Exploring causal correlations between inflammatory cytokines and varicose veins: A Mendelian randomization analysis.

This study aimed to investigate the causal relationship between inflammatory cytokines and the risk of varicose veins. The data were sourced from genome-wide association studies (GWAS) of European individuals. Multiple Mendelian randomization (MR) methods were used to evaluate the association between inflammatory cytokines and varicose veins. The study found significant associations between elevated levels of certain inflammatory biomarkers (e.g., CASP-8, Vascular endothelial growth factor A levels (VEGF_A)) and an increased risk of varicose veins, while others (e.g., 4EBP1, MMP-10) showed a protective effect. The MR-Egger Intercept and heterogeneity tests indicated no significant pleiotropy or heterogeneity. This comprehensive MR analysis identifies several cytokines as potential contributors to the pathogenesis of varicose veins, offering insights into novel therapeutic targets. Our findings underscore the importance of inflammation in varicose veins and suggest that targeting specific cytokines could be a promising strategy for the treatment and prevention of varicose veins.

Lessons from an elderly patient with pulmonary embolism caused by protein S deficiency: a case report.

BACKGROUND: Lower limb deep vein thrombosis (DVT) concurrent with pulmonary embolism (PE) is perilous, particularly in the elderly, exhibiting heterogeneity with thrombophilia mutations. Tailored treatment is essential, yet sudden deaths complicate causative factor elucidation. This report emphasizes genetic testing necessity in PE patients with thrombophilia indicators, facilitating cause identification, personalized treatment guidance, and family education. CASE PRESENTATION: This study details a 75-year-old Chinese woman with DVT and PE, where genetic testing identified thrombophilia, guiding personalized treatment decisions. RESULTS: Upon admission, the patient, after over 10 days of bed rest, presented chest tightness, shortness of breath, and unilateral leg swelling. Diagnostic measures revealed DVT and a substantial PE. Genetic testing identified a PROS1 gene C200A>C mutation, reducing protein S activity. Following 2 weeks of anticoagulation and inferior vena cava filter insertion, the patient, discharged, initiated lifelong anticoagulant therapy. A 1-year follow-up showed no recurrent thrombotic events. Family members carrying the mutation received informed and educational interventions. CONCLUSION: Genetic testing for thrombophilic predisposition post-PE is crucial, elucidating etiology, guiding individualized treatment, and playing a pivotal role in family education.

Design strategy and research progress of multifunctional nanoparticles in lung cancer therapy.

INTRODUCTION: Lung cancer is one of the cancer types with the highest mortality rate, exploring a more effective treatment modality that improves therapeutic efficacy while mitigating side effects is now an urgent requirement. Designing multifunctional nanoparticles can be used to overcome the limitations of drugs and conventional drug delivery systems. Nanotechnology has been widely researched, and through different needs, suitable nanocarriers can be selected to load anti-cancer drugs to improve the therapeutic effect. It is foreseeable that with the rapid development of nanotechnology, more and more lung cancer patients will benefit from nanotechnology. This paper reviews the merits of various multifunctional nanoparticles in the treatment of lung cancer to provide novel ideas for lung cancer treatment. AREAS COVERED: This review focuses on summarizing various nanoparticles for targeted lung cancer therapy and their advantages and disadvantages, using nanoparticles loaded with anti-cancer drugs, delivered to lung cancer sites, enhancing drug half-life, improving anti-cancer drug efficacy and reducing side effects. EXPERT OPINION: The delivery mode of nanoparticles with superior pharmacokinetic properties in the in vivo circulation enhances the half-life of the drug, and provides tissue-targeted selectivity and the ability to overcome biological barriers, bringing a revolution in the field of oncology.

Impact of insulin injection induced lipohypertrophy on multidimensional glucose variability in patients with type 1 diabetes mellitus / 中华内分泌代谢杂志

Objective:This study aimed to investigate the effect of lipohypertrophy induced by insulin injection on blood glucose fluctuation in patients with type 1 diabetes mellitus.Methods:A total of 80 patients with type 1 diabetes mellitus were recruited between June 2021 and December 2021 from the First Affiliated Hospital of Nanjing Medical University. And these patients all received insulin injection more than six months. Lipohypertrophy was assessed by ultrasound scanning, and blood glucose fluctuation was evaluated using the flash glucose monitoring system(FGM). Univariate analysis and multivariate linear regression were used to analyze the relationship of lipohypertrophy and and core indicators of blood glucose fluctuation.Results:Compared with patients without lipohypertrophy, patients with lipohypertrophy had higher mean amplitude of glycemic excursions(MAGE), coefficient of variation(CV), mean of daily differences(MODD), standard deviation(SD) of blood glucose, time above range(TAR), and high blood glucose index(HBGI; all P<0.05), while time in range(TIR) of glucose markedly become lower( P<0.01). Moreover, multivariate linear regression analysis showed that lipohypertrophy detected by ultrasound was an independent influencing factor of TIR( β=-9.423, P=0.032), MAGE( β=1.114, P=0.039), CV( β=4.304, P=0.041), MODD( β=0.717, P=0.046) after adjusting for age at diagnosis, duration of insulin injection, fasting C-peptide, and daily dose of insulin per unit weight. Conclusion:Lipohypertrophy increases glycemic variability and imposes negative impact on glycemic control rate in patients type 1 diabetes mellitus.

Association of Ego-depletion level with glycemic control and quality of life among adolescents with type 1 diabetes / 中国实用护理杂志

Objective:To investigate the ego-depletion level of adolescents with type 1 diabetes mellitus (T1DM) and to explore its association with glycemic control and quality of life.Methods:This was a cross-sectional survey study. A total of 195 adolescents with T1DM were recruited from the First Affiliated Hospital of Nanjing Medical University from March to September 2022 by convenient sampling method. The Self-Regulatory Fatigue Scale (SRF-S) and Short Form of the Chinese version Diabetes Quality of Life for Youth Scale (C-DQOLY-SF) and the general information questionnaire were collected and the glycated hemoglobin (HbA 1c) value was detected. Results:The total score of self-regulatory fatigue for 195 adolescents with T1DM was (42.23 ± 9.94) points, with a scoring rate of 52.79%, which was at a medium level. Pearson correlation analysis showed that the total score of self-regulatory fatigue was positively correlated with HbA1c ( r = 0.25, P<0.01), and negatively correlated with quality of life ( r = -0.61, P<0.01). The hierarchical linear regression results showed that after controlling for demographic sociolagy and disease variables, ego-depletion had a positive predictive effect on HbA1c ( t = 3.69, P<0.01), while ego-depletion had a negative predictive effect on Quality of life ( t = -8.48, P<0.01). Conclusions:Ego-depletion of adolescents with T1DM may affect their blood glucose control and quality of life, which should be noticed by medical workers.

Analysis on the current status and influence factors of professional quality of life among nurses in the department of hematology and oncology in tertiary grade A children′s hospitals / 中国实用护理杂志

Objective:To describe current status and analyze influencing factors of professional quality of life among nurses in the department of hematology and oncology in tertiary grade A children's hospitals, so as to provide some reference for improving the professional quality of life among nurses in the department of pediatric hematological oncology.Methods:This study was a cross-sectional study. By convenient sampling method, the General Data Scale, Professional Quality of Life Scale and Practice Environment Scale were used to investigate 205 nurses in the department of hematology and oncology in 4 tertiary grade A children's hospitals. Influencing factors of professional quality of life among nurses were analyzed by multiple linear regression.Results:Nurses in the department of pediatric hematological oncology with a low level of compassion satisfaction, high level of burnout and secondary trauma stress accounted for 21.0%(43/205), 26.3%(54/205) and 36.6%(75/205), respectively. Multiple linear regression analysis showed that mental health counseling, family supporting and nurses participating in hospital affairs entered into the multiple regression equation of compassion satisfaction ( t=2.08, 4.21, 2.34, all P<0.05), which explained 60.3% of the total variety. Family supporting, turnover intention, sufficient manpower and materials, age and number of children entered into the multiple regression equation of burnout ( t values were -4.42-2.33, all P<0.05), which explained 55.8% of the total variety. Family supporting, sufficient manpower and materials, age entered into the multiple regression equation of secondary trauma stress ( t=-2.37, -2.22, 2.82, all P<0.05), which explained 15.3% of the total variety. Conclusions:The professional quality of life among nurses in the department of pediatric hematology and oncology needs to be improved. Nursing managers should learn from advanced management concepts, actively improve the nursing practice environment of hematology and oncology department, increase the number of nurses, encourage nurses to participate in hospital affairs, attach importance to emotional support for nurses, and carry out regular mental health counseling, continuously improve the quality of professional life of nurses.

A Case Report of Blau Syndrome / 罕见病研究

Blau syndrome is a rare genetic disorder characterized by the a mix of granulomatous arthritis, uveitis, and dermatitis. Patients typically manifest multisystem involvement, including ocular, skin, and skeletal abnormalities. Blau syndrome is extremely rare, with a global incidence of less than one in a million among children. In this multidisciplinary consultation, we present a case of a 21-year-old young female patient having multisystemic involvement since early childhood. She was presented with multiple joint swelling, skin lesions, increased eye discharge, and accompanied by hypertension and arterial abnormalities, and received a diagnosis of uveitis. The patient had been receiving steroid treatment since the age of 6 and has tried various medications, with some improvement in joint swelling and ocular symptoms. Through this rare disease multidisciplinary consultation, we aim to provide guidance in the molecular diagnosis of the patient, multisystem assessment, and the selection and formulation of treatment plans. Additionally, we hope that by reporting this case, clinical physicians can gain a better understanding of the diagnosis and comprehensive treatment strategies for Blau syndrome, thereby improving the management and treatment of rare diseases.

Analysis of adverse events following immunization of adsorbed acellular DPT vaccine at different vaccination sites / 上海预防医学

ObjectiveTo analyze the occurrence of suspected adverse events following immunization （AEFI） after changing the priority vaccination sites of the adsorbed acellular diphtherior-pertussis-tetanus vaccine （hereinafter referred to as DPT vaccine）， so as to provide scientific basis for mass vaccination. MethodsMonitoring data of AEFI for the DPT vaccine in Wujiang District from September 2020 to August 2022 were collected from China's disease prevention and control information system， and the vaccination information of DPT vaccine in all children's vaccination clinics in Wujiang District during the same period was selected. The incidence of AEFI for the DPT vaccine was analyzed and compared. ResultsThe reported incidence of AEFI was significantly lower in the buttocks than that in other sites （P<0.05）. The reported incidence of AEFI was significantly higher in booster immunization than that in basic immunization （P<0.05）. After inoculation at different sites， the main clinical symptoms of AEFI were local redness and swelling. There were significant differences in the incidence of local redness and swelling， local induration， pruritus and other symptoms （lethargy， abnormal crying， etc.） （P<0.05）. There were significant differences in the severity of local redness and swelling in different sites （P<0.05）. The degree of redness and swelling in the anterolateral thigh was lower than that in other sites （P<0.05）. The local strong reaction of swelling （>5.0 cm） in the deltoid muscle of the upper arm was significantly higher than that in the buttocks （P<0.05）. ConclusionThe DPT vaccine is safe in different parts of the body and is worth popularizing.

Analysis of Forty-Two Psychoactive Substances in a Single Hair by Micro-Segmental Technique / 法医学杂志

OBJECTIVES@#To establish an LC-MS/MS method based on single hair micro-segmental technique, and verify the detection of 42 psychoactive substances in 0.4 mm hair segments.@*METHODS@#Each piece of single hair was cut into 0.4 mm segments and extracted by sonication and the segments were immersed in dithiothreitol-containing extraction medium. Mobile phase A was the aqueous solution containing 20 mmol/L ammonium acetate, 0.1% formic acid, and 5% acetonitrile. Mobile phase B was acetonitrile. An electrospray ionization source in positive ion mode was used for data acquisition in multiple reaction monitoring (MRM) mode.@*RESULTS@#The 42 psychoactive substances in hair had a good linear relationship within their respective linear ranges (r>0.99), the limits of detection were 0.2-10 pg/mm, the limits of quantification were 0.5-20 pg/mm, the intra-day and inter-day precisions were 1.5%-12.7%, the intra-day and inter-day accuracies were 86.5%-109.2%, the recovery rates were 68.1%-98.2%, and the matrix effects were 71.3%-111.7%. The method was applied to hair samples collected from one volunteer at 28 d after a single dose of zolpidem, with zolpidem detected in 5 hairs was 1.08-1.60 cm near the root tip, and the concentration range was 0.62-20.5 pg/mm.@*CONCLUSIONS@#The micro-segmental technique of single hair analysis can be applied to the investigation of drug-facilitated sexual assault cases.

Effects of Cytokines on Early Death in Patients with Newly Diagnosed Acute Promyelocytic Leukemia / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of cytokine levels on early death and coagulation function of patients with newly diagnosed acute promyelocytic leukemia (APL).@*METHODS@#Routine examination was performed on 69 newly diagnosed APL patients at admission. Meanwhile, 4 ml fasting venous blood was extracted from the patients. And then the supernatant was taken after centrifugation. The concentrations of cytokines, lactate dehydrogenase (LDH) and ferritin were detected by using the corresponding kits.@*RESULTS@#It was confirmed that cerebral hemorrhage was a major cause of early death in APL patients. Elevated LDH, decreased platelets (PLT) count and prolonged prothrombin time (PT) were high risk factors for early death (P <0.05). The increases of IL-5, IL-6, IL-10, IL-12p70 and IL-17A were closely related to the early death of newly diagnosed APL patients, and the increases of IL-5 and IL-17A also induced coagulation disorder in APL patients by prolonging PT (P <0.05). In newly diagnosed APL patients, ferritin and LDH showed a positive effect on the expression of IL-5, IL-10 and IL-17A, especially ferritin had a highly positive correlation with IL-5 (r =0.867) and IL-17A (r =0.841). Moreover, there was a certain correlation between these five high-risk cytokines, among which IL-5 and IL-17A (r =0.827), IL-6 and IL-10 (r =0.823) were highly positively correlated.@*CONCLUSION@#Elevated cytokine levels in newly diagnosed APL patients increase the risk of early bleeding and death. In addition to the interaction between cytokines themselves, ferritin and LDH positively affect the expression of cytokines, thus affecting the prognosis of APL patients.

The clinical journey and healthcare resources required for dialysis access of end-stage kidney disease patients during their first year of hemodialysis.

BACKGROUND: Creation and maintenance of dialysis vascular access (VA) is a major component of healthcare resource utilization and cost for patients newly started on hemodialysis (HD). Different VA format arises due to patient acceptance of anticipatory care versus late preparation, and clinical characteristics. This study reviews the clinical journey and resource utilization required for different VA formats in the first year of HD. METHOD: Data of patients newly commenced on HD between July 2015 and June 2016 were reviewed. Patients were grouped by their VA format: (A) pre-emptive surgically created VA (SCVA), (B) tunneled central venous catheter (CVC) followed by SCVA creation, (C) long-term tunneled CVC only. Clinical events, number of investigations and procedures, hospital admissions, and incurred costs of the three groups were compared. RESULTS: In the multivariable analysis, the cost incurred by the group A patients had no significant difference to that incurred in the group B patients (p = 0.08), while the cost of group C is significantly lower (p < 0.001). Both the 62.7% of group A with successful SCVA who avoided tunneled CVC usage, and those with a functionally matured SCVA in group B (66.1%), used fewer healthcare resources and incurred less cost for their access compared to those did not (p = 0.01, p = 0.02, respectively) during the first year of HD. CONCLUSION: With comparable cost, a pre-emptive approach enables avoidance of tunneled CVC. Tunneled CVC only access format incurred lower cost and is suitable for carefully selected patients. Successful maturation of SCVA greatly affects patients' clinical journey and healthcare cost.

A Suite of TMPRSS2 Assays for Screening Drug Repurposing Candidates as Potential Treatments of COVID-19

SARS-CoV-2 is the causative viral pathogen driving the COVID-19 pandemic that prompted an immediate global response to the development of vaccines and antiviral therapeutics. For antiviral therapeutics, drug repurposing allowed for rapid movement of existing clinical candidates and therapies into human clinical trials to be tested as COVID-19 therapies. One effective antiviral treatment strategy used early in symptom onset is to prevent viral entry. SARS-CoV-2 enters ACE2-expressing cells when the receptor-binding domain of the spike protein on the surface of SARS-CoV-2 binds to ACE2 followed by cleavage at two cut sites on the spike protein. TMPRSS2 has a protease domain capable of cleaving the two cut sites; therefore, a molecule capable of inhibiting the protease activity of TMPRSS2 could be a valuable antiviral therapy. Initially, we used a fluorogenic high-throughput screening assay for the biochemical screening of 6030 compounds in NCATS annotated libraries. Then, we developed an orthogonal biochemical assay that uses mass spectrometry detection of product formation to ensure that hits from the primary screen are not assay artifacts from the fluorescent detection of product formation. Finally, we assessed the hits from the biochemical screening in a cell-based SARS-CoV-2 pseudotyped particle entry assay. Of the six molecules advanced for further studies, two are approved drugs in Japan (camostat and nafamostat), two have entered clinical trials (PCI-27483 and otamixaban), while the other two molecules are peptidomimetic inhibitors of TMPRSS2 taken from the literature that have not advanced into clinical trials (compounds 92 and 114). This work demonstrates a suite of assays for the discovery and development of new inhibitors of TMPRSS2.

Application of a UPLC-MS/MS method for quantitative analysis of 29 synthetic cannabinoids and their metabolites, such as ADB-BUTINACA and MDMB-4en-PINACA in human hair in real cases.

In recent years, an increasing number of new synthetic cannabinoids have appeared on the drug trade market. Many of the new synthetic cannabinoids have not previously been reported. At present, there are relatively few methods available for detecting synthetic cannabinoids and their metabolites in hair matrices. Therefore, we established a simple and fast method to simultaneously identify 29 synthetic cannabinoids and their metabolites in human hair by UPLC-MS/MS. Twenty milligrams of hair was used and processed by cryo-grinding and extraction with methanol. A Waters Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 µm) was used for chromatographic separation. Mobile phase A was comprised of 20 mmol/L ammonium acetate, 0.1% formic acid, and 5% acetonitrile and water, and mobile phase B was acetonitrile. The method was fully validated and proved to have good selectivity, accuracy, precision, and satisfactory linearity within the calibrated range. The limit of detection (LOD) ranged from 0.5 to 5 pg/mg, and the lower limit of quantitation (LLOQ) ranged from 1 to 10 pg/mg. The extraction recovery was 36.1-93.3%, and the matrix effect was 19.1-110.0%. The validated method was successfully used to qualitatively and quantitatively analyze 29 synthetic cannabinoids and their metabolites in 59 actual hair samples. MDMB-4en-PINACA had the highest positive detection rate followed by ADB-BUTINACA, and there are multiple synthetic cannabinoid mixed ingestions. This methodology has great potential for the detection of 29 synthetic cannabinoids and their metabolites in forensic cases.

Factors associated with failure of trial of labor in primiparae with preeclampsia and establishment of risk prediction model / 中华围产医学杂志

Objective:To investigate the risk factors associated with failure of trial of labor in primiparous women with preeclampsia (PE) and to establish a risk prediction model.Methods:Primiparae with PE who underwent trial of labor in the Department of Obstetrics of Suzhou Ninth People's Hospital from February 2018 to July 2020 were retrospectively selected as the modeling set, and divided into two groups: the success group and the failure group. Various parameters were compared between the two groups and those data with statistically significant difference were analyzed with multivariate logistic regression analysis. Those factors related to vaginal delivery failure in primiparous women with PE were identified. Based on the results, a risk prediction model was established using R language. Its performance was assessed with receiver operating characteristic (ROC) curve and goodness-of-fit test. This study also retrospectively enrolled primiparae with PE who underwent trial of labor in the same hospital from August 2020 to December 2021 as the validation set. Bootstrap method was used for verification and a calibration chart was created.Results:A total of 312 PE patients were selected as the modeling set with 89 in the failure group and 223 in the success group. Another 146 primiparae with PE were selected as the validation set. Logistic regression analysis showed that older age ( OR=1.609, 95% CI: 1.251-2.483), higher body fat rate in early pregnancy ( OR=1.456, 95% CI: 1.209-2.159) and higher ratio of umbilical artery systolic to diastolic flow velocity within a week before delivery ( OR=1.799, 95% CI: 1.372-2.794) were risk factors for vaginal delivery failure in primiparae with PE, while more maternal education during pregnancy ( OR=0.233, 95% CI: 0.054-0.672) and higher Bishop score ( OR=0.395, 95% CI: 0.258-0.756) were protective factors. A nomogram model to predict the risks of vaginal delivery failure was constructed based on the above five factors. The area under the ROC curve (AUC) of the modeling set was 0.921 (95% CI: 0.847-0.963) with the cut-off value of 0.213, and the corresponding sensitivity and specificity were 0.871 and 0.852, respectively;goodness-of-fit test showed that the observed values matched with those expected ( χ2=7.69, P=0.464); and the calibration curve indicated that the consistency of the prediction model was good. The AUC of the validation set was 0.903 (95% CI: 0.835-0.942) with the sensitivity and specificity of 0.892 and 0.796, respectively; the discrepancy between the observed values and those expected was not significant as indicated by goodness-of-fit test ( χ2=6.82, P=0.512); calibration curve of the validation set showed that the predicted values of the model was consistent with the actual values. Conclusions:The failure of vaginal delivery in primiparae with PE is associated with maternal age, prenatal body fat percentage, ratio of fetal umbilical artery systolic to diastolic flow velocity within a week before delivery, maternal education during pregnancy and Bishop score. The nomogram model based on these five risk factors for prediction of vaginal delivery failure performs well.

An Adolescent with Recurrent Intracranial Hemorrhage, and Skin Lesion / 罕见病研究

We presented an adolescent with recurrent intracranial hemorrhage and skin lesion. The diagnosis was unclear and the treatment was difficult. Through a multidisciplinary effort type Ⅰ interferon disease was suspected and later, an interferon-stimulated gene was further detected. Considering the high morbidity and fatality rate of recurrent intracranial hemorrhage, tofacitinib and hydroxychloroquine were administered. After treatment, the livedo reticularis was significantly regressed. Unfortunately, the intracranial hemorrhage recurred due to a pre-existing cerebral aneurysm, leading to death of the patient. The diagnosis and treatment of this case highlight the importance of multidisciplinary collaboration in the diagnosis and treatment of difficult and rare diseases.

Strategies for Collection and Analysis of Samples in Simple Asphyxiant Gas Acute Poisoning Death Cases / 法医学杂志

At present, the death cases of simple asphyxiant gas acute poisoning are increasing sharply. Common asphyxiant gases in death cases include nitrogen, helium, carbon dioxide, methane, propane, laughing gas, etc. Simple asphyxiant gas has no affinity for biological matrices and escapes quickly, which puts forward new requirements for autopsy procedures, selection and collection of samples, laboratory analysis and identification. This paper reviews the research and development process of death cases caused by simple asphyxiant gas acute poisoning and put forwards the collection and analysis strategy of the samples in such cases. The most valuable biological samples in such cases should be lung tissues associated with the airways, followed by brain tissue and cardiac blood. Gaseous samples from the esophageal cavity, tracheal cavity, pulmonary bronchi, gastric and cardiac areas are also recommended as valuable samples. In the case of postmortem examination, the gas should be injected into gas sample bag directly. Biological materials such as tissue and blood should be directly sealed in head-space vials and analyzed by using the headspace gas chromatography-mass spectrometry.

Analysis of the Distribution of Total Phosphine and the Characteristics of Phosphine Poisoning in 29 Victims / 法医学杂志

OBJECTIVES@#To study the distribution of total phosphine in phosphine poisoning victims and summarize the characteristics of phosphine poisoning cases.@*METHODS@#The phosphine and its metabolites in the biological samples of 29 victims in 16 phosphine poisoning cases were qualified and quantified by headspace gas chromatography-mass spectrometry.@*RESULTS@#Five victims among 29 were poisoned by ingestion of aluminium phosphide and 24 by inhalation of phosphine gas. Phosphine metabolites were detected in the biological samples of 23 victims, and the concentrations of total phosphine in blood ranged 0.5-34.0 μg/mL. The total concentration of phosphine in liver tissue was up to 71.0 μg/g. Phosphine was not detected in the blood of the other six survived victims, which may be related to the small amount of phosphine exposure and the delay in blood sampling.@*CONCLUSIONS@#The total concentration of phosphine in blood and tissues caused by aluminum phosphine ingestion is higher than that caused by phosphine gas inhalation. The death cases of phosphine inhalation are characterized by long exposure time, repeated exposures and age susceptibility.

A Graph Convolutional Network-based screening strategy for rapid identification of SARS-CoV-2 cell-entry inhibitors

The cell entry of SARS-CoV-2 has emerged as an attractive drug development target. We previously reported that the entry of SARS-CoV-2 depends on the cell surface heparan sulfate proteoglycan (HSPG) and the cortex actin, which can be targeted by therapeutic agents identified by conventional drug repurposing screens. However, this drug identification strategy requires laborious library screening, which is time-consuming and often limited number of compounds can be screened. As an alternative approach, we developed and trained a graph convolutional network (GCN)-based classification model using information extracted from experimentally identified HSPG and actin inhibitors. This method allowed us to virtually screen 170,000 compounds, resulting in [~]2000 potential hits. A hit confirmation assay with the uptake of a fluorescently labeled HSPG cargo further shortlisted 256 active compounds. Among them, 16 compounds had modest to strong inhibitory activities against the entry of SARS-CoV-2 pseudotyped particles into Vero E6 cells. These results establish a GCN-based virtual screen workflow for rapid identification of new small molecule inhibitors against validated drug targets. Graphical TOC Entry O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/471787v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1632bc4org.highwire.dtl.DTLVardef@1ce912borg.highwire.dtl.DTLVardef@9cdc86org.highwire.dtl.DTLVardef@521712_HPS_FORMAT_FIGEXP M_FIG C_FIG

A high throughput screening assay for inhibitors of SARS-CoV-2 pseudotyped particle entry

Effective small molecule therapies to combat the SARS-CoV-2 infection are still lacking as the COVID-19 pandemic continues globally. High throughput screening assays are needed for lead discovery and optimization of small molecule SARS-CoV-2 inhibitors. In this work, we have applied viral pseudotyping to establish a cell-based SARS-CoV-2 entry assay. Here, the pseudotyped particles (PP) contain SARS-CoV-2 spike in a membrane enveloping both the murine leukemia virus (MLV) gag-pol polyprotein and luciferase reporter RNA. Upon addition of PP to HEK293-ACE2 cells, the SARS-CoV-2 spike protein binds to the ACE2 receptor on the cell surface, resulting in priming by host proteases to trigger endocytosis of these particles, and membrane fusion between the particle envelope and the cell membrane. The internalized luciferase reporter gene is then expressed in cells, resulting in a luminescent readout as a surrogate for spike-mediated entry into cells. This SARS-CoV-2 PP entry assay can be executed in a biosafety level 2 containment lab for high throughput screening. From a collection of 5,158 approved drugs and drug candidates, our screening efforts identified 7 active compounds that inhibited the SARS-CoV-2-S PP entry. Of these seven, six compounds were active against live replicating SARS-CoV-2 virus in a cytopathic effect assay. Our results demonstrated the utility of this assay in the discovery and development of SARS-CoV-2 entry inhibitors as well as the mechanistic study of anti-SARS-CoV-2 compounds. Additionally, particles pseudotyped with spike proteins from SARS-CoV-2 B.1.1.7 and B.1.351 variants were prepared and used to evaluate the therapeutic effects of viral entry inhibitors.

A hybrid in silico approach reveals novel inhibitors of multiple SARS-CoV-2 variants

The National Center for Advancing Translational Sciences (NCATS) has been actively generating SARS-CoV-2 high-throughput screening data and disseminates it through the OpenData Portal (https://opendata.ncats.nih.gov/covid19/). Here, we provide a hybrid approach that utilizes NCATS screening data from the SARS-CoV-2 cytophatic effect reduction assay to build predictive models, using both machine learning and pharmacophore-based modeling. Optimized models were used to perform two iterative rounds of virtual screening to predict small molecules active against SARS-CoV-2. Experimental testing with live virus provided 100 (~16% of predicted hits) active compounds (Efficacy > 30%, IC50 [≤] 15 M). Systematic clustering analysis of active compounds revealed three promising chemotypes which have not been previously identified as inhibitors of SARS-CoV-2 infection. Further analysis identified allosteric binders to host receptor angiotensin-converting enzyme 2, which were able to inhibit the entry of pseudoparticles bearing spike protein of wild type SARS-CoV-2 as well as South African B.1.351 and UK B.1.1.7 variants.