The dimerization interface of initiator RctB governs chaperone and enhancer dependence of Vibrio cholerae chromosome 2 replication.

Protein function often requires remodeling of protein structure. In the well-studied iteron-containing plasmids, the initiator of replication has a dimerization interface that undergoes chaperone-mediated remodeling. This remodeling reduces dimerization and promotes DNA replication, since only monomers bind origin DNA. A structurally homologs interface exists in RctB, the replication initiator of Vibrio cholerae chromosome 2 (Chr2). Chaperones also promote Chr2 replication, although both monomers and dimers of RctB bind to origin, and chaperones increase the binding of both. Here we report how five changes in the dimerization interface of RctB affect the protein. The mutants are variously defective in dimerization, more active as initiator, and except in one case, unresponsive to chaperone (DnaJ). The results indicate that chaperones also reduce RctB dimerization and support the proposal that the paradoxical chaperone-promoted dimer binding likely represents sequential binding of monomers on DNA. RctB is also activated for replication initiation upon binding to a DNA site, crtS, and three of the mutants are also unresponsive to crtS. This suggests that crtS, like chaperones, reduces dimerization, but additional evidence suggests that the remodelling activities function independently. Involvement of two remodelers in reducing dimerization signifies the importance of dimerization in limiting Chr2 replication.

Suitcase Lab for Rapid Detection of SARS-CoV-2 Based on Recombinase Polymerase Amplification Assay.

In March 2020, the SARS-CoV-2 virus outbreak was declared as a world pandemic by the World Health Organization (WHO). The only measures for controlling the outbreak are testing and isolation of infected cases. Molecular real-time polymerase chain reaction (PCR) assays are very sensitive but require highly equipped laboratories and well-trained personnel. In this study, a rapid point-of-need detection method was developed to detect the RNA-dependent RNA polymerase (RdRP), envelope protein (E), and nucleocapsid protein (N) genes of SARS-CoV-2 based on the reverse transcription recombinase polymerase amplification (RT-RPA) assay. RdRP, E, and N RT-RPA assays required approximately 15 min to amplify 2, 15, and 15 RNA molecules of molecular standard/reaction, respectively. RdRP and E RT-RPA assays detected SARS-CoV-1 and 2 genomic RNA, whereas the N RT-RPA assay identified only SARS-CoV-2 RNA. All established assays did not cross-react with nucleic acids of other respiratory pathogens. The RT-RPA assay's clinical sensitivity and specificity in comparison to real-time RT-PCR (n = 36) were 94 and 100% for RdRP; 65 and 77% for E; and 83 and 94% for the N RT-RPA assay. The assays were deployed to the field, where the RdRP RT-RPA assays confirmed to produce the most accurate results in three different laboratories in Africa (n = 89). The RPA assays were run in a mobile suitcase laboratory to facilitate the deployment at point of need. The assays can contribute to speed up the control measures as well as assist in the detection of COVID-19 cases in low-resource settings.

Female hospital workers in Morocco: factors influencing breastfeeding duration with self-reported experiences.

Health workers (HW) could be at risk of early weaning because of working conditions. Our aim was to determine factors influencing the duration of breastfeeding among Moroccan hospital workers, and to explore their breastfeeding (BF) experiences. A cross-sectional study was conducted in four hospitals in Rabat/Morocco (from November 2015 to April 2016), including each woman working in the hospital, with at least one living child and who accepted to be interviewed. Data of 203 hospital workers were analyzed. The median age was 39. The median duration of any breastfeeding was 8 months. Among different categories of HW, physicians had the lowest duration of breastfeeding. Factors significantly correlated to longer duration of breastfeeding were infant rank (p = 0.003), early initiation of breastfeeding (p < 0.001), access to milk storage generally (p = 0.04), husband's opinion on breastfeeding (p < 0.001) and category of hospital worker (p = 0.01). Three central themes emerged from the analysis of qualitative data: "Breastfeeding health worker has to assume her work duties as any other health worker", "the expression of need for support", and "the lack of knowledge on breastfeeding". In light of these results, we believe that physicians are a high-risk group of premature complete weaning; many actions should be taken for all HW to enhance their knowledge and giving them support.

Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC.

Most human coronaviruses cause mild upper respiratory tract disease but may be associated with more severe pulmonary disease in immunocompromised individuals. However, SARS coronavirus caused severe lower respiratory disease with nearly 10% mortality and evidence of systemic spread. Recently, another coronavirus (human coronavirus-Erasmus Medical Center (hCoV-EMC)) was identified in patients with severe and sometimes lethal lower respiratory tract infection. Viral genome analysis revealed close relatedness to coronaviruses found in bats. Here we identify dipeptidyl peptidase 4 (DPP4; also known as CD26) as a functional receptor for hCoV-EMC. DPP4 specifically co-purified with the receptor-binding S1 domain of the hCoV-EMC spike protein from lysates of susceptible Huh-7 cells. Antibodies directed against DPP4 inhibited hCoV-EMC infection of primary human bronchial epithelial cells and Huh-7 cells. Expression of human and bat (Pipistrellus pipistrellus) DPP4 in non-susceptible COS-7 cells enabled infection by hCoV-EMC. The use of the evolutionarily conserved DPP4 protein from different species as a functional receptor provides clues about the host range potential of hCoV-EMC. In addition, it will contribute critically to our understanding of the pathogenesis and epidemiology of this emerging human coronavirus, and may facilitate the development of intervention strategies.

Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia.

A previously unknown coronavirus was isolated from the sputum of a 60-year-old man who presented with acute pneumonia and subsequent renal failure with a fatal outcome in Saudi Arabia. The virus (called HCoV-EMC) replicated readily in cell culture, producing cytopathic effects of rounding, detachment, and syncytium formation. The virus represents a novel betacoronavirus species. The closest known relatives are bat coronaviruses HKU4 and HKU5. Here, the clinical data, virus isolation, and molecular identification are presented. The clinical picture was remarkably similar to that of the severe acute respiratory syndrome (SARS) outbreak in 2003 and reminds us that animal coronaviruses can cause severe disease in humans.

Human cell tropism and innate immune system interactions of human respiratory coronavirus EMC compared to those of severe acute respiratory syndrome coronavirus.

Infections with human coronavirus EMC (HCoV-EMC) are associated with severe pneumonia. We demonstrate that HCoV-EMC resembles severe acute respiratory syndrome coronavirus (SARS-CoV) in productively infecting primary and continuous cells of the human airways and in preventing the induction of interferon regulatory factor 3 (IRF-3)-mediated antiviral alpha/beta interferon (IFN-α/ß) responses. However, HCoV-EMC was markedly more sensitive to the antiviral state established by ectopic IFN. Thus, HCoV-EMC can utilize a broad range of human cell substrates and suppress IFN induction, but it does not reach the IFN resistance of SARS-CoV.

Accurate and reliable surface-enhanced Raman spectroscopy assay for early detection of SARS-CoV-2 RNA with exceptional sensitivity.

This research proposes a highly sensitive and simple surface-enhanced Raman spectroscopy (SERS) assay for the detection of SARS-CoV-2 RNA using suitably designed probes specific for RdRp and N viral genes attached to a Raman marker. The sensitivity of the assay was optimized through precise adjustments to the conditions of immobilization and hybridization processes of the target RNA, including modifications to factors such as time and temperature. The assay achieved a remarkable sensitivity down to 58.39 copies/mL, comparable to or lower than the sensitivities reported for commercial fluorescent polymerase chain reaction (PCR) based methods. It has good selectivity in discriminating SARS-CoV-2 RNA against other respiratory viruses, respiratory syncytial virus (RSV), and influenza A virus. The reliability of the assay was validated by testing 24 clinical samples, including 12 positive samples with varying cycle threshold (Ct) values and 12 negative samples previously tested using real-time PCR. The assay consistently predicted true results that were in line with the PCR results for all samples. Furthermore, the assay demonstrated a notable limit of detection (LOD) of Ct (38 for RdRp gene and 37.5 for N-gene), indicating its capability to detect low concentrations of the target analyte and potentially facilitating early detection of the pathogen.

Monitoring SARS-CoV-2 infection in different animal species and human in Egypt during 2020-2021.

Coronaviruses cause respiratory and intestinal infections in animals and humans. By the end of 2019, there was an epidemic of novel coronavirus (COVID-19), which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Coronaviruses have a highly mutable genome that makes them genetically and phenotypically modifiable with a potential transmission to new host species. Based on current sequence databases, all human coronaviruses have animal origins, so animals have important roles in virus spillover to humans. The aim of this study is to investigate the role of different animal species in the epidemiology of SARS-CoV-2 in Egypt. A pan-coronaviruses RT-PCR has been used for detection of possible coronaviruses infection in different species including bats, humans, birds, and dogs in Egypt during the period of November 2020 till June 2021. Ninety-two samples (46 from Rousettus aegyptiacus bats, 10 from human, 26 from wild birds, and 10 from dogs) were screened for SARS-CoV-2. Our results revealed that only human samples were SARS-CoV-2 positive for SARS-CoV-2 while all other animal and bird samples were negative. To recapitulate, our results suggest that animals may not actively transmit SARS-CoV-2 among people in Egypt during the current COVID-19 pandemic. Further structural surveillance and follow up screening for SARS-CoV-2 among domestic and wild animal populations in Egypt is crucially needed.

Computational modeling of cyanobacterial phytoconstituents against toll-like receptors of skin cancer.

Melanoma is an extremely dangerous disease. The diagnosis and treatment of it may be difficult because of its diversity and complexity. More than 90% of the marine biomass (microflora and microalgae) constitutes the natural biodiversity reserves. TLR-related research developments indicate possible cancer therapeutic possibilities. In addition to its significant function in innate immunity, TLR activation is connected to the start of pyroptosis, apoptosis, or autophagy in malignance cells. For these reasons, TLR agonists are appealing candidates for the production of cancer medications. From the web databases, the ternary structures of the receptors (TLR3 and TLR4) and ligands are extracted. Sixty-nine compounds were subjected to a drug likeness filter, but only twenty-two were screened further for evaluating ADMET criteria, in which only seven compounds satisfied the pharmacological properties. These compounds are further analyzed for docking parameters against TLRs (TLR3 and TLR4) and molecular simulation investigation of the best cluster to evaluate the complex stability. Molecular docking methodology discovered that Scytonmein has a significant binding potential energy of -5.21 and -7.92 kcal/mol against TLR3 and TLR4, respectively, in comparison to the redock co-crystal structure (-3.98 and -4.30 kcal/mol, respectively). The simulation analysis demonstrates the significant stability of the Scytonemin and TLR4 complexes in terms of average RMSD and RMSF compared to the redock complex, while criteria like solvent-accessible surface area (SASA), gyration (Rg) and hydrogen bonding have further supported the significant interaction and stability of the conformations.Communicated by Ramaswamy H. Sarma.

Identification of potential inhibitor molecule against MabA protein of Mycobacterium leprae by integrated in silico approach.

Leprosy is one of the chronic diseases with which humanity has struggled globally for millennia. The potent anti-leprosy medications rifampicin, clofazimine and dapsone, among others, are used to treat leprosy. Nevertheless, even in regions of the world where these drugs have been successfully implemented, resistance continues to be observed. Due to the problems with the current treatments, this disease should be fought at every level of society with new drugs. The purpose of this research was to identify natural candidates with the ability to inhibit MabA (gene-fabG1) with fewer negative effects. The work was accomplished through molecular docking, followed by a dynamic investigation of protein-ligand, which play a significant role in the design of pharmaceuticals. After modelling the protein structure with MODELLER 9.21v, AutoDock Vina was used to perform molecular docking with 13 3 D anti-leprosy medicines and a zinc library to determine the optimal protein-ligand interaction. In addition, the docking result was filtered based on binding energy, ADMET characteristics, PASS analysis and the most crucial binding residues. The ZINC08101051 chemical compound was prioritized for further study. Using an all-atom 100 ns MD simulation, the binding pattern and conformational changes in protein upon ligand binding were studied. Recommendation for subsequent validation based on deviation, fluctuation, gyration and hydrogen bond analysis, followed by main component and free energy landscape.Communicated by Ramaswamy H. Sarma.

Inhibitory effect of natural compounds on Dihydropteroate synthase of Mycobacterium leprae: Molecular dynamic study.

Leprosy is a chronic infectious disease caused by a bacillus, Mycobacterium leprae. According to official data from 139 countries in the 6 WHO Regions, there were 127558 new leprosy cases worldwide in 2020. Leprosy mainly affects the skin, the peripheral nerves, mucosa of the upper respiratory tract, and the eyes. If this disease is left untreated, can harm the skin, nerves, limbs, eyes, and skin permanently. The disease is curable with multidrug therapy. Over a period of time Mycobacterium leprae has become resistant to these drugs. Therefore, new therapeutic molecules are warranted. This study was aimed to carry out the in-silico analysis to determine the inhibitory effect of natural compounds on Dihydropteroate synthase (DHPS) of Mycobacterium leprae. The DHPS is a key enzyme in the folate biosynthesis pathway in M. leprae and acts as a competitive inhibitor of PABA. The 3D structure of DHPS protein was modeled using homology modeling and was validated. Molecular docking and simulation along with other in-silico methods were employed to determine the inhibitory effect of ligand molecules towards DHPS target protein. Results revealed ZINC03830554 molecule as a potential inhibitor of DHPS. Binding experiments and bioassays utilizing this strong inhibitor molecule against purified DHPS protein are necessary to validate these early findings.Communicated by Ramaswamy H. Sarma.

Skin colonization of Staphylococcus aureus harboring superantigen toxin genes and its correlation with serum IL-22 level in psoriasis patients.

Psoriasis is a chronic debilitating skin disease with an estimated prevalence reaching 2% of the worldwide population. Psoriatic disease is driven by the interactions among innate and adaptive immune systems with structural components of the skin. Interleukin (IL)-22 mediates keratinocyte proliferation and epidermal hyperplasia, and changes in the structure of skin flora can play a role in the secretion of IL-22. The aim of this study was to correlate serum levels of IL-22 and Staphylococcus aureus toxins with disease activity in plaque psoriasis. The study group included 50 patients with mild, moderate, and severe psoriasis. The control group comprised 20 sex- and age-matched apparently healthy volunteers. IL-22 concentration was assessed in sera of patients and the control group by using the ELISA technique. The serum levels of IL-22 in patients were higher than in the control group, but the difference was statistically insignificant (P=0.413). Serum IL-22 levels were positively correlated with the Psoriasis Area and Severity Index (PASI) score of psoriasis patients (P=0.0003). The IL-22 serum levels in patients colonized with toxigenic strains of S. aureus were significantly higher than in patients colonized with non-toxigenic strains (P= 0.028). In conclusion, IL-22 plays a role in the pathogenesis of psoriasis, and its secretion can be triggered by the toxins produced by S. aureus colonizing the skin of patients.

Crimean-Congo hemorrhagic fever virus in ticks collected from imported camels in Egypt.

Crimean-Congo hemorrhagic fever (CCHF) is one of the utmost broadly distributed tick-borne viruses, with an infection resulting in a fatality rate of up to 30%. During this study period, 25,000 hard adult ticks of Hyalomma species were collected from freshly slaughtered imported camels to determine the presence of Crimean-Congo hemorrhagic fever virus (CCHFV) and genetic lineage of the virus. Ticks were pooled and analyzed for the existence of CCHFV using nested RT- PCR and real-time reverse transcription PCR; the genome was detected in 18 (1.44%) tick pools. Partial genome sequences reveal an adjacent relationship with strains from South Africa to Namibia, Nigeria, Sudan, Senegal, and Mauritania, corresponding to the Africa I and III genotypes. This study indicates the presence of CCHFV in Egypt and illustrates the potential for tick-borne dissemination of the virus. Further studies focused on not only tick samples, but also human samples are epidemiologically valuable to obtain exact data in the region.

Tuberculosis in the middle of COVID-19 in Morocco: efforts, challenges and recommendations.

Tuberculosis (TB) is a deadly infectious disease that kills approximately 1.5 million people per year and is among the most frequent respiratory infections in developing countries. Morocco has made significant progress in the control and management of TB during the past 30 years thanks to its National Plan for Tuberculosis and the continuous support of national and international partners. While tremendous efforts were undertaken to tilt the balance against the COVID-19 pandemic, new challenges resurfaced with regard to long-standing health problems amongst which is TB. The spill-over effect of the COVID-19 pandemic disrupted health service delivery globally, threatening to reverse years of progress made on the TB control front. In Morocco, this crisis highlighted deep shortcomings within the national health system and in the adopted approach to TB control. This article discusses national efforts to get back on track with regard to TB management, the multitude of challenges that co-emerged with the onset of COVID-19 and lays down key recommendations to implement in order to build back a TB control plan that is resilient in the face of health hazards.

Antithyroid Peroxidase Antibodies and Histopathological Outcomes in Egyptian Patients Subjected to Total Thyroidectomy for Non-Malignant Nodular Goiter.

OBJECTIVE: The study aimed to assess antithyroid antibodies in patients with benign thyroid masses and the effect of total thyroidectomy on the antibodies titers. PATIENTS AND METHODS: This is a retrospective work of 112 cases managed with total thyroidectomy with positive antithyroid peroxidase antibodies (TPO-Ab), anti-thyroglobulin antibodies (Tg-Ab), or both. All patients were euthyroid before surgery. Thyroid function tests and thyroid antibodies levels were measured before and 6 and 12 months after surgery. RESULTS: Histopathological evaluation revealed Hashimoto thyroiditis (47.3%), colloid nodules (22.3%), and lymphocytic thyroiditis (30.4%). All patients were TPO-Ab positive, while 96 patients (85.7%) were Tg-Ab positive before surgery. There was no considerable change in TPO-Ab and Tg-Ab after surgery (p = 0.817, and p=0.560, respectively). Also, there was no significant difference between the three histopathological diagnoses in the levels of TPO-Ab (p = 0.086) or Tg-Ab (p = 0.673). CONCLUSION: Antithyroid antibodies are not valuable markers for diagnosis or prognosis of benign thyroid diseases subjected to total thyroidectomy. We do not recommend their use beyond supporting evidence of the possibility of the autoimmune nature of the illness if other criteria are confirmed.

Stability of MERS-CoV RNA on spin columns of RNA extraction kit at room temperature.

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging virus causing a highly fatal respiratory disease in humans. Confirmation of MERS-CoV infection and molecular study on the virus may require transportation of samples to specialized laboratories. While freezing at -80 °C is the gold standard method for RNA preservation, maintaining the integrity of viral RNA during transport will require additional precautions and, as a result, increase transport costs. We aimed at testing the stability of MERS-CoV RNA on spin columns of RNA extraction kit at room temperature for 16 weeks. Respiratory samples spiked with stock culture of MERS-CoV were extracted and loaded on QIAamp Viral RNA Mini Kit spin columns and preserved at room temperature. Amount of viral RNA was evaluated periodically by real-time quantitative reverse-transcription polymerase chain reaction. Minimal changes in cycle threshold values over the study period were noted, suggesting stability of viral RNA by this preservation method.

Susceptibility to hyperglycemia in patients with chronic kidney disease.

BACKGROUND: Patients with chronic kidney disease (CKD) are susceptible to hyperglycemia. AIM: To study the prevalence of pre-diabetes in CKD patients and determine the contribution of insulin resistance (IR) versus beta-cell dysfunction in patients with CKD. METHODS: 45 consecutive nondiabetic CKD patients and 40 healthy subjects were included. Patients were divided into a normoglycemic (NG) and a pre-diabetic (PDM) group. IR was assessed by homeostasis model assessment of insulin resistance (HOMA-IR) and beta-cell function was assessed by proinsulin/insulin ratio and beta-cell%. RESULTS: The prevalence of PDM was 40%. The prevalence of high HOMA-IR was 22.2 and 77.8% in the NG and PDM groups. Compared to NG patients, the PDM group showed higher fasting plasma glucose, HOMA-IR, insulin, and proinsulin, while the prevalence of beta-cell dysfunction of 22.2% was lower than the 37% present in the NG group. CONCLUSION: Increased IR, rather than beta-cell dysfunction, is the primary mechanism of PDM in CKD patients.

Phylogeny of dengue viruses circulating in Jeddah, Saudi Arabia: 1994 to 2006.

The nucleotide sequence of the 240 bp E/NS1 junction of 81 dengue viruses isolated from cases in Jeddah, Saudi Arabia was determined and used to serotype the viruses. The nucleotide sequences of the complete Envelope (E) genes of 19 isolates were used for a phylogenetic analysis of the dengue viruses circulating in Saudi Arabia from 1994 to 2006. Three of the four dengue serotypes (DENV-1, DENV-2 and DENV-3) were found to circulate, often with more than one serotype in each outbreak. There was a major outbreak caused by DENV-1 and DENV-2 in 1994 while DENV-3 emerged in 1997. In the summer of 2004, all three serotypes were isolated and this gave way to an extended outbreak of DENV-1 that stretched from the summer of 2005 through early 2006. In the 1994 outbreak, the DENV-1 circulating was from the America-Africa genotype (lineage India-2) while the most recent outbreak in 2005 and 2006 was caused by a different DENV-1 strain from genotype Asia (lineage Asia-2), suggesting a re-introduction of DENV-1 a decade after the first introduction in 1994. There has been no change in the genotypes of DENV-2 (cosmopolitan genotype) and DENV-3 (genotype III) circulating since introduction in 1994 and 1997, respectively.

Helicobacter pylori infection in hepatic encephalopathy: Relationship to plasma endotoxins and blood ammonia.

BACKGROUND/AIM: Hepatic encephalopathy (HE) is frequently observed in patients with advanced liver disease and manifests a wide variety of neuropsychiatric signs and symptoms. Ammonia toxicity and bacterial endotoxins have been suggested as key determinants of HE onset whereas a role for Helicobacter pylori infection has not been established. We investigated the correlation between H. pylori infection and HE severity (evaluated through functional tests) in 60 outpatients with established liver cirrhosis and 20 non-cirrhotic controls. METHODS: Fasting arterial blood ammonia, plasma endotoxins, and H. pylori infection status were investigated in all subjects. RESULTS: H. pylori infection was documented in 35/60 (58%) patients and in 6/20 (30%) controls (P = 0.039). Significant differences were observed between patients with and withoutHE for age, presence of ascites, fasting arterial blood ammonia, plasma endotoxin, and H. pylori infection. Further, a significant increase in fasting arterial blood ammonia and plasma endotoxin was associated with H. pylori infection in cirrhotic patients. Last, medical treatment of H. pylori infection led to a significant decrease in HE severity and fasting arterial blood ammonia levels. CONCLUSION: In conclusion, we submit that H. pylori infection might, in fact, play a role in increasing the circulating levels of ammonia and endotoxins in cirrhotic patients, thus facilitating the onset of HE.