Structural-Dynamics and Binding Analysis of RBD from SARS-CoV-2 Variants of Concern (VOCs) and GRP78 Receptor Revealed Basis for Higher Infectivity.

Glucose-regulated protein 78 (GRP78) might be a receptor for SARS-CoV-2 to bind and enter the host cell. Recently reported mutations in the spike glycoprotein unique to the receptor-binding domain (RBD) of different variants might increase the binding and pathogenesis. However, it is still not known how these mutations affect the binding of RBD to GRP78. The current study provides a structural basis for the binding of GRP78 to the different variants, i.e., B.1.1.7, B.1.351, B.1.617, and P.1 (spike RBD), of SARS-CoV-2 using a biomolecular simulation approach. Docking results showed that the new variants bound stronger than the wild-type, which was further confirmed through the free energy calculation results. All-atom simulation confirmed structural stability, which was consistent with previous results by following the global stability trend. We concluded that the increased binding affinity of the B.1.1.7, B.1.351, and P.1 variants was due to a variation in the bonding network that helped the virus induce a higher infectivity and disease severity. Consequently, we reported that the aforementioned new variants use GRP78 as an alternate receptor to enhance their seriousness.

Robust Three-Dimensional (3D) Expansion of Bovine Intestinal Organoids: An In Vitro Model as a Potential Alternative to an In Vivo System.

Intestinal organoids offer great promise for disease-modelling-based host-pathogen interactions and nutritional research for feed efficiency measurement in livestock and regenerative medicine for therapeutic purposes. However, very limited studies are available on the functional characterisation and three-dimensional (3D) expansion of adult stem cells in livestock species compared to other species. Intestinal crypts derived from intestinal organoids under a 3D culture system from the small intestine in adult bovine were successfully established and characterised for functionality testing, including the cellular potentials and genetic properties based on immunohistochemistry, immunocytochemistry, epithelial barrier permeability assay, QuantSeq 3' mRNA-Seq. data and quantitative reverse transcription-polymerase chain reaction. Intestinal organoids were long-term cultivated over several passages of culture without loss of the recapitulating capacity of crypts, and they had the specific expression of several specific markers involved in intestinal stem cells, intestinal epithelium, and nutrient absorption. In addition, they showed the key functionality with regard to a high permeability for compounds of up to FITC-dextran 4 kDa, while FITC-dextran 40 kDa failed to enter the organoid lumen and revealed that the genetic properties of bovine intestinal organoids were highly similar to those of in vivo. Collectively, these results provide a reliable method for efficient isolation of intestinal crypts from the small intestine and robust 3D expansion of intestinal organoids in adult bovine and demonstrate the in vitro 3D organoids mimics the in vivo tissue topology and functionality. Finally, intestinal organoids are potential alternatives to in vivo systems and will be facilitated as the practical model to replace animal experiments for various purposes in the fields of animal biotechnology.

The SARS-CoV-2 B.1.618 variant slightly alters the spike RBD-ACE2 binding affinity and is an antibody escaping variant: a computational structural perspective.

Continuing reports of new SARS-CoV-2 variants have caused worldwide concern and created a challenging situation for clinicians. The recently reported variant B.1.618, which possesses the E484K mutation specific to the receptor-binding domain (RBD), as well as two deletions of Tyr145 and His146 at the N-terminal binding domain (NTD) of the spike protein, must be studied in depth to devise new therapeutic options. Structural variants reported in the RBD and NTD may play essential roles in the increased pathogenicity of this SARS-CoV-2 new variant. We explored the binding differences and structural-dynamic features of the B.1.618 variant using structural and biomolecular simulation approaches. Our results revealed that the E484K mutation in the RBD slightly altered the binding affinity through affecting the hydrogen bonding network. We also observed that the flexibility of three important loops in the RBD required for binding was increased, which may improve the conformational optimization and consequently binding of the new variant. Furthermore, we found that deletions of Tyr145 and His146 at the NTD reduced the binding affinity of the monoclonal antibody (mAb) 4A8, and that the hydrogen bonding network was significantly affected consequently. This data show that the new B.1.618 variant is an antibody-escaping variant with slightly altered ACE2-RBD affinity. Moreover, we provide insights into the binding and structural-dynamics changes resulting from novel mutations in the RBD and NTD. Our results suggest the need for further in vitro and in vivo studies that will facilitate the development of possible therapies for new variants such as B.1.618.

Differential impact of some metal(loid)s on oxidative stress, antioxidant system, sulfur compounds, and protein profile of Indian mustard (Brassica juncea L.).

Levels of arsenic (As), chromium (Cr), and copper (Cu) are increasing in the soils worldwide. Such contaminants cause toxicity in the plant systems which adversely affects growth and productivity. The objective of the present investigation was to elucidate individual and combined effects of As, Cr, and Cu (100 µM each) stress in metal hyper-accumulator plant Indian mustard (Brassica juncea L.), exposed for a week. The highest accumulation was in the roots and in decreasing order viz. Cu > As > Cr. The magnitude of oxidative stress was maximal in combined stress, followed by As, Cr, and then Cu stress. Glutathione in conjunction with glutathione reductase, glutathione peroxidase, and glutathione S-transferase increased in all set of stress treatments, notably when exposed to Cr alone. In addition, the level of sulfur-rich compounds like cysteine, phytochelatins, and non-protein thiols increased under each stress indicating efficient coupling of the enzyme system and sulfur-containing compounds during stress conditions. The highest tolerance or growth index of plants was recorded for Cu. Protein profiling of leaf tissues showed modulation of protein patterns in each stress. Mediator of RNA polymerase II transcription subunit 1 isoform X1, RuBisCO (large subunit), and ribosomal protein S3 proteins were more abundant under Cr and Cu stress. Zinc finger A20/AN1 domain-containing stress-associated protein 5-like protein was more abundant under Cu stress. HSP (15.7 kDa) and autophagy protein 5-like were in higher abundance under As and combined stress. Our results suggest that Indian mustard has a differential mode of defense against a particular stressor at the level of protein expression profile.

Prevalence, predictors and outcomes of potential drug-drug interactions in left ventricular failure: considerable factors for quality use of medicines

Hospitalized patients with left ventricular failure (LVF) are at high risk for potential drug-drug interactions (pDDIs) and its related adverse effects owing to multiple risk factors such as old age, comorbidities and polypharmacy. This cross-sectional study conducted in two tertiary care hospitals aim to identify frequency, levels and predictors of pDDIs in LVF patients. Data about patients' demographic, hospital stay, medication therapy, sign/symptoms and laboratory test results were collected for 385 patients with LVF. Micromedex Drug-Reax® was used to screen patients' medication profiles for pDDIs. Overall prevalence and severity-wise prevalence of pDDIs were identified. Chi-square test was performed for comparative analysis of various variables. Logistic regression was applied to determine the odds-ratios (OR) for predictors of pDDIs. The prevalence of pDDIs was 96.4% (n=371). Overall 335 drug-interacting pairs were detected, which were presented in a total of 2870 pDDIs. Majority of pDDIs were of major- (48.9%) and moderate-severity (47.5%). Logistic regression analysis shows significant association of >6 all types of pDDIs with >12 drugs as compared with <8 drugs (OR=16.5; p=<0.001). Likewise, there was a significant association of >4 major-pDDIs with men as compared with female (OR=1.9; p=0.007) and >12 drugs as compared with <8 drugs (OR=10.9; p=<0.001). Hypotension (n=57), impaired renal function (23) and increased blood pressure (22) were the most frequent adverse outcomes associated with pDDIs. This study shows high prevalence of pDDIs in LVF patients. Majority of pDDIs were of major- and moderate-severity. Male patients and those prescribed greater number of medicines were more exposed to major-pDDIs

Potential drug-drug interactions in outpatient department of a tertiary care hospital in Pakistan: a cross-sectional study.

BACKGROUND: Potential drug-drug interactions (pDDIs) are one of the preventable drug related problems having the risk of serious adverse events or therapeutic failure. In developing countries like Pakistan, this issue remains poorly addressed. The objective of this study was to explore prevalence of pDDIs in the Outpatient Department (OPD) of a tertiary care hospital in Pakistan. The secondary aim was to describe the levels of reported pDDIs and develop a list of widespread clinically relevant interactions. METHODS: Prescriptions of 2400 OPD patients were analyzed for pDDIs through Micromedex Drug-Reax®. Prevalence, severity- and documentation-levels and widespread clinically relevant interactions were reported. RESULTS: Of total 2400 prescriptions, pDDIs were present in 22.3%. Whereas, moderate- and major-pDDIs were found in 377 (15.7%) and 225 (9.4%), respectively. PDDIs were more prevalent in Medicine (9.2%) and Cardiology (2.6%) as compared with other OPD specialties. Total 942 pDDIs were identified, of which, the majority were either moderate- (61.9%) or major-pDDIs (32.1%). Some of the most common interactions were ibuprofen + levofloxacin (n = 50), ciprofloxacin + diclofenac (32), aspirin + atenolol (24), and diclofenac + levofloxacin (19). The potential adverse outcomes of widespread interactions were seizures, bleeding, QT-interval prolongation, arrhythmias, tendon rupture, hypoglycemia/hyperglycemia, serotonin syndrome, drug toxicity, and decreased therapeutic response. CONCLUSIONS: OPD patients were at risk to pDDIs, particularly to major- and moderate-pDDIs. Screening of prescriptions for pDDIs and monitoring of pharmacotherapy in terms of response and associated adverse drug events will contribute to patient safety.

Potential drug-drug interactions in pediatric patients admitted to intensive care unit of Khyber Teaching Hospital, Peshawar, Pakistan: A cross-sectional study.

PURPOSE: To investigate frequencies, levels, clinical relevance and predictors of potential drug-drug interactions (pDDIs) in pediatric intensive care unit (PICU). METHODS: Case notes of 411 patients were reviewed for pDDIs through Micromedex. Frequencies, levels and clinical relevance of pDDIs were reported. Logistic regression was applied to calculate the odds-ratios for predictors of pDDIs. RESULTS: We recorded pDDIs in 59.4% patients. Major-pDDIs were found in 34.5% patients. Total 990 pDDIs were identified, of which, 37.8% were of moderate-severity and 30.6% of major-severity. Patient's case notes of top-ten pDDIs showed presence of signs/symptoms such as fever, jaundice, vomiting, anorexia, tachycardia, drowsiness, & lethargy; and abnormalities in labs such as total leukocytes count, blood urea nitrogen, alanine aminotransferase, & potassium-level. Odds of exposure to major-pDDIs were significantly higher in patients aged 6-12years (p=0.008); hospital stay of ≥7days (p=0.05); and ≥11 prescribed medicines (p<0.001). CONCLUSION: Substantial numbers of patients in PICU are exposed to pDDIs. Major-pDDIs are of particular concern. Timely identification of pDDIs, preferably with computerized source, is crucial point for their management. Monitoring of clinically relevant parameters and identification of various predictors are needed to minimize or prevent the associated negative consequences of pDDIs.

Potential drug-drug interactions in medical intensive care unit of a tertiary care hospital in Pakistan.

Background Patients admitted to intensive care unit (ICU) present with severe and life-threatening illnesses. Most of them suffer from various comorbidities. They usually receive complex pharmacotherapy with large number of medicines which increase the risk of drug-drug interactions (DDIs). Objective The present report aimed to investigate prevalence and levels of potential DDIs (pDDIs) in medical ICU. Methods Medications profiles of 416 patients were checked for pDDIs using Micromedex Drug-Reax(®). Prevalence, levels of severity and levels of documentation were reported. Results Of total 416 patients, 310 were exposed to pDDIs (overall prevalence = 74.5 %). Likewise, a prevalence rate of 13.9 % was recorded for contraindicated pDDIs, 52.2 % for major pDDI and 58.4 % for moderate pDDI. This study reported 740 interacting drug pairs that were presented in total 1686 pDDIs. Of 1686 pDDIs, 4.3 % were of contraindicated severity, 33.8 % of major severity and 49.6 % of moderate severity, whereas 45.5 % were of fair scientific evidence and 41.4 % of good scientific evidence. Conclusion In this study, pDDIs were found highly prevalent in ICU patients at a rate of 74.5 %. Most of the pDDIs had moderate severity; however, substantial number of interactions (38.1 %) had major and contraindicated severity.

Schools as potential vaccination venue for vaccines outside regular EPI schedule: results from a school census in Pakistan.

BACKGROUND: Vaccines are the most effective public health intervention. Expanded Program on Immunization (EPI) provides routine vaccination in developing countries. However, vaccines that cannot be given in EPI schedule such as typhoid fever vaccine need alternative venues. In areas where school enrolment is high, schools provide a cost effective opportunity for vaccination. Prior to start of a school-based typhoid vaccination program, interviews were conducted with staff of educational institutions in two townships of Karachi, Pakistan to collect baseline information about the school system and to plan a typhoid vaccination program. Data collection teams administered a structured questionnaire to all schools in the two townships. The administrative staff was requested information on school fee, class enrolment, past history of involvement and willingness of parents to participate in a vaccination campaign. RESULTS: A total of 304,836 students were enrolled in 1,096 public, private, and religious schools (Madrasahs) of the two towns. Five percent of schools refused to participate in the school census. Twenty-five percent of schools had a total enrolment of less than 100 students whereas 3% had more than 1,000 students. Health education programs were available in less than 8% of public schools, 17% of private schools, and 14% of Madrasahs. One-quarter of public schools, 41% of private schools, and 43% of Madrasahs had previously participated in a school-based vaccination campaign. The most common vaccination campaign in which schools participated was Polio eradication program. Cost of the vaccine, side effects, and parents' lack of information were highlighted as important limiting factors by school administration for school-based immunization programs. Permission from parents, appropriateness of vaccine-related information, and involvement of teachers were considered as important factors to improve participation. CONCLUSIONS: Health education programs are not part of the regular school curriculum in developing countries including Pakistan. Many schools in the targeted townships participated in immunization activities but they were not carried out regularly. In the wake of low immunization coverage in Pakistan, schools can be used as a potential venue not only for non-EPI vaccines, but for a catch up vaccination of routine vaccines.

A study of micronutrient status in pregnancy.

The present study was performed, to analyse the inter-relationship among trace elements: Iron, copper and zinc in pregnancy. Eighty-four pregnant women were compared with 30 age matched nonpregnant healthy controls. Fasting blood samples were analysed for heamoglobin, iron, copper and zinc. On the basis of Hb concentration (<10g/dl) and iron levels(<50microg/dl), the pregnant women were sub-grouped as anaemic, non-anaemic, Iron deficient anaemic and non-iron deficient anaemic. The level of copper was found to be significantly higher in iron deficiency anaemia, when compared to non-iron deficiency anaemia (p<0.05), and in non-anaemic pregnant women, compared to non-anaemic non-pregnant women (controls).The level of zinc is also significantly lower in Iron deficiency anaemic pregnancy, when compared to the other groups. There is evidence of influence of pregnancy, on the level of trace elements in blood. This could be a result of competitive inhibition in the absorption of trace elements in the intestine, or an effect of hormonal changes (insulin, oestrogen), during pregnancy. A judicious supplementation of micronutrients, during pregnancy, especially in iron deficiency anaemia, is essential.

Pseudoxanthoma elasticum and nephrolithiasis: a rare association.

Pseudoxanthoma elasticum (PXE) is a multi-systemic, genetic disorder of connective tissue characterized by progressive calcification and fragmentation of elastic fibers in skin, retina and cardiovascular system. A young patient reported to urologist in emergency with azotemia secondary to acute bilateral obstructive uropathy. Upon successful management, he was found to have full-blown skin and eye manifestations and was evaluated thoroughly regarding extent and potential systemic complications of Pseudoxanthoma elasticum.