SARS-CoV-2 spike S1 mediated lung tumor regression in a mouse model

Lung cancer is the leading cause of cancer related deaths worldwide, with a relatively low 5-year survival rate. Although there are some therapies against lung cancer, new effective treatment options are urgently required. Recently during the COVID-19 pandemic, we have seen that SARSCoV-2 binds to its receptor angiotensin-converting enzyme 2 (ACE2) via spike S1 to enter the cells. This study underlines the importance of SARS-CoV-2 spike S1 in inducing death in human lung cancer cells. Interestingly, we have seen that recombinant spike S1 treatment at very low doses led to death of human A549 lung cancer cells. On the other hand, boiled recombinant SARS-CoV-2 spike S1 remained unable to induce death, suggesting that the induction of cell death in A549 cells was due to native SARS-CoV-2 spike S1 protein. SARS-CoV-2 spike S1-induced A549 cell death was also inhibited by neutralizing antibodies against spike S1 and ACE2. Moreover, our newly designed wild type ACE2-interacting domain of SARS-CoV-2 (wtAIDS), but not mAIDS, peptide also attenuated SARS-CoV-2 spike S1-induced cell death, suggesting that SARS-CoV-2 spike S1- induced death in lung cancer cells depends on its interaction with ACE2 receptor. Similarly, recombinant spike S1 treatment also led to death of H1299 and H358 human lung cancer cells. Finally, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) intoxication led to the formation tumors in lungs of A/J mice and alternate day intranasal treatment with low dose of recombinant SARS-CoV-2 spike S1 from 22-weeks of NNK insult (late stage) led to induced apoptosis and tumor regression in the lungs. These studies indicate that recombinant SARS-CoV-2 Spike S1 protein may have implications in the treatment of lung cancer.

Oncolytic virus TG6002 safety and activity after intrahepatic artery administration in patients with liverdominant metastatic colorectal cancer

The TG6002.03 trial is a dose-escalation phase 1 clinical trial of TG6002 infusion via the hepatic artery in patients with liver-dominant colorectal cancer metastases. TG6002 is an engineered Copenhagen strain oncolytic Vaccinia virus, deleted of thymidine kinase and ribonucleotide reductase to enhance tumor selective viral replication and expressing FCU1, an enzyme converting the non-cytotoxic prodrug 5-fluorocytosine (5-FC) into the chemotherapeutic compound 5-fluorouracil (5-FU). In this trial, patients with advanced unresectable liver-dominant metastatic colorectal cancer who had failed previous oxaliplatin and irinotecan-based chemotherapy were treated with up to 2 cycles of TG6002 infusion 6 weeks apart via the hepatic artery on day 1 combined with oral 5-FC on days 5 to 14 (where day 1 = TG6002 infusion). TG6002 infusion was performed over 30 minutes via selective catheterization of the hepatic artery proper. 5-FC oral dosing was 50mg/kg x4 daily. Blood was sampled for TG6002 pharmacokinetics and 5-FC and 5-FU measurements. Sampling of liver metastases was performed at screening and on day 4 or day 8 for virus detection and 5-FC and 5-FU quantification. In total, 15 patients (median age 61 years, range 37-78) were treated in 1 UK centre and 2 centres in France and received a dose of TG6002 of 1 x 106 (n=3), 1 x 107 (n=3), 1 x 108 (n=3), or 1 x 109 pfu (n=6). Fourteen of the 15 patients received a single cycle of treatment, including one patient who did not received 5-FC, and one patient received two cycles. TG6002 was transiently detected in plasma following administration, suggesting a strong tissue selectivity for viral replication. In the highest dose cohort, a virus rebound was observed on day 8, concordant with replication time of the virus. In serum samples, 5-FU was present on day 8 in all patients with a high variability ranging from 0.8 to 1072 ng/mL and was measurable over several days after initiation of therapy. Seven of the 9 patients evaluable showed the biodistribution of the virus in liver lesions by PCR testing on day 4 or day 8. Translational blood samples showed evidence for T-cell activation and immune checkpoint receptor-ligand expression. At 1 x 109 pfu, there was evidence for T-cell proliferation and activation against tumour-associated antigens by ELISpot and for immunogenic cell death. In terms of safety, a total of 34 TG6002-related adverse events were reported, of which 32 were grade 1-2 and 2 were grade 3. The maximum tolerated dose was not reached, and a single dose-limiting toxicity was observed consisting of a myocardial infarction in a context of recent Covid-19 infection in a 78-year-old patient. These results indicate that TG6002 infused via the hepatic artery in combination with oral 5-FC was well tolerated, effectively localized and replicated in the tumor tissues, expressed its therapeutic payload and showed anti-tumoral immunological activity.

PRE-EXPOSURE PROPHYLAXIS OF COVID-19 WITH TIXAGEVIMAB AND CILGAVIMAB IN RHEUMATOLOGIC PATIENTS TREATED WITH RITUXIMAB: DATA FROM A SINGLE-CENTRE COHORT

BackgroundRheumatologic patients treated with Rituximab (RTX) are at higher risk of severe COVID-19 and death. The B-cell depletive treatment significantly affects B cell functions involved in anti-SARS-CoV-2 response, leading to relevant impacts on the clinical and serological course of infection, long-term immunity, and vaccine responses. In light of these observations, pre-exposure prophylaxis (PrEP) of COVID-19 with Tixagevimab and Cilgavimab (TGM/CGM) was recently approved in Italy for all patients treated with RTX in the previous year, independently of their serological status against SARS-CoV-2.ObjectivesWe aimed to evaluate the efficacy and safety of TGM/CGM in a single-centre cohort of rheumatologic patients treated with RTX.MethodsFrom October 2022, all patients who had been treated with RTX in the previous 12 months and who underwent clinical assessment at our rheumatologic tertiary centre were screened for eligibility to PrEP of COVID-19 with TGM/CGM. According to the indications of the Italian Medicines Agency (AIFA), we excluded subjects with major cardiovascular risk factors and/or coagulation abnormalities;those who reported a previous allergic reaction to any anti-COVID19 vaccine were referred to an allergologist for an evaluation before TGM/CGM administration. Patients who agreed to be treated with TGM/CGM signed an informed consent. Clinical and demographic features were collected at baseline, and follow-up phone assessment was performed the day after and 1 month after TGM/CGM administration, to assess treatment tolerability and new COVID-19- related events. A descriptive analysis was performed.ResultsFrom 1 October 2022 to 31 December 2022, 90 subjects were screened for eligibility to TGM/CGM. Among them, 11 were excluded for contraindications due to comorbidities and 55 refused TGM/CGM administration. Among patients who agreed to receive PrEP of COVID-19, 21 received TGM/CGM before 31 December 2022 and 3 were scheduled for January2023. Patients treated with TGM/CGM had a mean age of 54 years (standard deviation: 17) and 19 (90.5%) were female;9 were affected by rheumatoid arthritis and 12 by other rheumatologic diseases (3 systemic lupus erythematosus, 2 systemic sclerosis, 1 Sjögren syndrome, 1 juvenile idiopathic arthritis, 3 anti-synthetase syndrome, 2 vasculitides). Most of them had completed the vaccination schedule against COVID-19 (19, 90.5%) and 9 (42.8%) reported an infectious event by SARS-CoV-2 in the previous year. One month after TGM/CGM administration, no patient reported adverse events related to TGM/CGM nor COVID-19 related symptoms.ConclusionPrEP of COVID-19 with TGM/CGM was well tolerated in our population of rheumatologic patients treated with RTX in the previous year and no COVID-19 related symptoms were observed in the month of follow-up after TGM/CGM administration. Future observations may provide further data on long-term efficacy of TGM/CGM in preventing COVID-19.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of InterestsMaria Manara Speakers bureau: Novartis, Angelini, Consultant of: Lilly, MSD, Manuel Sette: None declared, Laura Giudice: None declared, Martina Biggioggero: None declared, Nicoletta Del Papa Speakers bureau: Janssen, Boehringer-Ingelheim, Actelion, Ennio Giulio Favalli Speakers bureau: AbbVie, BMS, Celltrion, Galapagos, Janssen, Lilly, MSD, Novartis, Pfizer, UCB, Consultant of: AbbVie, BMS, Celltrion, Galapagos, Janssen, Lilly, MSD, Novartis, Pfizer, UCB, Maria Gerosa: None declared, Francesca Ingegnoli: None declared, Roberto Caporali Speakers bureau: AbbVie, Amgen, BMS, Celltrion, Fresenius, Galapagos, Janssen, Lilly, Novartis, Pfizer, UCB, Consultant of: AbbVie, Fresenius, Galapagos, Lilly, Novartis, Pfizer, UCB.

Exosomes/EVs: BIOXOME: A NATURAL LIPOSOME MIMETIC AS DRUG DELIVERY SYSTEM

Background & Aim: Liposomes are spherical-shaped vesicles composed of one or more lipid bilayers. The ability of liposomes to encapsulate hydro- or lipophilic drugs allowed these vesicles to become a useful drug delivery system. Natural cell membranes, such as Bioxome, have newly emerged as new source of materials for molecular delivery systems. Bioxome are biocompatible and GMP-compliant liposome-like membrane that can be produced from more than 200 cell types. Bioxome self-assemble, with in-process self-loading capacity and can be loaded with a variety of therapeutic compounds. Once close to the target tissue, Bioxome naturally fuse with the cell membrane and release the inner compound. Orgenesis is interested in evaluating the potential of Bioxome as new drug delivery system for treatment of several diseases, including skin repair, local tumour or COVID19. Methods, Results & Conclusion(s): Bioxome were obtained from adipose- derived Mesenchymal Stem Cells, with a process of organic- solvent lipid extraction, followed by lyophilization and sonication assemblage. During the sonication process, Bioxome were charged or not with several cargos. Size distribution of empty Bioxome was detected by Particle Size Analyzer (NanoSight). Electron Microscopy (EM) was performed to assess Bioxome morphology. Lipid content was evaluated by electrospray ionization system. Dose response in vitro test on human lung fibroblasts treated or not with Bioxome encapsulating a specific cargo (API) against COVID19 were performed. NanoSight analysis showed that nanoparticle size in Bioxome samples ranged between 170+/-50 nm, with a concentration ranging between 109-1010+/-106 particles/mL. EM clearly showed the double phospholipid layers that composes the Bioxome. Stability study demonstrated that Bioxome are stable in size and concentration up to 90 days at +4Cdegree or even at RT. No change in size between encapsulated Bioxome with small size (~340 Da) cargo vs empty Bioxome was observed up to 30 days storage. Lipidomic analysis approach revealed that the yield of lipids and their composition are satisfactory for a therapeutic product using Bioxome. Lastly, in the in vitro model of COVID19, Bioxome encapsulating API effectively saved cells from death (20x vs untreated cells) and at lower doses of API than these of non-encapsulated cargo (0.005 microM vs 0.1 microM). Bioxome seems to be an excellent candidate for liposome mimetic tool as drug delivery system for targeting specific organs and diseases treatment.Copyright © 2023 International Society for Cell & Gene Therapy

Discussed Poster Abstracts – PM1

B PM1-030 b B Adverse drug reaction profile of drug interactions involving a protein kinase inhibitor indicated in chronic myeloid leukemia from pharmacovigilance databases b M. C. Pajiep, M. Lapeyre-Mestre and F. Despas I Centre Hospitalier Universitaire (CHU) de Toulouse, France i B Introduction: b The introduction of protein kinase inhibitors (PKIs) for chronic myeloid leukemia (CML) has considerably improved prognosis of the disease but has also demonstrated a great potential for drug-drug interactions. Service de Médecine Interne et Infectiologie, Groupe Hospitalier Diaconesses-Croix Saint-Simon, Paris, France i B Introduction: b Despite an important drug-drug interaction, it was previously suggested the clindamycin-rifampicin combination could be used in patients with bone and joints infections (BJIs) provided clindamycin is administered by continuous infusion. Most of eligible patients to the antiviral drug can benefit from it despite the risk of drug-drug interaction. Twenty patients received clindamycin without rifampicin, 19 patients received clindamycin concomitantly with rifampicin and the remaining 85 received clindamycin successively without and with rifampicin. B Results: b Among 957 patients treated with anti-PD-1/PD-L1 during the data collection period, 686 patients were included: 430 new users of a SD regimen, 161 patients who started with SD and switched to ED regimen during follow-up, and 95 new users of an ED regimen. [Extracted from the article] Copyright of Fundamental & Clinical Pharmacology is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

SARS-CoV-2 E and 3a Proteins Are Inducers of Pannexin Currents.

Controversial reports have suggested that SARS-CoV E and 3a proteins are plasma membrane viroporins. Here, we aimed at better characterizing the cellular responses induced by these proteins. First, we show that expression of SARS-CoV-2 E or 3a protein in CHO cells gives rise to cells with newly acquired round shapes that detach from the Petri dish. This suggests that cell death is induced upon expression of E or 3a protein. We confirmed this by using flow cytometry. In adhering cells expressing E or 3a protein, the whole-cell currents were not different from those of the control, suggesting that E and 3a proteins are not plasma membrane viroporins. In contrast, recording the currents on detached cells uncovered outwardly rectifying currents much larger than those observed in the control. We illustrate for the first time that carbenoxolone and probenecid block these outwardly rectifying currents; thus, these currents are most probably conducted by pannexin channels that are activated by cell morphology changes and also potentially by cell death. The truncation of C-terminal PDZ binding motifs reduces the proportion of dying cells but does not prevent these outwardly rectifying currents. This suggests distinct pathways for the induction of these cellular events by the two proteins. We conclude that SARS-CoV-2 E and 3a proteins are not viroporins expressed at the plasma membrane.

Immunogenic cell death-led discovery of COVID-19 biomarkers and inflammatory infiltrates.

Immunogenic cell death (ICD) serves a critical role in regulating cell death adequate to activate an adaptive immune response, and it is associated with various inflammation-related diseases. However, the specific role of ICD-related genes in COVID-19 remains unclear. We acquired COVID-19-related information from the GEO database and a total of 14 ICD-related differentially expressed genes (DEGs) were identified. These ICD-related DEGs were closely associated with inflammation and immune activity. Afterward, CASP1, CD4, and EIF2AK3 among the 14 DEGs were selected as feature genes based on LASSO, Random Forest, and SVM-RFE algorithms, which had reliable diagnostic abilities. Moreover, functional enrichment analysis indicated that these feature genes may have a potential role in COVID-19 by being involved in the regulation of immune response and metabolism. Further CIBERSORT analysis demonstrated that the variations in the immune microenvironment of COVID-19 patients may be correlated with CASP1, CD4, and EIF2AK3. Additionally, 33 drugs targeting 3 feature genes had been identified, and the ceRNA network demonstrated a complicated regulative association based on these feature genes. Our work identified that CASP1, CD4, and EIF2AK3 were diagnostic genes of COVID-19 and correlated with immune activity. This study presents a reliable diagnostic signature and offers an overview to investigate the mechanism of COVID-19.

Research highlights of renal transplantation in 2020: voice from China

Renal transplantation is the optimal approach to improve the quality of life and restore normal life for patients with end-stage renal diseases. With the development of medical techniques and immunosuppressants, the short-term survival of renal graft has been significantly prolonged, whereas the long-term survival remains to be urgently solved. Renal ischemia-reperfusion injury (IRI), acute rejection, chronic renal allograft dysfunction, renal fibrosis and other factors are still the major problems affecting the survival of renal graft. Relevant researches have always been hot spots in the field of renal transplantation. Meantime, 2020 is an extraordinary year. The novel coronavirus pneumonia (COVID-19) pandemic severely affects the development of all walks of life. Researches related to renal transplantation have also sprung up. In this article, the frontier hotspots of clinical and basic studies related to renal transplantation and the COVID-19 related researches in the field of renal transplantation in China were reviewed, aiming to provide novel therapeutic ideas and strategies.Copyright © 2021 Journal of Zhongshan University. All Rights Reserved.

Classification of tuberculosis-related programmed cell death-related patient subgroups and associated immune cell profiling.

Background: Tuberculosis (TB) is the deadliest communicable disease in the world with the exception of the ongoing COVID-19 pandemic. Programmed cell death (PCD) patterns play key roles in the development and progression of many disease states such that they may offer value as effective biomarkers or therapeutic targets that can aid in identifying and treating TB patients. Materials and methods: The Gene Expression Omnibus (GEO) was used to gather TB-related datasets after which immune cell profiles in these data were analyzed to examine the potential TB-related loss of immune homeostasis. Profiling of differentially expressed PCD-related genes was performed, after which candidate hub PCD-associated genes were selected via a machine learning approach. TB patients were then stratified into two subsets based on the expression of PCD-related genes via consensus clustering. The potential roles of these PCD-associated genes in other TB-related diseases were further examined. Results: In total, 14 PCD-related differentially expressed genes (DEGs) were identified and highly expressed in TB patient samples and significantly correlated with the abundance of many immune cell types. Machine learning algorithms enabled the selection of seven hub PCD-related genes that were used to establish PCD-associated patient subgroups, followed by the validation of these subgroups in independent datasets. These findings, together with GSVA results, indicated that immune-related pathways were significantly enriched in TB patients exhibiting high levels of PCD-related gene expression, whereas metabolic pathways were significantly enriched in the other patient group. Single cell RNA-seq (scRNA-seq) further highlighted significant differences in the immune status of these different TB patient samples. Furthermore, we used CMap to predict five potential drugs for TB-related diseases. Conclusion: These results highlight clear enrichment of PCD-related gene expression in TB patients and suggest that this PCD activity is closely associated with immune cell abundance. This thus indicates that PCD may play a role in TB progression through the induction or dysregulation of an immune response. These findings provide a foundation for further research aimed at clarifying the molecular drivers of TB, the selection of appropriate diagnostic biomarkers, and the design of novel therapeutic interventions aimed at treating this deadly infectious disease.

Ferroptosis Is a Potential Therapeutic Target for Pulmonary Infectious Diseases

Ferroptosis is a new type of iron-dependent cell death caused by lipid peroxide (LPO) accumulation and involved in disease of pulmonary infection. The dysregulation of iron metabolism, the accumulation of LPO, and the inactivation and consumption of glutathione peroxidase 4 (GPX4) are the crucial cause of ferroptosis. Pulmonary infectious diseases caused by Pseudomonas aeruginosa (PA), Mycobacterium tuberculosis (MTB), and severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) are associated with ferroptosis. Ferroptosis may be a potential therapeutic target for pulmonary infectious diseases. However, the mechanisms by which these infections are involved in ferroptosis and whether pulmonary infectious diseases caused by Staphylococcus aureus, Klebsiella pneumoniae, and Leishmania spp are related to ferroptosis are unclear. Accordingly, more researches are needed.Copyright © 2023 Yurong Zhang et al.

Covid-19 Vaccination Impacts Functional Immune Responses and Plasma Proteome in Plwh

Background: The impact of COVID-19 infection or COVID-19 vaccination on the immune system of people living with HIV (PLWH) is unclear. We therefore studied the effects of COVID-19 infection or vaccination on functional immune responses and systemic inflammation in PLWH. Method(s): Between 2019 and 2021, 1985 virally suppressed, asymptomatic PLWH were included in the Netherlands in the 2000HIV study (NCT039948350): 1514 participants enrolled after the start of the COVID-19 pandemic were separated into a discovery and validation cohort. PBMCs were incubated with different stimuli for 24 hours: cytokine levels were measured in supernatants. ~3000 targeted plasma proteins were measured with Olink Explore panel. Past COVID-19 infection was proven when a positive PCR was reported or when serology on samples from inclusion proved positive. Compared were unvaccinated PLWH with and without past COVID-19 infection, and PLWH with or without anti-COVID-19 vaccination excluding those with past COVID-19 infection. Result(s): 471 out of 1514 participants were vaccinated (median days since vaccination: 33, IQR 16-66) and 242 had a past COVID-19 infection (median days since +PCR: 137, IQR 56-206). Alcohol, smoking, drug use, BMI, age, latest CD4 count and proportion with viral blips were comparable between groups. Systemic inflammation as assessed by targeted proteomics showed 89 upregulated and 43 downregulated proteins in the vaccinated participants. In contrast, individuals with a past COVID-19 infection display lower levels of 138 plasma proteins compared to the uninfected group (see figure). 'Innate immune system' and 'cell death' were upregulated in pathway analysis in vaccinated PLWH, but downregulated in COVID-19 infected participants. The increased systemic inflammation of the COVID-19 vaccinated group was accompanied by lower TNF-alpha and IL-1beta production capacity upon restimulation with a range of microbial stimuli, while production of IL-1Ra was increased. In COVID-19 infected PLWH only a reduced production of TNF-alpha to S. pneumonia was significant. Vaccinated PLWH also showed upregulation of platelet aggregation pathways. Conclusion(s): COVID-19 vaccination in PLWH leads to an increased systemic inflammation, but less effective cytokine production capacity of its immune cells upon microbial stimulation. This pattern is different from that of COVID-19 infection that leads to a decreased inflammatory profile and only minimal effects on cytokine production capacity. (Figure Presented).

Activated CD8 T-cell hepatitis in children: a single institution experience

Background: Pediatric acute liver failure is a rare and serious life-threatening situation, principally for the 30 to 50% of children in whom the etiology of their liver failure is unclear or indeterminate. Treating these patients is challenging, requiring constant assessment over time with regular evaluation for possible liver transplantation. Children with pediatric acute liver failure of undetermined etiology have lower spontaneous survival and higher rates of transplantation and death than other diagnostic groups. Emerging evidence suggests that a subgroup of patients with indeterminate pediatric acute liver failure have clinical, laboratory, and liver biopsy features of immune dysregulation with a dense infiltration of CD8 T cells. Method(s): In 2022, we received percutaneous liver biopsies from three children with acute hepatic dysfunction that showed an increased number of lymphocytes including CD8 T cells. For each case, routine H&E stains with levels, special stains and immunostains were performed. The first biopsy was from an 18-month-old male who presented with COVID infection, pancytopenia, elevated transaminases, and synthetic liver dysfunction (elevated INR). The second was from a 9-year-old female with a history of elevated liver enzymes with no clear cause. The third case was from a 2-year-old male with elevated liver enzymes, coagulopathy, and cholestasis. Result(s): The three cases showed similar histopathologic findings;an acute liver injury pattern with lobular architectural disarray, giant cell formation, reactive changes, single cell necrosis, cholestasis and marked mixed lymphocytic infiltrates. The infiltrates were predominantly composed of CD8-positive T-lymphocytes with scattered neutrophils, eosinophils and rare plasma cells. Portal areas were mildly expanded with mild bile ductular proliferation and mild to moderate lymphocytic infiltrates. Immunostains for CD8 demonstrated that the infiltrates were predominantly composed of CD8-positive T-lymphocytes. All three patients received steroids and responded to treatment evidenced by normalization of liver enzymes and function. Conclusion(s): Dense hepatic CD8 T-cell infiltration is a major finding inactivated CD8 T-cell hepatitis. However, the percentage distribution of lymphocyte subtypes in the setting of hepatitis is not well established, and CD8 T-cell infiltration has also been described in cases of drug-induced hypersensitivity reactions, viral hepatitis, hemophagocytic lymphohistiocytosis, and macrophage activation syndrome, as well as autoimmune hepatitis. Further investigation is needed to better understand the diagnostic criteria in this disease.

Towards New Antimicrobials - Targeting Bacterial Response Regulators

Purpose of Study: Antibiotic resistance remains one of the largest healthcare and public health challenges. Several studies have documented that the spread of antibiotic resistant bacteria in nosocomial settings has been exacerbated worldwide due to increased rates of hospitalization and intubation in the wake of the COVID-19 pandemic. One way to address antibiotic resistance is to identify novel compounds that inhibit essential microbial processes. Two-component regulatory systems are important mediators of signal transduction that allow bacteria to communicate with and respond to changes in their environment. The WalRK system is a two-component system that is conserved and essential for viability in many Gram-positive human pathogens. We hypothesize that a ligand that specifically binds with the DNA-interaction surface of the WalR protein can lead to cell death and can serve as a lead compound for future drug development efforts. Methods Used: We describe the development process of an assay to identify WalR binding compounds. In silico molecular dynamics docking approaches were utilized to identify potential WalR binding compounds from virtual compound libraries. To assess their WalR-binding capacity in vitro, overexpression strains for several WalR recombinant constructs were engineered and protein constructs were purified to homogenicity. Isothermal titration calorimetry (ITC) is a technique that measures heat release or absorption when two molecules interact. A MicroCal PEAQ ITC instrument was utilized to develop a WalR-binding assay. Summary of Results: WalR is a two-domain protein featuring a regulatory and a DNA-binding domain. Two constructs, a truncated DNA-binding domain and a full-length protein construct proved soluble, and pure quantities necessary to conduct ITC measurements could be successfully obtained (12 mg full-length protein and 23 mg truncated protein). These proteins were amenable to ITC experiments. We found that experiments were best run with at least a two-fold increase of ligand concentration to protein concentration supplied in identical buffer conditions over nineteen injections. We are currently assessing the binding affinities of our in silico hit compounds. Conclusion(s): Our results show that ITC enables the detailed, rapid, and reproducible characterization of the binding relationship between the DNA-binding domain of the WalR protein and any potential ligands. The protocol discussed herein will enable further drug discovery studies on the WalR response regulator protein to identify and characterize inhibitors, providing insight towards the development of novel antimicrobial compound.

Using ex vivo bioengineered lungs to model pathologies and screening therapeutics

Introduction: More than 8 million lives are claimed annually by various respiratory diseases including lung cancer. While therapeutics is the first line of defence, treatment failure always remains challenging and research studies face a lag of transition from preclinical to clinical phase. This is partly due to the inadequate representation of the preclinical models in clinical trials. In this proof-of-concept study, we sought to use an ex-vivo model to identify lung pathologies and therapeutically screen them in a rodent model. Method(s): Briefly, the heart-lung tissues were extracted and decellularized using a detergent-based decellularization technique. Subsequently, lungs were seeded and cultured (6-10 days) with human cell lines: BEAS-2B, A549, and Calu3, demonstrating healthy lung, cancerous state, and congenital pathologies (cystic fibrosis), respectively. By altering the cultural conditions and exploiting the unique characteristics of these cell lines, we were able to model a variety of novel pathological models in ex vivo, such as advanced-stage solid tumours and the primary phase of infection via SARS-COV2. We also validated the above-mentioned observations by histology and immunofluorescence staining. Another novel part of our study includes a qualitative screening of efficacy and impact of important Therapeutics (anti-neoplastic)- Cisplatin and Wogonin, in our cancer models. Result(s): Using A549 and BEAS-2B cells, we were able to model different stages of Non-small cell lung cancer, qualitatively validated the resemblance to clinical samples and monitor the impact of different therapeutics on these models. The qualitative assessment also demonstrated different levels of cell death depending on the efficacy of the drugs. Contribution to research : Collectively this study demonstrates the remarkable versatility and strength of the ex vivo model in representing important lung pathologies and screening therapeutics in the preclinical phase.

Platelets and SARS-CoV-2 During COVID-19: Immunity, Thrombosis, and Beyond.

COVID-19 is characterized by dysregulated thrombosis and coagulation that can increase mortality in patients. Platelets are fast responders to pathogen presence, alerting the surrounding immune cells and contributing to thrombosis and intravascular coagulation. The SARS-CoV-2 genome has been found in platelets from patients with COVID-19, and its coverage varies according to the method of detection, suggesting direct interaction of the virus with these cells. Antibodies against Spike and Nucleocapsid have confirmed this platelet-viral interaction. This review discusses the immune, prothrombotic, and procoagulant characteristics of platelets observed in patients with COVID-19. We outline the direct and indirect interaction of platelets with SARS-CoV-2, the contribution of the virus to programmed cell death pathway activation in platelets and the consequent extracellular vesicle release. We discuss platelet activation and immunothrombosis in patients with COVID-19, the effect of Spike on platelets, and possible activation of platelets by classical platelet activation triggers as well as contribution of platelets to complement activation. As COVID-19-mediated thrombosis and coagulation are still not well understood in vivo, we discuss available murine models and mouse adaptable strains.

Comparison of disinfectants-induced gene expression profile: Potential adverse effects.

Despite their importance in combating the spread of the COVID-19 pandemic, adverse effects of disinfectants on human health, especially the respiratory system, have been of continuing concern to researchers. Considering that bronchi are the main target of sprayed disinfectants, we here treated the seven major active ingredients in disinfectant products accepted by the US EPA to human bronchial epithelial cells and determined the subtoxic levels. Then, we performed microarray analysis using total RNA obtained at the subtoxic level and designed a network representing disinfectant-induced cellular response using the KEGG pathway analysis technique. Polyhexamethylguanidine phosphate, a lung fibrosis inducer, was used as a reference material to verify the relationship between cell death and pathology. The derived results reveal potential adverse effects along with the need for an effective application strategy for each chemical.

Antioxidant and Anti-Breast Cancer Properties of Hyaluronidase from Marine Staphylococcus aureus (CASMTK1)

This work studied the antioxidant and anti-breast cancer properties of hyaluronidase, extracted from a potential marine strain, Staphylococcus aureus (CASMTK1), isolated from Parangipettai coastal waters in southeast coast of India. The Staphylococcal enzyme production was tested under different carbon and nitrogen sources;and recorded the maximum production when the microbial strain was cultured with starch as the carbon source and ammonium sulphate as the inorganic nitrogen source with the enzyme production of 92.5 U/mL and 95.0 U/mL, respectively. The hyaluronidase enzyme production was also tested in different pH and temperature;and recorded the maximum yield of 102.5 U/mL in pH 5 and that of 95.5 U/mL in 45 °C. The partially purified enzyme was subjected to FTIR and FT Raman technique and found the presence of the amide- I and II, Carboxyl, N-H bending, C-H stretching and α-helices and β-sheet proteins between wave number 1500–1700 cm−1. The partially purified enzyme also exhibited strong antioxidant and in-vitro breast cancer properties. The enzyme showed the highest hydroxyl radical scavenging activity of 79% at the 50 µg/mL concentration, and this activity increased in a dose-dependent manner. The enzyme inhibited proliferation of the breast cancer cell line of MCF-7, and it caused 100% cell death at the concentration of 80 µg/mL. The enzyme generated capacity of producing free radicles that damage the cancer cells, and this effect was very nearer to the standard drug, paclitaxel. The enzyme damaged the cancer cells and induced apoptosis in 78% of cancer cells as evident by condensed or fragmented chromatin at 40 µg/mL. Further purification of the enzyme, analysis of its molecular aspects, and elucidation of exact mechanisms of its biological activities will throw new light on the utility of staphylococcal hyaluronidase in anticancer chemotherapy.

Synthesis, characterization, cytotoxicity evaluation, and molecular docking with DNA, bovine serum albumin, and SARS-Cov-2 of copper complexes bearing tris-(2-pyridyl)-pyrazolyl borate ligand

Four copper (II) complexes bearing tris-(2-pyridyl)-pyrazolyl borate (Tppy) ligand with corresponding chloride (Cu-1), aqua (Cu-2), azide (Cu-3), and thiocyanide (Cu-4) substitutions were synthesized and characterized by spectroscopic and analytical methods. Spectroscopic and molecular docking studies were employed to investigate the interactions of these complexes with calf thymus (CT) DNA and bovine serum albumin (BSA). The results inferred intercalation binding mode of the complexes with DNA. All the complexes exhibited good binding with BSA as well. In addition, the binding efficacy of the Cu (II) complexes with SARS-Cov-2 was tested in silico. Further, in vitro anticancer activity of the complexes was investigated against the HeLa-cervical, HepG2-liver and A549-lung cancer, and one normal (L929-fibroblast) cell line. IC50 values unveiled that the complexes were more active than cisplatin against all three cancer cells. It was understood that complex Cu-3 containing azide substitution displayed the highest activity on the HeLa cell line (IC50 = 6.3 μM). More importantly, TppyCu (II) complexes were not active against the normal cell line. Lastly, the acridine orange/ethidium bromide (AO/EB) and 4′,6-diamidino-2-phenylindole staining assays indicated that Cu-3 induced cell death in HeLa cells at the late apoptotic stage. This complex also efficiently generated ROS in HeLa cells promoting apoptosis as understood from the DCFH-DA assay. © 2023 John Wiley & Sons, Ltd.

Update of cellular responses to the efferocytosis of necroptosis and pyroptosis.

Cell death is a basic physiological process that occurs in all living organisms. A few key players in these mechanisms, as well as various forms of cell death programming, have been identified. Apoptotic cell phagocytosis, also known as apoptotic cell clearance, is a well-established process regulated by a number of molecular components, including 'find-me', 'eat-me' and engulfment signals. Efferocytosis, or the rapid phagocytic clearance of cell death, is a critical mechanism for tissue homeostasis. Despite having similar mechanism to phagocytic clearance of infections, efferocytosis differs from phagocytosis in that it induces a tissue-healing response and is immunologically inert. However, as field of cell death has rapid expanded, much attention has recently been drawn to the efferocytosis of additional necrotic-like cell types, such as necroptosis and pyroptosis. Unlike apoptosis, this method of cell suicide allows the release of immunogenic cellular material and causes inflammation. Regardless of the cause of cell death, the clearance of dead cells is a necessary function to avoid uncontrolled synthesis of pro-inflammatory molecules and inflammatory disorder. We compare and contrast apoptosis, necroptosis and pyroptosis, as well as the various molecular mechanisms of efferocytosis in each type of cell death, and investigate how these may have functional effects on different intracellular organelles and signalling networks. Understanding how efferocytic cells react to necroptotic and pyroptotic cell uptake can help us understand how to modulate these cell death processes for therapeutic purposes.

The Relationship Between Ferroptosis and Diseases.

Ferroptosis is an iron-dependent mode of cell death. It can occur through two major pathways, exogenous (or transporter-dependent) and endogenous (or enzyme-regulated) pathways are activated by biological or chemical inducers, and glutathione peroxidase activity is inhibited, which causes intracellular iron accumulation and lipid Peroxidation. Ferroptosis is closely related to the pathological process of many diseases. How to intervene in the occurrence and development of related diseases by regulating ferroptosis has become a hot research topic. At present, studies have shown that ferroptosis is found in common diseases such as tumors, inflammatory diseases, bacterial infections, pulmonary fibrosis, hepatitis, inflammatory bowel disease, neurodegenerative diseases, kidney injury, ischemia-reperfusion injury and skeletal muscle injury. This article reviews the characteristics and mechanism of ferroptosis, and summarizes how ferroptosis participates in the pathophysiological process in various systemic diseases of the body, which may provide new references for the treatment of clinical diseases in the future.