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Alveolar Type II (AT2) epithelial cell dysfunction is a hallmark of the pathogenesis of Idiopathic Pulmonary Fibrosis (IPF), a progressive interstitial lung disease. Mutations in the surfactant protein C (SP-C) gene (SFTPC) are a high effect size etiological cause of PF in a subset of these patients. Prior in vitro studies from our lab showed that SFTPC mutations located within the BRICHOS domain of the SP-C proprotein result in production of ER retained and aggregation prone conformers. A major aspect of the proteostasis network is the unfolded protein response (UPR), which is initiated when misfolded proteins activate one or more of 3 ER transmembrane sensing proteins (Protein kinase R-like ER kinase (PERK), the Inositol-Requiring kinase 1 (IRE1) and Activating Transcription Factor 6 (ATF6). Consistent with this, we published that clinical IPF associated BRICHOS SFTPC mutations activate all 3 arms of UPR/ER stress signaling in mice in vivo. The goal of this study was to define AT2 proteostatic responses to SFTPC expression and the contribution of ATF6 to the UPR. For this, we generated stable doxycycline-inducible Mouse Lung Epithelial (MLE-12) cell lines expressing either Wild Type (SP-CWT) or an aggregating mutant (SP-CC121G) which were interrogated using cycloheximide chase and / or treatments with activators or inhibitors of ATF6, IRE1, and PERK as well as ERAD (vp97) or autophagy. Readouts included qPCR, luciferase reporter assays, and immunoblotting to assess UPR signaling and apoptosis, coupled with fluorescence microscopy to localize SP-C expression. Using this model system we found that SP-CC121G was ER retained and activated the UPR within 8 hours of Dox induction expression as measured by BIP expression. Commensurate with this both IRE1a (as spliced XBP1 (sXBP1)) and ATF6 were activated by SP-CC121G. The clearance of both mutant SP-CC121G as well as a portion of proSP-CWT was dependent on ERAD as its disruption using the vp97 inhibitor Eeyarestatin I induced formation of higher molecular weight aggregates while bafilomycin failed to induce SP-CC121G aggregates or block their clearance. ATF6 and IRE1 activation during ERAD inhibition accelerated aggregate protein degradation. When SP-CC121G MLE12 cells were treated with either an ATF6 inhibitor or an IRE1 activator, marked increases in levels of C/EBP Homologous Protein (CHOP) occur while either ATF6 agonism or IRE1 inhibition decreased CHOP expression. Collectively, these our data demonstrate a primary role for ERAD in the proteostatic response to SP-C expression while ATF6 agonism appears to provide protection from SFTPC mutant expression in part by accelerating degradation of SP-C aggregates in the face of IRE1 UPR signaling. NIH R01 HL145408 (MFB), VA Merit Review 2I01BX001176 (MFB) and NIH F32HL160011 (LRR) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Idiopathic Pulmonary Fibrosis (IPF) is a chronic progressive fibrotic interstitial lung disease (ILD). A barrier to developing more effective therapies for IPF is the dearth of preclinical models that recapitulate the early pathobiology of this disease. Intratracheal bleomycin, the conventional preclinical murine model of IPF, fails to reproduce the intrinsic dysfunction to the alveolar epithelial type 2 cell (AEC2) that is thought to be a proximal event in the pathogenesis of IPF. Murine fibrosis models based on Surfactant Protein C gene (

Many human acid tolerant bacterial and fungal pathogens can be transmitted through the consumption of the contaminated fruit juices. We aim to formulate essential oil nanoemulsions (basil, black seed, turmeric, clove & cinnamon), determine their ability to clear contamination by food borne bacterial pathogens from fruit juices. The antibacterial activity of the optimised formulations was tested in the fruit juices against bacterial pathogens causing gastrointestinal tract infections. The minimum bactericidal concentration (MBC) of clove emulsions ranged from 15.6 to 25 μL mL⁻¹. Cinnamon oil emulsion had an MBC ranging between 15 and 31 μL mL⁻¹. At MBC, cinnamon oil emulsions caused a 6log₁₀ decrease in viable counts by 8 h and maintained the sterility of fruit juices for 7 days at ambient temperature. Thus, clove and cinnamon microemulsions can be used as juice additives to control food borne bacterial pathogens and maintain the bacterial sterility of fruit juices.

The establishment of cardiac function in the developing embryo is essential to ensure blood flow and, therefore, growth and survival of the animal. The molecular mechanisms controlling normal cardiac rhythm remain to be fully elucidated. From a forward genetic screen, we identified a unique mutant, grime, that displayed a specific cardiac arrhythmia phenotype. We show that loss-of-function mutations in tmem161b are responsible for the phenotype, identifying Tmem161b as a regulator of cardiac rhythm in zebrafish. To examine the evolutionary conservation of this function, we generated knockout mice for Tmem161b. Tmem161b knockout mice are neonatal lethal and cardiomyocytes exhibit arrhythmic calcium oscillations. Mechanistically, we find that Tmem161b is expressed at the cell membrane of excitable cells and live imaging shows it is required for action potential repolarization in the developing heart. Electrophysiology on isolated cardiomyocytes demonstrates that Tmem161b is essential to inhibit Ca2+ and K+ currents in cardiomyocytes. Importantly, Tmem161b haploinsufficiency leads to cardiac rhythm phenotypes, implicating it as a candidate gene in heritable cardiac arrhythmia. Overall, these data describe Tmem161b as a highly conserved regulator of cardiac rhythm that functions to modulate ion channel activity in zebrafish and mice.

T cells recognize tumor neoantigens (TNag) in the context of MHC and eliminate cells which express these antigens, leaving those lacking immunodominant epitopes. Central to investigating tumor immunoediting is the ability to define immunogenic TNag. Differential aggretope index (DAI) is a measure of the difference in MHC binding affinity between mutated and unmutated peptides and has emerged as a highly efficient way of identifying immunogenic TNag. Our previous studies have demonstrated that in mice, TNag with a DAI >8 are considered immunogenic and elicit T cells that lead to the tumor elimination. Thus, tumors expressing TNag of DAI >8 are only found in immunodeficient mice. Given these observations, we investigated the mutational burden and immune phenotype of bone proximal tumors in 122 patients. Tumors included are of bone, surrounding tissue and distal metastases, and were analyzed by flow cytometry, whole exome and TCR-Vβ sequencing. We hypothesized that tumors with high DAI TNag would be immunoedited in patients with robust T cell responses as measured by T cell clonality. Similar to observations in mice, this correlation was only observed in TNag with DAI >8. In contrast, there was no correlation between DAI and frequency of CD8 TILs indicating that the repertoire of TILs is a better indicator of immune reactivity than their frequency. Among tumor types analyzed, osteosarcomas were the least immunoedited, suggesting greatest potential for immunotherapy. We confirmed this potential in a mouse model of osteosarcoma, where we show that therapeutic HSP-mediated responses, augmented by PD-1 blockade, conferred the best survival. These studies provide compelling rationale for novel immunotherapies in patients with osteosarcoma. Supported by NIH R01 CA233803 and an HCC UPMC Developmental Award

Background Tropomyosins are highly conserved proteins, an attribute that forms the molecular basis for their IgE antibody cross‐reactivity. Despite sequence similarities, their allergenicity varies greatly between ingested and inhaled invertebrate sources. In this study, we investigated the relationship between the structural stability of different tropomyosins, their endolysosomal degradation patterns, and T‐cell reactivity. Methods We investigated the differences between four tropomyosins—the major shrimp allergen Pen m 1 and the minor allergens Der p 10 (dust mite), Bla g 7 (cockroach), and Ani s 3 (fish parasite)—in terms of IgE binding, structural stability, endolysosomal degradation and subsequent peptide generation, and T‐cell cross‐reactivity in a BALB/c murine model. Results Tropomyosins displayed different melting temperatures, which did not correlate with amino acid sequence similarities. Endolysosomal degradation experiments demonstrated differential proteolytic digestion, as a function of thermal stability, generating different peptide repertoires. Pen m 1 (Tm 42°C) and Der p 10 (Tm 44°C) elicited similar patterns of endolysosomal degradation, but not Bla g 7 (Tm 63°C) or Ani s 3 (Tm 33°C). Pen m 1–specific T‐cell clones, with specificity for regions highly conserved in all four tropomyosins, proliferated weakly to Der p 10, but did not proliferate to Bla g 7 and Ani s 3, indicating lack of T‐cell epitope cross‐reactivity. Conclusions Tropomyosin T‐cell cross‐reactivity, unlike IgE cross‐reactivity, is dependent on structural stability rather than amino acid sequence similarity. These findings contribute to our understanding of cross‐sensitization among different invertebrates and design of suitable T‐cell peptide‐based immunotherapies for shrimp and related allergies., Allergenic tropomyosins have dissimilar structural stabilities despite having high amino acid sequence similarity. Tropomyosins are differentially degraded in the endolysosomal compartment of antigen‐presenting cells due to differences in their structural stability. Tropomyosin T‐cell cross‐reactivity, unlike IgE antibody cross‐reactivity, is dependent on structural stability rather than amino acid sequence similarity.

Shrimp is one of the predominant causes of food allergy among adults, often presenting with severe reactions. Current in vitro diagnostics are based on quantification of patient specific-IgE (sIgE) to shrimp extract. Tropomyosin is the known major shrimp allergen, but IgE sensitisation to other allergens is poorly characterised. In this study, the binding of IgE to various shrimp allergens, additional to tropomyosin, was investigated using sera from 21 subjects who had clinical reactions to one or more shellfish species. Total shrimp-sIgE was quantified using ImmunoCAP, while allergen-sIgEs were quantified using immunoblotting and mass spectrometry, and immuno-PCR to recombinant shrimp tropomyosin. Sixty-two percent of subjects (13/21) were positive to shrimp by ImmunoCAP. IgE from 43% of subjects (9/21) bound tropomyosin, while an additional 29% of subjects (6/21) demonstrated IgE-binding solely to other shrimp allergens, including sarcoplasmic calcium-binding protein, arginine kinase and hemocyanin. Furthermore, IgE sensitisation to other shrimp allergens was demonstrated in 50% of subjects (4/8) who were ImmunoCAP negative. The lack of standardised shrimp allergens and inadequacy of current extracts for shrimp allergy diagnosis is highlighted by this study. Comprehensive knowledge of less studied allergens and their inclusion in component-resolved diagnostics will improve diagnostic accuracy, benefitting the wider population suffering from shellfish allergy.

Adhesiveness of the endometrial epithelium to an embryo plays a critical role in the initiation of pregnancy. Loss or gain of adhesiveness also dictates the potential of endometrial epithelial cells to metastasize, an event that can result from certain genetic insults. A proteomics-based exploration of the "adhesiveness" these epithelial cells was employed that could identify targets that could disrupt embryo-endometrium interactions and/or metastasis of endometrial cancer cells. The present study defined the surfactomes of two human endometrial epithelial cell lines known for their differential adhesiveness to embryonic cells. Comparative, two-dimensional electrophoretic analysis of the surfactomes of RL95-2 (exhibiting higher adhesiveness to the embryonic cell line JAr) and HEC-1A (exhibiting reduced adhesiveness to JAr cells) revealed 55 differentially enriched proteins. Of these, 10 proteins were identified by MALDI-TOF/TOF or LC-MS/MS. TUBB2C, ADAMTS3, and elongation factor beta were more abundant on the HEC-1A cell surface whereas HSP27, HSPA9, GP96, CRT, Tapasin-ERP57, PDI, and β-actin were more abundant on the RL95-2 cell surface. Nano LC-MS/MS was also employed to generate a more comprehensive surfactomes of RL95-2 and HEC-1A. The study also demonstrated a pro-adhesive role of CRT and HSPA9 and an anti-adhesive role of TUBB2C populations found on the cell surface. In brief, this study identifies the cell-surface protein complements of two human endometrial epithelial cell lines, and reveals the role of three proteins in heterotypic cell adhesion.

To overcome the societal and financial burden of cardiovascular disease, a thorough understandingof the molecular and cellular programmes governing cardiac and coronary vascular development isvital. Cre-mediated genetic tracing in various models has provided valuable insight into cell-lineagecontribution and inductive cues required for cardiac morphogenesis. Using mouse models, we haveidentified the transmembrane protein Cysteine-Rich Transmembrane BMP Regulator-1 (Crim1) asan essential protein for numerous aspects of heart development. The presence of six Cysteine-RichRepeats (CRRs) in Crim1 render it to be a versatile molecule – these have been shown to bind awide range of growth factors when Crim1 is co-expressed in the same cell as the growth factor(Wilkinson et al., 2003, Wilkinson et al., 2007). The main objective of this thesis is to examine therole that Crim1 plays both temporally and spatially during heart development, and how it functionsin tandem with other factors to ensure the progression of this critical process.Crim1 is strongly expressed in the proepicardium (PE), epicardium, coronary vascular smoothmuscle cells and, to a weaker extent, in coronary endothelial cells (Pennisi et al., 2007).Interestingly, Crim1 mutant homozygotes die perinatally on a C57BL6 background (Chiu et al.,2012). Loss of Crim1 function leads to a reduced ventricular size and hypoplastic ventricularcompact myocardium, and abnormal epicardial morphology including blebbing and a loss of theregular, cobblestone appearance of the epicardium. Furthermore, epicardium-restricted deletion ofCrim1 resulted in increased epicardial epithelial-to-mesenchymal transition (EMT) and invasion ofthe myocardium, while primary epicardial cells lacking Crim1 displayed an increased migration.Growth factors like TGFβs are known to promote epicardial EMT; however, we observed aparadoxical reduction in epicardial TGFβ signalling in Crim1 mutant embryos. Interestingly, thereappeared to be an accumulation of β-catenin, a cell-adhesion molecule present at cadherindependentjunctional complexes in epithelial cells, at epicardial cell-cell junctions, suggesting aninteraction with Crim1 to promote stabilization of these junctions. There was also an increase in thelevel of phospho-ERK1/2 in the ventricular compact myocardium of mutants, though phospho-AKTlevels remained unchanged. This indicates a cell-autonomous role for Crim1 in controllingepicardial migration, EMT and invasion, and a potential paracrine role in regulating myocardialdevelopment, likely through regulation of epicardium derived factors.Another notable defect observed in the absence of Crim1 is that the coronary vasculature alsoappears to be malformed. We found a reduction in coronary vascular endothelial cell endowment in both ventricles, in Crim1Δflox/Δflox homozygotes and in endothelium-restricted mutants, identifyinga cell-autonomous role for Crim1. To further study Crim1 function in vitro, lentiviral small hairpinribonucleic acid (shRNA) targeting the CRIM1 gene was transduced into Human CardiacMicrovascular Endothelial Cells (HCMVECs), with a knockdown of more than 70% achieved at thetranscript level. Trypan Blue viability assays in both HCMVECs and Human Umbilical VeinEndothelial Cells (HUVECs) indicate reduced viability in the absence of CRIM1. Protein lysatesobtained from both Crim1-knockdown HCMVECs and HUVECs indicate a reduction in phosphoSMAD1/5levels, but no change in phospho-ERK1/2 and phospho-AKT levels.We also sought to understand the role of Crim1 in growth factor modulation in endothelial cells invivo. There was a decrease in phospho-SMAD1/5 activity in endothelial cells from ventricles ofCrim1Δflox/Δflox homozygotes, suggesting a dysregulation of TGFβ/BMP signalling in these cells,confirming our in vitro data. However, there was an increase in endothelial phospho-SMAD1/5levels in ventricles of Crim1Flox/Flox; Tie2-Cre hearts, indicating a possible paracrine role for theother resident cells of the ventricular myocardium that retain Crim1 function.cDNA synthesised from HCMVECs that had lost Crim1 was then used in a human endothelial cellbiology array. This revealed an upregulation of genes involved in increased inflammatory responseand cell death, and, an increased probability of disease states including vascular lesions, infarctionand heart failure, upon Ingenuity Pathway Analysis. Collectively, we illustrate a cell-autonomousrole for Crim1 in the development of coronary vascular endothelial cells, in vivo and in vitro.We further report for the first time, that CRIM1 is able to bind Insulin-like Growth Factors (IGFs).This was shown by means of co-immunoprecipitation experiments, and reveals that the IGFBPdomain of CRIM1, and CRRs 3-6 are responsible for this binding. Moreover, in HCMVECs lackingCRIM1, phosphorylation of the IGF-1 receptor was decreased, indicating the ability of CRIM1 toregulate IGF signalling.Our findings thus provide a platform for understanding how Crim1 functions in both the early andlate stages of embryonic heart development in vivo. Many of these findings are mimicked in vitro,most significantly in the context of growth factor modulation in human cardiac microvascularendothelial cells, and aids in understanding the pathogenesis of disease states. This indicatestranslational relevance to humans, making the study of CRIM1 an exciting new avenue for futureresearch in the field of cardiac development.

Introduction Metropolitan cities in India,producing half a kilogram of waste per household, are virtual garbage producing factories. Thus rag-picking has emerged as an alternative occupation adopted by underprivileged communities. Rag-pickers randomly sort and collect garbage and sell to scrap-dealers, gaining money in return based on the weight and type of garbage. Risk in the occupation outweighs the economic benefits. The multi-factorial implication in the sustenance of this occupation in urban underprivileged communities provokes epidemiologists to examine determinants influencing this occupation Aims and objectives To assess the socio demographic and morbidity profile of women rag-pickers To assess socio-epidemiological factors correlated with the occupational pattern of rag- pickers Methodology A cross sectional study covering 1500 women rag-pickers with the sampling frame of 10% study subjects. One to One interview with predetermined questionnaire followed by focus group discussion with stake holders and study subjects was done. Result 1) Emergence of adverse social determinants namely misery, disease, hunger,lack of dignity and inhuman treatment was found 2) Hand to mouth survival instincts compromising basic human needs of clothing, shelter and food 3) Predominance of adverse cultural influences hampering the quality of life 4) Vicious cycle of social determinants and inevitable health risks emerged 5) Social exclusion/caste system were observed as root cause. Conclusion 1) Rag-picking community and occupation be organised, registered and recognised under the Factory Act and ESI Act 2) Work safety measures should be promoted and provided 3) Local municipal governance may identify their role and develop work safety protocol 4) Alternative skills development training be given for empowering rag-picker community 5) Socio-political intervention and policy implementation for elimination of caste system 6) Regulation of trade business related to sale of garbage.

Endothelial cells form a critical component of the coronary vasculature, yet the factors regulating their development remain poorly defined. Here we reveal a novel role for the transmembrane protein CRIM1 in mediating cardiac endothelial cell development. In the absence of Crim1 in vivo, the coronary vasculature is malformed, the number of endothelial cells reduced, and the canonical BMP pathway dysregulated. Moreover, we reveal that CRIM1 can bind IGFs, and regulate IGF signalling within endothelial cells. Finally, loss of CRIM1 from human cardiac endothelial cells results in misregulation of endothelial genes, predicted by pathway analysis to be involved in an increased inflammatory response and cytolysis, reminiscent of endothelial cell dysfunction in cardiovascular disease pathogenesis. Collectively, these findings implicate CRIM1 in endothelial cell development and homeostasis in the coronary vasculature.

Within the adult mammalian brain, neurogenesis persists within two main discrete locations, the subventricular zone lining the lateral ventricles and the hippocampal dentate gyrus. Neurogenesis within the adult dentate gyrus contributes to learning and memory and deficiencies in neurogenesis have been linked to cognitive decline. Neural stem cells within the adult dentate gyrus reside within the subgranular zone (SGZ) and proteins intrinsic to stem cells and factors within the niche microenvironment, are critical determinants for development and maintenance of this structure. Our understanding of the repertoire of these factors, however, remains limited. The deubiquitylating enzyme USP9X has recently emerged as a mediator of neural stem cell identity. Furthermore, mice lacking Usp9x exhibit a striking reduction in the overall size of the adult dentate gyrus. Here we reveal that the development of the postnatal SGZ is abnormal in mice lacking Usp9x. Usp9x conditional knockout mice exhibit a smaller hippocampus and shortened dentate gyrus blades from as early as P7. Moreover, the analysis of cellular populations within the dentate gyrus revealed reduced stem cell, neuroblast and neuronal numbers and abnormal neuroblast morphology. Collectively, these findings highlight the critical role played by USP9X in the normal morphological development of the postnatal dentate gyrus.

The regulation of growth factor localization, availability and activity is critical during embryogenesis to ensure appropriate organogenesis. This process is regulated through the coordinated expression of growth factors and their cognate receptors, as well as via proteins that can bind, sequester or localize growth factors to distinct locations. One such protein is the transmembrane protein Crim1. This protein has been shown to be expressed broadly within the developing embryo, and to regulate organogenesis within the eye, kidney and placenta. Mechanistically, Crim1 has been revealed to mediate organogenesis via its interaction with growth factors including TGFβs, BMPs, VEGFs and PDFGs. More recently, Crim1 has been shown to influence cardiac development, providing further insights into the function of this protein. This review will provide an overview of the role of Crim1 in organogenesis, largely focusing on how this protein regulates growth factor signaling in the nascent heart. Moreover, we will address the challenges ahead relating to further elucidating how Crim1 functions during development.

The epicardium has a critical role during embryonic development, contributing epicardium-derived lineages to the heart, as well as providing regulatory and trophic signals necessary for myocardial development. Crim1 is a unique trans-membrane protein expressed by epicardial and epicardially-derived cells but its role in cardiogenesis is unknown. Using knockout mouse models, we observe that loss of Crim1 leads to congenital heart defects including epicardial defects and hypoplastic ventricular compact myocardium. Epicardium-restricted deletion of Crim1 results in increased epithelial-to-mesenchymal transition and invasion of the myocardium in vivo, and an increased migration of primary epicardial cells. Furthermore, Crim1 appears to be necessary for the proliferation of epicardium-derived cells (EPDCs) and for their subsequent differentiation into cardiac fibroblasts. It is also required for normal levels of cardiomyocyte proliferation and apoptosis, consistent with a role in regulating epicardium-derived trophic factors that act on the myocardium. Mechanistically, Crim1 may also modulate key developmentally expressed growth factors such as TGFβs, as changes in the downstream effectors phospho-SMAD2 and phospho-ERK1/2 are observed in the absence of Crim1. Collectively, our data demonstrates that Crim1 is essential for cell-autonomous and paracrine aspects of heart development.

Background: India is a major player in the field of IT industry and employs more than half million people today who spend long stressful hours using the computer. Due to urbanization stress has now became an inescapable part of life, workplace has become a stress factory and it is rightly known as the age of anxiety. Hence, the aim of present study was to determine the level of stress using a simple tool like the World Health Organization (WHO) 5 well-being index and its effects on blood pressure (BP) at an early stage among software professionals. Aims and Objectives: To assess and correlate the WHO 5 well-being index and BP in software professionals. Materials and Methods: It was a cross-sectional study carried out among 60 software professionals within the age group of 25–35 years with 3 years of working experience in the same, working for 8–10 h a day. Known hypertensive and people with a family history of hypertension were excluded. BP was recorded using a sphygmomanometer, and the WHO 5 well-being index was calculated. Results: It was observed that there was a negative correlation though statistically not significant (P > 0.05) between BP and WHO 5 well-being index. Conclusion: As the level of stress increases, an increase in BP was noted with a subsequent decline in the well-being of the individual.

Adhesiveness of the endometrial epithelium to an embryo plays a critical role in the initiation of pregnancy. Loss or gain of adhesiveness also dictates the potential of endometrial epithelial cells to metastasize, an event that can result from certain genetic insults. A proteomics-based exploration of the "adhesiveness" these epithelial cells was employed that could identify targets that could disrupt embryo-endometrium interactions and/or metastasis of endometrial cancer cells. The present study defined the surfactomes of two human endometrial epithelial cell lines known for their differential adhesiveness to embryonic cells. Comparative, two-dimensional electrophoretic analysis of the surfactomes of RL95-2 (exhibiting higher adhesiveness to the embryonic cell line JAr) and HEC-1A (exhibiting reduced adhesiveness to JAr cells) revealed 55 differentially enriched proteins. Of these, 10 proteins were identified by MALDI-TOF/TOF or LC-MS/MS. TUBB2C, ADAMTS3, and elongation factor beta were more abundant on the HEC-1A cell surface whereas HSP27, HSPA9, GP96, CRT, Tapasin-ERP57, PDI, and β-actin were more abundant on the RL95-2 cell surface. Nano LC-MS/MS was also employed to generate a more comprehensive surfactomes of RL95-2 and HEC-1A. The study also demonstrated a pro-adhesive role of CRT and HSPA9 and an anti-adhesive role of TUBB2C populations found on the cell surface. In brief, this study identifies the cell-surface protein complements of two human endometrial epithelial cell lines, and reveals the role of three proteins in heterotypic cell adhesion.