SARS-CoV-2 NSP12 associates with TRiC and the P323L substitution acts as a host adaption.

ImportanceSARS-CoV-2 has caused a worldwide health and economic crisis. During the course of the pandemic, genetic changes occurred in the virus, which have resulted in new properties of the virus-particularly around gains in transmission and the ability to partially evade either natural or vaccine-acquired immunity. Some of these viruses have been labeled Variants of Concern (VoCs). At the root of all VoCs are two mutations, one in the viral spike protein that has been very well characterized and the other in the virus polymerase (NSP12). This is the viral protein responsible for replicating the genome. We show that NSP12 associates with host cell proteins that act as a scaffold to facilitate the function of this protein. Furthermore, we found that different variants of NSP12 interact with host cell proteins in subtle and different ways, which affect function

Causes of elective cesarean delivery on maternal request in Aljouf, Saudi Arabia

Background: Recently observed there is a steadily higher rate of cesarean delivery worldwide mostly due to the increasing number of women requesting an elective cesarean section on maternal request without valid indication. The aim of the study was to determine the causes of elective cesarean delivery on maternal requests in Aljouf Saudi Arabia.Methods: This was a descriptive cross-sectional study and data was evaluated by completing seven questionnaires and interviews with laboratory reports who were admitted for cesarean delivery at the Obstetrics department of Maternity and Children Hospital (MCH) Aljouf, Saudi Arabia from January 2020 to December 2020. A total of 141 Saudi women of age between 18 and over 35 years were enrolled, including those who have singleton pregnancy, no complications during pregnancy, and no medical indication for cesarean delivery.Results: 141 women reported willingness to request cesarean delivery. The mean systolic 120±6.23, diastolic 75±2.45 blood pressure mm of Hg, and fasting blood sugar level 4.1±1.1 mmol/l have been found within the normal limit. The ultrasound (US) confirmed singleton pregnancy without any abnormalities.  Data reveals that common causes of elective cesarean section on maternal request to avoid the episiotomy 77.3%, fear of labor pain 69.5%, trauma to the vagina 79.4%, uncertainty about timing 61.7%, losing a baby during vaginal delivery 54.6%, experience other members 41.8%, the risk for baby 39%, prolapse or incontinence24.1%, unsatisfactory sexual intercourse 17.7% and the undesirable experience of the previous vaginal delivery 12%.Conclusions: Maternal request for cesarean delivery is considered one of the reasons for increasing the rate of cesarean delivery in Saudi Arabia. To avoid the episiotomy and fear of labor pain may strong causes for choosing cesarean delivery

A Comparison of Whole Genome Sequencing of SARS-CoV-2 Using Amplicon-Based Sequencing, Random Hexamers, and Bait Capture

Genome sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is increasingly important to monitor the transmission and adaptive evolution of the virus. The accessibility of high-throughput methods and polymerase chain reaction (PCR) has facilitated a growing ecosystem of protocols. Two differing protocols are tiling multiplex PCR and bait capture enrichment. Each method has advantages and disadvantages but a direct comparison with different viral RNA concentrations has not been performed to assess the performance of these approaches. Here we compare Liverpool amplification, ARTIC amplification, and bait capture using clinical diagnostics samples. All libraries were sequenced using an Illumina MiniSeq with data analyzed using a standardized bioinformatics workflow (SARS-CoV-2 Illumina GeNome Assembly Line; SIGNAL). One sample showed poor SARS-CoV-2 genome coverage and consensus, reflective of low viral RNA concentration. In contrast, the second sample had a higher viral RNA concentration, which yielded good genome coverage and consensus. ARTIC amplification showed the highest depth of coverage results for both samples, suggesting this protocol is effective for low concentrations. Liverpool amplification provided a more even read coverage of the SARS-CoV-2 genome, but at a lower depth of coverage. Bait capture enrichment of SARS-CoV-2 cDNA provided results on par with amplification. While only two clinical samples were examined in this comparative analysis, both the Liverpool and ARTIC amplification methods showed differing efficacy for high and low concentration samples. In addition, amplification-free bait capture enriched sequencing of cDNA is a viable method for generating a SARS-CoV-2 genome sequence and for identification of amplification artifacts

Amplicon-Based Detection and Sequencing of SARS-CoV-2 in Nasopharyngeal Swabs from Patients With COVID-19 and Identification of Deletions in the Viral Genome That Encode Proteins Involved in Interferon Antagonism

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions

The P323L substitution in the SARS-CoV-2 polymerase (NSP12) confers a selective advantage during infection

Background The mutational landscape of SARS-CoV-2 varies at the dominant viral genome sequence and minor genomic variant population. During the COVID-19 pandemic, an early substitution in the genome was the D614G change in the spike protein, associated with an increase in transmissibility. Genomes with D614G are accompanied by a P323L substitution in the viral polymerase (NSP12). However, P323L is not thought to be under strong selective pressure. Results Investigation of P323L/D614G substitutions in the population shows rapid emergence during the containment phase and early surge phase during the first wave. These substitutions emerge from minor genomic variants which become dominant viral genome sequence. This is investigated in vivo and in vitro using SARS-CoV-2 with P323 and D614 in the dominant genome sequence and L323 and G614 in the minor variant population. During infection, there is rapid selection of L323 into the dominant viral genome sequence but not G614. Reverse genetics is used to create two viruses (either P323 or L323) with the same genetic background. L323 shows greater abundance of viral RNA and proteins and a smaller plaque morphology than P323. Conclusions These data suggest that P323L is an important contribution in the emergence of variants with transmission advantages. Sequence analysis of viral populations suggests it may be possible to predict the emergence of a new variant based on tracking the frequency of minor variant genomes. The ability to predict an emerging variant of SARS-CoV-2 in the global landscape may aid in the evaluation of medical countermeasures and non-pharmaceutical interventions

The Impact of Cytokines and HSV-1 on Rab5 Protein Expression, F-actin Cytoskeleton Rearrangement, and Cell Viability of Uninfected and Virus-Infected M0, M1, and M2 RAW264.7 Murine Macrophages

The endocytic pathway in all eukaryotic cells is necessary to maintain cellular functions, such as initiating transport of intracellular cargos and ingesting pathogens. The main regulator of this process is a member of small GTPase family, Rab5 protein. Rab5 protein plays a key role in the endocytic dynamic delivery of molecules, receptors, and pathogens from the cell membrane to cytoplasmic vesicles.as well as in the exocytic delivery of cellular products to the cell\u27s exterior (Bonifacino & Glick, 2004). Many pathogens have exploited this protein to enter cells. Herpes Simplex Virus Type 1 (HSV-1) enters most cells by fusion or utilizes the endocytic pathway using Rab5 protein (Spearman, 2017). Also, HSV-1 depends on Rab5 in the enveloping process to produce mature viral particles (Hollinshead, 2012). F-actin is a major microfilament of the cell\u27s cytoskeleton, aiding migration to the site of infection, muscles contraction, and cell division (Khadijeh, Amir, & Maryam, 2015). As a barrier, F-actin also protects the cellular organelles within the cytoplasm (Vitale, Seward, & Trifaro, 1995). The goals of this study were to determine the effect of the HSV-1 infection on Rab5 expression in RAW 264.7 murine macrophages and on F-actin distribution at 2, 4, 6, and 24 hours or late time at 24 hours after polarization of the macrophages to M1, M2a, and M2c phenotypes. M1 macrophages were polarized by interferon gamma (IFN-¿) and lipopolysaccharides (LPS). Unpolarized cells (M0) were converted to M2a phenotype by treating them with interleukin 4 (IL-4). M2c phenotype was polarized with interleukin 10 (IL-10). HSV-1 infection upregulated Rab5 protein expression at 2-6 hours in both M2a and M2c phenotypes but not in M1 polarized macrophages. The Impact of Cytokine Treatments and HSV-1 on Rab5 Protein Expression, F-actin Cytoskeleton Rearrangement, and Cell Viability of Uninfected and Virus- Infected M0, M1, and M2 RAW 264.7 Murine Macrophages. increase in Rab5 expression was seen at 24 hours after virus infection in any of the polarized macrophages but was seen in HSV-1 infected unpolarized M0 cells. Both M1 and M2 polarizing agents caused an upregulation in Rab5 expression from 2 to 6 hours after polarization. HSV-1 infection caused a decrease F-actin distribution (staining intensity) levels among test groups at most time points. Polarization caused a decrease in cell viability of M1 macrophage; HSV-1 infection did not enhance this decrease in cell viability after 24 hours. M2 phenotypes, uninfected or HSV-1-infected, did not exhibit any decrease in cell viability at 24 hours. Treatment of HSV-1 phenotypes with M2 polarizing anti-inflammatory cytokines, Il-4 and Il-10, as well as with SOCS3, an inducer of IL-10 expression, enhanced expression of Rab5 and F-actin distribution

Brain Tumor/Mass Classification Framework Using Magnetic-Resonance-Imaging-Based Isolated and Developed Transfer Deep-Learning Model

With the advancement in technology, machine learning can be applied to diagnose the mass/tumor in the brain using magnetic resonance imaging (MRI). This work proposes a novel developed transfer deep-learning model for the early diagnosis of brain tumors into their subclasses, such as pituitary, meningioma, and glioma. First, various layers of isolated convolutional-neural-network (CNN) models are built from scratch to check their performances for brain MRI images. Then, the 22-layer, binary-classification (tumor or no tumor) isolated-CNN model is re-utilized to re-adjust the neurons’ weights for classifying brain MRI images into tumor subclasses using the transfer-learning concept. As a result, the developed transfer-learned model has a high accuracy of 95.75% for the MRI images of the same MRI machine. Furthermore, the developed transfer-learned model has also been tested using the brain MRI images of another machine to validate its adaptability, general capability, and reliability for real-time application in the future. The results showed that the proposed model has a high accuracy of 96.89% for an unseen brain MRI dataset. Thus, the proposed deep-learning framework can help doctors and radiologists diagnose brain tumors early

Female Healthcare Workers’ Knowledge, Attitude towards Breast Cancer, and Perceived Barriers towards Mammogram Screening: A Multicenter Study in North Saudi Arabia

Breast cancer is the most commonly diagnosed cancer among women in the Kingdom of Saudi Arabia and other Middle East countries. This analytical cross-sectional study assessed knowledge, attitude towards breast cancer, and barriers to mammogram screening among 414 randomly selected female healthcare workers from multiple healthcare facilities in northern Saudi Arabia. Of the studied population, 48.6% had low knowledge, and 16.1% had a low attitude towards breast cancer risk factors and symptoms. The common barriers to mammogram screening were fear to discover cancer (57.2%) and apprehension regarding radiation exposure (57%). Logistic regression analysis found that lack of awareness regarding mammogram was significantly associated with age (p = 0.030) and healthcare workers category (ref: physicians: p = 0.016). In addition, we found a significant negative correlation between knowledge and barrier scores (Spearman’s rho: −0.315, p < 0.001). It is recommended to develop target-oriented educational programs for the healthcare workers, which would empower them to educate the community regarding the risk factors and the importance of mammogram screening. Furthermore, a prospective study is warranted in other regions of the Kingdom of Saudi Arabia to understand the region-specific training needs for the healthcare workers

Amplicon based MinION sequencing of SARS-CoV-2 and metagenomic characterisation of nasopharyngeal swabs from patients with COVID-19

COVID-19 is a complex disease phenotype where the underlying microbiome could influence morbidity and mortality. Amplicon and metagenomic MinION based sequencing was used to rapidly (within 8 hours) identify SARS-CoV-2 and assess the microbiome in nasopharyngeal swabs obtained from patients with COVID-19 by the ISARIC 4C consortium