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Abstract Background Ischemic stroke elicits a complex and sustained immune response in the brain. Immunomodulatory treatments have long held promise for improving stroke outcomes, yet none have succeeded in the clinical setting. This lack of success is largely due to our incomplete understanding of how immune cells respond to stroke. The objective of the current study was to dissect the effect of permanent stroke on microglia, the resident immune cells within the brain parenchyma. Methods A permanent middle cerebral artery occlusion (pMCAO) model was used to induce ischemic stroke in young male and female mice. Microglia were sorted from fluorescence reporter mice after pMCAO or sham surgery and then subjected to single-cell RNA sequencing analysis. Various methods, including flow cytometry, RNA in situ hybridization, immunohistochemistry, whole-brain imaging, and bone marrow transplantation, were also employed to dissect the microglial response to stroke. Stroke outcomes were evaluated by infarct size and behavioral tests. Results First, we showed the morphologic and spatial changes in microglia after stroke. We then performed single-cell RNA sequencing analysis on microglia isolated from sham and stroke mice of both sexes. The data indicate no major sexual dimorphism in the microglial response to permanent stroke. Notably, we identified seven potential stroke-associated microglial clusters, including four major clusters characterized by a disease-associated microglia-like signature, a highly proliferative state, a macrophage-like profile, and an interferon (IFN) response signature, respectively. Importantly, we provided evidence that the macrophage-like cluster may represent the long-sought stroke-induced microglia subpopulation with increased CD45 expression. Lastly, given that the IFN-responsive subset constitutes the most prominent microglial population in the stroke brain, we used fludarabine to pharmacologically target STAT1 signaling and found that fludarabine treatment improved long-term stroke outcome. Conclusions Our findings shed new light on microglia heterogeneity in stroke pathology and underscore the potential of targeting specific microglial populations for effective stroke therapies.

Abstract Glioblastoma (GBM) remains an untreatable malignant tumor with poor patient outcomes, characterized by palisading necrosis and microvascular proliferation. While single-cell technology made it possible to characterize different lineage of glioma cells into neural progenitor-like (NPC-like), oligodendrocyte-progenitor-like (OPC-like), astrocyte-like (AC-like) and mesenchymal like (MES-like) states, it does not capture the spatial localization of these tumor cell states. Spatial transcriptomics empowers the study of the spatial organization of different cell types and tumor cell states and allows for the selection of regions of interest to investigate region-specific and cell-type-specific pathways. Here, we obtained paired 10x Chromium single-nuclei RNA-sequencing (snRNA-seq) and 10x Visium spatial transcriptomics data from three GBM patients to interrogate the GBM microenvironment. Integration of the snRNA-seq and spatial transcriptomics data reveals patterns of segregation of tumor cell states. For instance, OPC-like tumor and NPC-like tumor significantly segregate in two of the three samples. Our differentially expressed gene and pathway analyses uncovered significant pathways in functionally relevant niches. Specifically, perinecrotic regions were more immunosuppressive than the endogenous GBM microenvironment, and perivascular regions were more pro-inflammatory. Our gradient analysis suggests that OPC-like tumor cells tend to reside in areas closer to the tumor vasculature compared to tumor necrosis, which may reflect increased oxygen requirements for OPC-like cells. In summary, we characterized the localization of cell types and tumor cell states, the gene expression patterns, and pathways in different niches within the GBM microenvironment. Our results provide further evidence of the segregation of tumor cell states and highlight the immunosuppressive nature of the necrotic and perinecrotic niches in GBM.

Abstract The performance of an ejector-assisted condenser outlet split dual-evaporator cycle is compared with a conventional dual-evaporator cycle albeit consisting a pressure reducing valve. The cycles do not employ any separator due to its inability to efficiently separate the liquid and the vapor phases. The comparison of both the cycles has been made for the same cooling capacity in low-temperature evaporator and unit flow rate of R134a and R1234yf as refrigerants. The impacts of changing the operating temperatures of evaporator and condenser have been examined in the current investigation. The study reveals that with the increase in temperature of the high-temperature evaporator, the cooling capacity of the high-temperature evaporator yields, while that of the low-temperature evaporator plummets in both the cycles. Further, the compressor work is allayed in the ejector-assisted cycle; thus, the COP is enhanced considerably. The percentage COP improvement over the basic cycle is obtained from 14.7 to 17.53% for the refrigerant R1234yf and from 14.45 to 17.32% for R134a; however, the COP of both the cycles with R12134yf is slightly lower than with R134a. The ejector has been modeled assuming a constant pressure theory. The observed trend indicates that the entrainment ratio is improved with the rise in the temperature of low-temperature evaporator, whereas it is decreased with the rise in the temperature of high-temperature evaporator.

Abstract Gangliogliomas are brain tumors composed of neuron-like and macroglia-like components that occur in children and young adults. Gangliogliomas are often characterized by a rare population of immature astrocyte-appearing cells expressing CD34, a marker expressed in the neuroectoderm (neural precursor cells) during embryogenesis. New insights are needed to refine tumor classification and to identify therapeutic approaches. We evaluated five gangliogliomas with single nucleus RNA-seq, cellular indexing of transcriptomes and epitopes by sequencing, and/or spatially-resolved RNA-seq. We uncovered a population of CD34+ neoplastic cells with mixed neuroectodermal, immature astrocyte, and neuronal markers. Gene regulatory network interrogation in these neuroectoderm-like cells revealed control of transcriptional programming by TCF7L2/MEIS1-PAX6 and SOX2, similar to that found during neuroectodermal/neural development. Developmental trajectory analyses place neuroectoderm-like tumor cells as precursor cells that give rise to neuron-like and macroglia-like neoplastic cells. Spatially-resolved transcriptomics revealed a neuroectoderm-like tumor cell niche with relative lack of vascular and immune cells. We used these high resolution results to deconvolute clinically-annotated transcriptomic data, confirming that CD34+ cell-associated gene programs associate with gangliogliomas compared to other glial brain tumors. Together, these deep transcriptomic approaches characterized a ganglioglioma cellular hierarchy—confirming CD34+ neuroectoderm-like tumor precursor cells, controlling transcription programs, cell signaling, and associated immune cell states. These findings may guide tumor classification, diagnosis, prognostication, and therapeutic investigations.

Abstract A promising strategy to cure HIV‐infected individuals is to use latency reversing agents (LRAs) to reactivate latent viruses, followed by host clearance of infected reservoir cells. However, reactivation of latent proviruses within infected cells is heterogeneous and often incomplete. This fact limits strategies to cure HIV which may require complete elimination of viable virus from all cellular reservoirs. For this reason, understanding the mechanism(s) of reactivation of HIV within cellular reservoirs is critical to achieve therapeutic success. Methodologies enabling temporal tracking of single cells as they reactivate followed by sorting and molecular analysis of those cells are urgently needed. To this end, microraft arrays were adapted to image T‐lymphocytes expressing mCherry under the control of the HIV long terminal repeat (LTR) promoter, in response to the application of LRAs (prostratin, iBET151, and SAHA). In response to prostratin, iBET151, and SAHA, 30.5%, 11.2%, and 12.1% percentage of cells, respectively. The arrays enabled large numbers of single cells (>25,000) to be imaged over time. mCherry fluorescence quantification identified cell subpopulations with differing reactivation kinetics. Significant heterogeneity was observed at the single‐cell level between different LRAs in terms of time to reactivation, rate of mCherry fluorescence increase upon reactivation, and peak fluorescence attained. In response to prostratin, subpopulations of T lymphocytes with slow and fast reactivation kinetics were identified. Single T‐lymphocytes that were either fast or slow reactivators were sorted, and single‐cell RNA‐sequencing was performed. Different genes associated with inflammation, immune activation, and cellular and viral transcription factors were found.

Genes associated with increased susceptibility to multiple sclerosis (MS) have been identified, but their functions are incompletely understood. One of these genes codes for the RNA helicase DExD/H-Box Polypeptide 39B (DDX39B), which shows genetic and functional epistasis with interleukin-7 receptor-α gene (IL7R) in MS-risk. Based on evolutionary and functional arguments, we postulated that DDX39B enhances immune tolerance thereby decreasing MS risk. Consistent with such a role we show that DDX39B controls the expression of many MS susceptibility genes and important immune-related genes. Among these we identified Forkhead Box P3 (FOXP3), which codes for the master transcriptional factor in CD4+/CD25+ T regulatory cells. DDX39B knockdown led to loss of immune-regulatory and gain of immune-effector expression signatures. Splicing of FOXP3 introns, which belong to a previously unrecognized type of introns with C-rich polypyrimidine tracts, was exquisitely sensitive to DDX39B levels. Given the importance of FOXP3 in autoimmunity, this work cements DDX39B as an important guardian of immune tolerance.

Technology is constantly evolving, and chronic health issues are on the rise. It is essential to have affordable and easy access to remote biomedical measurements. This makes flexible sensors a more attractive choice owing to their high sensitivity and flexibility along with low cost and ease of use. As an additional advantage, 3D printing has become increasingly popular in areas such as biomedicine, environment, and industry. This study demonstrates 3D-printed flexible sensors for tactile sensing. A biocompatible silicone elastomer such as polydimethylsiloxane (PDMS) with low elastic modulus and high stretchability makes an excellent wearable sensor material. Incorporating CNTs at varying concentrations (0.5, 1, 2)wt% enhances the sensor’s mechanical strength, conductivity, and responsiveness to mechanical strain. In addition to enhancing the thermal stability of the composite by 44%, multi-walled carbon nanotubes (MWCNTs) also enhanced the breaking strength by 57% with a 2 wt% CNT loading. Moreover, the contact angle values improved by 15%, making it a biomedical-grade hydrophobic surface. The electrical characteristics of these sensors reveal excellent strain sensitivity, making them perfect for monitoring finger movements and biomedical measurements. Overall, 2 wt% CNT-PDMS sensors exhibit optimal performance, paving the way for advanced tactile sensing in biomedical and industrial settings.

Abstract Background Ischemic stroke is a medical emergency that primarily affects the elderly. A complex immune response in the post-stroke brain constitutes a key component of stroke pathophysiology. This study aimed to determine how stroke affects immune cell populations in the aged brain based on molecular profiles of individual cells. Methods Single-cell RNA sequencing and a new transient ischemic stroke mouse model with late reperfusion were used. Results We generated, for the first time, a composite picture of immune cell populations in the stroke aged brain at single-cell resolution. We discovered at least 6 microglial subsets in the stroke aged brain, including a potentially stroke-specific subtype. Moreover, we identified major cell subpopulations formed by infiltrated myeloid cells after stroke, and revealed their unique molecular profiles. Conclusions This study provided the first scRNA-seq data set for immune cells in the stroke aged brain, and offered novel insights into post-stroke immune cell heterogeneity.

Lysosomal inhibition elicited by palmitoyl-protein thioesterase 1 (PPT1) inhibitors such as DC661 can produce cell death, but the mechanism for this is not completely understood. Programmed cell death pathways (autophagy, apoptosis, necroptosis, ferroptosis, and pyroptosis) were not required to achieve the cytotoxic effect of DC661. Inhibition of cathepsins, or iron or calcium chelation, did not rescue DC661-induced cytotoxicity. PPT1 inhibition induced lysosomal lipid peroxidation (LLP), which led to lysosomal membrane permeabilization and cell death that could be reversed by the antioxidant N-acetylcysteine (NAC) but not by other lipid peroxidation antioxidants. The lysosomal cysteine transporter MFSD12 was required for intralysosomal transport of NAC and rescue of LLP. PPT1 inhibition produced cell-intrinsic immunogenicity with surface expression of calreticulin that could only be reversed with NAC. DC661-treated cells primed naive T cells and enhanced T cell–mediated toxicity. Mice vaccinated with DC661-treated cells engendered adaptive immunity and tumor rejection in “immune hot” tumors but not in “immune cold” tumors. These findings demonstrate that LLP drives lysosomal cell death, a unique immunogenic form of cell death, pointing the way to rational combinations of immunotherapy and lysosomal inhibition that can be tested in clinical trials.

Chronic lung allograft dysfunction (CLAD) is the leading cause of death in lung transplant recipients. CLAD is characterized clinically by a persistent decline in pulmonary function and histologically by the development of airway-centered fibrosis known as bronchiolitis obliterans. There are no approved therapies to treat CLAD, and the mechanisms underlying its development remain poorly understood. We performed single-cell RNA-Seq and spatial transcriptomic analysis of explanted tissues from human lung recipients with CLAD, and we performed independent validation studies to identify an important role of Janus kinase–signal transducer and activator of transcription (JAK-STAT) signaling in airway epithelial cells that contributes to airway-specific alloimmune injury. Specifically, we established that activation of JAK-STAT signaling leads to upregulation of major histocompatibility complex 1 (MHC-I) in airway basal cells, an important airway epithelial progenitor population, which leads to cytotoxic T cell–mediated basal cell death. This study provides mechanistic insight into the cell-to-cell interactions driving airway-centric alloimmune injury in CLAD, suggesting a potentially novel therapeutic strategy for CLAD prevention or treatment.

Abstract Background COVID-19 pneumonia has been associated with severe acute hypoxia, sepsis-like states, thrombosis and chronic sequelae including persisting hypoxia and fibrosis. The molecular hypoxia response pathway has been associated with such pathologies and our recent observations on anti-hypoxic and anti-inflammatory effects of whole aqueous extract of Adhatoda Vasica (AV) prompted us to explore its effects on relevant preclinical mouse models. Methods In this study, we tested the effect of whole aqueous extract of AV, in murine models of bleomycin induced pulmonary fibrosis, Cecum Ligation and Puncture (CLP) induced sepsis, and siRNA induced hypoxia-thrombosis phenotype. The effect on lung of AV treated naïve mice was also studied at transcriptome level. We also determined if the extract may have any effect on SARS-CoV2 replication. Results Oral administration AV extract attenuates increased airway inflammation, levels of transforming growth factor-β1 (TGF-β1), IL-6, HIF-1α and improves the overall survival rates of mice in the models of pulmonary fibrosis and sepsis and rescues the siRNA induced inflammation and associated blood coagulation phenotypes in mice. We observed downregulation of hypoxia, inflammation, TGF-β1, and angiogenesis genes and upregulation of adaptive immunity-related genes in the lung transcriptome. AV treatment also reduced the viral load in Vero cells infected with SARS-CoV2. Conclusion Our results provide a scientific rationale for this ayurvedic herbal medicine in ameliorating the hypoxia-hyperinflammation features and highlights the repurposing potential of AV in COVID-19-like conditions.

There is an urgent need to discover blood-based biomarkers to predict response to immune checkpoint inhibitors (ICI). Here the authors show that effective ICI therapy correlates with increased frequency of circulating CX3CR1+CD8+ T cells in preclinical tumor models and in a cohort of patients with non-small cell lung cancer treated with anti-PD-1.

Human red blood cells (RBCs), or erythrocytes, are the most abundant blood cells responsible for gas exchange. RBC diseases affect hundreds of millions of people and impose enormous financial and personal burdens. One well-recognized, but poorly understood feature of RBC populations within the same individual are their phenotypic heterogeneity. The granular characterization of phenotypic RBC variation in normative and disease states may allow us to identify the genetic determinants of red cell diseases and reveal novel therapeutic approaches for their treatment. Previously, we discovered diverse RNA transcripts in RBCs that has allowed us to dissect the phenotypic heterogeneity and malaria resistance of sickle red cells. However, these analyses failed to capture the heterogeneity found in RBC sub-populations. To overcome this limitation, we have performed single cell RNA-Seq to analyze the transcriptional heterogeneity of RBCs from three adult healthy donors which have been stored in the blood bank conditions and assayed at day 1 and day 15. The expression pattern clearly separated RBCs into seven distinct clusters that include one RBC cluster that expresses HBG2 and a small population of RBCs that express fetal hemoglobin (HbF) that we annotated as F cells. Almost all HBG2-expessing cells also express HBB, suggesting bi-allelic expression in single RBC from the HBG2/HBB loci, and we annotated another cluster as reticulocytes based on canonical gene expression. Additional RBC clusters were also annotated based on the enriched expression of NIX, ACVR2B and HEMGN, previously shown to be involved in erythropoiesis. Finally, we found the storage of RBC was associated with an increase in the ACVR2B and F-cell clusters. Collectively, these data indicate the power of single RBC RNA-Seq to capture and discover known and unexpected heterogeneity of RBC population.

A novel idea to create new alloys for Additive manufacturing (AM) by mixing a small addition of nanoparticles with bulk material was put forward. Integrated Computational Material Engineering (ICME) may be used to guide the AM process, predict thermal behaviour, optimize process parameters, secure net-shape, and qualify parts. Modelling is needed to predict the effect of defects and the effect of inclusions on mechanical properties, build quality and in-service performance. ICME can reduce trial-and-error fabrication and establish an AM digital twin, thereby accelerating part qualification and AM adoption. AM creates complex thermal processes which impact material microstructure and result in changes to mechanical properties in terms of strength and plasticity. In this study, Grain-Boundary Engineering (GBE) and multi-scale modelling are performed to expedite qualification of new and existing AM polycrystalline alloys.AM parts exhibit cracks, low toughness, low plasticity, and high residual stresses. To mitigate these characteristics, a methodology for GBE and microstructural control was developed. Depending on part requirements a low angle, coincident site lattice, or high angle grain boundary (LAGB,CSL, HAGB) might be the preferred option. For example, HAGB occurs in equiaxed grain microstructure, prevents precipitation and crystallization formation, and inhibits cracks and corrosion. Some polycrystalline materials may be non-weldable and exhibit cracks. Microstructural control can be used to address this phenomenon. AM offers unique advantages over casting, including material composition change and unique thermal processing. By utilizing nano-inclusions, cracks may be mitigated and AM parts improved.ICME physics-based approach is used to implement GBE modelling to improve the microstructure of an AM polycrystalline material from Equiaxed/HAGB to LAGB and CSL considering nano-scale inclusions. The ICME tool integrates meltpool engineering; thermal transport; nano-micro-mechanics; material characterization; calibration and qualification; Voronoi-tessellation, finite element modelling, and design of experiment optimization. With this background, ICME predicts time-temperature transformation, thermal properties, process map, meltpool size, precipitation, porosity, and effects of nano inclusions. In this manner, ICME helps determine ideal AM machine parameters that secure qualified parts as a consequence of the AM process.In this study, mechanical properties and fracture toughness were validated against test. Predictions included creep, crack growth, and the effect of precipitates and voids under in-service crack growth behaviour. Stainless steel (SS316L) dog bones specimens were printed and stress relieved by air and water cooling supported by fractography and imaging. The objective of this effort was to develop technologies and processes in AM design and engineering that could predetermine the microstructure of AM parts. Future efforts will employ the tool to tailor grain boundaries to produce predictable mechanical properties - including mode of failure.

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease characterized by recurrent abscesses, nodules, and sinus tracts in areas of high hair follicle and sweat gland density. These sinus tracts can present with purulent drainage and scar formation. Dysregulation of multiple immune pathways drives the complexity of HS pathogenesis and may account for the heterogeneity of treatment response in HS patients. Using transcriptomic approaches, including single-cell sequencing and protein analysis, we here characterize the innate inflammatory landscape of HS lesions. We identified a shared upregulation of genes involved in interferon (IFN) and antimicrobial defense signaling through transcriptomic overlap analysis of differentially expressed genes (DEGs) in datasets from HS skin, diabetic foot ulcers (DFUs), and the inflammatory stage of normal healing wounds. Overlap analysis between HS- and DFU-specific DEGs revealed an enrichment of gene signatures associated with monocyte/macrophage functions. Single-cell RNA sequencing further revealed monocytes/macrophages with polarization toward a pro-inflammatory M1-like phenotype and increased effector function, including antiviral immunity, phagocytosis, respiratory burst, and antibody-dependent cellular cytotoxicity. Specifically, we identified the STAT1/IFN-signaling axis and the associated IFN-stimulated genes as central players in monocyte/macrophage dysregulation. Our data indicate that monocytes/macrophages are a potential pivotal player in HS pathogenesis and their pathways may serve as therapeutic targets and biomarkers in HS treatment.

Myoepithelial tumor of lung is a rare tumor; the histopathological findings resemble the myoepithelial tumors of the salivary gland. Distinguishing low-grade nonmetastatic myoepithelial carcinoma from benign myoepithelioma can be challenging both radiologically and histomorphologically. We present a case report of a low-grade myoepithelial carcinoma of lung with contralateral lung metastasis which was treated with low-dose external beam radiotherapy

Background: Despite the significant risk of cardiovascular mortality after tetralogy of Fallot (TOF) repair, there are limited data about left ventricular (LV) cardiomyopathy in this population, thus creating important knowledge gaps. This study aims to address some of these knowledge gaps by describing the risk and prognostic implications of LV systolic dysfunction (LVD) after TOF repair. Methods: We performed a cohort study of adult patients after TOF repair with an echocardiographic assessment of LV ejection fraction (LVEF) to determine the association between LVD and cardiovascular events, defined as sustained ventricular tachycardia, aborted sudden death, heart transplantation, or death. Prevalent and incidence LVD were defined as LVEF < 50% at baseline or new decrease in LVEF to

New strategies are needed to enhance the efficacy of anti–programmed cell death protein antibody (anti–PD-1 Ab) in cancer. Here, we report that inhibiting palmitoyl-protein thioesterase 1 (PPT1), a target of chloroquine derivatives like hydroxychloroquine (HCQ), enhances the antitumor efficacy of anti–PD-1 Ab in melanoma. The combination resulted in tumor growth impairment and improved survival in mouse models. Genetic suppression of core autophagy genes, but not Ppt1, in cancer cells reduced priming and cytotoxic capacity of primed T cells. Exposure of antigen-primed T cells to macrophage-conditioned medium derived from macrophages treated with PPT1 inhibitors enhanced melanoma-specific killing. Genetic or chemical Ppt1 inhibition resulted in M2 to M1 phenotype switching in macrophages. The combination was associated with a reduction in myeloid-derived suppressor cells in the tumor. Ppt1 inhibition by HCQ, or DC661, induced cyclic GMP-AMP synthase/stimulator of interferon genes/TANK binding kinase 1 pathway activation and the secretion of interferon-β in macrophages, the latter being a key component for augmented T cell–mediated cytotoxicity. Genetic Ppt1 inhibition produced similar findings. These data provide the rationale for this combination in melanoma clinical trials and further investigation in other cancers.

Cardiac magnetic resonance imaging derived right ventricular (RV) volumes are often necessary for optimal timing of pulmonary valve replacement in patients with tetralogy of Fallot (TOF). This practice is based on previous studies that reported preoperative RV volumetric thresholds that predicted postoperative RV remodeling. As a result, pulmonary valve replacements are being performed even in asymptomatic patients based on RV volumetric thresholds that predict complete postoperative RVOT remodeling. Hence, RV volumes are now being used as surrogate markers/endpoints for future cardiovascular outcomes. Unfortunately, there are no studies showing survival benefit for performing pulmonary valve replacement at smaller RV volumes. This review underscores some of the limitations of using RV volumes as surrogate markers for clinical outcomes, and also highlights knowledge gaps about the pathophysiologic mechanism of cardiovascular death in the TOF population. Keywords: Right ventricular volumes, Tetralogy of fallot, Cardiovascular mortality

Objective To evaluate the results of operative management of subaxial spine injuries managed with 2-level anterior cervical corpectomy and fusion with a cervical locking plate and autologous bone–filled titanium mesh cage. Methods This study included 23 patients with a subaxial spine injury who matched the inclusion criteria, underwent 2-level anterior cervical corpectomy and fusion at our institution between 2013 and 2016, and were followed up for neurological recovery, axial pain, fusion, pseudarthrosis, and implant failure. Results According to Allen and Ferguson classification, there were 9 cases of distractive extension; 4 of compressive extension; 3 each of compressive flexion, vertical compression, and distractive flexion; and 1 of lateral flexion. Sixteen patients had a score of 6 on the Subaxial Injury Classification system, and the rest had a score of more than 6. The mean follow-up period was 19 months (range, 12–48 months). Neurological recovery was observed in most of the patients (78.21%). All patients experienced relief of axial pain. None of the patients received a blood transfusion. Twenty-one patients (91.3%) showed solid fusion and 2 (8.69%) showed possible pseudarthrosis, with no complications related to the cage or plate. Conclusion Two-level anterior cervical corpectomy and fusion, along with stabilization with a cervical locking plate and autologous bone graft-filled titanium mesh cage, can be considered a feasible and safe method for treating specific subaxial spine injuries, with the benefits of high primary stability, anatomical reduction, and direct decompression of the spinal cord.

Purpose: To compare the diagnostic ability of the ganglion cell analysis (GCA) and retinal nerve fiber layer (RNFL) protocol on optical coherence tomography (OCT), to diagnose preperimetric glaucoma. Methods: A prospective, cross-sectional study of 275 adult patients including 47 early glaucoma (mean deviation better than -6.0 D), 150 glaucoma suspects (106 with suspicious discs and 44 ocular hypertensive (OHT), and 78 normal controls was done. Eligible participants were scanned with the spectral domain CirrusTM OCT (Carl Zeiss Meditec, Dublin, CA). Average peripapillary RNFL thickness and GCA measurements were obtained. Area under receiver operating characteristic (AROC) curves were used to evaluate discriminant value of both protocols to diagnose likely preperimetric glaucoma among glaucoma suspects. Results: Average RNFL and GCA were significantly thinner in glaucoma patients compared to glaucoma suspects and normal controls (P < 0.001). The RNFL was 92.26 ± 8.8 μ in normal controls, 87.9 ± 12.12 μ in glaucoma suspects and significantly thinner in POAG (70.29 ± 10.18 μ; P < 0.001). The GCA was 81.94 ± 6.17 μ in normal controls, 77.69 ± 9.03 μ in glaucoma suspects, and significantly thinner in POAG (69.36 ± 11.06 μ; P < 0.001). AROCs for discriminating glaucoma suspects from normal were modest, with no difference in AROC of average RNFL or GCA measurements (DeLong; P = 0.93). Average RNFL thickness had significantly greater AROC values than average GCA for discriminating glaucoma suspects (both suspicious discs and OHT) from glaucoma (P = 0.03 and 0.05, respectively. AROC for diagnosing glaucoma was significantly better (P = 0.02) for RNFL (0.88 ± 0.03) than GCA (0.77 ± 0.04). Conclusion: In the present time, GCA measurements, as provided by the SD-OCT, do not appear to outperform RNFL measurements in the diagnosis of preperimetric glaucoma.

Alternative splicing coupled to nonsense-mediated decay (AS-NMD) is a conserved mechanism for post-transcriptional gene regulation. Here, the authors provide evidence that AS-NMD is enhanced during dietary restriction (DR) and is required for DR-mediated longevity assurance in C. elegans.

In a present study, the performance of a vapour compression–absorption cascaded refrigeration system (CRS) under fouled conditions was analysed. The main effect of fouling is to decrease the effectiveness of the heat exchanger. Thus, the overall conductance (UA) of the heat exchanger is decreased. Hence, another interpretation of fouling is to reduce the effective size of the heat exchanger. In the present work, the percentage decrease in the overall conductance value (UA) of evaporator and condenser due to their fouling is varied from 0 to 50% and its consequences on various aspects of CRS are generated to ascertain any possible patterns. The detailed first law analysis reveals that for a clean evaporator and condenser, the electricity consumption is 67.5% less than vapour compression system (VCS) for the same cooling capacity. CRS is able to save only 61.3% of electrical energy when evaporator and condenser conductance is reduced by 50% due to fouling. Evaporator and condenser fouling decreased the COP and rational efficiency of the system by 4.7% and 10.5% respectively. It is also important to note that irreversibility in the evaporator and condenser is increased by 42.4% and 62.1% respectively, when their individual performance is degraded by 50% due to fouling.

Kaposi's sarcoma (KS) is an AIDS-defining cancer caused by the KS-associated herpesvirus (KSHV). Unanswered questions regarding KS are its cellular ontology and the conditions conducive to viral oncogenesis. We identify PDGFRA(+)/SCA-1(+) bone marrow-derived mesenchymal stem cells (Pα(+)S MSCs) as KS spindle-cell progenitors and found that pro-angiogenic environmental conditions typical of KS are critical for KSHV sarcomagenesis. This is because growth in KS-like conditions generates a de-repressed KSHV epigenome allowing oncogenic KSHV gene expression in infected Pα(+)S MSCs. Furthermore, these growth conditions allow KSHV-infected Pα(+)S MSCs to overcome KSHV-driven oncogene-induced senescence and cell cycle arrest via a PDGFRA-signaling mechanism; thus identifying PDGFRA not only as a phenotypic determinant for KS-progenitors but also as a critical enabler for viral oncogenesis.

Ovarian cancer (OC) has an overall modest number of mutations that facilitate a functional immune infiltrate able to recognize tumor mutated antigens, or neoantigens. Although patient-derived xenografts (PDXs) can partially model the tumor mutational load and mimic response to chemotherapy, no study profiled a neoantigen-driven response in OC PDXs. Here we demonstrate that the genomic status of the primary tumor from an OC patient can be recapitulated in vivo in a PDX model, with the goal of defining autologous T cells activation by neoantigens using in silico, in vitro and in vivo approaches. By profiling the PDX mutanome we discovered three main clusters of mutations defining the expansion, retraction or conservation of tumor clones based on their variant allele frequencies (VAF). RNASeq analyses revealed a strong functional conservation between the primary tumor and PDXs, highlighted by the upregulation of antigen presenting pathways. We tested in vitro a set of 30 neoantigens for recognition by autologous T cells and identified a core of six neoantigens that define a potent T cell activation able to slow tumor growth in vivo. The pattern of recognition of these six neoantigens indicates the pre-existence of anti-tumor immunity in the patient. To evaluate the breadth of T cell activation, we performed single cell sequencing profiling the TCR repertoire upon stimulation with neoantigenic moieties and identified sequence motifs that define an oligoclonal and autologous T cell response. Overall, these results indicate that OC PDXs can be a valid tool to model OC response to immunotherapy.

Aim: To study the functional outcome in unstable Hangman s fracture managed with anterior decompression and stabilization with cervical locking plate and tricortical bone graft. Materials and Methods: Between 2010 and 2016, 44 patients (range: 19-75 years) with unstable Hangman's fracture underwent anterior decompression and stabilization with cervical locking plate and tricortical bone graft in our institution. Result: According to the Levine and Edwards classification, all patients were unstable with Type IA 6 (13.6), Type IIA 35 (79.5%), Type II (0), and Type III (6.8). The mean period of follow-up was 17 months (range: 6-48 months). Neurological recovery was observed in all nine patients. All patients were relieved from axial pain. None of the patients received blood transfusion. All patients showed solid fusion with no complication related to bone graft and plate. Conclusion: The anterior C2/C3 discectomy, fusion, and stabilization with cervical locking plate and tricortical bone graft are feasible and safe method in treating HangmanÊs fracture, with the benefit of high primary stability, anatomical reduction, and direct decompression of the spinal cord.

Acute cholangitis manifest as jaundice, abdominal pain and fever. Patients of severe acute cholangitis may develop septic shock, altered sensorium and later may progress to multiorgan failure. Septic arthritis of shoulder joint usually develops due to septicaemia, direct inoculation or because of direct spread of infection from adjacent tissue. There is no reported case of septic arthritis of shoulder joint, secondary to acute cholangitis in literature. Herein, we are reporting a first of its kind case of septic arthritis of left shoulder joint secondary to complication of acute cholangitis, which was managed successfully with antibiotics and arthroscopic debridement.

Memory T and B lymphocyte numbers are thought to be regulated by recent and cumulative microbial exposures. We report here that memory-phenotype lymphocyte frequencies in B, CD4 and CD8 T-cells in 3-monthly serial bleeds from healthy young adult humans were relatively stable over a 1-year period, while Plasmablast frequencies were not, suggesting that recent environmental exposures affected steady state levels of recently activated but not of memory lymphocyte subsets. Frequencies of memory B and CD4 T cells were not correlated, suggesting that variation in them was unlikely to be determined by cumulative antigenic exposures. Immunophenotyping of adult siblings showed high concordance in memory, but not of recently activated lymphocyte subsets. To explore the possibility of cell-intrinsic regulation of T cell memory, we screened effector memory-phenotype T cell (TEM) frequencies in common independent inbred mice strains. Using two pairs from these strains that differed predominantly in either CD4 TEM and/or CD8 TEM frequencies, we constructed bi-parental bone marrow chimeras in F1 recipient mice, and found that memory T cell frequencies in recipient mice were determined by donor genotypes. Together, these data suggest cell-autonomous determination of memory T niche size, and suggest mechanisms maintaining immune variability.

Kaposi's sarcoma (KS) is a highly prevalent cancer in AIDS patients, especially in sub-Saharan Africa. Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of KS and other cancers like Primary Effusion Lymphoma (PEL). In KS and PEL, all tumors harbor latent KSHV episomes and express latency-associated viral proteins and microRNAs (miRNAs). The exact molecular mechanisms by which latent KSHV drives tumorigenesis are not completely understood. Recent developments have highlighted the importance of aberrant long non-coding RNA (lncRNA) expression in cancer. Deregulation of lncRNAs by miRNAs is a newly described phenomenon. We hypothesized that KSHV-encoded miRNAs deregulate human lncRNAs to drive tumorigenesis. We performed lncRNA expression profiling of endothelial cells infected with wt and miRNA-deleted KSHV and identified 126 lncRNAs as putative viral miRNA targets. Here we show that KSHV deregulates host lncRNAs in both a miRNA-dependent fashion by direct interaction and in a miRNA-independent fashion through latency-associated proteins. Several lncRNAs that were previously implicated in cancer, including MEG3, ANRIL and UCA1, are deregulated by KSHV. Our results also demonstrate that KSHV-mediated UCA1 deregulation contributes to increased proliferation and migration of endothelial cells.

Extraction of maxillary third molar always comes as a challenge for any dental practitioner owing to its limited accessibility and minimal space for instrument placement. Moreover, the complications related to faulty extraction technique can never be ignored. We have here described a simple and easy method of extraction of the maxillary third molar tooth using hockey stick elevators which can solve most of the problems that are faced by dental surgeons in the extraction of maxillary third molars. This technique has proved its efficacy and has been time tested for removal of a maxillary third molar providing a good alternative for routine extraction techniques of maxillary third molars.

A double orifice mitral valve (DOMV) represents a rare congenital malformation characterized by two valve orifices with two separate subvalvular apparatus. We present an interesting case of an asymptomatic 1 year and 8 months old male child with DOMV who successfully underwent surgery.

Asthma is a complex disease of airways, where the interactions of immune and structural cells result in disease outcomes with airway remodeling and airway hyper-responsiveness. Polyamines, which are small-sized, natural super-cations, interact with negatively charged intracellular macromolecules, and altered levels of polyamines and their interactions have been associated with different pathological conditions including asthma. Elevated levels of polyamines have been reported in the circulation of asthmatic patients as well as in the lungs of a murine model of asthma. In various studies, polyamines were found to potentiate the pathogenic potential of inflammatory cells, such as mast cells and granulocytes (eosinophils and neutrophils), by either inducing the release of their pro-inflammatory mediators or prolonging their life span. Additionally, polyamines were crucial in the differentiation and alternative activation of macrophages, which play an important role in asthma pathology. Importantly, polyamines cause airway smooth muscle contraction and thus airway hyper-responsiveness, which is the key feature in asthma pathophysiology. High levels of polyamines in asthma and their active cellular and macromolecular interactions indicate the importance of the polyamine pathway in asthma pathogenesis; therefore, modulation of polyamine levels could be a suitable approach in acute and severe asthma management. This review summarizes the possible roles of polyamines in different pathophysiological features of asthma.

KSHV is a DNA tumor virus that causes Kaposi's sarcoma. Upon KSHV infection, only a limited number of latent genes are expressed. We know that KSHV infection regulates host gene expression, and hypothesized that latent genes also modulate the expression of host miRNAs. Aberrant miRNA expression contributes to the development of many types of cancer. Array-based miRNA profiling revealed that all six miRNAs of the oncogenic miR-17-92 cluster are up-regulated in KSHV infected endothelial cells. Among candidate KSHV latent genes, we found that vFLIP and vCyclin were shown to activate the miR-17-92 promoter, using luciferase assay and western blot analysis. The miR-17-92 cluster was previously shown to target TGF-β signaling. We demonstrate that vFLIP and vCyclin induce the expression of the miR-17-92 cluster to strongly inhibit the TGF-β signaling pathway by down-regulating SMAD2. Moreover, TGF-β activity and SMAD2 expression were fully restored when antagomirs (inhibitors) of miR-17-92 cluster were transfected into cells expressing either vFLIP or vCyclin. In addition, we utilized viral genetics to produce vFLIP or vCyclin knock-out viruses, and studied the effects in infected TIVE cells. Infection with wildtype KSHV abolished expression of SMAD2 protein in these endothelial cells. While single-knockout mutants still showed a marked reduction in SMAD2 expression, TIVE cells infected by a double-knockout mutant virus were fully restored for SMAD2 expression, compared to non-infected TIVE cells. Expression of either vFLIP or vCycIin was sufficient to downregulate SMAD2. In summary, our data demonstrate that vFLIP and vCyclin induce the oncogenic miR-17-92 cluster in endothelial cells and thereby interfere with the TGF-β signaling pathway. Manipulation of the TGF-β pathway via host miRNAs represents a novel mechanism that may be important for KSHV tumorigenesis and angiogenesis, a hallmark of KS.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a γ-herpesvirus associated with KS and two lymphoproliferative diseases. Recent studies characterized epigenetic modification of KSHV episomes during latency and determined that latency-associated genes are associated with H3K4me3 while most lytic genes are associated with the silencing mark H3K27me3. Since the latency-associated nuclear antigen (LANA) (i) is expressed very early after de novo infection, (ii) interacts with transcriptional regulators and chromatin remodelers, and (iii) regulates the LANA and RTA promoters, we hypothesized that LANA may contribute to the establishment of latency through epigenetic control. We performed a detailed ChIP-seq analysis in cells of lymphoid and endothelial origin and compared H3K4me3, H3K27me3, polII, and LANA occupancy. On viral episomes LANA binding was detected at numerous lytic and latent promoters, which were transactivated by LANA using reporter assays. LANA binding was highly enriched at H3K4me3 peaks and this co-occupancy was also detected on many host gene promoters. Bioinformatic analysis of enriched LANA binding sites in combination with biochemical binding studies revealed three distinct binding patterns. A small subset of LANA binding sites showed sequence homology to the characterized LBS1/2 sequence in the viral terminal repeat. A large number of sites contained a novel LANA binding motif (TCCAT)3 which was confirmed by gel shift analysis. Third, some viral and cellular promoters did not contain LANA binding sites and are likely enriched through protein/protein interaction. LANA was associated with H3K4me3 marks and in PEL cells 86% of all LANA bound promoters were transcriptionally active, leading to the hypothesis that LANA interacts with the machinery that methylates H3K4. Co-immunoprecipitation demonstrated LANA association with endogenous hSET1 complexes in both lymphoid and endothelial cells suggesting that LANA may contribute to the epigenetic profile of KSHV episomes.

Kaposi’s sarcoma-associated herpesvirus (KSHV) encodes 12 viral microRNAs (miRNAs) that are expressed during latency. Research into KSHV miRNA function has suffered from a lack of genetic systems to study viral miRNA mutations in the context of the viral genome. We used the Escherichia coli Red recombination system together with a new bacmid background, BAC16, to create mutants for all known KSHV miRNAs. The specific miRNA deletions or mutations and the integrity of the bacmids have been strictly quality controlled using PCR, restriction digestion, and sequencing. In addition, stable viral producer cell lines based on iSLK cells have been created for wildtype KSHV, for 12 individual miRNA knock-out mutants (ΔmiR-K12-1 through -12), and for mutants deleted for 10 of 12 (ΔmiR-cluster) or all 12 miRNAs (ΔmiR-all). NGS, in combination with SureSelect technology, was employed to sequence the entire latent genome within all producer cell lines. qPCR assays were used to verify the expression of the remaining viral miRNAs in a subset of mutants. Induction of the lytic cycle leads to efficient production of progeny viruses that have been used to infect endothelial cells. Wt BAC16 and miR mutant iSLK producer cell lines are now available to the research community.

DNA methylation is crucial for gene regulation and maintenance of genomic stability. Rat has been a key model system in understanding mammalian systemic physiology, however detailed rat methylome remains uncharacterized till date. Here, we present the first high resolution methylome of rat liver generated using Methylated DNA immunoprecipitation and high throughput sequencing (MeDIP-Seq) approach. We observed that within the DNA/RNA repeat elements, simple repeats harbor the highest degree of methylation. Promoter hypomethylation and exon hypermethylation were common features in both RefSeq genes and expressed genes (as evaluated by proteomic approach). We also found that although CpG islands were generally hypomethylated, about 6% of them were methylated and a large proportion (37%) of methylated islands fell within the exons. Notably, we obeserved significant differences in methylation of terminal exons (UTRs); methylation being more pronounced in coding/partially coding exons compared to the non-coding exons. Further, events like alternate exon splicing (cassette exon) and intron retentions were marked by DNA methylation and these regions are retained in the final transcript. Thus, we suggest that DNA methylation could play a crucial role in marking coding regions thereby regulating alternative splicing. Apart from generating the first high resolution methylome map of rat liver tissue, the present study provides several critical insights into methylome organization and extends our understanding of interplay between epigenome, gene expression and genome stability.