestation lethality prohibiting analysis of the role of TFPI as a regulator of the late-gestation wave of intrauterine trophoblast cell invasion. In vivo trophoblast-specific TFPI knockdown was compatible with pregnancy but had profound effects at the uterine-placental interface, including restriction of the depth of intrauterine trophoblast cell invasion while leading to the accumulation of natural killer cells and increased fibrin deposition. Collectively, the experimentation implicates TFPI as a conserved regulator of invasive/EVT cell development, uterine spiral artery remodeling, and hemostasis at the maternal-fetal interface.

The hepatic MAPK cascade leading to JNK activation has been implicated in the pathogenesis of nonalcoholic fatty liver /non-alcoholic steatohepatitis (NAFL/NASH). In acute hepatotoxicity we previously identified a pivotal role for mitochondrial SH3BP5 (SAB) as a target of JNK which sustains its activation through promotion of reactive oxygen species (ROS) production.Assess the role of hepatic SAB in experimental NASH and metabolic syndrome.In mice fed high-fat, high-calorie, high-fructose (HFHC) diet, SAB expression progressively increased through a sustained JNK/ATF2 activation loop. Inducible deletion of hepatic SAB markedly decreased sustained JNK activation and improved systemic energy expenditure at 8 weeks followed by decreased body fat at 16 weeks of HFHC diet. After 30 weeks mice treated with control-ASO developed steatohepatitis and fibrosis which was prevented by Sab-ASO treatment. P-JNK and P-ATF2 were markedly attenuated by Sab-ASO treatment. After 52 weeks of HFHC feeding control N-acetylgalactosamine antisense oligonucleotide (GalNAc-Ctl-ASO) treated mice fed the HFHC diet exhibited progression of steatohepatitis and fibrosis but GalNAc-Sab-ASO treatment from weeks 40 to 52 reversed these findings while decreasing hepatic SAB, P-ATF2, and P-JNK to chow fed levels.Hepatic SAB expression increases in HFHC diet fed mice. Deletion or knockdown of SAB inhibited sustained JNK activation and steatohepatitis, fibrosis, and systemic metabolic effects, suggesting that induction of hepatocyte Sab is an important driver of the interplay between the liver and the systemic metabolic consequences of overfeeding. In established NASH, hepatocyte targeted GalNAc-Sab-ASO treatment reversed steatohepatitis and fibrosis.This article is protected by

Parkinson's disease (PD), the most common degenerative movement disorder, is clinically manifested with various motor and non-motor symptoms. Degeneration of midbrain substantia nigra pas compacta (SNc) dopaminergic neurons (DANs) is generally attributed to the motor syndrome. The underlying neuronal mechanisms of non-motor syndrome are largely unexplored. Besides SNc, midbrain ventral tegmental area (VTA) DANs also produce and release dopamine and modulate movement, reward, motivation, and memory. Degeneration of VTA DANs also occurs in postmortem brains of PD patients, implying an involvement of VTA DANs in PD-associated non-motor symptoms. However, it remains to be established that there is a distinct segregation of different SNc and VTA DAN subtypes in regulating different motor and non-motor functions, and that different DAN subpopulations are differentially affected by normal ageing or PD. Traditionally, the distinction among different DAN subtypes was mainly based on the location of cell bodies and axon terminals. With the recent advance of single cell RNA sequencing technology, DANs can be readily classified based on unique gene expression profiles. A combination of specific anatomic and molecular markers shows great promise to facilitate the identification of DAN subpopulations corresponding to different behavior modules under normal and disease conditions. In this review, we first summarize the recent progress in characterizing genetically, anatomically, and functionally diverse midbrain DAN subtypes. Then, we provide perspectives on how the preclinical research on the connectivity and functionality of DAN subpopulations improves our current understanding of cell-type and circuit specific mechanisms of the disease, which could be critically informative for designing new mechanistic treatments.

Non-invasive electrical stimulation methods are often used in experimental settings to investigate the possible modulation of antinociceptive mechanisms. Studies using transcranial direct current stimulation (tDCS) typically incorporate a fade-in, short-stimulation, fade-out sham (placebo) protocol, which is assumed to be indistinct from a 10-30min active protocol on the scalp. However, many studies report that participants can dissociate active stimulation from sham, even during low-intensity 1mA currents. In the present study we assessed whether delivery of a high-intensity 2mA current would exacerbate differences in the perception of active and sham protocols. Two protocols were delivered to 32 healthy, pain-free adults in a double-blinded, within-subjects design (active: 10min of 2mA, and sham: 20s of 2mA), with the anode over the left primary motor cortex and the cathode on the right forehead. Participants were asked “Is the stimulation on?” and “How sure are you?” at 30s intervals during and after stimulation. The differences between active and sham were more consistent and sustained during 2mA than during 1mA. We then quantified how well participants were able to track the presence and absence of stimulation (i.e. their sensitivity) during the experiment using cross-correlations. A good classifier of sensitivity during active tDCS was current strength, but exhibited only moderate specificity during sham. The accuracy of the end-of-study guess was no better than chance at predicting sensitivity. Our results from this methodological approach indicate that the traditional end-of-study guess poorly reflects the sensitivity of participants to stimulation, and may not be a valid method of assessing sham blinding. Further research should be carried out into inter-individual responses to sham-blinding and assessment methods in pain studies and the broader neurostimulation field. Wellcome Trust.

Gastric-type mucinous adenocarcinoma (GAS) is an uncommon cervical adenocarcinoma, which is not associated with human papillomavirus (HPV) infection. Compared with HPV-associated cervical adenocarcinoma, GAS has characteristics of larger volume, deep invasion, and easy to metastasize to distant sites. Also, GAS is typically resistant to chemo/radiotherapy. Few studies have reported the molecular genetic characteristics of GAS. In order to investigate the molecular genetic characteristics of GAS and reveal its possible pathogenesis, 15 GAS patients were enrolled from Peking University People's Hospital (2009-2019) and examined with next-generation sequencing (NGS). Based on the clinicopathologic feature analysis, we found that the presence of lymph node metastasis and extensive lymphovascular invasion were associated with poor survival outcomes of GAS (p = 0.0042 and p = 0.005, respectively). Based on the NGS testing, our results showed that the most frequently mutated gene was TP53 (8/15, 53.3%), followed by STK11, CDKN2A, and ARID1A. STK11 mutations were more frequent in well-differentiated GAS (33.3% vs. 0.0%, p = 0.026) and patients with extensive lymphovascular invasion (33.3% vs. 0.0%, p = 0.044). Survival analysis revealed that STK11 mutations were significantly associated with the poor prognosis of GAS (p = 0.01). Our results also showed that mutations in the target drug were detected in 53.3% of GAS patients. Patients with ERBB2 amplification (13.3%) presented the highest level of evidence according to OncoKB recommendations. These results indicate that the genomic alterations of GAS mainly involved the cell cycle and PI3K/AKT signaling pathways, and some therapeutic candidates were identified in GAS patients.

Modelling cell infection in-a-dish can represent a useful tool to understand the susceptibility of different cell types towards severe acute respiratory coronavirus-2 (SARS-CoV-2) and to decipher its neurotropism. In this perspective, retinoic acid (RA)-differentiated neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2) and glioblastoma cell lines, U-87 MG and U-373 MG, were infected with a SARS-CoV-2 strain, at various multiplicity-of-infection (MOI). We first demonstrated that the common entry genes - needed for invading epithelial cells - were expressed. RA-differentiation induced an upregulation of ace2 and tmprss2 gene expression while inducing downregulation of ctsb and ctsl. Using in situ hybridization and confocal analysis, SARS-CoV-2 gene S RNA was detected intracellularly at MOI 5.0, and localized in both soma and neuritic-like or glial-like processes. The infection was confirmed by quantification of viral gene E RNA and showed a dose-dependency, with few infected cells at MOI 0.1. After 24 h of infection, no cytopathic effect was observed in SH-SY5Y abilities to maintain neuritic processes or in U-373 MG for the uptake of glutamate. Unlike the permissive Vero E6 cells, no significant apoptosis death was detected following SARS-CoV-2 infection of neuroblastoma or glioblastoma cells. This study demonstrates the susceptibility of neuronal- and glial-like cell lines towards SARS-CoV-2 infection at high MOIs. Once inside the cells, the virus does not seem to rapidly replicate nor exert major cytopathic effect. Overall, our results strengthen the idea that SARS-CoV-2 has a tropism for nervous cells that express commonly described entry genes.

Purpose : The human cornea has been defined as our “external window” to the visual world that serves as a barrier against the outside environment and as the main refractive lens to focus light into the retina. The histological structure is defined by three layers of cellular elements (epithelium, stroma, endothelium) and two layers of extracellular membranes. Here, we used a combination of single-cell RNA sequencing and in-situ hybridization to characterize the transcriptomic features of different cells and their localization in the human cornea Methods : Six adult human corneas from healthy donors were processed by sequential tissue digestion and cell sorting. Single-cell suspensions were profiled using 10x Genomics Chromium Single Cell 3′ (v2) Gene Expression workflow. Spatial distribution of cell markers was localized by RNA in situ hybridization (RNAscope) in human cornea cross-sections. Results : Unsupervised clustering of individual cell transcriptomes based on overall gene expression similarity identified 16 transcriptionally distinct clusters within corneal cells, including stromal keratocytes, endothelium, several subtypes of corneal epithelium, and supportive cells in the limbal stem cell niche. Epithelial cells represent the most diverse cell type with eleven sub-clusters. By combining pseudotime bioinformatic analysis and RNAscope we mapped the epithelial cell fate trajectory and location including their initial generation in the limbal region, differentiation, and migration to superficial epithelial layers. Conclusions : Our study reveals the single-cell map of the adult human cornea and expands the knowledge of the molecularly define cellular subsets of the cornea on a whole genome transcriptional level. This information can be applied to better understand normal corneal biology, serve as a reference to study corneal diseases, and provide potential insights into disease pathology and therapeutics.

Current in vitro systems are powerful tools for studying early heart specification but lack the ability to model morphological events. Reporting in this issue of Cell Stem Cell, Rossi et al. (2021) present a patterned embryonic organoid model (gastruloid) that mimics aspects of early cardiogenesis.

The antitumor efficacies of immune checkpoint inhibitors (ICIs) and the usefulness of potential predictive markers such as programmed death-ligand 1 (PD-L1) expression, density of tumor-infiltrating lymphocytes (TILs) and microsatellite instability (MSI) in sinonasal squamous cell carcinoma (SNSCC) have not been fully elucidated. We retrospectively analyzed 131 SNSCCs with immunohistochemistry for PD-L1 expression, TIL subpopulations and loss of mismatch repair (MMR) proteins as a surrogate for MSI-high. We also comprehensively evaluated the mutual relationships among these immuno-markers, high-risk human papillomavirus (HPV) infection, epidermal growth factor receptor (EGFR) gene status, and KRAS mutation. PD-L1 expression (tumor proportion score ≥ 1%) was detected in 60 (45.8%) SNSCC cases and was significantly associated with worse overall survival (OS) (p = 0.0240). High density of cluster of differentiation 8 (CD8)-positive TILs was significantly associated with better progression-free survival (PFS) (p = 0.0368), and high density of forkhead box protein P3-positive TILs was significantly associated with better PFS and OS (p = 0.0007 and 0.0143, respectively). With respect to the combination of CD8 + TIL and PD-L1 expression, the high-CD8/PD-L1-negative group showed the most favorable prognosis, whereas the low-CD8/PD-L1-positive group showed the worst prognosis. MMR loss was detected in 3 (2.3%) of the 131 cases. HPV infection (6.1%), EGFR mutation (14.5%), EGFR copy number gain (26%), and MMR loss were essentially mutually exclusive; patients in these molecular groups showed significant differences in prognosis but not in the degree of PD-L1 expression or TILs. Among the nine ICI-treated patients, three (33.3%) were responders, and the EGFR-wild type cases (n = 7) showed better clinical responses to an ICI compared to the EGFR-mutant cases (n = 2). Among the patients with residual/recurrent EGFR-wild type tumors (n = 43), ICI treatment significantly improved OS (p = 0.0281). The results suggest that the evaluation of immuno-markers and molecular subclassification may be helpful for prognostic prediction and selecting an individualized therapeutic strategy for patients with SNSCC.

The growth plates are key engines of skeletal development and growth and contain a top reserve zone followed by maturation zones of proliferating, pre-hypertrophic and hypertrophic/mineralizing chondrocytes. Trauma or drug treatment of certain disorders can derange the growth plates and cause accelerated maturation and premature closure, one example being anti-hedgehog drugs such as LDE225 (Sonidegib) used against pediatric brain malignancies. Here we tested whether such acceleration and closure in LDE225-treated mice could be prevented by co-administration of a selective retinoid antagonist, based on previous studies showing that retinoid antagonists can slow down chondrocyte maturation rates. Treatment of juvenile mice with an experimental dose of LDE225 for 2 days (100 mg/kg by gavage) initially caused a significant shortening of long bone growth plates, with concomitant decreases in: chondrocyte proliferation; expression of Indian hedgehog, Sox9 and other key genes; and surprisingly, the number of reserve progenitors. Growth plate involution followed with time leading to impaired long bone lengthening. Mechanistically, LDE225 treatment markedly decreased the expression of retinoid catabolic enzyme Cyp26b1 within growth plate whereas it increased and broadened the expression of retinoid synthesizing enzyme Raldh3, thus subverting normal homeostatic retinoid circuitries and in turn accelerating maturation and closure. All such severe skeletal and molecular changes were prevented when LDE-treated mice were co-administered the selective retinoid antagonist CD2665 (1.5 mg/kg/daily), a drug targeting retinoid acid receptor γ most abundantly expressed in growth plate. When given alone, CD2665 elicited the expected maturation delay and growth plate expansion. In vitro data showed that LDE225 acted directly to dampen chondrogenic phenotypic expression, a response fully reversed by CD2665 co-treatment. In sum, our proof-of-principle data indicate that drug-induced premature growth plate closures can be prevented or delayed by targeting a separate phenotypic regulatory mechanism in chondrocytes. The translation applicability of the findings remains to be studied. This article is protected by

Immunohistochemical staining demonstrated cytoplasmic and nuclear expression of OCT4 on the endothelium and the media of the lesional vessels with nuclear staining of the cells within the stroma (Figure 3A). Cytoplasmic and nuclear expression of SOX2 was present on the endothelium of the lesional vessels and the cells within the stroma with strong expression in the media of the lesional vessels (Figure 3B). Weak cytoplasmic expression of KLF4 was observed on the endothelium of the lesional vessels (Figure 3C). Nuclear expression of c-MYC was demonstrated on the endothelium and the media of the lesional vessels and within the cells in the stroma (Figure 3D). NANOG was not expressed in any of the 15 samples (Figure 3E). [Figure 3.]Figure 3.: Representative immunohistochemical-stained images of hypertrophic port-wine stain tissue samples, demonstrating the expression of OCT4 (A, brown), SOX2 (B, brown), KLF4 (C, brown), and c-MYC (D, brown). Cytoplasmic staining of the endothelium was demonstrated for SOX2, OCT4, and KLF4. Nuclear staining of SOX2, OCT4, and c-MYC was demonstrated on the endothelium and the cells within the stroma. E, NANOG was not expressed in any of the 15 samples. Nuclei were counterstained with hematoxylin (A–E, blue). Original magnification: 200×. Positive staining was demonstrated on human control tissues: seminoma for OCT4 (Supplemental Digital Content Figure 1A, http://links.lww.com/JV9/A2), skin epidermis for SOX2 (Supplemental Digital Content Figure 1B, http://links.lww.com/JV9/A2), breast carcinoma for KLF4 (Supplemental Digital Content Figure 1C, http://links.lww.com/JV9/A2), normal colon mucosa for c-MYC (Supplemental Digital Content Figure 1D, http://links.lww.com/JV9/A2), and seminoma for NANOG (Supplemental Digital Content Figure 1E, http://links.lww.com/JV9/A2). Immunohistochemical staining of normal skin showed no expression of OCT4 (Supplemental Digital Content Figure 1F, http://links.lww.com/JV9/A2) and NANOG (Supplemental Digital Content Figure 1G, http://links.lww.com/JV9/A2). SOX2 (Supplemental Digital Content Figure 1B, http://links.lww.com/JV9/A2), KLF4 (Supplemental Digital Content Figure 1H, http://links.lww.com/JV9/A2), and c-MYC (Supplemental Digital Content Figure 1I, http://links.lww.com/JV9/A2) were present in the epidermis of the normal skin. In addition, SOX2 (Supplemental Digital Content Figure 1B, http://links.lww.com/JV9/A2) and KLF4 (Supplemental Digital Content Figure 1H, http://links.lww.com/JV9/A2) were expressed by some cells but not blood vessels within the stroma. A negative stain using combined Flex Negative Control Mouse and Flex Negative Control Rabbit on a section of HPWS (Supplemental Digital Content Figure 1J, http://links.lww.com/JV9/A2) showed no staining, confirming the specificity of the primary antibodies.