Multilineage murine stem cells generate complex organoids to model distal lung development and disease.

Organoids derived from mouse and human stem cells have recently emerged as a powerful tool to study organ development and disease. We here established a three-dimensional (3D) murine bronchioalveolar lung organoid (BALO) model that allows clonal expansion and self-organization of FACS-sorted bronchioalveolar stem cells (BASCs) upon co-culture with lung-resident mesenchymal cells. BALOs yield a highly branched 3D structure within 21 days of culture, mimicking the cellular composition of the bronchioalveolar compartment as defined by single-cell RNA sequencing and fluorescence as well as electron microscopic phenotyping. Additionally, BALOs support engraftment and maintenance of the cellular phenotype of injected tissue-resident macrophages. We also demonstrate that BALOs recapitulate lung developmental defects after knockdown of a critical regulatory gene, and permit modeling of viral infection. We conclude that the BALO model enables reconstruction of the epithelial-mesenchymal-myeloid unit of the distal lung, thereby opening numerous new avenues to study lung development, infection, and regenerative processes in vitro.

Molecular Understanding of Adhesion of Epoxy Resin to Graphene and Graphene Oxide Surfaces in Terms of Orbital Interactions.

The adhesion mechanism of epoxy resin (ER) cured material consisting of diglycidyl ether of bisphenol A (DGEBA) and 4,4'-diaminodiphenyl sulfone (DDS) to pristine graphene and graphene oxide (GO) surfaces is investigated on the basis of first-principles density functional theory (DFT) with dispersion correction. Graphene is often used as a reinforcing filler incorporated into ER polymer matrices. The adhesion strength is significantly improved by using GO obtained by the oxidation of graphene. The interfacial interactions at the ER/graphene and ER/GO interfaces were analyzed to clarify the origin of this adhesion. The contribution of dispersion interaction to the adhesive stress at the two interfaces is almost identical. In contrast, the DFT energy contribution is found to be more significant at the ER/GO interface. Crystal orbital Hamiltonian population (COHP) analysis suggests the existence of hydrogen bonding (H-bonding) between the hydroxyl, epoxide, amine, and sulfonyl groups of the ER cured with DDS and the hydroxyl groups of the GO surface, in addition to the OH-π interaction between the benzene rings of ER and the hydroxyl groups of the GO surface. The H-bond has a large orbital interaction energy, which is found to contribute significantly to the adhesive strength at the ER/GO interface. The overall interaction at the ER/graphene is much weaker due to antibonding type interactions just below the Fermi level. This finding indicates that only dispersion interaction is significant when ER is adsorbed on the graphene surface.

UltraSound Axillary Vein Access (USAA): Learning curve and randomized comparison to traditional venous access for cardiac device implantation.

BACKGROUND: Many techniques exist for venous access (VA) during cardiac implantable electronic device (CIED) implantation. OBJECTIVE: We sought to evaluate the learning curve with ultrasound (US) guided axillary vein access (USAA). METHODS: Single-center prospective randomized controlled trial of patients undergoing CIED implantation. Patients were randomized in a 2:1 fashion to USAA versus conventional VA techniques. The primary outcomes were the success rates, VA times and 30-day complication rates. RESULTS: The study included 100 patients (age 68 ± 14 years, BMI 27 ± 4 kg/m2 ). USAA was successful in 66/70 implants (94%). Initial attempts at conventional VA included 47% axillary (n = 14), 30% (n = 9) cephalic, and 23% (n = 7) subclavian. The median access time was longer for USAA than conventional access (8.3 IQR 4.2-15.3 min vs. 5.2 IQR 3.4-8.6 min, p = .009). Among the five inexperienced USAA implanters, there was a significant improvement in median access time from first to last tertile of USAA implants (17.0 IQR 7.0-21.0 min to 8.6 IQR 4.5-10.8 min, p = .038). The experienced USAA implanter had similar access times with USAA compared with conventional access (4.0 IQR 3.3-4.7 min vs. 5.2 IQR 3.4-8.6 min, p = .15). Venograms were less common with USAA than conventional access (2% vs. 33%, p < .0001). The 30-day complication rate was similar with USAA (n = 4/70, 6%) versus conventional (n = 3/30, 10%, p = .44). CONCLUSION: Although the success rate with USAA was high, there was a significant learning curve. Once experienced with the USAA technique, there is the potential for reduced complications without adding to the procedure duration.

A critical role for miR-142 in alveolar epithelial lineage formation in mouse lung development.

The respiratory epithelium arises from alveolar epithelial progenitors which differentiate into alveolar epithelial type 1 (AT1) and type 2 (AT2) cells. AT2 cells are stem cells in the lung critical for the repair process after injury. Mechanisms regulating AT1 and AT2 cell maturation are poorly defined. We report that the activation of the glucocorticoid pathway in an in vitro alveolar epithelial lineage differentiation assay led to increased AT2 marker Sftpc and decreased miR-142 expression. Using miR-142 KO mice, we demonstrate an increase in the AT2/AT1 cell number ratio. Overexpression of miR-142 in alveolar progenitor cells in vivo led to the opposite effect. Examination of the KO lungs at E18.5 revealed enhanced expression of miR-142 targets Apc, Ep300 and Kras associated with increased ß-catenin and p-Erk signaling. Silencing of miR-142 expression in lung explants grown in vitro triggers enhanced Sftpc expression as well as increased AT2/AT1 cell number ratio. Pharmacological inhibition of Ep300-ß-catenin but not Erk in vitro prevented the increase in Sftpc expression triggered by loss of miR-142. These results suggest that the glucocorticoid-miR-142-Ep300-ß-catenin signaling axis controls pneumocyte maturation.

Evidence for the involvement of fibroblast growth factor 10 in lipofibroblast formation during embryonic lung development.

Lipid-containing alveolar interstitial fibroblasts (lipofibroblasts) are increasingly recognized as an important component of the epithelial stem cell niche in the rodent lung. Although lipofibroblasts were initially believed merely to assist type 2 alveolar epithelial cells in surfactant production during neonatal life, recent evidence suggests that these cells are indispensable for survival and growth of epithelial stem cells during adulthood. Despite increasing interest in lipofibroblast biology, little is known about their cellular origin or the molecular pathways controlling their formation during embryonic development. Here, we show that a population of lipid-droplet-containing stromal cells emerges in the developing mouse lung between E15.5 and E16.5. This is accompanied by significant upregulation, in the lung mesenchyme, of peroxisome proliferator-activated receptor gamma (master switch of lipogenesis), adipose differentiation-related protein (marker of mature lipofibroblasts) and fibroblast growth factor 10 (previously shown to identify a subpopulation of lipofibroblast progenitors). We also demonstrate that although only a subpopulation of total embryonic lipofibroblasts derives from Fgf10(+) progenitor cells, in vivo knockdown of Fgfr2b ligand activity and reduction in Fgf10 expression lead to global reduction in the expression levels of lipofibroblast markers at E18.5. Constitutive Fgfr1b knockouts and mutants with conditional partial inactivation of Fgfr2b in the lung mesenchyme reveal the involvement of both receptors in lipofibroblast formation and suggest a possible compensation between the two receptors. We also provide data from human fetal lungs to demonstrate the relevance of our discoveries to humans. Our results reveal an essential role for Fgf10 signaling in the formation of lipofibroblasts during late lung development.

Fgf10 deficiency is causative for lethality in a mouse model of bronchopulmonary dysplasia.

Inflammation-induced FGF10 protein deficiency is associated with bronchopulmonary dysplasia (BPD), a chronic lung disease of prematurely born infants characterized by arrested alveolar development. So far, experimental evidence for a direct role of FGF10 in lung disease is lacking. Using the hyperoxia-induced neonatal lung injury as a mouse model of BPD, the impact of Fgf10 deficiency in Fgf10+/- versus Fgf10+/+ pups was investigated. In normoxia, no lethality of Fgf10+/+ or Fgf10+/- pups was observed. By contrast, all Fgf10+/- pups died within 8 days of hyperoxic injury, with lethality starting at day 5, whereas Fgf10+/+ pups were all alive. Lungs of pups from the two genotypes were collected on postnatal day 3 following normoxia or hyperoxia exposure for further analysis. In hyperoxia, Fgf10+/- lungs exhibited increased hypoalveolarization. Analysis by FACS of the Fgf10+/- versus control lungs in normoxia revealed a decreased ratio of alveolar epithelial type II (AECII) cells over total Epcam-positive cells. In addition, gene array analysis indicated reduced AECII and increased AECI transcriptome signatures in isolated AECII cells from Fgf10+/- lungs. Such an imbalance in differentiation is also seen in hyperoxia and is associated with reduced mature surfactant protein B and C expression. Attenuation of the activity of Fgfr2b ligands postnatally in the context of hyperoxia also led to increased lethality with decreased surfactant expression. In summary, decreased Fgf10 mRNA levels lead to congenital lung defects, which are compatible with postnatal survival, but which compromise the ability of the lungs to cope with sub-lethal hyperoxic injury. Fgf10 deficiency affects quantitatively and qualitatively the formation of AECII cells. In addition, Fgfr2b ligands are also important for repair after hyperoxia exposure in neonates. Deficient AECII cells could be an additional complication for patients with BPD. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

MicroRNA-142 is a multifaceted regulator in organogenesis, homeostasis, and disease.

Over the past decade, microRNA-142 (miR-142) is emerging as a major regulator of cell fate decision in the hematopoietic system. However, miR-142 is expressed in many other tissues, and recent evidence suggests that it may play a more pleiotropic role during embryonic development. In addition, miR-142 has been shown to play important functions in disease. miR-142 displays a functional role in cancer, virus infection, inflammation, and immune tolerance. Both a guide strand (miR-142-3p) and passenger strand (miR-142-5p) are generated from the miR-142 hairpin. miR-142-3p and -5p display overlapping but also independent target genes. Loss of function mouse models (genetrap, global knock out [KO], and conditional KO) have been reported and support the important role of miR-142 in different biological processes. This review will summarize the abundant literature already available for miR-142 and will lay the foundation for future works on this important microRNA. Developmental Dynamics 246:285-290, 2017. © 2016 Wiley Periodicals, Inc.

miR-142-3p balances proliferation and differentiation of mesenchymal cells during lung development.

The regulation of the balance between proliferation and differentiation in the mesenchymal compartment of the lung is largely uncharacterized, unlike its epithelial counterpart. In this study, we determined that miR-142-3p contributes to the proper proliferation of mesenchymal progenitors by controlling the level of WNT signaling. miR-142-3p can physically bind to adenomatous polyposis coli mRNA, functioning to regulate its expression level. In miR-142-3p loss-of-function experiments, proliferation of parabronchial smooth muscle cell progenitors is significantly impaired, leading to premature differentiation. Activation of WNT signaling in the mesenchyme, or Apc loss of function, can both rescue miR-142-3p knockdown. These findings show that in the embryonic lung mesenchyme, the microRNA machinery modulates the level of WNT signaling, adding an extra layer of control in the feedback loop between FGFR2C and ß-catenin-mediated WNT signaling.

Segawa Syndrome, a Dramatic Response to Dopamine.

Segawa syndrome usually manifests as dystonia, disturbance of gait with fatigue, and may be confused with spasticity. Also known as dopamine-responsive dystonia (DRD), it should be considered in any child who presents with paroxysmal or progressive hypertonia of unknown etiology, which responds dramatically to levodopa. It is a clinical diagnosis, but the level of pterins in cerebrospinal fluid and guanosine triphosphate cyclohydrolase-1 (GTCH 1) gene mutation testing done by molecular genetic testing are confirmatory. Our case is a 45-year female with a family history of similar illness expressed as autosomal recessive inheritance pattern. She had symptoms onset at an early age of 13 years with features of dystonia of predominantly lower limbs, hence the inability to maintain posture and walk. Dramatic improvement with levodopa but sudden deterioration to dystonia due to noncompliance was evident in our patient with troublesome features of concomitant adjustment disorder during presentation.

Knowledge and Attitude about Labor Epidural Analgesia among Pregnant Women Attending Antenatal Clinic.

BACKGROUND: Labour pain is one of the most painful experiences that a woman faces in her lifetime. There are various options for labour pain management. Epidural analgesia is the gold standard method for pain management during labour. In developing and under-developed countries there are many challenges to provide this service, a very important one being knowledge about epidural analgesia among the parturients and the physicians. METHODS: A prospective cross-sectional study was carried out among 384 pregnant women attending Antenatal clinic of a tertiary level teaching hospital using pretested structured questionnaire prepared by a team of anaesthesiologists and obstetricians. Non-probability purposive sampling technique was used. The data was analysed by using SPSS version 20.0. Descriptive and inferential statistical methods were used for analysis. RESULTS: Of the 384 pregnant women, only 29.4% had knowledge about some form of labour analgesia techniques and only 16% of the participants were aware of labour epidural analgesia. Also, only 6.2% were aware that epidural service is available at our hospital. Regarding acceptance, only 42.4% were willing to use epidural analgesia in their present pregnancy. CONCLUSIONS: The present study findings revealed that a significant number of participants had very limited knowledge about labour epidural analgesia and were also unaware of the availability of the service in our hospital. Educational programs need to be provided to all the pregnant women for enhancement of knowledge and awareness about epidural labour analgesia to increase its acceptability among them.

Sustained cancer-relevant alternative RNA splicing events driven by PRMT5 in high-risk neuroblastoma.

Protein arginine methyltransferase 5 (PRMT5) is over-expressed in a wide variety of cancers and is implicated as having a key oncogenic role, achieved in part through its control of the master transcription regulator E2F1. We investigated the relevance of PRMT5 and E2F1 in neuroblastoma (NB) and found that elevated expression of PRMT5 and E2F1 occurs in poor prognosis high-risk disease and correlates with an amplified Myelocytomatosis viral-related oncogene, neuroblastoma-derived (MYCN) gene. Our results show that MYCN drives the expression of splicing factor genes that, together with PRMT5 and E2F1, lead to a deregulated alternative RNA splicing programme that impedes apoptosis. Pharmacological inhibition of PRMT5 or inactivation of E2F1 restores normal splicing and renders NB cells sensitive to apoptosis. Our findings suggest that a sustained cancer-relevant alternative RNA splicing programme desensitises NB cells to apoptosis, and identify PRMT5 as a potential therapeutic target for high-risk disease.

Evaluation of resazurin phenoxazine dye as a highly sensitive cell viability potency assay for natural killer cell-derived extracellular vesicle-based cancer biotherapeutics.

Natural killer cell-derived extracellular vesicles (NK-EVs) are candidate biotherapeutics against various cancers. However, standardised potency assays are necessary for a reliable assessment of NK-EVs' cytotoxicity. This study aims to thoroughly evaluate a highly sensitive resazurin phenoxazine-based cell viability potency assay (measurement of the cellular redox metabolism) for quantifying the cytotoxicity of NK-EVs against leukaemia K562 cells (suspension model) and breast cancer MDA-MB-231 cells (adherent model) in vitro. The assay was evaluated based on common analytical parameters setforth by regulatory guidelines, including specificity, selectivity,accuracy, precision, linearity, range and stability. Our results revealed that this resazurin-based cell viability potency assay reliably and reproducibly measured a dose-response of NK-EVs' cytotoxic activity against both cancer models. The assay showed precision with 5% and 20% variation for intra-run and inter-run variability. The assay signal showed specificity and selectivity of NK-EVs against cancer target cells, as evidenced by the diminished viability of cancer cells following a 5-hour treatment with NK-EVs, without any detectable interference or background. The linearity analysis of target cancer cells revealed strong linearity for densities of 5000 K562 and 1000 MDA-MB-231 cells per test with a consistent range. Importantly, NK-EVs' dose-response for cytotoxicity showed a strong correlation (|ρ| ∼ 0.8) with the levels of known cytotoxic factors associated with the NK-EVs' corona (FasL, GNLY, GzmB, PFN and IFN-γ), thereby validating the accuracy of the assay. The assay also distinguished cytotoxicity changes in degraded NK-EVs, indicating the ability of the assay to detect the potential loss of sample integrity. Compared to other commonly reported bioassays (i.e., flow cytometry, cell counting, lactate dehydrogenase release assay, DNA-binding reporter assay and confluence assay), our results support this highly sensitive resazurin-based viability potency assay as a high-throughput and quantitative method for assessing NK-EVs' cytotoxicity against both suspension and adherent cancer models for evaluating NK-EVs' biotherapeutics.

Progeria-based vascular model identifies networks associated with cardiovascular aging and disease.

Hutchinson-Gilford Progeria syndrome (HGPS) is a lethal premature aging disorder caused by a de novo heterozygous mutation that leads to the accumulation of a splicing isoform of Lamin A termed progerin. Progerin expression deregulates the organization of the nuclear lamina and the epigenetic landscape. Progerin has also been observed to accumulate at low levels during normal aging in cardiovascular cells of adults that do not carry genetic mutations linked with HGPS. Therefore, the molecular mechanisms that lead to vascular dysfunction in HGPS may also play a role in vascular aging-associated diseases, such as myocardial infarction and stroke. Here, we show that HGPS patient-derived vascular smooth muscle cells (VSMCs) recapitulate HGPS molecular hallmarks. Transcriptional profiling revealed cardiovascular disease remodeling and reactive oxidative stress response activation in HGPS VSMCs. Proteomic analyses identified abnormal acetylation programs in HGPS VSMC replication fork complexes, resulting in reduced H4K16 acetylation. Analysis of acetylation kinetics revealed both upregulation of K16 deacetylation and downregulation of K16 acetylation. This correlates with abnormal accumulation of error-prone nonhomologous end joining (NHEJ) repair proteins on newly replicated chromatin. The knockdown of the histone acetyltransferase MOF recapitulates preferential engagement of NHEJ repair activity in control VSMCs. Additionally, we find that primary donor-derived coronary artery vascular smooth muscle cells from aged individuals show similar defects to HGPS VSMCs, including loss of H4K16 acetylation. Altogether, we provide insight into the molecular mechanisms underlying vascular complications associated with HGPS patients and normative aging.

Targeting CD73 with flavonoids inhibits cancer stem cells and increases lymphocyte infiltration in a triple-negative breast cancer mouse model.

Introduction: Chemotherapy remains the mainstay treatment for triple-negative breast cancer (TNBC) due to the lack of specific targets. Given a modest response of immune checkpoint inhibitors in TNBC patients, improving immunotherapy is an urgent and crucial task in this field. CD73 has emerged as a novel immunotherapeutic target, given its elevated expression on tumor, stromal, and specific immune cells, and its established role in inhibiting anti-cancer immunity. CD73-generated adenosine suppresses immunity by attenuating tumor-infiltrating T- and NK-cell activation, while amplifying regulatory T cell activation. Chemotherapy often leads to increased CD73 expression and activity, further suppressing anti-tumor immunity. While debulking the tumor mass, chemotherapy also enriches heterogenous cancer stem cells (CSC), potentially leading to tumor relapse. Therefore, drugs targeting both CD73, and CSCs hold promise for enhancing chemotherapy efficacy, overcoming treatment resistance, and improving clinical outcomes. However, safe and effective inhibitors of CD73 have not been developed as of now. Methods: We used in silico docking to screen compounds that may be repurposed for inhibiting CD73. The efficacy of these compounds was investigated through flow cytometry, RT-qPCR, CD73 activity, cell viability, tumorsphere formation, and other in vitro functional assays. For assessment of clinical translatability, TNBC patient-derived xenograft organotypic cultures were utilized. We also employed the ovalbumin-expressing AT3 TNBC mouse model to evaluate tumor-specific lymphocyte responses. Results: We identified quercetin and luteolin, currently used as over-the-counter supplements, to have high in silico complementarity with CD73. When quercetin and luteolin were combined with the chemotherapeutic paclitaxel in a triple-drug regimen, we found an effective downregulation in paclitaxel-enhanced CD73 and CSC-promoting pathways YAP and Wnt. We found that CD73 expression was required for the maintenance of CD44highCD24low CSCs, and co-targeting CD73, YAP, and Wnt effectively suppressed the growth of human TNBC cell lines and patient-derived xenograft organotypic cultures. Furthermore, triple-drug combination inhibited paclitaxel-enriched CSCs and simultaneously improved lymphocyte infiltration in syngeneic TNBC mouse tumors. Discussion: Conclusively, our findings elucidate the significance of CSCs in impairing anti-tumor immunity. The high efficacy of our triple-drug regimen in clinically relevant platforms not only underscores the importance for further mechanistic investigations but also paves the way for potential development of new, safe, and cost-effective therapeutic strategies for TNBC.

Levetiracetam-Induced Acute Psychosis in an Adolescent.

Levetiracetam (LEV), a second-generation antiepileptic, is used as an adjunct therapy in primary generalized tonic-clonic seizures, refractory partial-onset seizures, and seizure prophylaxis after brain surgery. It is well tolerated, effective and has a convenient dosing regimen. As any other drugs, it has some adverse drug effects, including neuropsychiatric adverse effects ranging from agitation and mood symptoms to psychosis and suicide. Strong diagnostics guidelines are yet to be formulated for LEV-induced psychosis; however, complete recovery from psychotic symptoms after stopping LEV supports the possible adverse reaction from Naranjo's algorithm and, hence, the diagnosis. This case report presents a 16 years boy with focal onset generalized tonic-clonic seizure, whose drug regimen was switched to LEV, following which he had the delusion of persecution, second-person auditory hallucination, and aggressive behavior, which decreased on the 2nd day of cessation of LEV.

Citrullination and the protein code: crosstalk between post-translational modifications in cancer.

Post-translational modifications (PTMs) of proteins are central to epigenetic regulation and cellular signalling, playing an important role in the pathogenesis and progression of numerous diseases. Growing evidence indicates that protein arginine citrullination, catalysed by peptidylarginine deiminases (PADs), is involved in many aspects of molecular and cell biology and is emerging as a potential druggable target in multiple diseases including cancer. However, we are only just beginning to understand the molecular activities of PADs, and their underlying mechanistic details in vivo under both physiological and pathological conditions. Many questions still remain regarding the dynamic cellular functions of citrullination and its interplay with other types of PTMs. This review, therefore, discusses the known functions of PADs with a focus on cancer biology, highlighting the cross-talk between citrullination and other types of PTMs, and how this interplay regulates downstream biological events. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Correlation of Body Mass Index and Oxygen Saturation in Chronic Obstructive Pulmonary Disease Patients at a Tertiary Care Center in Nepal: A Cross-Sectional Study.

Purpose: Chronic obstructive pulmonary disease (COPD) is a major cause of mortality and morbidity worldwide. COPD is associated with clinically relevant pulmonary and extrapulmonary manifestations, including hypoxemia and weight loss. The correlation of body mass index (BMI) and oxygen saturation (SpO2) with COPD grades may provide a useful additional marker for understanding and managing the disease. The aim of the study was to study the correlation of BMI and oxygen saturation with COPD in patients presenting to a tertiary care center in Nepal. Patients and Methods: A descriptive cross-sectional study was conducted among 145 COPD patients visiting the Department of Medicine in Shree Birendra Hospital between 1 March 2019 and 28 February 2020. A non-probability purposive sampling method was used and data were analyzed using SPSS version 21. A p-value of <0.05 was considered significant. Results: Out of 145 COPD patients, 58 (40%) were underweight, 53 (36.55%) were of normal weight, 20 (13.79%) were overweight, and 14 (9.6%) were obese. The number of underweight patients was highest in COPD grade 4 and lowest in COPD grade 1. The proportion of subjects with underweight BMI and hypoxia increased with COPD severity, and both were statistically significant (p-values <0.01). Conclusion: Our study shows that BMI and oxygen saturation have an inverse association with COPD severity. The correlation of BMI and oxygen saturation with COPD grade could provide a supplementary marker of disease severity, which could be useful in the understanding of the disease process and subsequent management of COPD.

Mean Brixia Severity Scores among Symptomatic COVID-19 Patients in a Tertiary Care Centre: A Descriptive Cross-sectional Study.

Introduction: COVID-19 is a highly contagious viral disease which escalated into a global pandemic since its outbreak on 31 December 2019. Chest X-rays are the most common investigation in suspected cases to diagnose and manage pneumonia. The aim of this study was to find out the mean Brixia severity scores among symptomatic COVID-19 patients in a tertiary care centre. Methods: A descriptive cross-sectional study was conducted among the chest X-rays of symptomatic COVID-19-positive patients of a tertiary care centre. Data from 1 May 2021 to 31 July 2021 were collected between 1 August 2022 and 1 January 2023 from the hospital records. Ethical approval was taken from Institutional Review Committee (Reference number: 01-079/080). Patients with reverse transcriptase polymerase chain reaction with symptoms of COVID-19 were included in this study. Convenience sampling method was used. Point estimate and 95% Confidence Interval were calculated. Results: Among the total of 300 patients, the mean Brixia severity score was 7.15±5.07 and out of 235 patients with abnormal chest X-ray, the mean Brixia severity score was 9.13±3.84. A total of 68 (22.66%) patients had mild, 115 (38.33%) had moderate and 52 (17.33%) had severe scores. Conclusions: The mean Brixia severity score among symptomatic COVID-19 patients was found to be higher than the other studies done in similar settings. Keywords: COVID-19; Nepal; pneumonia; prevalence; x-ray.

The HDAC inhibitor zabadinostat is a systemic regulator of adaptive immunity.

Protein acetylation plays a key role in regulating cellular processes and is subject to aberrant control in diverse pathologies. Although histone deacetylase (HDAC) inhibitors are approved drugs for certain cancers, it is not known whether they can be deployed in other therapeutic contexts. We have explored the clinical HDAC inhibitor, zabadinostat/CXD101, and found that it is a stand-alone regulator of the adaptive immune response. Zabadinostat treatment increased expression of MHC class I and II genes in a variety of cells, including dendritic cells (DCs) and healthy tissue. Remarkably, zabadinostat enhanced the activity of DCs, and CD4 and CD8 T lymphocytes. Using an antigenic peptide presented to the immune system by MHC class I, zabadinostat caused an increase in antigen-specific CD8 T lymphocytes. Further, mice immunised with covid19 spike protein and treated with zabadinostat exhibit enhanced covid19 neutralising antibodies and an increased level of T lymphocytes. The enhanced humoral response reflected increased activity of T follicular helper (Tfh) cells and germinal centre (GC) B cells. Our results argue strongly that zabadinostat has potential to augment diverse therapeutic agents that act through the immune system.

Analysis of conditions affecting auto-phosphorylation of human kinases during expression in bacteria.

Bacterial over-expression of kinases is often associated with high levels of auto-phosphorylation resulting in heterogeneous recombinant protein preparations or sometimes in insoluble protein. Here we present expression systems for nine kinases in Escherichia coli and, for the most heavily phosphorylated, the characterisation of factors affecting auto-phosphorylation. Experiments showed that the level of auto-phosphorylation was proportional to the rate of expression. Comparison of phosphorylation states following in vitro phosphorylation with phosphorylation states following expression in E. coli showed that the non-physiological 'hyper-phosphorylation' was occurring at sites that would require local unfolding to be accessible to a kinase active site. In contrast, auto-phosphorylation on unphosphorylated kinases that had been expressed in bacteria overexpressing λ-phosphatase was only observed on distinct exposed sites. Remarkably, the Ser/Thr kinase PLK4 auto-phosphorylated on a tyrosine residue (Tyr177) located in the activation segment. The results give support to a mechanism in which auto-phosphorylation occurs before or during protein folding. In addition, the expression systems and protocols presented will be a valuable resource to the research community.