Screening and identification of potential MERS-CoV papain-like protease (PLpro) inhibitors; Steady-state kinetic and Molecular dynamic studies.

MERS-CoV belongs to the coronavirus group. Recent years have seen a rash of coronavirus epidemics. In June 2012, MERS-CoV was discovered in the Kingdom of Saudi Arabia, with 2,591 MERSA cases confirmed by lab tests by the end of August 2022 and 894 deaths at a case-fatality ratio (CFR) of 34.5% documented worldwide. Saudi Arabia reported the majority of these cases, with 2,184 cases and 813 deaths (CFR: 37.2%), necessitating a thorough understanding of the molecular machinery of MERS-CoV. To develop antiviral medicines, illustrative investigation of the protein in coronavirus subunits are required to increase our understanding of the subject. In this study, recombinant expression and purification of MERS-CoV (PLpro), a primary goal for the development of 22 new inhibitors, were completed using a high throughput screening methodology that employed fragment-based libraries in conjunction with structure-based virtual screening. Compounds 2, 7, and 20, showed significant biological activity. Moreover, a docking analysis revealed that the three compounds had favorable binding mood and binding free energy. Molecular dynamic simulation demonstrated the stability of compound 2 (2-((Benzimidazol-2-yl) thio)-1-arylethan-1-ones) the strongest inhibitory activity against the PLpro enzyme. In addition, disubstitutions at the meta and para locations are the only substitutions that may boost the inhibitory action against PLpro. Compound 2 was chosen as a MERS-CoV PLpro inhibitor after passing absorption, distribution, metabolism, and excretion studies; however, further investigations are required.

Simple fabrication of an electrospun polystyrene microfiber filter that meets N95 filtering facepiece respirator filtration and breathability standards

During the global spread of COVID‐19, high demand and limited availability of melt‐blown filtration material led to a manufacturing backlog of N95 Filtering Facepiece Respirators (FFRs). This shortfall prompted the search for alternative filter materials that could be quickly mass produced while meeting N95 FFR filtration and breathability performance standards. Here, an unsupported, nonwoven layer of uncharged polystyrene (PS) microfibers was produced via electrospinning that achieves N95 performance standards based on physical parameters (e.g., filter thickness) alone. PS microfibers 3–6 μm in diameter and deposited in an ~5 mm thick filter layer are favorable for use in FFRs, achieving high filtration efficiencies (≥97.5%) and low pressure drops (≤15 mm H2O). The PS microfiber filter demonstrates durability upon disinfection with hydroxyl radicals (•OH), maintaining high filtration efficiencies and low pressure drops over six rounds of disinfection. Additionally, the PS microfibers exhibit antibacterial activity (1‐log removal of E. coli) and can be modified readily through integration of silver nanoparticles (AgNPs) during electrospinning to enhance their activity (≥3‐log removal at 25 wt% AgNP integration). Because of their tunable performance, potential reusability with disinfection, and antimicrobial properties, these electrospun PS microfibers may represent a suitable, alternative filter material for use in N95 FFRs.

Unglycosylated Soluble SARS-CoV-2 Receptor Binding Domain (RBD) Produced in E. coli Combined with the Army Liposomal Formulation Containing QS21 (ALFQ) Elicits Neutralizing Antibodies against Mismatched Variants.

The emergence of novel potentially pandemic pathogens necessitates the rapid manufacture and deployment of effective, stable, and locally manufacturable vaccines on a global scale. In this study, the ability of the Escherichia coli expression system to produce the receptor binding domain (RBD) of the SARS-CoV-2 spike protein was evaluated. The RBD of the original Wuhan-Hu1 variant and of the Alpha and Beta variants of concern (VoC) were expressed in E. coli, and their biochemical and immunological profiles were compared to RBD produced in mammalian cells. The E. coli-produced RBD variants recapitulated the structural character of mammalian-expressed RBD and bound to human angiotensin converting enzyme (ACE2) receptor and a panel of neutralizing SARS-CoV-2 monoclonal antibodies. A pilot vaccination in mice with bacterial RBDs formulated with a novel liposomal adjuvant, Army Liposomal Formulation containing QS21 (ALFQ), induced polyclonal antibodies that inhibited RBD association to ACE2 in vitro and potently neutralized homologous and heterologous SARS-CoV-2 pseudoviruses. Although all vaccines induced neutralization of the non-vaccine Delta variant, only the Beta RBD vaccine produced in E. coli and mammalian cells effectively neutralized the Omicron BA.1 pseudovirus. These outcomes warrant further exploration of E. coli as an expression platform for non-glycosylated, soluble immunogens for future rapid response to emerging pandemic pathogens.

Atmospheric Pressure Plasma Polymerisation of D-Limonene and Its Antimicrobial Activity.

Antibacterial coating is necessary to prevent biofilm-forming bacteria from colonising medical tools causing infection and sepsis in patients. The recent coating strategies such as immobilisation of antimicrobial materials and low-pressure plasma polymerisation may require multiple processing steps involving a high-vacuum system and time-consuming process. Some of those have limited efficacy and durability. Here, we report a rapid and one-step atmospheric pressure plasma polymerisation (APPP) of D-limonene to produce nano-thin films with hydrophobic-like properties for antibacterial applications. The influence of plasma polymerisation time on the thickness, surface characteristic, and chemical composition of the plasma-polymerised films was systematically investigated. Results showed that the nano-thin films deposited at 1 min on glass substrate are optically transparent and homogenous, with a thickness of 44.3 ± 4.8 nm, a smooth surface with an average roughness of 0.23 ± 0.02 nm. For its antimicrobial activity, the biofilm assay evaluation revealed a significant 94% decrease in the number of Escherichia coli (E. coli) compared to the control sample. More importantly, the resultant nano-thin films exhibited a potent bactericidal effect that can distort and rupture the membrane of the treated bacteria. These findings provide important insights into the development of bacteria-resistant and biocompatible coatings on the arbitrary substrate in a straightforward and cost-effective route at atmospheric pressure.

Geo-referenced simulations of E. coli in a sub-catchment of the Vecht River using a probabilistic approach.

The proportion of wild swimmers at non-official bathing sites has increased during the Covid-19 pandemic. Bathing water quality at designated sites is monitored through analysis of the concentration of fecal indicator bacteria such as E. coli. However, non-official sites are generally not monitored. In a previous work, steady state modelling of E. coli was achieved at catchment scale, enabling a comparison of expected concentrations along an entire catchment for longtime average. However, E. coli concentrations can vary over several orders of magnitude at the same monitoring site throughout the year. To capture the temporal variability of E. coli concentrations on the catchment scale, we extended the existing deterministic E. coli sub-module of the GREAT-ER (Geo-referenced Exposure Assessment tool for European Rivers) model for probabilistic Monte-Carlo simulations. Here, selected model parameters are represented by probability distributions instead of fixed values. Wastewater treatment plant (WWTP) emissions and diffuse emissions were parameterized using selected data from a previous monitoring campaign (calibration data set) and in-stream processes were modeled using literature data. Comparison of simulation results with monitoring data (evaluation data set) indicates that predicted E. coli concentrations well-represent median measured concentrations, although the range of predicted concentrations is slightly larger than the observed concentration variability. The parameters with the largest influence on the range of predicted concentrations are flow rate and E. coli removal efficiency in WWTPs. A comparison of predicted 90th percentiles with the threshold for sufficient bathing water quality (according to the EU Bathing Water Directive) indicates that year-round swimming at sites influenced by WWTP effluents is advisable almost nowhere in the study area. A refinement of the model can be achieved if quantitative relationships between the WWTP removal efficiency and both, the treatment technologies as well as the operating parameters are further established.

Case report: Diarrhea-associated hemolytic uremic syndrome in the Era of COVID-19.

Over the past two years, a growing number of SARS-CoV-2 infection-associated clinical pediatric phenotypes have been identified, including a hemolytic uremic syndrome (HUS) form of thrombotic microangiopathy. Oregon's high prevalence of Shiga toxin-producing Escherichia coli (STEC) infections gives it a unique perspective to discuss the impact of COVID-19 and HUS. We seek to highlight SARS-CoV-2 as a potential new infectious etiology of severe diarrhea-associated HUS, based on two cases from Portland, Oregon, occurring in non-COVID-19 immunized children. The first case is a previously healthy ten-year-old who presented with SARS-CoV-2 infection and bloody diarrhea after an appendectomy, followed by full-blown oligo-anuric HUS. Second is a previously healthy six-year-old who presented with short-lived bloody diarrhea, rapidly evolving to HUS, and who tested positive for COVID-19 via polymerase chain reaction and STEC toxins one and two. These two cases highlight two main points. First, SARS-CoV-2 must be included in the differential diagnosis of diarrhea-associated HUS, either as the sole agent or concurrent with a STEC infection. Second, when managing STEC gastroenteritis the recommendation has been to maintain excellent hydration as a strategy to prevent the progression to oligo-anuric acute kidney injury and HUS. This strategy may need to be re-evaluated in a patient with SARS-CoV-2 infection or co-infection.

Functional Expression of the Recombinant Spike Receptor Binding Domain of SARS-CoV-2 Omicron in the Periplasm of Escherichia coli.

A new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant known as Omicron has caused a rapid increase in recent global patients with coronavirus infectious disease 2019 (COVID-19). To overcome the COVID-19 Omicron variant, production of a recombinant spike receptor binding domain (RBD) is vital for developing a subunit vaccine or a neutralizing antibody. Although bacterial expression has many advantages in the production of recombinant proteins, the spike RBD expressed in a bacterial system experiences a folding problem related to disulfide bond formation. In this study, the soluble Omicron RBD was obtained by a disulfide isomerase-assisted periplasmic expression system in Escherichia coli. The Omicron RBD purified from E. coli was very well recognized by anti-SARS-CoV-2 antibodies, sotrovimab (S309), and CR3022, which were previously reported to bind to various SARS-CoV-2 variants. In addition, the kinetic parameters of the purified Omicron RBD upon binding to the human angiotensin-converting enzyme 2 (ACE2) were similar to those of the Omicron RBD produced in the mammalian expression system. These results suggest that an E. coli expression system would be suitable to produce functional and correctly folded spike RBDs of the next emerging SARS-CoV-2 variants quickly and inexpensively.

Emphysematous Pyelonephritis, Emphysematous Cystitis, and Emphysematous Ureteritis: A Case Report.

Emphysematous urinary tract infections (EUTIs) are rare, severe, and suppurative infections affecting various parts of the urinary tract. We report a case of a 75-year-old male presenting with hematuria and generalized weakness with uncontrolled diabetes mellitus (DM) and hypertension. He tested positive for COVID-19 on the second day of hospital admission. A non-contrast-enhanced CT of the abdomen and pelvis revealed gas within the left renal parenchyma, walls of the left ureter, and urinary bladder, establishing the diagnosis of EUTIs. The patient was treated using intravenous antibiotics without any surgical intervention, and four weeks later was stable and transported to long-term acute care (LTAC) facility. DM is the most common risk factor for the development of EUTIs and Escherichia coli is the most common causative pathogen.

Synthesis of ZnO/Au Nanocomposite for Antibacterial Applications.

Annually, antimicrobial-resistant infections-related mortality worldwide accelerates due to the increased use of antibiotics during the coronavirus pandemic and the antimicrobial resistance, which grows exponentially, and disproportionately to the current rate of development of new antibiotics. Nanoparticles can be an alternative to the current therapeutic approach against multi-drug resistance microorganisms caused infections. The motivation behind this work was to find a superior antibacterial nanomaterial, which can be efficient, biocompatible, and stable in time. This study evaluated the antibacterial activity of ZnO-based nanomaterials with different morphologies, synthesized through the solvothermal method and further modified with Au nanoparticles through wet chemical reduction. The structure, crystallinity, and morphology of ZnO and ZnO/Au nanomaterials have been investigated with XRD, SEM, TEM, DLS, and FTIR spectroscopy. The antibacterial effect of unmodified ZnO and ZnO/Au nanomaterials against Escherichia coli and Staphylococcus aureus was investigated through disc diffusion and tetrazolium/formazan (TTC) assays. The results showed that the proposed nanomaterials exhibited significant antibacterial effects on the Gram-positive and Gram-negative bacteria. Furthermore, ZnO nanorods with diameters smaller than 50 nm showed better antibacterial activity than ZnO nanorods with larger dimensions. The antibacterial efficiency against Escherichia coli and Staphylococcus aureus improved considerably by adding 0.2% (w/w) Au to ZnO nanorods. The results indicated the new materials' potential for antibacterial applications.

Complementary Sets of Autoantibodies Induced by SARS-CoV-2, Adenovirus and Bacterial Antigens Cross-React with Human Blood Protein Antigens in COVID-19 Coagulopathies.

COVID-19 patients often develop coagulopathies including microclotting, thrombotic strokes or thrombocytopenia. Autoantibodies are present against blood-related proteins including cardiolipin (CL), serum albumin (SA), platelet factor 4 (PF4), beta 2 glycoprotein 1 (ß2GPI), phosphodiesterases (PDE), and coagulation factors such as Factor II, IX, X and von Willebrand factor (vWF). Different combinations of autoantibodies associate with different coagulopathies. Previous research revealed similarities between proteins with blood clotting functions and SARS-CoV-2 proteins, adenovirus, and bacterial proteins associated with moderate-to-severe COVID-19 infections. This study investigated whether polyclonal antibodies (mainly goat and rabbit) against these viruses and bacteria recognize human blood-related proteins. Antibodies against SARS-CoV-2 and adenovirus recognized vWF, PDE and PF4 and SARS-CoV-2 antibodies also recognized additional antigens. Most bacterial antibodies tested (group A streptococci [GAS], staphylococci, Escherichia coli [E. coli], Klebsiella pneumoniae, Clostridia, and Mycobacterium tuberculosis) cross-reacted with CL and PF4. while GAS antibodies also bound to F2, Factor VIII, Factor IX, and vWF, and E. coli antibodies to PDE. All cross-reactive interactions involved antibody-antigen binding constants smaller than 100 nM. Since most COVID-19 coagulopathy patients display autoantibodies against vWF, PDE and PF4 along with CL, combinations of viral and bacterial infections appear to be necessary to initiate their autoimmune coagulopathies.