Fangchinoline Exerts Anticancer Effects on Colorectal Cancer Cells by Evoking Cell Apoptosis via Endoplasmic Reticulum Stress.

We studied the anti-tumor effect of fangchinoline (FAN) against human colorectal cancer cell lines CCL-244 and SW480 and analyzed the mechanism of FAN action. The cell viability and apoptosis were assessed by MTT test and Annexin V-PI staining; caspase-3 activity was measured by Western blotting. The expression of endoplasmic reticulum stress-related proteins was assessed by real-time PCR, Western blotting, and gene transfection. It was found that FAN inhibited cell growth and induced apoptosis in human colorectal cancer cell lines CCL-244 and SW480 in a dose-dependent manner. The caspase-3 inhibitor Ac-DEVD-CHO could reverse the inhibitory effect of FAN. Moreover, FAN significantly increased the expression of endoplasmic reticulum stress-related proteins p-PERK, p-eIF2α, ATF4, and CHOP in CCL-244 and SW480 cells. In addition, endoplasmic reticulum stress inhibitor 4-phenylbutyric acid or CHOP knockdown could prevent FAN-induced apoptosis. Thus, FAN induced apoptosis of human colorectal cancer through activation of endoplasmic reticulum stress.

Is There any Alternative Receptor for SARS-CoV-2?

Angiotensin-converting enzyme II (ACE2) in association with type II transmembrane serine protease (TMPRSS2) is considered the main receptor of SARS-CoV-2. However, considering the clinical complications of COVID-19 in different organs, there is no strong association between the abundance of ACE2/TMPRSS2 co-expression and clinical features of the disease and the severity of complications. Since SARS-CoV-2 affects certain organs that lack or have low expression of ACE2/TMPRSS2, it may be possible that the virus employs other receptors for colonization and entry. Based on recent studies, glucose-regulated protein 78 (GRP78) can be a potential alternative receptor for SARS-CoV-2 entry. In this letter, supporting evidence proposed GRP78 as an alternative receptor in SARS-CoV-2 infection.

Coagulopathies after vaccination against SARS-CoV-2: The sole solution might lead to another problem

The common reported adverse impacts of COVID-19 vaccination include the injection site's local reaction followed by various non-specific flu-like symptoms. Nevertheless, uncommon cases of vaccine-induced immune thrombotic thrombocytopenia (VITT) and cerebral venous sinus thrombosis (CVST) following viral vector vaccines (ChAdOx1 nCoV-19 vaccine, Ad26.COV2 vaccine) have been reported. This literature review was performed using PubMed and Google Scholar databases using appropriate keywords and their combinations: SARS-CoV-2, adenovirus, spike protein, thrombosis, thrombocytopenia, vaccine-induced immune thrombotic thrombocytopenia (VITT), NF-kappaB, adenoviral vector, platelet factor 4 (PF4), COVID-19 Vaccine, AstraZeneca COVID vaccine, ChAdOx1 nCoV-19 COVID vaccine, AZD1222 COVID vaccine, coagulopathy. The s and titles of each article were assessed by authors for screening and inclusion English reports about post-vaccine CVST and VITT in humans were also collected. Some SARS-CoV-2 vaccines based on viral vector, mRNA, or inactivated SARS-CoV-2 virus have been accepted and are being pragmatic global. Nevertheless, the recent augmented statistics of normally very infrequent types of thrombosis associated with thrombocytopenia have been stated, predominantly in the context of the adenoviral vector vaccine ChAdOx1 nCoV-19 from Astra Zeneca. The numerical prevalence of these side effects seems to associate with this particular vaccine type, i.e., adenoviral vector-based vaccines, but the meticulous molecular mechanisms are still not clear. The present review summarizes the latest data and hypotheses for molecular and cellular mechanisms into one integrated hypothesis demonstrating that coagulopathies, including thromboses, thrombocytopenia, and other associated side effects, are correlated to an interaction of the two components in the COVID-19 vaccine.Copyright © 2022, Iranian Pediatric Hematology and Oncology Society. All rights reserved.

Advance in human coronaviruses research of host interactions.

2019-novel coronavirus (2019-nCoV) is a highly pathogenic human CoV that first emerged in Wuhan in 2019. 2019-nCoV has a zoonotic origin and poses a major threat to public health. However, little is known about the viral factors contributing to the high virulence of 2019-nCoV. Many animal viruses, including CoVs, encode proteins that interfere with host gene expression, including those involved in antiviral immune responses, and these viral proteins are often major virulence factors. Human coronaviruses (HCoVs) are known respiratory pathogens associated with a range of respiratory infection. In the past 17 years, the onset of 2019-nCoV, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) have thrust HCoVs into spotlight of the research community due to their high pathogenicity in humans. The recent study of HCoVs-host interactions has contributed extensively to our understanding of infection pathogenesis of 2019-nCoV. This review discuss various host physiopathologic mechanism, such as apoptosis, innate immunity, endoplasmic reticulum (ER) stress response, mitogen-activated protein kinase (MAPK) pathway and nuclear factor kappa B (NF-kappaB) pathway that may be modulated by HCoVs and provides evidence for the intensive investigate of 2019-nCoV infection.Copyright © 2020 by the Chinese Medical Association.

Apoptosis, Autophagy, and Unfolded Protein Response and Cerebellar Development

Development is an evolutionary process that is tightly regulated in mammalian species. Several different cascades are involved in various stages of development. Among these mechanisms, apoptosis, autophagy, and unfolded protein response play critical roles in regulating development by affecting cell fate. All of these pathways are involved in the regulation of cell numbers via determining the life and death cycles of the cells. In this chapter, we first explain the brief mechanisms that are involved in the regulation of apoptosis, autophagy, and unfolded protein response, and later, we briefly describe how these mechanisms play roles in general development. We next address the critical role of these pathways in cerebellar development regulation and how they will aid in our knowledge of the processes behind neurodevelopmental disorders. Additionally, we summarize the present findings on neurological symptoms and disorders related to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and their linkage to autophagy pathways in the cerebellum. © 2023, The Author(s), under exclusive license to Springer Nature Switzerland AG.

SARS-CoV-2 ORF8 Protein Induces Endoplasmic Reticulum Stress-like Responses and Facilitates Virus Replication by Triggering Calnexin: an Unbiased Study.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the worldwide coronavirus disease 2019 (COVID-19) pandemic. The novel SARS-CoV-2 ORF8 protein is not highly homologous with known proteins, including accessory proteins of other coronaviruses. ORF8 contains a 15-amino-acid signal peptide in the N terminus that localizes the mature protein to the endoplasmic reticulum. Oligomannose-type glycosylation has been identified at the N78 site. Here, the unbiased molecular functions of ORF8 are also demonstrated. Via an immunoglobulin-like fold in a glycan-independent manner, both exogenous and endogenous ORF8 interacts with human calnexin and HSPA5. The key ORF8-binding sites of Calnexin and HSPA5 are indicated on the globular domain and the core substrate-binding domain, respectively. ORF8 induces species-dependent endoplasmic reticulum stress-like responses in human cells exclusively via the IRE1 branch, including intensive HSPA5 and PDIA4 upregulation, with increases in other stress-responding effectors, including CHOP, EDEM and DERL3. ORF8 overexpression facilitates SARS-CoV-2 replication. Both stress-like responses and viral replication induced by ORF8 have been shown to result from triggering the Calnexin switch. Thus, ORF8 serves as a key unique virulence gene of SARS-CoV-2, potentially contributing to COVID-19-specific and/or human-specific pathogenesis. IMPORTANCE Although SARS-CoV-2 is basically regarded as a homolog of SARS-CoV, with their genomic structure and the majority of their genes being highly homologous, the ORF8 genes of SARS-CoV and SARS-CoV-2 are distinct. The SARS-CoV-2 ORF8 protein also shows little homology with other viral or host proteins and is thus regarded as a novel special virulence gene of SARS-CoV-2. The molecular function of ORF8 has not been clearly known until now. Our results reveal the unbiased molecular characteristics of the SARS-CoV-2 ORF8 protein and demonstrate that it induces rapidly generated but highly controllable endoplasmic reticulum stress-like responses and facilitates virus replication by triggering Calnexin in human but not mouse cells, providing an explanation for the superficially known in vivo virulence discrepancy of ORF8 between SARS-CoV-2-infected patients and mouse.

Leukocyte surface expression of the endoplasmic reticulum chaperone GRP78 is increased in severe COVID-19.

Hyperinflammation present in individuals with severe COVID-19 has been associated with an exacerbated cytokine production and hyperactivated immune cells. Endoplasmic reticulum stress leading to the unfolded protein response has been recently reported as an active player in inducing inflammatory responses. Once unfolded protein response is activated, GRP78, an endoplasmic reticulum-resident chaperone, is translocated to the cell surface (sGRP78), where it is considered a cell stress marker; however, its presence has not been evaluated in immune cells during disease. Here we assessed the presence of sGRP78 on different cell subsets in blood samples from severe or convalescent COVID-19 patients. The frequency of CD45+sGRP78+ cells was higher in patients with the disease compared to convalescent patients. The latter showed similar frequencies to healthy controls. In patients with COVID-19, the lymphoid compartment showed the highest presence of sGRP78+ cells versus the myeloid compartment. CCL2, TNF-α, C-reactive protein, and international normalized ratio measurements showed a positive correlation with the frequency of CD45+sGRP78+ cells. Finally, gene expression microarray data showed that activated T and B cells increased the expression of GRP78, and peripheral blood mononuclear cells from healthy donors acquired sGRP78 upon activation with ionomycin and PMA. Thus, our data highlight the association of sGRP78 on immune cells in patients with severe COVID-19.

Exercise as a non-pharmacological intervention to protect pancreatic beta cells in individuals with type 1 and type 2 diabetes.

AIMS/HYPOTHESIS: Diabetes is characterised by progressive loss of functional pancreatic beta cells. None of the therapeutic agents used to treat diabetes arrest this process; preventing beta cell loss remains a major unmet need. We have previously shown that serum from eight young healthy male participants who exercised for 8 weeks protected human islets and insulin-producing EndoC-ßH1 cells from apoptosis induced by proinflammatory cytokines or the endoplasmic reticulum (ER) stressor thapsigargin. Whether this protective effect is influenced by sex, age, training modality, ancestry or diabetes is unknown. METHODS: We enrolled 82 individuals, male or female, non-diabetic or diabetic, from different origins, in different supervised training protocols for 8-12 weeks (including training at home during the COVID-19 pandemic). EndoC-ßH1 cells were treated with 'exercised' serum or with the exerkine clusterin to ascertain cytoprotection from ER stress. RESULTS: The exercise interventions were effective and improved [Formula: see text] values in both younger and older, non-obese and obese, non-diabetic and diabetic participants. Serum obtained after training conferred significant beta cell protection (28% to 35% protection after 4 and 8 weeks of training, respectively) from severe ER stress-induced apoptosis. Cytoprotection was not affected by the type of exercise training or participant age, sex, BMI or ancestry, and persisted for up to 2 months after the end of the training programme. Serum from exercised participants with type 1 or type 2 diabetes was similarly protective. Clusterin reproduced the beneficial effects of exercised sera. CONCLUSIONS/INTERPRETATION: These data uncover the unexpected potential to preserve beta cell health by exercise training, opening a new avenue to prevent or slow diabetes progression through humoral muscle-beta cell crosstalk.

Therapeutic Effects of Renin-Angiotensin Systeminhibiting Drugs Losartan and Enalapril on Sars-Cov-2 Infection in Ex Vivo Cultured Human Precisioncut Lung Slices

The role of the renin-angiotensin system (RAS) in coronavirus disease 2019 (COVID-19) has received much attention, because the angiotensin-converting enzyme 2 (ACE2) has been identified as the main receptor for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has been speculated that, in COVID-19, RAS dysregulation in favor of angiotensin II (Ang-II)-mediated signaling might result in severe tissue inflammation and lung injury. Likewise, the role of a pre-existing therapy with angiotensin-converting enzyme 1 inhibitors (ACEi) or Ang- II type 1 receptor blockers (ARBs) in COVID-19 is largely unclear. We evaluated the effects of the ACEi enalapril (ENA) and the ARB losartan (LOS) on SARS-CoV-2 infection in human ex vivo-cultured, precision-cut lung slices (PCLS) obtained from normal human lung tissue. The PCLS were pretreated for 5 days with vehicle, LOS or ENA (300 muM), followed by mock infection or infection with SARS-CoV-2 and (continued) incubation with vehicle, LOS or ENA for 1 or 2 days. Thereafter, PCLS were harvested for analysis of viral replication, inflammatory responses, endoplasmic reticulum (ER) stress and apoptosis pathways. Both LOS and ENA significantly reduced viral replication in PCLS, with ENA being more potent. LOS was more efficient than ENA in reducing the expression of IL1B, CCL2, CXCL2 and TNFA, but not of IL6, whereas ENA preferentially caused a reduction of IL6 and CCL2 in SARS-CoV-2-infected PCLS. Furthermore, ENA, but not LOS, significantly decreased the expression of viral entry factors, ACE2 and transmembrane serine protease 2 (TMPRSS2), in infected PCLS, both of which were found to be robustly induced upon SARS-CoV-2 infection. Importantly, LOS or ENA did not exert apoptosis or other cytotoxic effects. Renin-angiotensin system-antagonizing drugs do not seem to exert detrimental effects during SARS-CoV-2 infection.On the contrary, in an ex vivo model of human PCLS, such treatment was found to dampen SARS-CoV-2 infection and consecutive inflammation.

AKI in Patients With SARS-CoV-2 Is Significantly Associated With Mitochondrial Dysfunction and ER Stress

Background: AKI is a common complication of COVID-19. The peripheral blood molecular signatures are unknown and could unveil potential therapeutic targets. Method(s): We enrolled a prospective patient cohort of 283 patients with COVID-19 (Mar 24-Aug 26, 2020), with blood samples from Mount Sinai Biobank. We determined AKI severity using KDIGO criteria on admission parameters. 31 patients with severe AKI (AKI 2-3) were defined as cases. We then performed bulk peripheral RNA sequencing and fit a multivariate linear regression model adjusting for key covariates. We also performed cell-type deconvolution following to adjust for neutrophils, and whole blood cells. We considered a significant p-value (0.05) after Bonferroni correction and then used ingenuity pathway analysis (IPA) to analyze differentially expressed genes. Result(s): Patients who developed AKI were significantly older (67 vs. 60 yrs.) and had a greater prevalence of type 2 diabetes (37% vs 20%), and chronic kidney disease (20% vs 4%) vs. controls. Of the 18539 genes in the analysis, 1597 were upregulated and 1267 were downregulated after Bonferroni correction. Top canonical pathways (Fig 1) showed significantly downregulated genes including EIF2, eIF4, and p70S6K via activation of ATF6, a marker of ER stress. Potential mechanisms displayed by our analyses include upregulation of the NF-KB inhibitor and IL6 pathways. Genes involved in oxidative Phosphorylation and mitochondrial dysfunction were heavily downregulated and there was upregulation of markers of kidney cell necrosis. In contrast, upregulated genes CRK and TIMP2 have been previously implicated in kidney injury and progression. Downregulated mTOR pathway is responsible for the activation of the ER stress response via the eIF2/4 complex which is also supported by our finding of upregulated NRF2- transcriptional pathway. Conclusion(s): Transcriptomic analysis of AKI in COVID-19 revealed evidence of mitochondrial dysfunction driven by ER stress and immune-mediated pathways. Addressing these pathways could aide development of targeted therapies. (Figure Presented).

Porcine epidemic diarrhea virus E protein inhibits type I interferon production through endoplasmic reticulum stress response (ERS)-mediated suppression of antiviral proteins translation.

Porcine epidemic diarrhea virus (PEDV) envelope protein (E) is recognized as a viroporin that plays important functions in virus budding, assembly and virulence. Our previous study found that PEDV E protein induces endoplasmic reticulum stress (ERS), as well as suppresses the type I interferon (IFN) response, but their link and underlying mechanism remain obscure. To better understand this relationship, we investigated the roles of PEDV E protein-induced ERS in regulating cellular type I IFN production. Our results showed that PEDV E protein localized in the ER and triggered ERS through activation of PERK/eIF2α branch, as revealed by the up-regulated phosphorylation of PERK and eIF2α. PEDV E protein also significantly inhibited both poly(I:C)-induced and RIG-I signaling-mediated type I interferon production. The PERK/eIF2α branch of ERS activated by PEDV E protein led to the translation attenuation of RIG-I signaling-associated antiviral proteins, resulting in the suppression of type I IFN production. However, PEDV E protein had no effect on the mRNA transcription of RIG-I-associated molecules. Moreover, suppression of ERS with 4-PBA, a widely used ERS inhibitor, restored the expression of RIG-I-signaling-associated antiviral proteins and mRNA transcription of IFN-ß and ISGs genes to their normal levels, suggesting that PEDV E protein blocks the production of type I IFN through inhibiting expression of antiviral proteins caused by ERS-mediated translation attenuation. This study elucidates the mechanism by which PEDV E protein specifically modulates the ERS to inhibit type I IFN production, which will augment our understanding of PEDV E protein-mediated virus evasion of host innate immunity.

IDENTIFYING A NOVEL SURFACTANT PROTEIN MUTATION IN A FAMILY WITH PULMONARY FIBROSIS

SESSION TITLE: Rare Genetic Mutations and Anatomical Variants SESSION TYPE: Rapid Fire Case Reports PRESENTED ON: 10/18/2022 12:25 pm - 01:25 pm INTRODUCTION: Idiopathic pulmonary fibrosis (IPF) is a fatal disease affecting older adults that results in progressive scarring of the lung parenchyma. Familial IPF (FPF), defined by disease in two or more first-degree relatives, is estimated to occur in 2–20% of all IPF cases and can present with varying phenotypes which may be difficult to diagnose. Inherited gene variation as well as environmental factors predispose a patient to disease development. Additionally, rare genetic variants in the genes encoding surfactant A (SFTPA1, and SFTPA2) that affect alveolar stability and endoplasmic reticulum stress have been reported in less than 1% of FPF cases. Understanding these genetic variants is essential in the diagnosis and management of patients with FPF. CASE PRESENTATION: A 47-year-old Hispanic male with a history of COVID-19 one year ago (not requiring hospitalization) presented to the hospital for a two-day history of subjective fever and shortness of breath. He was hypoxic requiring oxygen via high flow nasal cannula. He was admitted four months ago for shortness of breath and treated for pneumonia. Since then, he has had chronic dyspnea with exertion. Computed tomography of the chest showed extensive ground glass opacities, worse in the right lung, with basilar and upper lobe honeycombing, and air bronchograms in the bilateral lower lobes. Family history was significant for a mother, maternal aunt, maternal grandfather, and maternal cousin who all died from pulmonary fibrosis. His maternal cousin was treated at our facility, in which genetic sequencing revealed a mutation in SFTPA2, c.697T>C. Our patient was found to have the same genetic mutation. DISCUSSION: The genetic basis of IPF remains poorly understood. Prior studies suggest only 20-30% of FPF cases harbor an identifiable causative genetic variant. Rare variants in two biologic pathways contribute to the known heritability of FPF including pathologic variants in surfactant related genes which cause improper protein trafficking leading to endoplasmic reticulum stress, defects in autophagy, and type II alveolar cell toxicity. SFTPA1 and SFTPA2 variants have been associated with FPF and lung adenocarcinoma in a small number of families and there are few reported cases. While currently the SFTPA2, c.697T>C mutation, previously reported by our group in 2016, is considered a variant of unknown significance, its occurrence in two relatives with serious progressive interstitial lung diseases suggests that it is indeed pathogenic. CONCLUSIONS: Gene sequencing should be considered for all patients with a family history of pulmonary fibrosis as identification of a rare genetic variant may offer guidance to diagnosis, prognostication, and risk stratification when considering lung transplantation as well as identify additional relatives who may be affected by IPF. Reference #1: Kropski JA, Young LR, Cogan JD, et al. Genetic Evaluation and Testing of Patients and Families with Idiopathic Pulmonary Fibrosis. Am J Respir Crit Care Med. 2017;195(11):1423-1428. doi:10.1164/rccm.201609-1820PP Reference #2: Wang Y, Kuan PJ, Xing C, Cronkhite JT, Torres F, Rosenblatt RL, DiMaio JM, Kinch LN, Grishin NV, Garcia CK. Genetic defects in surfactant protein A2 are associated with pulmonary fibrosis and lung cancer. Am J Hum Genet. 2009 Jan;84(1):52-9. doi: 10.1016/j.ajhg.2008.11.010. Epub 2008 Dec 18. PMID: 19100526;PMCID: PMC2668050. Reference #3: Pulmonary Fibrosis Due to a Novel Surfactant Protein Mutation R.A. Arciniegas Flores, I.A. Vital, K. Medepalli, D. DeMarzo, M.K. Glassberg Csete, R.A. Alvarez. https://doi.org/10.1164/ajrccm-conference.2019.199.1_Meetings.A5437 DISCLOSURES: No relevant relationships by Roger Alvarez No relevant relationships by Eduardo Lopez Gonzalez No relevant relationships by Anita Singh

The Role of Endoplasmic Reticulum Stress in Differentiation of Cells of Mesenchymal Origin.

Endoplasmic reticulum (ER) is a multifunctional membrane-enclosed organelle. One of the major ER functions is cotranslational transport and processing of secretory, lysosomal, and transmembrane proteins. Impaired protein processing caused by disturbances in the ER homeostasis results in the ER stress. Restoration of normal ER functioning requires activation of an adaptive mechanism involving cell response to misfolded proteins, the so-called unfolded protein response (UPR). Besides controlling protein folding, UPR plays a key role in other physiological processes, in particular, differentiation of cells of connective, muscle, epithelial, and neural tissues. Cell differentiation is induced by the physiological levels of ER stress, while excessive ER stress suppresses differentiation and can result in cell death. So far, it remains unknown whether UPR activation induces cell differentiation or if UPR is initiated by the upregulated synthesis of secretory proteins during cell differentiation. Cell differentiation is an important stage in the development of multicellular organisms and is tightly controlled. Suppression or excessive activation of this process can lead to the development of various pathologies in an organism. In particular, impairments in the differentiation of connective tissue cells can result in the development of fibrosis, obesity, and osteoporosis. Recently, special attention has been paid to fibrosis as one of the major complications of COVID-19. Therefore, studying the role of UPR in the activation of cell differentiation is of both theoretical and practical interest, as it might result in the identification of molecular targets for selective regulation of cell differentiation stages and as well as the potential to modulate the mechanisms involved in the development of various pathological states.

Donepezil Beyond Alzheimer’s Disease? A Narrative Review of Therapeutic Potentials of Donepezil in Different Diseases

Donepezil hydrochloride is an acetylcholine esterase inhibitor studied and approved to treat Alzheimer’s disease (AD). However, this drug can have positive therapeutic potential in treating different conditions, including various neurodegenerative disorders such as other types of dementia, multiple sclerosis, Parkinson’s disease, psychiatric and mood disorders, and even infectious diseases. Hence, this study reviewed the therapeutic potential of this drug in treating Alzheimer’s and other diseases by reviewing the articles from databases including Web of Science, Scopus, PubMed, Cochrane, and Science Direct. It was shown that donepezil could affect the pathophysiology of these diseases via mechanisms such as increasing the concentration of acetylcholine, modulating local and systemic inflammatory processes, affecting acetylcholine receptors like nicotinic and muscarinic receptors, and activating various cellular signaling via receptors like sigma-1 receptors. Despite many therapeutic potentials, this drug has not yet been approved for treating non-Alzheimer’s diseases, and more comprehensive studies are needed.

ENTERIC NEURONAL VASOACTIVE INTESTINAL PEPTIDE MEDIATES SARS-COV-2 ASSOCIATED DIARRHEA

Background: Diarrhea is present in up to 36.6% of patients with COVID-19. The mechanism of SARS-CoV-2-induced diarrhea remains unclear. We hypothesized that enterocyte-enteric neuron interactions were important in SARS-CoV-2-induced diarrhea. SARS-CoV-2 induces endoplasmic reticulum (ER) stress in enterocytes causing the release of Damage Associated Molecular Patterns (DAMPs). The DAMPs then stimulate the release of enteric neurotransmitters that disrupt gut electrolyte homeostasis. The influence of ER stress and enteric neuronderived vasoactive intestinal peptide (VIP) on the expression of Na+/H+ exchanger 3 (NHE3), an important transporter that mediates intestinal Na+/fluid absorption, was further examined. Methods: SARS-CoV-2 propagated in Vero-E6 cells was used to infect Caco-2, a human colon epithelial cell line that expresses SARS-CoV-2 entry receptor ACE2. The expression of ER stress markers, phospho-PERK, Xbp1s, and DAMP proteins, was examined by Western blotting. Primary mouse enteric neurons were treated with a conditioned medium of Caco- 2 cells that were infected with SARS-CoV-2 or treated with tunicamycin. VIP expression by cultured enteric neurons was assessed by RT-qPCR, Western blotting, and ELISA. Membrane expression of NHE3 was determined by surface biotinylation. Results: SARS-CoV-2 infection of Caco-2 cells led to increased expression of phospho-PERK and Xbp1s indicating increased ER stress. Infected Caco-2 cells secreted DAMP proteins, including HSP70 and calreticulin, as revealed by proteomic and Western analyses. The expression of VIP mRNA in enteric neurons was up-regulated after treatment with a conditioned medium of SARS-CoV-2- infected Caco-2 cells (Mock, 1 ± 0.0885;and SARS-CoV-2, 1.351 ± 0.020, P=.005). CD91, a receptor for HSP70 and calreticulin, is abundantly expressed in cultured mouse and human enteric neurons and was up-regulated by a conditioned medium of SARS-CoV-2-infected Caco-2 cells. Tunicamycin, an inducer of ER stress, also induced the secretion of HSP70 and calreticulin, mimicking SARS-CoV-2 infection. Moreover, co-culture of enteric neurons with tunicamycin-treated Caco-2 cells stimulated VIP production as determined by ELISA. Co-treatment of Caco-2 cells with tunicamycin (apical) and VIP (basolateral) induced a synergistic decrease in the membrane expression of NHE3. Conclusions: Our findings demonstrate that SARS-CoV-2 infection of enterocytes leads to ER stress and the release of DAMPs that up-regulate the expression and release of VIP by enteric neurons. The presence of ER stress together with the secreted VIP, in turn, inhibits fluid absorption through the downregulation of brush-border membrane expression of NHE3 in the enterocytes. These data highlight epithelial-neuronal crosstalk in COVID-19 related diarrhea. (Figure Presented)

Potential Targets and Mechanisms of Phellodendrine in the Treatment of Diabetes Mellitus Based on Network Pharmacology and Molecular Docking

Phellodendrine is a Phellodendri Cortex-derived isoquinoline alkaloids, has been shown to have various activities, especially hypoglycemic effect in mice, predicting its medicinal value on diabetes mellitus. To further understand the pharmacological effect of phellodendrine on diabetes mellitus, network pharmacological techniques have been used to elaborate the involved mechanisms. 84 common target molecules were screened, based on the chemical structure of phellodendrine molecule and disease database. These proteins were enriched in insulin resistance, insulin secretion and inflammatory response, mainly focus on the phosphatidylinositol 3-kinase/protein kinase B signaling pathway, mitogen-activated protein kinase signaling pathway and interleukin-17 signaling pathway. Moreover, enrichment analysis suggested that the targets of phellodendrine such as phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha and mitogen-activated protein kinases 8 were associated with coronavirus disease 2019. To verify the results, molecular docking technique was used to evaluate the interaction between phellodendrine and key targets in the signaling pathway. The calculated binding energy indicates that phellodendrine can form stable complex with insulin receptor, mitogen-activated protein kinases 8, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha and glycogen synthase kinase 3 beta. These data suggest that phellodendrine should be beneficial for treatment of diabetes mellitus.

PRE-EXPOSURE TO ALCOHOL IMPAIRS ANTI-COVID DRUGS TREATMENTASSOCIATED CELLULAR STRESS RESPONSES IN LIVER CELLS

As the delta and omicron SARS-CoV-2 variants spread across the world, more tools to fight off serious infection have been developed. COVID antiviral drugs that can be taken orally at home could cut serious illness and reduce the risk of hospitalization and death. However, significant population of people consume alcohol before the infection and use of the antiviral drugs, which could potentiate side effects of the drugs on the liver. We investigated the role of alcohol in anti-Covid drug-induced stress responses in live cells. METHODS: HepG2 cells or primary mouse hepatocytes (PMH) were pre-treated with alcohol (50 mMlow dose or 100 mMhigh dose) for 6-24 hours and then treated with the newly developed oral anti-Covid drugs: nirmatrelvir, ritonavir, molnupiravir, and remdesivir at 10- 30 lg/ml for 6-24 hours. Unfolded protein response (UPR)/ER stress molecular markers (e.g. IRE1 GRP78, PERK, Xbp1 and CHOP), Golgi stress response (GSR) markers of GCP60, HSP47 and TFE3, and STAT3 were measured after the treatments. Cell death was assessed through double staining the liver cells with Syntox Green and Hoesche's Blue. RESULTS: ER stress response as indicated by IRE1, Xbp1 and CHOP was insignificant or mild in either HepG2 or PMH treated individually with alcohol at the low dose, nirmatrelvir, ritonavir, molnupiravir, or remdesivir. Alcohol or remdesivir induced moderate GSR based on mRNA increase of GCP60, HSP47 and TFE3, which was accompanied with apparent Golgi fragmentation in either HepG2 or PMH. Cell death rates in HepG2 treated with alcohol, nirmatrelvir, ritonavir, molnupiravir, or remdesivir individually were less than 5%. Pre-exposure to alcohol combined with subsequent treatment with nirmatrelvir, ritonavir molnupiravir, or remdesivir significantly increased both ER stress and GSR markers and expression of phosphorylated STAT3 (p-STAT3). Most significantly, cell death rates in HepG2 or PMH were increased by 2- to 5-fold by pre-alcohol exposure plus ritonavir, nirmatrelvir, molnupiravir, or remdesivir. The organelle stress markers, p-STAT3 and cell death were all further increased in alcoholand anti-Covid drug-treated HepG2 or primary mouse hepatocytes that were pre-infected with the lentiviruses that were pseudotyped with the SARS-CoV-2 spike protein. CONCLUSION: Our results indicate that pre-exposure to alcohol potentiates the liver cells to anti-Covid-19 drugs induced stress responses and cell death.

IRE1A/ XBP1s Axis of ER Stress Promotes DRP1 Phosphorylation at Specific Serine Residue (S616) and Induces Mitochondrial Fragmentation During Airway Inflammation

RATIONALE: Airway inflammation plays a role in airway diseases such as asthma, chronic obstructive pulmonary disease (COPD), chronic bronchitis, and COVID-19 that affect millions of people worldwide. Previously, we showed that acute (24-h) exposure to the pro-inflammatory cytokine tumor necrosis factor α (TNFα) triggers an endoplasmic reticulum (ER) stress response in human airway smooth muscle (hASM) cells. In hASM cells, TNFα selectively activates the inositol requiring enzyme 1α (IRE1α) ER stress pathway with downstream splicing of X-box binding protein 1 (XBP1s), which transcriptionally activates expression of target genes that include proteins mediating phosphorylation of dynamin-related protein 1 (pDRP1) at the Ser616 (S616) residue. Increased pDRP1 at S616 is associated with mitochondrial fission (fragmentation);however, DRP1 is also phosphorylated at Ser637 (S637) residue, and the balance between phosphorylation at S616 and S637 regulates the translocation of DRP1 from cytosol to mitochondria and subsequent fragmentation of mitochondria. In the present study, we hypothesized that TNFα induces ER stress leading to XBP1s mediated increase in the expression of specific kinases that phosphorylate DRP1 at S616 and promote mitochondrial fragmentation. METHODS: hASM cells, dissociated from bronchial tissue obtained from patients with no history of respiratory diseases, were exposed to TNFα (20 ng/ ml for 6-h). As an inhibitor of fragmentation, cells were treated with Mdivi1 (50 μM for 6-h), GTPase inhibitor of DRP1. The expression and phosphorylation status of IRE1α, DRP1, XBP1, cyclin dependent kinases (CDK1, CDK5) and cyclin B1 were quantified by Western blot and immunohistochemistry. Mitochondrial morphology was assessed by 3D confocal microscopy using MitoTracker. XBP1-targets were confirmed by chromatin immunoprecipitation (ChIP) and quantitative PCR. RESULTS: Bioinformatics analysis predicted putative binding sites of XBP1 in the promoter region of CDK1, CDK5 and cyclin B1 genes that are reported to phosphorylate DRP1 at S616. Consistent with our previous findings, we found that TNFα increases IRE1α phosphorylation and XBP1 splicing. The TNFα induced increase in XBP1s transcriptionally activated expression of CDK1, CDK5 and cyclin B1 and leads to subsequent phosphorylation of DRP1 at S616 with no change in S637 phosphorylation. As a result, TNFα mediated increase in the ratio of S616/ S637 phosphorylation, which promoted translocation of DRP1 from cytosol to mitochondria and mitochondrial fragmentation. We also showed that Mdivi1 mediated inhibition of DRP1-GTPase activity ameliorated phosphorylation at S616 residue and significantly reduced mitochondrial fragmentation. CONCLUSIONS: The present study elucidates the mechanism underlying TNFα induced ER stress and mitochondrial fragmentation.

The PERK/PKR-eIF2α Pathway Negatively Regulates Porcine Hemagglutinating Encephalomyelitis Virus Replication by Attenuating Global Protein Translation and Facilitating Stress Granule Formation.

The replication of coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is closely associated with the endoplasmic reticulum (ER) of infected cells. The unfolded protein response (UPR), which is mediated by ER stress (ERS), is a typical outcome in coronavirus-infected cells and is closely associated with the characteristics of coronaviruses. However, the interaction between virus-induced ERS and coronavirus replication is poorly understood. Here, we demonstrate that infection with the betacoronavirus porcine hemagglutinating encephalomyelitis virus (PHEV) induced ERS and triggered all three branches of the UPR signaling pathway both in vitro and in vivo. In addition, ERS suppressed PHEV replication in mouse neuro-2a (N2a) cells primarily by activating the protein kinase R-like ER kinase (PERK)-eukaryotic initiation factor 2α (eIF2α) axis of the UPR. Moreover, another eIF2α phosphorylation kinase, interferon (IFN)-induced double-stranded RNA-dependent protein kinase (PKR), was also activated and acted cooperatively with PERK to decrease PHEV replication. Furthermore, we demonstrate that the PERK/PKR-eIF2α pathways negatively regulated PHEV replication by attenuating global protein translation. Phosphorylated eIF2α also promoted the formation of stress granules (SGs), which in turn repressed PHEV replication. In summary, our study presents a vital aspect of the host innate response to invading pathogens and reveals attractive host targets (e.g., PERK, PKR, and eIF2α) for antiviral drugs. IMPORTANCE Coronavirus diseases are caused by different coronaviruses of importance in humans and animals, and specific treatments are extremely limited. ERS, which can activate the UPR to modulate viral replication and the host innate response, is a frequent occurrence in coronavirus-infected cells. PHEV, a neurotropic betacoronavirus, causes nerve cell damage, which accounts for the high mortality rates in suckling piglets. However, it remains incompletely understood whether the highly developed ER in nerve cells plays an antiviral role in ERS and how ERS regulates viral proliferation. In this study, we found that PHEV infection induced ERS and activated the UPR both in vitro and in vivo and that the activated PERK/PKR-eIF2α axis inhibited PHEV replication through attenuating global protein translation and promoting SG formation. A better understanding of coronavirus-induced ERS and UPR activation may reveal the pathogenic mechanism of coronavirus and facilitate the development of new treatment strategies for these diseases.

AACR Virtual Special Conference: Tumor Immunology and Immunotherapy

The proceedings contain 100 papers. The topics discussed include: unconventional er stress response pro-tumorigenic polarization and survival in TAMs;tumor microenvironment metabolism in T cell differentiation and dysfunction;CAR NK cells: the future of cancer immunotherapy;chimeric antigen receptor macrophages for the treatment of solid tumors;impact of immunotherapy on COVID-19 immunity: insights from checkpoint blockade in cancer;adaptive immune dysregulation in cancer patients with SARS-CoV-2 infection;mapping myeloid programs that control tumor immunity;and cancer evolution: chromosomal instability and immune evasion.