Probes for INS

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.

PRION DISEASES are a group of incurable neurodegenerative diseases caused by the prions, affecting both humans and animals. The atypical folding and aggregation of the soluble cellular prion proteins (PrPC) into scrapie isoform (PrPSc) in the CNS, results in brain damage and other symptoms associated with it. Different therapeutic approaches ranging from organic compounds to antibodies have been proposed, including stalling PrPCto PrPScconversion, increasing PrPScremoval, and/or PrPCstabilization using different research methodologies like cell-therapy, immunotherapy, pharmacotherapy and compounds ranging from chemicals to proteins have been studied to target the disease with special attention to PrPScaggregation inhibition. Compounds destabilizing PrPScand reducing infection have also been identified. A few important CHEMICAL COMPOUNDS (Sulfated polyanions, diazo dyes, Phenothiazine derivative, Cyclic Tetrapyrroles, Diphenylmethane derivatives, Diphenylpyrazole derivatives, Indole-3-Glyoxylamides, 2-Aminothiazoles, Carbazole derivatives, Benzoxazole derivative, Ethanolamine, Dimethyl sulfoxide); REPURPOSED DRUGS (Quinacrine, _Chlorpromazine_, Celecoxib, Flupirtine, Imatinib, Efavirenz, Simvastatin, Glimepiride, Doxycycline); NATURAL PRODUCTS (Polydatin, Curcumin, Resveratrol, Epigallocatechin gallate, Cannabidiol, Baicalein, Hinokitiol, Ginsenoside, Bile acids) and ANTI-PRION ANTIBODIES (6H4, D13, D18, 8B4, 8H4, ICSM18, ICSM35, POM1-2, 4H11, 44B1). All these compounds have displayed anti-prion activity _in vitro_, but only a few were effective _in vivo_. Recently, encouraging results of a PRION PROTEIN MONOCLONAL ANTIBODY (an IgG4κ isotype; PNR100) in a clinical trial study on CJD patients have been announced which stabilized PrPCand increased the survival in infected mice. After such promising results, PRN100 will be evaluated for Phase-II trials. The success story doesn’t end here as another promising molecule MC and GN8 are ready for human clinical trials on prion-diseases while Anle138b is in Phase 1b for PD patients. The ineffectiveness of most of the molecules tested was either due to inability to cross BBB, toxicity or transitory accumulation of drug resistant prions. Hence, it is incredibly important to learn from the backstory. By understanding what all has already been done, what is the mechanism of drug action, why the clinical trials failed, we can comprehend prion diseases better and chalk out the right direction leading to more effective treatment. Also, targeting more than one pathway involved in prion diseases may provide synergistic benefits.

Hypokalemic periodic paralysis is a rare neuromuscular genetic disorder due to defect of ion channels and subsequent function impairment. It belongs to a periodic paralyses group including hyperkalemic periodic paralysis (HEKPP), hypokalemic periodic paralysis (HOKPP) and Andersen-Tawil syndrome (ATS). Clinical presentations are mostly characterized by episodes of flaccid generalized weakness with transient hypo- or hyperkalemia.A teenage boy presented to Emergency Department (ED) for acute weakness and no story of neurological disease, during the anamnestic interview he revealed that he had a carbohydrates-rich meal the previous evening. Through a focused diagnostic work-up the most frequent and dangerous causes of paralysis were excluded, but low serum potassium concentration and positive family history for periodic paralyses raised the diagnostic suspicion of HOKPP. After the acute management in ED, he was admitted to Pediatric Department where a potassium integration was started and the patient was counselled about avoiding daily life triggers. He was discharged in few days. Unfortunately, he presented again because of a new paralytic attack due to a sugar-rich food binge the previous evening. Again, he was admitted and treated by potassium integration. This time he was strongly made aware of the risks he may face in case of poor adherence to therapy or behavioral rules. Currently, after 15 months, the boy is fine and no new flare-ups are reported.HOKPP is a rare disease but symptoms can have a remarkable impact on patients' quality of life and can interfere with employment and educational opportunities. The treatment aims to minimize the paralysis attacks by restoring normal potassium level in order to reduce muscle excitability but it seems clear that a strong education of the patient about identification and avoidance triggering factors is essential to guarantee a benign clinical course. In our work we discuss the typical clinical presentation of these patients focusing on the key points of the diagnosis and on the challenges of therapeutic management especially in adolescence. A brief discussion of the most recent knowledge regarding this clinical condition follows.

Axon regenerative failure in the mature CNS contributes to functional deficits following many traumatic injuries, ischemic injuries and neurodegenerative diseases. The complement cascade of the innate immune system responds to pathogen threat through inflammatory cell activation, pathogen opsonization, and pathogen lysis, and complement is also involved in CNS development, neuroplasticity, injury, and disease. Here, we investigated the involvement of the classical complement cascade and microglia/monocytes in CNS repair using the mouse optic nerve injury (ONI) model, in which axons arising from retinal ganglion cells (RGCs) are disrupted. We report that central complement C3 protein and mRNA, classical complement C1q protein and mRNA, and microglia/monocyte phagocytic complement receptor CR3 all increase in response to ONI, especially within the optic nerve itself. Importantly, genetic deletion of C1q, C3, or CR3 attenuates RGC axon regeneration induced by several distinct methods, with minimal effects on RGC survival. Local injections of C1q function-blocking antibody revealed that complement acts primarily within the optic nerve, not retina, to support regeneration. Moreover, C1q opsonizes and CR3+ microglia/monocytes phagocytose growth-inhibitory myelin debris after optic nerve injury, a likely mechanism through which complement and myeloid cells support axon regeneration. Collectively, these results indicate that local optic nerve complement-myeloid phagocytic signaling is required for CNS axon regrowth, emphasizing the axonal compartment and highlighting a beneficial neuro-immune role for complement and microglia/monocytes in CNS repair.SIGNIFICANCE STATEMENTDespite the importance of achieving axon regeneration after CNS injury and the inevitability of inflammation after such injury, the contributions of complement and microglia to CNS axon regeneration are largely unknown. Whereas inflammation is commonly thought to exacerbate the effects of CNS injury, we find that complement proteins C1q and C3 and microglia/monocyte phagocytic complement receptor CR3 are each required for retinal ganglion cell axon regeneration through the injured mouse optic nerve. Also, whereas studies of optic nerve regeneration generally focus on the retina, we show that the regeneration-relevant role of complement and microglia/monocytes likely involves myelin phagocytosis within the optic nerve. Thus, our results point to the importance of the innate immune response for CNS repair.

The origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing the global coronavirus disease 19 (COVID-19) pandemic, remains a mystery. Current evidence suggests a likely spillover into humans from an animal reservoir. Understanding the host range and identifying animal species that are susceptible to SARS-CoV-2 infection may help to elucidate the origin of the virus and the mechanisms underlying cross-species transmission to humans. Here we demonstrated that white-tailed deer (Odocoileus virginianus), an animal species in which the angiotensin converting enzyme 2 (ACE2) - the SARS-CoV-2 receptor - shares a high degree of similarity to humans, are highly susceptible to infection. Intranasal inoculation of deer fawns with SARS-CoV-2 resulted in established subclinical viral infection and shedding of infectious virus in nasal secretions. Notably, infected animals transmitted the virus to non-inoculated contact deer. Viral RNA was detected in multiple tissues 21 days post-inoculation (pi). All inoculated and indirect contact animals seroconverted and developed neutralizing antibodies as early as day 7 pi. The work provides important insights into the animal host range of SARS-CoV-2 and identifies white-tailed deer as a susceptible wild animal species to the virus.IMPORTANCEGiven the presumed zoonotic origin of SARS-CoV-2, the human-animal-environment interface of COVID-19 pandemic is an area of great scientific and public- and animal-health interest. Identification of animal species that are susceptible to infection by SARS-CoV-2 may help to elucidate the potential origin of the virus, identify potential reservoirs or intermediate hosts, and define the mechanisms underlying cross-species transmission to humans. Additionally, it may also provide information and help to prevent potential reverse zoonosis that could lead to the establishment of a new wildlife hosts. Our data show that upon intranasal inoculation, white-tailed deer became subclinically infected and shed infectious SARS-CoV-2 in nasal secretions and feces. Importantly, indirect contact animals were infected and shed infectious virus, indicating efficient SARS-CoV-2 transmission from inoculated animals. These findings support the inclusion of wild cervid species in investigations conducted to assess potential reservoirs or sources of SARS-CoV-2 of infection.

Obesity represents the single greatest ongoing roadblock to improving cardiovascular health. Prolonged obesity is associated with fundamental changes in the integrative control of energy balance, including the development of selective leptin resistance, which is thought to contribute to obesity-associated hypertension, and adaptation of resting metabolic rate (RMR) when excess weight is reduced. Leptin and the melanocortin system within the hypothalamus contribute to the control of both energy balance and blood pressure. While the development of drugs to stimulate RMR and thereby reverse obesity through activation of the melanocortin system has been pursued, most of the resulting compounds simultaneously cause hypertension. Evidence supports the concept that although feeding behaviors, RMR, and blood pressure are controlled through mechanisms that utilize similar molecular mediators, these mechanisms exist in anatomically dissociable networks. New evidence supports a major change in molecular signaling within AgRP (Agouti-related peptide) neurons of the arcuate nucleus of the hypothalamus during prolonged obesity and the existence of multiple distinct subtypes of AgRP neurons that individually contribute to control of feeding, RMR, or blood pressure. Finally, ongoing work by our laboratory and others support a unique role for AT1 (angiotensin II type 1 receptor) within one specific subtype of AgRP neuron for the control of RMR. We propose that understanding the unique biology of the AT1-expressing, RMR-controlling subtype of AgRP neurons will help to resolve the selective dysfunctions in RMR control that develop during prolonged obesity and potentially point toward novel druggable antiobesity targets that will not simultaneously cause hypertension.