A call for an integrated biology

The Song of the Cell tells the story of how we came to understand ourselves and other complex living organisms as mosaics of these atoms of life. There is the "dividing cell”, which takes us to the 2001 Nobel Prize in Physiology or Medicine that Nurse won, alongside Leland Hartwell and Tim Hunt, for elucidating the roles of cyclin and cyclin-dependent kinase proteins in regulating the cell cycle, and to the invention of reproductive in-vitro fertilisation by Patrick Steptoe and Robert Edwards during the 1970s. Quite what the "new human” of the subtitle refers to is never fully clear, but the ability to reprogramme cell states, as for example, in the method devised by stem-cell researcher Shinya Yamanaka to induce mature somatic cells back to pluripotency, might have the potential to grow organs in vitro, to regenerate tissues in vivo, and even to create synthetic embryo-like structures without the involvement of fertilisation. Mukherjee rightly admits that this view goes too far, but it might at least be understood as suggesting that trying to attack cancer at the genetic level is like hoping to stop traffic jams by fixing the faulty brakes of the car that caused the last one.

COVID-19: Using high-throughput flow cytometry to dissect clinical heterogeneity.

Here we consider how high-content flow cytometric methodology at appropriate scale and throughput rapidly provided meaningful biological data in our recent studies of COVID-19, which we discuss in the context of other similar investigations. In our work, high-throughput flow cytometry was instrumental to identify a consensus immune signature in COVID-19 patients, and to investigate the impact of SARS-CoV-2 exposure on patients with either solid or hematological cancers. We provide here some examples of our 'holistic' approach, in which flow cytometry data generated by lymphocyte and myelomonocyte panels were integrated with other analytical metrics, including SARS-CoV-2-specific serum antibody titers, plasma cytokine/chemokine levels, and in-depth clinical annotation. We report how selective differences between T cell subsets were revealed by a newly described flow cytometric TDS assay to distinguish actively cycling T cells in the peripheral blood. By such approaches, our and others' high-content flow cytometry studies collectively identified overt abnormalities and subtle but critical changes that discriminate the immuno-signature of COVID-19 patients from those of healthy donors and patients with non-COVID respiratory infections. Thereby, these studies offered several meaningful biomarkers of COVID-19 severity that have the potential to improve the management of patients and of hospital resources. In sum, flow cytometry provides an important means for rapidly obtaining data that can guide clinical decision-making without requiring highly expensive, sophisticated equipment, and/or "-omics" capabilities. We consider how this approach might be further developed.

A New CURE in Molecular Biology: Testing a Novel Pyrrolidine Compound to Determine Mechanism of Action in an Upper-Division Molecular Biology Course

The COVID-19 pandemic shut down forced introductory biology and chemistry laboratory courses online at DePauw University from March 2020-June 2021, leaving multiple classes of students without the opportunity to learn basic laboratory skills that are essential for the molecular biology laboratory. In an effort to provide students with both basic laboratory skills and advanced molecular biology skills, a new course-based undergraduate research experience (CURE) was developed for the 2022-23 academic year. In collaboration with Dr. Jeff Hansen in the Chemistry and Biochemistry department, novel compounds with potential anti-tumor properties were identified. The CURE in Molecular Biology was designed to have students use Saccharomyces cerevisiae as a model system to evaluate possible cellular pathways affected by the compound, including: cytoskeleton and cell migration, nucleotide biosynthesis, glucose metabolism, apoptosis, and cell cycle regulation. Students learned techniques DNA isolation and PCR, transformation, RNA isolation, cDNA synthesis, qPCR, and Western Blotting, while contributing to an active research project. At the conclusion of the project, students were surveyed about their comfort with molecular techniques and data analysis.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Central Nervous System Distribution of the Ataxia-Telangiectasia Mutated Kinase Inhibitor AZD1390: Implications for the Treatment of Brain Tumors

Effective drug delivery to the brain is critical for the treatment of glioblastoma (GBM), an aggressive and invasive primary brain tumor that has a dismal prognosis. Radiation therapy, the mainstay of brain tumor treatment, works by inducing DNA damage. Therefore, inhibiting DNA damage response (DDR) pathways can sensitize tumor cells to radiation and enhance cytotoxicity. AZD1390 is an inhibitor of ataxia-telangiectasia mutated kinase, a critical regulator of DDR. Our in vivo studies in the mouse indicate that delivery of AZD1390 to the central nervous system (CNS) is restricted due to active efflux by P-glycoprotein (P-gp). The free fraction of AZD1390 in brain and spinal cord were found to be low, thereby reducing the partitioning of free drug to these organs. Coadministration of an efflux inhibitor significantly increased CNS exposure of AZD1390. No differences were observed in distribution of AZD1390 within different anatomic regions of CNS, and the functional activity of P-gp and breast cancer resistance protein also remained the same across brain regions. In an intracranial GBM patient-derived xenograft model, AZD1390 accumulation was higher in the tumor core and rim compared with surrounding brain. Despite this heterogenous delivery within tumor-bearing brain, AZD1390 concentrations in normal brain, tumor rim, and tumor core were above in vitro effective radiosensitizing concentrations. These results indicate that despite being a substrate of efflux in the mouse brain, sufficient AZD1390 exposure is anticipated even in regions of normal brain. SIGNIFICANCE STATEMENT Given the invasive nature of glioblastoma (GBM), tumor cells are often protected by an intact blood-brain barrier, requiring the development of brain-penetrant molecules for effective treatment. We show that efflux mediated by P-glycoprotein (P-gp) limits central nervous system (CNS) distribution of AZD1390 and that there are no distributional differences within anatomical regions of CNS. Despite efflux by P-gp, concentrations effective for potent radiosensitization are achieved in GBM tumor-bearing mouse brains, indicating that AZD1390 is an attractive molecule for clinical development of brain tumors.Copyright © 2022 American Society for Pharmacology and Experimental Therapy. All rights reserved.

Long COVID in relation to severity of acute COVID-19

Introduction: Many patients suffer from persistent and prolonged effects after acute COVID-19. The aim of our study is to describe the prevalence of Long COVID in survivors after discharge and to determine the relation between the severity of acute COVID-19 infection and Long COVID. Method(s): Retrospective study of hospitalized patients with COVID-19 in pneumology department II of Abderahman Mami hospital. We compared 2 groups: a moderate COVID-19 group (G1) and a severe COVID-19 group (G2). We compared persistent symptoms, Chest CT and pulmonary function tests assessments at 3 months after discharge in the two groups. Result(s): A total of 150 patients were initially admitted. Median age was 64 years. Patients in G2 were older and had more comorbidities than those in the moderate group. The main comorbidities were hypertension (38.6%) and diabetes (33.3%). Common symptoms of acute COVID 19 revealed were: breathlessness (86%), cough (68%) and fatigue (63%). Ninety patients were followed up for a mean of 3 months. The Prevalence of Long COVID was 54.4% (n=49). G2 had a higher Prevalence compared to G1: 35.7%G1 (n=15) vs 70.8%G2 (n=34), p=0.001. The most frequent persistent symptoms revealed were: breathlessness (27.2% G1 vs 67.5% 2, p=0.001) fatigue (21.2% G1 s 42.5%, p=0.05) and cough (12.1% G1 vs 18.4% G2, p=0.205). Pulmonary diffusion impairment was the most common pulmonary dysfunction (45% G1 vs 69.2% G2 p=0.19). Abnormal CT findings are predominately found in patients of G2. Lung fibrotic-like changes were observed in 25% of the patients in G2 vs 14.2% in G1 within 3 months. Conclusion(s): A spectrum of sequelae has been reported among COVID-19 survivors. Patients with high-risk of postacute COVID-19 should be identified.

Immunohistochemical profile of cell cycle control, proliferation and differentiation proteins of the pulmonary atypical alveolar epithelium with COVID-19-induced diffuse alveolar damage as compared to pulmonary [lepidic-growth] adenocarcinoma.

We aim aim to compare immunophenotypic charac-teritics of atypical epithelium (AE) with COVID-19-induced diffuse alveolar damage (DAD) and pulmonary lepidic-growth adenocarcinoma, accounting for cell cycle control, proliferation and differentiation]. Methods. We examined pulmonary tissue specimens from twenty-four fatal cases of CO VID-19-induced acute respiratory damage syndrome confirmed by autopsy (Group 1) and four cases of pulmonary lepidic-growth adenocarcinoma (Group 2). Perpendicular dimensions of 10 nuclei were measured on the H&E slides, means of their sums of products (SPNM) were calculated. We have used p53, Ki67, pi6, p63 antibodies for immunohistochemical staining in each case. We evaluate colour intensity, rate of stained cells of AE and the product of these parameters. We evaluated separately Nuclear and cyto-plasmic staining (couple) and only cytoplasmic staining (cyt) for pi6 expression. We measured proliferative index only at KI-67 stained slides. U-test and Spearman rank correlation test were used for statistical analysis. Results. Expression of p63 was higher in group 1 (p=0.001), while pi6 was more frequently expressed in group 2 (p=0.002). We have found no statistically significant differences (p>0.1) in the p53 and Ki67 expression. Group 1 showed There was negative correlation between the number of days from onset of symptoms and the following variables: Ki67 (r=M).587, p=0.003);SPNM (r 0.406, p=0.049). Conclusion. The present study has shown heterogeneity in levels of cell cycle control expression, proliferation and differentiation of atypical epithelium in the pulmonary lep-idic-growth adenocarcinoma and CO VID-19-induced diffuse alveolar damage.Copyright © 2022 Izdatel'stvo Meditsina. All rights reserved.

Oxygen home therapy after discharge of hospitalized patients with COVID-19

Introduction: Patients hospitalized with COVID-19 may have many healthcare needs after hospital discharge. Providing transitional care in pandemic circumstances, must adapt to specific needs and limitations. Aim(s): Define the profile of patients with COVID-19 who needed oxygen home therapy after discharge. Method(s): Retrospective study including patients infected with COVID-19 ;confirmed either by RT-PCR or Rapid Antigen Test and hospitalized in COVID-19 unit from September 2020 to June2021. Patients who deceded and those who were transferred to ICU without any data about evolution, were excluded. We defined 2 groups: G0: Patients discharged without oxygen home therapy G1 : Patients who required oxygen home therapy Results: A total of 528 patients were included: G0: 491 patients (93%), G1: 37 patients (7%). The mean age was significantly different between both groups (G0: 61,4 ;G1:68,0 p=0,003). There was no significant correlation regarding the extent of the lesions on the chest CT scan (G0:45%, G1:47% p=0.698), however maximum oxygen needs were higher in G1 (G0:7L, G1: 11L;p=0.001). Smoking, COPD, and hypercapnia were significantly related to prolonged oxygen need (G0: 30%, G1: 50%;p=0,026);(G03,7%, G1:24,3%;p=0,000) and (G0:34, G1:43;p=0.003) respectively. Conclusion(s): Chronic hypoventilation seems to be a predictive factor of prolonged oxygen needs. Oxygen home therapy can be a transitional solution to shorten hospital stay.

Human induced pluripotent stem cell-derived alveolar type 2 cells mature at air-liquid interface and respond to airborne stimuli

Human alveolar type II cells (AT2s) are progenitors of the alveolar epithelium and are among the pulmonary cells that are directly exposed to inhaled stimuli. Primary human AT2s can be cultured in three-dimensional alveolospheres, but are difficult to culture in the physiologically relevant air-liquid interface (ALI) format. Human induced pluripotent stem cells (iPSCs) can be directed to differentiate to iPSC-derived AT2s (iAT2s) in alveolospheres, where they transcriptomically resemble fetal lung. Here we report the successful adaptation of iAT2s to ALI culture, which promotes their maturation and permits exposure to inhaled stimuli. We transcriptomically profile iAT2s cultured at ALI and find that they mature as they downregulate cell cycle-associated transcripts. We then evaluate the extent of iAT2 maturation at ALI within the developmental context by comparison to primary AT2s. We find that iAT2s at ALI are more similar to primary AT2s than iAT2s cultured as spheres, and that differences are driven by primary AT2s' response to immune stimuli. We then test the capacity of iAT2s to respond to immune stimuli by infecting with SARSCoV-2. We find that iAT2s mount an epithelial-intrinsic interferon and inflammatory response to SARS-CoV-2 infection, and can serve as a platform for testing antiviral therapeutics. Finally, we demonstrate that iAT2s at ALI respond to cigarette smoke and electronic cigarette vapor, enabling the direct comparison of these common inhaled stimuli. Overall, we describe a novel disease modeling platform that will enable exploration of gene-environment interactions unique to inhaled exposures of the alveolar epithelium.

Bioinformatics Analysis of Candidate Genes and Pathways Related to Hepatocellular Carcinoma in China: A Study Based on Public Databases.

Background and Objective: Hepatocellular carcinoma (HCC) is a highly aggressive malignant tumor of the digestive system worldwide. Chronic hepatitis B virus (HBV) infection and aflatoxin exposure are predominant causes of HCC in China, whereas hepatitis C virus (HCV) infection and alcohol intake are likely the main risk factors in other countries. It is an unmet need to recognize the underlying molecular mechanisms of HCC in China. Methods: In this study, microarray datasets (GSE84005, GSE84402, GSE101685, and GSE115018) derived from Gene Expression Omnibus (GEO) database were analyzed to obtain the common differentially expressed genes (DEGs) by R software. Moreover, the gene ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed by using Database for Annotation, Visualization and Integrated Discovery (DAVID). Furthermore, the protein-protein interaction (PPI) network was constructed, and hub genes were identified by the Search Tool for the Retrieval of Interacting Genes (STRING) and Cytoscape, respectively. The hub genes were verified using Gene Expression Profiling Interactive Analysis (GEPIA), UALCAN, and Kaplan-Meier Plotter online databases were performed on the TCGA HCC dataset. Moreover, the Human Protein Atlas (HPA) database was used to verify candidate genes' protein expression levels. Results: A total of 293 common DEGs were screened, including 103 up-regulated genes and 190 down-regulated genes. Moreover, GO analysis implied that common DEGs were mainly involved in the oxidation-reduction process, cytosol, and protein binding. KEGG pathway enrichment analysis presented that common DEGs were mainly enriched in metabolic pathways, complement and coagulation cascades, cell cycle, p53 signaling pathway, and tryptophan metabolism. In the PPI network, three subnetworks with high scores were detected using the Molecular Complex Detection (MCODE) plugin. The top 10 hub genes identified were CDK1, CCNB1, AURKA, CCNA2, KIF11, BUB1B, TOP2A, TPX2, HMMR and CDC45. The other public databases confirmed that high expression of the aforementioned genes related to poor overall survival among patients with HCC. Conclusion: This study primarily identified candidate genes and pathways involved in the underlying mechanisms of Chinese HCC, which is supposed to provide new targets for the diagnosis and treatment of HCC in China.

Utility of urinary biomarkers for diagnosis of acute kidney injury (AKI) in COVID-19

Introduction: Incidence of AKI reported varies from 0.5% to 37%.These incidence cannot be extrapolated in our patients as the severity of COVID-19 infection, the ethnicity of the patients l, the clinical profile and the healthcare delivery system is different.The aim of this study was to explore whether urinary cell cycle arrest markers and other renal biomarkers have a role in predicting AKI in critically ill patients with COVID-19 and acute respiratory disease Methods: This prospective, longitudinal cohort study included critically ill COVID-19 patients without AKI at study entry. Patients aged more than 18 years with moderate or severe respiratory disease as defined by Berlin criteria were subsequently recruited from November 2020 to May 2021. Urine samples were collected on admission to critical care areas for determination of KIM1, NGAL, IL-18,IGF-BP-7, TIMP -2 at the time point of study inclusion, 12h, 24h, 48h, after inclusion. The demographic information, comorbidities, clinical, and laboratory data were recorded. The study outcomes were the development of AKI and mortality during hospitalization. Result(s): [Formula presented] ROC analysis was done to determine the diagnostic performance of the various urinary biomarkers;AUC was 0.655 for normalised IL-18, 0.685 for normalised NGAL, 0.658 for normalised TIM-1, and so on Conclusion(s): AKI was common in critically ill COVID-19 patients. Raised values of urinary biomarkers with clinical information, are useful for the identification of AKI in critically ill COVID-19 patients. No conflict of interestCopyright © 2023

Persistent dyspnea six months after SARS-CoV-2 pneumonia : what are the contributing factors ?

Introduction: Dyspnea is a common symptom in post Covid-19 Syndrome. The aim of this study was to evaluate the frequency of persistent dyspnea and assess the factors associated with persistent dyspnea in patients 6 months after discharge for covid-19 pneumonia. Method(s): A retrospective comparative study was performed between november 2020 and december 2021. Sixty - two patients were included from the post-COVID follow-up consultation 6 months after hospitalization for the acute episode. Patients were divided into 2 groups: G1 (n=19) with persistent dyspnea and G2 (n=43) patients without persistent dyspnea. Result(s): The frequency of persistent dyspnea was 30.64%. Compared to G2, the G1 have also more persistent dray cough (42.1% vs 9.3 % ;p=0.005) and memory loss (10.5% vs 0% ;p=0.03) at 6 months. G1 had more severe cases of COVID-19 pneumonia (47.4% vs 41.9%,p=0.01) and non-invasive ventilation (22.2% vs 2.4%,p=0.02) and less anosmia (0% vs 20.9%,p=0.04) in the acute phase of Covid-19 infection than G2. Asthma (21,1% vs 2,3% p=0,02) was more prevalent in G1. The age (p=0.6), female sex (p=0.53), cardiovascular (p=0.5) and metabolic (p=0.8) diseases and extensive radiological damage greater than 50% in the acute phase (p=0.4) were similar in both groups. The mean of FEV1(77.6% vs 104,2%,p=0.01) and DLCO (57% vs 78.72%,p=0.01) were lower in G1 than G2. Radiological cleaning (p=0.1), the mean of FVC (p=0.09), TLC(p=0.32) and 6MWT (p=0.8) were similar in both groups. Conclusion(s): The severity of COVID-19 pneumonia and impairment of DLCO and FEV1 are factors associated with persistent dyspnea in patients at 6 months post Covid -19 infection.

Hypoxia induces dichotomous and reversible attenuation of T cell responses through reactive oxygen species-dependent phenotype redistribution and delay in lymphoblast proliferation.

As T cells transit between blood, lymphoid organs, and peripheral tissues, they experience varied levels of oxygen/hypoxia in inflamed tissues, skin, intestinal lining, and secondary lymphoid organs. Critical illness among COVID-19 patients is also associated with transient hypoxia and attenuation of T cell responses. Hypoxia is the fulcrum of altered metabolism, impaired functions, and cessation of growth of a subset of T cells. However, the restoration of normal T cell functions following transient hypoxia and kinetics of their phenotype-redistribution is not completely understood. Here, we sought to understand kinetics and reversibility of dichotomous T cell responses under sustained and transient hypoxia. We found that a subset of activated T cells accumulated as lymphoblasts under hypoxia. Further, T cells showed the normal expression of activation markers CD25 and CD69 and inflammatory cytokine secretion but a subset exhibited delayed cell proliferation under hypoxia. Increased levels of reactive oxygen species (ROS) in cytosol and mitochondria were seen during dichotomous and reversible attenuation of T cell response under hypoxia. Cell cycle analysis revealed maximum levels of cytosolic and mitochondrial ROS in dividing T cells (in S, G2, or M phase). Hypoxic T cells also showed specific attenuation of activation induced memory phenotype conversion without affecting naïve and activated T cells. Hypoxia-related attenuation of T cell proliferation was also found to be reversible in an allogeneic leukocyte specific mixed lymphocyte reaction assay. In summary, our results show that hypoxia induces a reversible delay in proliferation of a subset of T cells which is associated with obliteration of memory phenotype and specific increase in cytosolic/mitochondrial ROS levels in actively dividing subpopulation. Thus, the transient reoxygenation of hypoxic patients may restore normal T cell responses.

Polyoxometalates Impact as Anticancer Agents.

Polyoxometalates (POMs) are oxoanions of transition metal ions, such as V, Mo, W, Nb, and Pd, forming a variety of structures with a wide range of applications. Herein, we analyzed recent studies on the effects of polyoxometalates as anticancer agents, particularly their effects on the cell cycle. To this end, a literature search was carried out between March and June 2022, using the keywords "polyoxometalates" and "cell cycle". The effects of POMs on selected cell lines can be diverse, such as their effects in the cell cycle, protein expression, mitochondrial effects, reactive oxygen species (ROS) production, cell death and cell viability. The present study focused on cell viability and cell cycle arrest. Cell viability was analyzed by dividing the POMs into sections according to the constituent compound, namely polyoxovanadates (POVs), polyoxomolybdates (POMos), polyoxopaladates (POPds) and polyoxotungstates (POTs). When comparing and sorting the IC50 values in ascending order, we obtained first POVs, then POTs, POPds and, finally, POMos. When comparing clinically approved drugs and POMs, better results of POMs in relation to drugs were observed in many cases, since the dose required to have an inhibitory concentration of 50% is 2 to 200 times less, depending on the POMs, highlighting that these compounds could become in the future an alternative to existing drugs in cancer therapy.

Immunohistochemical profile of cell cycle control, proliferation and differentiation proteins of the pulmonary atypical alveolar epithelium with COVID-19-induced diffuse alveolar damage as compared to pulmonary [lepidic-growth] adenocarcinoma

We aim aim to compare immunophenotypic charac-teritics of atypical epithelium (AE) with COVID-19-induced diffuse alveolar damage (DAD) and pulmonary lepidic-growth adenocarcinoma, accounting for cell cycle control, proliferation and differentiation]. Methods. We examined pulmonary tissue specimens from twenty-four fatal cases of CO VID-19-induced acute respiratory damage syndrome confirmed by autopsy (Group 1) and four cases of pulmonary lepidic-growth adenocarcinoma (Group 2). Perpendicular dimensions of 10 nuclei were measured on the H&E slides, means of their sums of products (SPNM) were calculated. We have used p53, Ki67, pi6, p63 antibodies for immunohistochemical staining in each case. We evaluate co¬lour intensity, rate of stained cells of AE and the product of these parameters. We evaluated separately Nuclear and cyto-plasmic staining (couple) and only cytoplasmic staining (cyt) for pi6 expression. We measured proliferative index only at KI-67 stained slides. U-test and Spearman rank correlation test were used for statistical analysis. Results. Expression of p63 was higher in group 1 (p=0.001), while pi6 was more frequently expressed in group 2 (p=0.002). We have found no statistically significant differences (p>0.1) in the p53 and Ki67 expression. Group 1 showed There was negative correlation between the number of days from onset of symptoms and the following variables: Ki67 (r=M).587, p=0.003);SPNM (r 0.406, p=0.049). Conclusion. The present study has shown heterogeneity in levels of cell cycle control expression, proliferation and differentiation of atypical epithelium in the pulmonary lep-idic-growth adenocarcinoma and CO VID-19-induced diffuse alveolar damage.

CDK4/6 inhibitor palbociclib promotes SARS-CoV-2 cell entry by down-regulating SKP2 dependent ACE2 degradation.

Coronavirus disease 2019 (COVID-19) outbreak has become a global pandemic. CDK4/6 inhibitor palbociclib was reported to be one of the top-scored repurposed drugs to treat COVID-19. As the receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry, expression level of angiotensin-converting enzyme 2 (ACE2) is closely related to SARS-CoV-2 infection. In this study, we demonstrated that palbociclib and other methods could arrest cells in G0/G1 phase and up-regulate ACE2 mRNA and protein levels without altering its subcellular localization. Palbociclib inhibited ubiquitin-proteasome and lysosomal degradation of ACE2 through down-regulating S-phase kinase-associated protein 2 (SKP2). In addition, increased ACE2 expression induced by palbociclib and other cell cycle arresting compounds facilitated pseudotyped SARS-CoV-2 infection. This study suggested that ACE2 expression was down-regulated in proliferating cells. Cell cycle arresting compounds could increase ACE2 expression and facilitate SARS-CoV-2 cell entry, which may not be suitable therapeutic agents for the treatment of SARS-CoV-2 infection.

Impact of the Covid-19 pandemic on the care of patients with chronic arterial disease. A nuclear medicine physician's perspective

Aim/Introduction: The health reorganisation measures, imposed after the serious health emergency resulting from the SARS-CoV-2 pandemic, may have had a negative impact on the follow-up of patients with chronic pathologies. Our intention was to assess their repercussion in our environment, observing what happened in patients with a history of coronary artery disease (CAD) in terms of accesibility and diagnostic performance. Material(s) and Method(s): Retrospective observational study of 756 patients consecutively referred to our Department for myocardial perfusion (MP) studies. Two time groups were established (G1 and G2), both lasting one year, taking as a cut-off point the establishment of the state of health alarm in our country. From the total number of records reviewed 131 patients were selected (mean age 65.74 > 11.03 SD), all with a history of CAD prior to the performance of the MP (G1 56.49%;G2 43.52%). Delay times between test request and test performance were obtained: G1 (17.89 days > 9.85 SD), G2 (16.32 > 20.81 SD) and study results based on two variables, stability or inducible ischaemia. Result(s): In the pre-alarm period (G1) there were more requests for PM studies than in G2 (419 vs 337), with a similar percentage of CAD to total patients (17.66% vs 16.91%). Typical clinical presentation, following the traditional clinical classification, predominated and was statistically significant (p=0.001) for this first group (78.38% vs 52.63%). However, G1 showed a lower number of positive PM results than G2 (37.83% vs 43.85%), and the percentage of total catheterisations performed was also lower (57.14% vs 68%). In relation to the mean delay time between positive PM and catheterisation, a shorter duration was observed for G1 (65.64 days > 46.19 SD) than for G2 (95.41 days > 71.73 SD). No statistically significant differences (p=0.768) were found between the mean ages of patients in the two groups. Conclusion(s): In our health care system, the follow-up of patients with CAD after the establishment of the alarm status was affected by a lower percentage of patients referred to our Department and a longer delay between positive PM and catheterisation. In this period, it is remarkable the higher number of positive PM in patients without typical clinical features. We are unaware of the possible involvement of covid-19 in this situation, currently being investigated by our working group.

Coronavirus RNA-dependent RNA polymerase interacts with the p50 regulatory subunit of host DNA polymerase delta and plays a synergistic role with RNA helicase in the induction of DNA damage response and cell cycle arrest in the S phase.

Disruption of the cell cycle is a common strategy shared by many viruses to create a conducible cellular microenvironment for their efficient replication. We have previously shown that infection of cells with gammacoronavirus infectious bronchitis virus (IBV) activated the theataxia-telangiectasia mutated (ATM) Rad3-related (ATR)/checkpoint kinase 1 (Chk1) pathway and induced cell cycle arrest in S and G2/M phases, partially through the interaction of nonstructural protein 13 (nsp13) with the p125 catalytic subunit of DNA polymerase delta (pol δ). In this study, we show, by GST pulldown, co-immunoprecipitation and immunofluorescent staining, that IBV nsp12 directly interacts with the p50 regulatory subunit of pol δ in vitro and in cells overexpressing the two proteins as well as in cells infected with a recombinant IBV harbouring an HA-tagged nsp12. Furthermore, nsp12 from severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 was also able to interact with p50. These interactions play a synergistic role with nsp13 in the induction of S phase arrest. The fact that subunits of an essential cellular DNA replication machinery physically associate with two core replication enzymes from three different coronaviruses highlights the importance of these associations in coronavirus replication and virus-host interaction, and reveals the potential of targeting these subunits for antiviral intervention.

Integrative systems immunology uncovers molecular networks of the cell cycle that stratify COVID-19 severity.

Several perturbations in the number of peripheral blood leukocytes, such as neutrophilia and lymphopenia associated with Coronavirus disease 2019 (COVID-19) severity, point to systemic molecular cell cycle alterations during severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. However, the landscape of cell cycle alterations in COVID-19 remains primarily unexplored. Here, we performed an integrative systems immunology analysis of publicly available proteome and transcriptome data to characterize global changes in the cell cycle signature of COVID-19 patients. We found significantly enriched cell cycle-associated gene co-expression modules and an interconnected network of cell cycle-associated differentially expressed proteins (DEPs) and genes (DEGs) by integrating the molecular data of 1469 individuals (981 SARS-CoV-2 infected patients and 488 controls [either healthy controls or individuals with other respiratory illnesses]). Among these DEPs and DEGs are several cyclins, cell division cycles, cyclin-dependent kinases, and mini-chromosome maintenance proteins. COVID-19 patients partially shared the expression pattern of some cell cycle-associated genes with other respiratory illnesses but exhibited some specific differential features. Notably, the cell cycle signature predominated in the patients' blood leukocytes (B, T, and natural killer cells) and was associated with COVID-19 severity and disease trajectories. These results provide a unique global understanding of distinct alterations in cell cycle-associated molecules in COVID-19 patients, suggesting new putative pathways for therapeutic intervention.

Cell cycle block by p53 activation reduces SARS-CoV-2 release in infected alveolar basal epithelial A549-hACE2 cells.

SARS-CoV viruses have been shown to downregulate cellular events that control antiviral defenses. They adopt several strategies to silence p53, key molecule for cell homeostasis and immune control, indicating that p53 has a central role in controlling their proliferation in the host. Specific actions are the stabilization of its inhibitor, MDM2, and the interference with its transcriptional activity. The aim of our work was to evaluate a new approach against SARS-CoV-2 by using MDM2 inhibitors to raise p53 levels and activate p53-dependent pathways, therefore leading to cell cycle inhibition. Experimental setting was performed in the alveolar basal epithelial cell line A549-hACE2, expressing high level of ACE2 receptor, to allow virus entry, as well as p53 wild-type. Cells were treated with several concentrations of Nutlin-3 or RG-7112, two known MDM2 inhibitors, for the instauration of a cell cycle block steady-state condition before and during SARS-CoV-2 infection, and for the evaluation of p53 activation and impact on virus release and related innate immune events. The results indicated an efficient cell cycle block with inhibition of the virion release and a significant inhibition of IL-6, NF-kB and IFN-λ expression. These data suggest that p53 is an efficient target for new therapies against the virus and that MDM2 inhibitors deserve to be further investigated in this field.