Folic Acid and Leucovorin Have Potential to Prevent SARS-CoV-2-Virus Internalization by Interacting with S-Glycoprotein/Neuropilin-1 Receptor Complex

The interaction of the SARS-CoV-2 spike (S) glycoprotein receptor-binding domain with the host-cell ACE2 receptor is a well-known step in virus infection. Neuropilin-1 (NRP-1) is another host factor involved in virus internalization. The interaction between S-glycoprotein and NRP-1 has been identified as a potential COVID-19 treatment target. Herein, the effectiveness of folic acid and leucovorin in preventing contact between S-glycoprotein and NRP-1 receptors was investigated using in silico studies and then confirmed in vitro. The results of a molecular docking study showed that leucovorin and folic acid had lower binding energies than EG01377, a well-known NRP-1 inhibitor, and lopinavir. Two hydrogen bonds with Asp 320 and Asn 300 residues stabilized the leucovorin, while interactions with Gly 318, Thr 349, and Tyr 353 residues stabilized the folic acid. The molecular dynamic simulation revealed that the folic acid and leucovorin created very stable complexes with the NRP-1. The in vitro studies showed that the leucovorin was the most active inhibitor of the S1-glycoprotein/NRP-1 complex formation, with an IC75 value of 185.95 µg/mL. The results of this study suggest that folic acid and leucovorin could be considered as potential inhibitors of the S-glycoprotein/NRP-1 complex and, thus, could prevent the SARS-CoV-2 virus' entry into host cells.

Folic Acid and Leucovorin Have Potential to Prevent SARS-CoV-2-Virus Internalization by Interacting with S-Glycoprotein/Neuropilin-1 Receptor Complex.

The interaction of the SARS-CoV-2 spike (S) glycoprotein receptor-binding domain with the host-cell ACE2 receptor is a well-known step in virus infection. Neuropilin-1 (NRP-1) is another host factor involved in virus internalization. The interaction between S-glycoprotein and NRP-1 has been identified as a potential COVID-19 treatment target. Herein, the effectiveness of folic acid and leucovorin in preventing contact between S-glycoprotein and NRP-1 receptors was investigated using in silico studies and then confirmed in vitro. The results of a molecular docking study showed that leucovorin and folic acid had lower binding energies than EG01377, a well-known NRP-1 inhibitor, and lopinavir. Two hydrogen bonds with Asp 320 and Asn 300 residues stabilized the leucovorin, while interactions with Gly 318, Thr 349, and Tyr 353 residues stabilized the folic acid. The molecular dynamic simulation revealed that the folic acid and leucovorin created very stable complexes with the NRP-1. The in vitro studies showed that the leucovorin was the most active inhibitor of the S1-glycoprotein/NRP-1 complex formation, with an IC75 value of 185.95 µg/mL. The results of this study suggest that folic acid and leucovorin could be considered as potential inhibitors of the S-glycoprotein/NRP-1 complex and, thus, could prevent the SARS-CoV-2 virus' entry into host cells.

Antibiotic Utilization during COVID-19: Are We Over-Prescribing?

The aims of this study were to analyze the utilization of antibiotics before (2018, 2019) and during the COVID-19 pandemic (2020) and the practice of prescribing antibiotics in outpatient settings for COVID-19 patients during the 2020-2022 period. The Anatomical Therapeutic Chemical Classification/Defined Daily Dose methodology was used for the analysis of outpatient antibiotic utilization in the Republic of Srpska. The data was expressed in DDD/1000 inhabitants/day. The rate of antibiotics prescribed to COVID-19 outpatients was analyzed using medical record data from 16,565 patients registered with B34.2, U07.1, and U07.2 World Health Organization International Classification of Diseases 10th revision codes. During 2020, outpatient antibiotic utilization increased by 53.80% compared to 2019. At least one antibiotic was prescribed for 91.04%, 83.05%, and 73.52% of COVID-19 outpatients during 2020, 2021, and the first half of 2022, respectively. On a monthly basis, at least one antibiotic was prescribed for more than 55% of COVID-19 outpatients. The three most commonly prescribed antibiotics were azithromycin, amoxicillin/clavulanic acid, and doxycycline. The trend of repurposing antibiotics for COVID-19 and other diseases treatment might be a double-edged sword. The long-term effect of this practice might be an increase in antimicrobial resistance and a loss of antibiotic effectiveness.

Dynamic changes in coagulation, hematological and biochemical parameters as predictors of mortality in critically ill COVID-19 patients: A prospective observational study.

INTRODUCTION: This study was created to analyze dynamic alterations in coagulation, hematological and biochemical parameters and their association with mortality of COVID-19 patients. To identify the most sensitive biomarkers as predictors of mortality more research is required. METHODS: The present study was a prospective, one-year-long observational study conducted on all critically ill, COVID-19 patients with respiratory failure. The following data were collected: demographic and clinical characteristics of the study population, comorbidities, coagulation, biochemical and hematological parameters. The primary outcome was the proportion of patients who died. RESULTS: 91 patients with median age 60 (50-67), 76.9% male, met the acute respiratory distress syndrome criteria. It was tested whether dynamic change (delta-Δ) of parameters that were found to be predictors of mortality is independently associated with poor outcome. Adjusted (multivariate) analysis was used, where tested parameters were corrected for basic and clinical patients characteristics. The only inflammatory parameter which dynamic change had statistically significant odds ratio was ΔCRP (p < 0.005), while among coagulation parameters statistically significant OR was found for Δ fibrinogen (p < 0.005) in predicting mortality. CONCLUSION: Monitoring of coagulation, hematological and biochemical parameters abnormalities and their dynamical changes can potentially improve management and predict mortality in critically ill COVID -19 patients.

Boosting ICU capacity during the COVID-19 pandemic in the western Balkan region, The Republic of Srpska experience.

Background: Response to the outbreak of poliomyelitis in mid-1950 led to recognition and consequent development of critical care. Seventy years later the humankind was struck by COVID-19, another major challenge for critical care medicine which was especially big in Low-Resources-Settings where more than two thirds of the world population live, including the population of the Republic of Srpska (RS). Design and methods: The main aim was to show an overview of all interventions in order to boost hospitals' capacities to the level which is sufficient to manage high amount of critically ill COVID-19 patients in the RS. A before-after cohort study design was conducted to evaluate the effects of interventions for increase in preparedness and capacity of ICUs for admission and treatment of COVID-19 critically ill patients in nine hospitals in the RS. Results: Following interventions, the biggest and university affiliated hospital in the RS has increased ICU capacities: total number of ICU beds increased by 38% and number of ventilators by 114%. Availability of machines for veno-venous extracorporeal membrane oxygenation (vvECMO) increased by 100%. Number of doctors who were involved in treatment of critically ill patients increased by 47% and nurse/patient's ratio reached 1:2.5. Similarly, all other hospitals experienced boosting of ICU beds by 189% and ventilators by 373% while number of doctors increased by 108% and nurse/patient's ratio reached 1:4. Conclusion: All interventions implemented during COVID-19 pandemic outbreak in the RS resulted in increasing capacity for treatment of critically ill patients, but the education of health care professionals was identified as the most important conducted intervention.

Boosting ICU capacity during the COVID-19 pandemic in the western Balkan region, The Republic of Srpska experience

Background: Response to the outbreak of poliomyelitis in mid-1950 led to recognition and consequent development of critical care. Seventy years later the humankind was struck by COVID-19, another major challenge for critical care medicine which was especially big in Low-Resources-Settings where more than two thirds of the world population live, including the population of the Republic of Srpska (RS). Design and methods: The main aim was to show an overview of all interventions in order to boost hospitals' capacities to the level which is sufficient to manage high amount of critically ill COVID-19 patients in the RS. A before-after cohort study design was conducted to evaluate the effects of interventions for increase in preparedness and capacity of ICUs for admission and treatment of COVID-19 critically ill patients in nine hospitals in the RS. Results: Following interventions, the biggest and university affiliated hospital in the RS has increased ICU capacities: total number of ICU beds increased by 38% and number of ventilators by 114%. Availability of machines for veno-venous extracorporeal membrane oxygenation (vvECMO) increased by 100%. Number of doctors who were involved in treatment of critically ill patients increased by 47% and nurse/patient's ratio reached 1:2.5. Similarly, all other hospitals experienced boosting of ICU beds by 189% and ventilators by 373% while number of doctors increased by 108% and nurse/patient's ratio reached 1:4. Conclusion: All interventions implemented during COVID-19 pandemic outbreak in the RS resulted in increasing capacity for treatment of critically ill patients, but the education of health care professionals was identified as the most important conducted intervention.

Characteristics and Outcomes of Critically Ill Pregnant/Postpartum Women with COVID-19 Pneumonia in Western Balkans, The Republic of Srpska Report.

Background and Objectives: Coronavirus disease 2019 (COVID-19) is a novel infectious disease that has spread worldwide. As of 5 March 2020, the COVID-19 pandemic has resulted in approximately 111,767 cases and 6338 deaths in the Republic of Srpska and 375,554 cases and 15,718 deaths in Bosnia and Herzegovina. Our objective in the present study was to determine the characteristics and outcomes of critically ill pregnant/postpartum women with COVID-19 in the Republic of Srpska. Materials and Methods: The retrospective observational study of prospectively collected data included all critically ill pregnant/postpartum women with COVID-19 in a university-affiliated hospital between 1 April 2020 and 1 April 2022. Infection was confirmed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) from nasopharyngeal swab specimens and respiratory secretions. Patients' demographics, clinical and laboratory data, pharmacotherapy, and neonatal outcomes were analysed. Results: Out of the 153 registered pregnant women with COVID-19 treated at the gynaecology department of the University Clinical Centre of the Republic of Srpska, 19 (12.41%) critically ill pregnant/postpartum women (median age of 36 (IQR, 29-38) years) were admitted to the medical intensive care unit (MICU). The mortality rate was 21.05% (four patients) during the study period. Of all patients (19), 14 gave birth (73.68%), and 4 (21.05%) were treated with veno-venous extracorporeal membrane oxygenation (vvECMO). Conclusions: Fourteen infants were born prematurely and none of them died during hospitalisation. A high mortality rate was detected among the critically ill pregnant/postpartum patients treated with mechanical ventilation and vvECMO in the MICU. The preterm birth rate was high in patients who required a higher level of life support (vvECMO and ventilatory support).

In Silico and In Vitro Studies of Alchemilla viridiflora Rothm-Polyphenols' Potential for Inhibition of SARS-CoV-2 Internalization.

Since the outbreak of the COVID-19 pandemic, it has been obvious that virus infection poses a serious threat to human health on a global scale. Certain plants, particularly those rich in polyphenols, have been found to be effective antiviral agents. The effectiveness of Alchemilla viridiflora Rothm. (Rosaceae) methanol extract to prevent contact between virus spike (S)-glycoprotein and angiotensin-converting enzyme 2 (ACE2) and neuropilin-1 (NRP1) receptors was investigated. In vitro results revealed that the tested samples inhibited 50% of virus-receptor binding interactions in doses of 0.18 and 0.22 mg/mL for NRP1 and ACE2, respectively. Molecular docking studies revealed that the compounds from A. viridiflora ellagitannins class had a higher affinity for binding with S-glycoprotein whilst flavonoid compounds more significantly interacted with the NRP1 receptor. Quercetin 3-(6″-ferulylglucoside) and pentagalloylglucose were two compounds with the highest exhibited interfering potential for selected target receptors, with binding energies of -8.035 (S-glycoprotein) and -7.685 kcal/mol (NRP1), respectively. Furthermore, computational studies on other SARS-CoV-2 strains resulting from mutations in the original wild strain (V483A, N501Y-K417N-E484K, N501Y, N439K, L452R-T478K, K417N, G476S, F456L, E484K) revealed that virus internalization activity was maintained, but with different single compound contributions.

Amelioration of Endotoxin-Induced Acute Lung Injury and Alveolar Epithelial Cells Apoptosis by Simvastatin Is Associated with Up-Regulation of Survivin/NF-kB/p65 Pathway.

Disruption of the alveolar-endothelial barrier caused by inflammation leads to the progression of septic acute lung injury (ALI). In the present study, we investigated the beneficial effects of simvastatin on the endotoxin lipopolysaccharide (LPS)-induced ALI and its related mechanisms. A model of ALI was induced within experimental sepsis developed by intraperitoneal injection of a single non-lethal LPS dose after short-term simvastatin pretreatment (10-40 mg/kg orally). The severity of the lung tissue inflammatory injury was expressed as pulmonary damage scores (PDS). Alveolar epithelial cell apoptosis was confirmed by TUNEL assay (DNA fragmentation) and expressed as an apoptotic index (AI), and immunohistochemically for cleaved caspase-3, cytochrome C, and anti-apoptotic Bcl-xL, an inhibitor of apoptosis, survivin, and transcriptional factor, NF-kB/p65. Severe inflammatory injury of pulmonary parenchyma (PDS 3.33 ± 0.48) was developed after the LPS challenge, whereas simvastatin significantly and dose-dependently protected lung histology after LPS (p < 0.01). Simvastatin in a dose of 40 mg/kg showed the most significant effects in amelioration alveolar epithelial cells apoptosis, demonstrating this as a marked decrease of AI (p < 0.01 vs. LPS), cytochrome C, and cleaved caspase-3 expression. Furthermore, simvastatin significantly enhanced the expression of Bcl-xL and survivin. Finally, the expression of survivin and its regulator NF-kB/p65 in the alveolar epithelium was in strong positive correlation across the groups. Simvastatin could play a protective role against LPS-induced ALI and apoptosis of the alveolar-endothelial barrier. Taken together, these effects were seemingly mediated by inhibition of caspase 3 and cytochrome C, a finding that might be associated with the up-regulation of cell-survival survivin/NF-kB/p65 pathway and Bcl-xL.

Seroprevalence of SARS-CoV-2 antibodies among primary healthcare workers in the Republic of Srpska, Bosnia & Herzegovina: A cross-sectional study.

Healthcare workers (HCW) in primary healthcare centres in the Republic of Srpska, Bosnia and Herzegovina, are on the first combat line with COVID-19. This study aimed to assess the seroprevalence of SARS-CoV-2 among HCW at the primary healthcare centres and to analyse the risk exposure to COVID-19, clinical signs and vaccination status. A cross-sectional study was conducted among HCW at the selected primary healthcare centres between 19 March and 30 April 2021. Antibodies against the SARS-CoV-2 virus were detected by enzyme-linked immunosorbent assay (ELISA). A total of 1,023 HCW (mean age 45 years; 71% female) were included in the study. The anti-SARS-CoV-2 antibodies were detected in 69.5% of all participants. There was a significant difference in seropositivity among primary healthcare centres from different geographical regions. As many as 432 (42%) of all participants had confirmed COVID-19 symptoms before the study and, 84.8% of them were seropositive. This study showed that 702 primary HCW were vaccinated with any of these vaccines: Sputnik V, Sinopharm, Pfizer/Biontech. High titre of SARS-CoV-2 antibodies was found amongst those who received one (92.6%) or both (97.2%) doses of vaccines. In this study, we report high prevalence of SARS-CoV-2 antibody among HCW in primary healthcare in the Republic of Srpska, Bosnia and Herzegovina during the third pandemic wave.

Pomegranate peel extract polyphenols attenuate the SARS-CoV-2 S-glycoprotein binding ability to ACE2 Receptor: In silico and in vitro studies.

The novel coronavirus disease (Covid-19) has become a major health threat globally. The interaction of SARS-CoV-2 spike (S) glycoprotein receptor-binding domain (RBD) with ACE2 receptor on host cells was recognized as the first step of virus infection and therefore as one of the primary targets for novel therapeutics. Pomegranate extracts are rich sources of bioactive polyphenols that were already recognized for their beneficial health effects. In this study, both in silico and in vitro methods were employed for evaluation of pomegranate peel extract (PoPEx), their major polyphenols, as well as their major metabolite urolithin A, to attenuate the contact of S-glycoprotein RBD and ACE2. Our results showed that PoPEx, punicalin, punicalagin and urolithin A exerted significant potential to block the S-glycoprotein-ACE2 contact. These in vitro results strongly confirm the in silico predictions and provide a valuable insight in the potential of pomegranate polyphenols for application in SARS-CoV-2 infection.

Computational study of pomegranate peel extract polyphenols as potential inhibitors of SARS-CoV-2 virus internalization.

The search for effective coronavirus disease (COVID-19) therapy has attracted a great deal of scientific interest due to its unprecedented health care system overload worldwide. We have carried out a study to investigate the in silico effects of the most abundant pomegranate peel extract constituents on the multi-step process of serious acute respiratory syndrome coronavirus 2 (SARS-CoV-2) internalization in the host cells. Binding affinities and interactions of ellagic acid, gallic acid, punicalagin and punicalin were studied on four selected protein targets with a significant and confirmed role in the process of the entry of virus into a host cell. The protein targets used in this study were: SARS-CoV-2 spike glycoprotein, angiotensin-converting enzyme 2, furin and transmembrane serine protease 2. The results showed that the constituents of pomegranate peel extracts, namely punicalagin and punicalin had very promising potential for significant interactions with the selected protein targets and were therefore deemed good candidates for further in vitro and in vivo evaluation.