Probes for INS

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.

Aberrant ETV1 overexpression arising from gene rearrangements or mutations occur frequently in prostate cancer, round cell sarcomas, gastrointestinal stromal tumors, gliomas, and other malignancies. The absence of specific monoclonal antibodies (mAb) has limited its detection and our understanding of its oncogenic function.An ETV1 specific rabbit mAb (29E4) was raised using an immunogenic peptide. Key residues essential for its binding were probed by ELISA and its binding kinetics were measured by surface plasmon resonance imaging (SPRi). Its selective binding to ETV1 was assessed by immunoblots and immunofluorescence assays (IFA), and by both single and double-immuno-histochemistry (IHC) assays on prostate cancer tissue specimens.Immunoblot results showed that the mAb is highly specific and lacked cross-reactivity with other ETS factors. A minimal epitope with two phenylalanine residues at its core was found to be required for effective mAb binding. SPRi measurements revealed an equilibrium dissociation constant in the picomolar range, confirming its high affinity. ETV1 (+) tumors were detected in prostate cancer tissue microarray cases evaluated. IHC staining of whole-mounted sections revealed glands with a mosaic staining pattern of cells that are partly ETV1 (+) and interspersed with ETV1 (-) cells. Duplex IHC, using ETV1 and ERG mAbs, detected collision tumors containing glands with distinct ETV1 (+) and ERG (+) cells.The selective detection of ETV1 by the 29E4 mAb in immunoblots, IFA, and IHC assays using human prostate tissue specimens reveals a potential utility for the diagnosis, the prognosis of prostate adenocarcinoma and other cancers, and the stratification of patients for treatment by ETV1 inhibitors.

Previous studies have demonstrated effects of racialized minority status on thermal pain sensitivity, sensibility, and tolerance. However, there is limited evidence demonstrating effects of minority status on painful punctate mechanical stimuli and self-report pain. We analyzed the effects of racialized minority status on heat pain sensitivity, sensibility to painful heat and punctate mechanical stimuli, and Pain Sensitivity Questionnaire (PSQ) scores. Our secondary purpose was to test face validity of the PSQ in a US population. Using quantitative sensory testing for painful heat and punctate mechanical stimuli (forces: 64, 128, 256 and 512 mN), and self-report PSQ, we evaluated pain sensitivity and sensibility in 134 healthy participants (34 Asian, 25 Black, and 75 White). We used linear mixed models to analyze outcomes allowing maximal inclusion of incomplete data sets. Racialized minority status was associated with greater heat pain sensitivity (F=7.63; p=0.00074) and PSQ scores (F=15.45; p=9.84 × 10-7) but had no effect on painful suprathreshold heat (model improvement by addition of race: Χ2=2.199; p=0.333) or punctate mechanical stimuli (F=1.50; p=0.229). Face validity of the PSQ in racialized minorities was limited by differential experience of individual items (F=19.87; p=3.28 × 10-8). Ratings of painful suprathreshold heat (R=0.204; p=0.00020) and punctate mechanical stimuli (R=0.333; p=0.00062) positively correlated with PSQ scores. Consistent with previous research, sensitivity to painful heat was affected by racialized minority status. In contrast, there was no significant effect of racialized minority status on suprathreshold painful heat or punctate mechanical stimuli. Certain items of the PSQ are inapplicable to healthy participants from racialized minority groups. NIH National Institute of Nursing Research P30NR014129.

Sudden, unpredictable, severe acute pain episodes are the most common sickle cell disease (SCD) complication. Some SCD patients experience frequent pain episodes while others experience rare episodes. Knowledge of the biology driving this variability is limited. Using gene transcription analyses, we previously showed an elevated inflammatory response is associated with increased SCD pain episode frequency. We sought to replicate these findings in a larger SCD cohort and identify hub genes closely associated with increased pain frequency. We conducted plasma-induced transcription analyses in 132 SCD patients (baseline health) and 60 Black controls (4-21 years, both groups). 3028 differentially expressed genes between SCD patients and controls were retained for subsequent analyses with Weighted Gene Co-Expression Network Analysis (WGCNA). WGCNA was used to define modules (functionally grouped genes) and we correlated these modules with number of pain episodes requiring health care utilization in prior three years. Of 11 identified modules, four showed significant correlation with number of pain episodes. Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used for ontological analysis of the four significant modules and key biological processes identified were inflammatory response and cellular response to lipopolysaccharide. Cytoscape was used to construct a protein-protein interaction network and the 10 top hub genes identified in hierarchical order were: TNF, CCR5, CCR1, CCL2, CXCL2, ITGAM, CCL7, CXCL3, TLR2 and MMP9. These genes, as part of the inflammatory response, support the immune system contributes to increased pain episode frequency. Identified hub genes may be leveraged as therapeutic targets for reducing SCD pain episodes. 1R61NS114954-01.

Background: HIV infection persists predominantly within follicular helper CD4+ T cell-rich B cell follicles of lymphoid tissues. Cytotoxic CD8+ T cells, which are associated with natural control of HIV infection in peripheral blood, are relatively excluded from this niche, representing a potential barrier to cellular immunity and HIV cure. To better understand the mechanisms of HIV control within lymph nodes (LN), we investigated functionality, clonotypic compartmentalization, spatial localization, phenotypic characteristics and transcriptional profiles of LN-resident virus-specific and CXCR5-expressing follicular CD8+ T cells (fCD8) in persons who control HIV without medications. Methods: We obtained paired excisional inguinal LN biopsies and peripheral blood (PB) from 19 spontaneous HIV controllers and 17 HIV+ individuals on long-term ART. HIV-specific CD8+ T cell responses were identified by IFN-γ ELISpot and functional response to antigenic stimulation was measured by flow cytometry and CFSE-based proliferation assay. Clonotypic compartmentalization and transcriptional signatures associated with localization of HIV-specific CD8+ T cells were assessed via TCR and RNA-sequencing. Spatial relationships between ongoing viral replication and fCD8 cytotoxic effector potential in GCs were measured by HIV gagpol RNAscope and immunofluorescence on fixed LN sections. Results: Antigen-induced HIV-specific CD8+ T cell proliferation and cytolytic effector upregulation consistently distinguished spontaneous controllers from noncontrollers in PB (p=0.03) and LN (p=0.04). HIV-specific CD8+ T cells from both compartments shared TCR clonotypic composition (Morisita-Horn Similarity Index 0.8-1.0), consistent with ongoing infiltration from circulation. Migration into LNs was associated with gene signatures of inflammatory chemotaxis and antigen-induced effector function. The cytolytic effectors perforin and granzyme B were elevated among virus-specific CXCR5 + fCD8 s (p

Inner ear development requires the complex interaction of numerous cell types arising from multiple embryologic origins. Current knowledge of inner ear organogenesis is limited primarily to animal models. Although most mechanisms of cellular development show conservation between vertebrate species, there are uniquely human aspects of inner ear development which remain unknown. Our group recently described a model of _in vitro_ human inner ear organogenesis using pluripotent stem cells in a 3D organoid culture system. This method promotes the formation of an entire sensorineural circuit, including hair cells, inner ear neurons, and Schwann cells. Our past work has characterized certain aspects of this culture system, however we have yet to fully define all the cell types which contribute to inner ear organoid assembly. Here, our goal was to reconstruct a time-based map of _in vitro_ development during inner ear organoid induction to understand the developmental elements captured in this system. We analyzed inner ear organoid development using single-cell RNA sequencing at ten time points during the first 36 days of induction. We reconstructed the on-target progression of undifferentiated pluripotent stem cells to surface ectoderm, pre-placodal, and otic epithelial cells, including supporting cells, hair cells, and neurons, following treatment with FGF, BMP, and WNT signaling modulators. Our data revealed endogenous signaling pathway-related gene expression that may influence the course of on-target differentiation. In addition, we classified a diverse array of off-target ectodermal cell types encompassing the neuroectoderm, neural crest, and mesenchymal lineages. Our work establishes the Inner ear Organoid Developmental Atlas (IODA), which can provide insights needed for understanding human biology and refining the guided differentiation of in vitro inner ear tissue.