A chromatin-dependent role of the fragile X mental retardation protein FMRP in the DNA damage response.

Fragile X syndrome, a common form of inherited intellectual disability, is caused by loss of the fragile X mental retardation protein FMRP. FMRP is present predominantly in the cytoplasm, where it regulates translation of proteins that are important for synaptic function. We identify FMRP as a chromatin-binding protein that functions in the DNA damage response (DDR). Specifically, we show that FMRP binds chromatin through its tandem Tudor (Agenet) domain in vitro and associates with chromatin in vivo. We also demonstrate that FMRP participates in the DDR in a chromatin-binding-dependent manner. The DDR machinery is known to play important roles in developmental processes such as gametogenesis. We show that FMRP occupies meiotic chromosomes and regulates the dynamics of the DDR machinery during mouse spermatogenesis. These findings suggest that nuclear FMRP regulates genomic stability at the chromatin interface and may impact gametogenesis and some developmental aspects of fragile X syndrome.

Whole genome sequencing of families diagnosed with cardiac channelopathies reveals structural variants missed by whole exome sequencing.

Cardiac channelopathies are a group of heritable disorders that affect the heart's electrical activity due to genetic variations present in genes coding for ion channels. With the advent of new sequencing technologies, molecular diagnosis of these disorders in patients has paved the way for early identification, therapeutic management and family screening. The objective of this retrospective study was to understand the efficacy of whole-genome sequencing in diagnosing patients with suspected cardiac channelopathies who were reported negative after whole exome sequencing and analysis. We employed a 3-tier analysis approach to identify nonsynonymous variations and loss-of-function variations missed by exome sequencing, and structural variations that are better resolved only by sequencing whole genomes. By performing whole genome sequencing and analyzing 25 exome-negative cardiac channelopathy patients, we identified 3 pathogenic variations. These include a heterozygous likely pathogenic nonsynonymous variation, CACNA1C:NM_000719:exon19:c.C2570G:p. P857R, which causes autosomal dominant long QT syndrome in the absence of Timothy syndrome, a heterozygous loss-of-function variation CASQ2:NM_001232.4:c.420+2T>C classified as pathogenic, and a 9.2 kb structural variation that spans exon 2 of the KCNQ1 gene, which is likely to cause Jervell-Lange-Nielssen syndrome. In addition, we also identified a loss-of-function variation and 16 structural variations of unknown significance (VUS). Further studies are required to elucidate the role of these identified VUS in gene regulation and decipher the underlying genetic and molecular mechanisms of these disorders. Our present study serves as a pilot for understanding the utility of WGS over clinical exomes in diagnosing cardiac channelopathy disorders.

Left atrial appendage closure using WATCHMAN device in chronic kidney disease and end-stage renal disease patients.

BACKGROUND: Chronic kidney disease (CKD) and end-stage renal disease are considered independent risk factors for developing atrial fibrillation (AF). Percutaneous occlusion of left atrial appendage (LAAC) using WATCHMAN device is a widely accepted alternative to anticoagulation therapy to prevent ischemic stroke in AF in patients who are not candidates for anticoagulation. There is limited data regarding the utilization and periprocedural safety of this intervention in patients with CKD/ESRD. METHODS: We retrospectively reviewed all hospitalizations from 2016 to 2017 with (ICD-10) procedure diagnosis code of LAA closure using WATCHMAN procedure with and without a secondary diagnosis of CKD/ESRD in acute-care hospitals across the United States using the national inpatient sample. Demographic variables (gender, race, income, hospital characteristics, medical comorbidities) were collected and compared. The primary outcomes were inpatient mortality, hospital length, and cost of stay. RESULTS: There were over 71 million discharges included in the combined 2016 and 2017 NIS database. Sixteen thousand five hundred five hospitalizations were for adult patients with a procedure code for LAA closure via watchman procedure. Of 16,505 patients, 3245 (19.66%) had CKD and ESRD. There was no statistically significant difference in mortality, length, and cost of stay in patients with and without CKD/ESRD. There were no statistically significant differences in periprocedural cerebrovascular accidents in both groups. CONCLUSION: Patients with and without ESRD/CKD who undergo LAA occlusion with Watchman have similar procedure related, in-hospital mortality, and complications.

Improving Postoperative Acute Kidney Injury Rates Following Primary Total Joint Arthroplasty.

BACKGROUND: Perioperative hip and knee arthroplasty complications remain a significant clinical and financial burden. Our institution has shifted to developing protocols to decrease these perioperative complications. This study focuses on acute kidney injury (AKI) rate status post primary total joint arthroplasty (TJA). Current literature demonstrates a 2%-15% incidence of AKI following TJA. However, there is a paucity of published literature on protocols that have effectively reduced AKI rates following TJA. The purpose of this study is to evaluate the effect that our institutionally developed perioperative renal protocol had on the postoperative AKI rates. METHODS: A retrospective cohort study was performed. Patient demographics, baseline creatinine, and postoperative creatinine values during the patient's hospitalization were collected and analyzed. The preintervention cohort data contained all patients at our institution who underwent a primary TJA from November 1, 2016 to January 1, 2018. The postintervention cohort included all primary TJA patients from July 1, 2018 to February 2, 2020. AKI was defined using the AKI Network classification system comparing baseline and postoperative creatinine values. A multivariate analysis was performed to determine the statistical significance of our results. RESULTS: Before intervention 1013 patients underwent a primary TJA with 68 patients developing an AKI postoperatively. After intervention 2169 patients underwent primary TJA with 90 patients developing an AKI (6.71% vs 4.15%; P = .0015, odds ratio = 0.59, 95% confidence interval = 0.42-0.82). CONCLUSION: This study demonstrated that implementation of a perioperative renal protocol can significantly reduce AKI rates. A reduction in AKI rates following TJA will result in improved outcomes and secondarily decrease the financial impact of postoperative complications seen following TJA.

Inappropriate sinus tachycardia: a review.

Inappropriate sinus tachycardia (IST) has been defined as a resting heart rate of >100 beats per minute and an average 24-hour heart rate >90 bpm with distressing symptoms resulting from the persistent tachycardia. IST is prevalent in 1% of the middle-aged population, mostly females. Rarely can elderly patients also present with IST. Possible mechanisms of IST include intrinsic sinus node abnormality, beta-adrenergic receptor stimulating autoantibody, beta-adrenergic receptor supersensitivity, muscarinic receptor autoantibody, or hyposensitivity, impaired baroreflex control, depressed efferent parasympathetic/vagal function, nociceptive stimulation, central autonomic overactivity, aberrant neurohumoral modulation, etc. Symptoms associated with IST are palpitations, chest pain, fatigue, shortness of breath, presyncope, and syncope. Despite these distressing symptoms, IST has not been associated with tachycardia-associated cardiomyopathy or increased major cardiovascular events. Various treatment options for patients with IST are ivabradine, beta-adrenergic blockers, calcium channel blockers, psychiatric evaluation, and exercise training. Although, endocardial radiofrequency ablation targeting the sinus node has been used as a treatment modality for otherwise treatment-refractory IST, the results have been dismal. The other modalities used for refractory IST treatment are endocardial modification of the sinus node using radiofrequency energy, combined endo and epicardial ablation of the sinus node, thoracoscopic epicardial ablation of the sinus node, sinus node sparing thoracoscopic and endocardial hybrid ablation. The goal of this review is to provide the readership with the pathophysiological basis of IST and its management options.

Management strategies for vasovagal syncope.

Vasovagal syncope (VVS) (or neurocardiogenic syncope) is a common clinical condition that is challenging to both physicians and patients alike. Severe episodes of recurrent syncope can result in physical injury as well as psychological distress. This article provides a brief overview of current management strategies and a detailed overview of therapeutic modalities such as closed loop stimulation (CLS) and cardioneuroablation (CNA).

An unusual case of Adrenocortical Adenocarcinoma with Biochemical Masquerade of Pheochromocytoma.

Adrenocortical carcinoma (ACC) is a rare malignancy that arises from the adrenal cortex and often presents as adrenal incidentaloma on abdominal scans with rise in the use of imaging modalities. ACC often presents as Cushing's syndrome or virilization. On the other hand, pheochromocytoma is an adrenal medullary tumor. It is rare for ACC to present as pheochromocytoma even though both may coexist. Moreover, ACC tumors have radiological and histological features suggestive of aggressive nature of the disease. We present a case of a 65-year-old lady who initially presented with a 3cm left adrenal incidentaloma. All of her adrenal hormones were in normal range. She was lost to follow up for several years and returned with a much enlarged lesion. Biochemical work up showed mildly increased catecholamines and metanephrines suggestive of pheochromocytoma. She didn't have any signs or symptoms of pheochromocytoma. She was treated with alpha blockers before surgery as a prophylactic measure. Surgical pathology was consistent with the diagnosis of primary adrenal adenocarcinoma. We recommend that adrenal incidentalomas should be followed annually for up to five years as per American association of Endocrinology and the Endocrine Society guidelines to prevent morbidity and mortality in patients.

Different risk factors are associated with alcohol use versus problematic use in male Pakistani adolescents.

Substance abuse is a significant problem in Pakistan (Hussain, 2017), yet, there are few studies of risk factors in this population, particularly in remote areas. Male adolescents (N = 243; M = 16.9 years, SD = 1.3 years) were recruited from schools in Gilgit-Baltistan, and completed questions on demographics, substance use, and psychosocial functioning. As a Muslim country, there are strict prohibitions against alcohol use, yet 44.9% of the sample reported lifetime alcohol use. The Alcohol Use Disorders Identification Test (Babor, Higgins-Biddle, Saunders, & Monteiro, 2001), was used to quantify level of risk associated with use, and 22.6% of the sample had scores indicating significant risk levels. Logistic regression analyses indicated that lifetime alcohol use was associated with internalising problems (e.g., depression, anxiety), while risky alcohol use was associated with externalising problems (e.g., aggressive and delinquent behaviour). Attention problems were related to both outcomes. Implications for prevention are discussed.

Avian Influenza A(H9N2) Virus in Poultry Worker, Pakistan, 2015.

Avian influenza A(H9N2) virus isolated from a poultry worker in Pakistan in 2015 was closely related to viruses detected in poultry farms. Observed mutations in the hemagglutinin related to receptor-binding affinity and antigenicity could affect cross-reactivity with prepandemic H9N2 vaccine strains.

Circulation of multiple subtypes (A, G and CRFs 02_AG) of human immunodeficiency virus type 1 (HIV-1) in selected districts of Punjab province, Pakistan.

Owing to consistent genetic mutation and recombination, various escape mutants and/or drug-resistant mutants of human immunodeficiency virus (HIV-1) are now emerging worldwide. Therefore, an understanding of the genetic characteristics of prevailing strains, particularly with regard to drug-resistance-associated substitutions, is essential for devising and implementing treatments and disease control interventions in endemic settings such as Pakistan. We processed a total of 130 plasma samples originating from HIV-treatment centers in selected districts of Punjab province, Pakistan. The samples were first screened using an HIV-1 Ag/Ab Combo test followed by amplification of the pol gene (1084 bp) from samples that were positive either for the antigen or for both the antigen and antibodies simultaneously. Screening revealed that a total of 45 samples were positive (34.62%; 95% CI: 26.99-43.13) for either antigen or both antigen and antibodies (n = 18, 40%; 95% CI: 27.02-54.55) or for antibodies alone (n = 27, 60%; 95% CI: 45.45-72.98). A largest number of positive samples was from the district of Lahore (n = 19/43, 44.18%; 95% CI: 30.44-58.9) followed by Faisalabad (n= 12/36, 33.33%; 95% CI: 20.21-49.66), Gujranwala (n = 05/23, 21.7%; 95% CI: 9.66-41.9) and Sargodha (n = 09/28, 32.1%; 95% CI: 17.93-50.66). The probability of occurrence of HIV infection was significantly associated with individuals having a history of injecting drug use (68.08%; OR = 11.15; 95% CI: 53.84-79.61, p = 0.0001). Phylogenetic analysis based on the pol gene showed that the sequences from this study clustered into three distinct clades representing recombinant form 02_AG (n = 14, 77.0%; 95% CI: 54.79-91.00), and subtypes A (n = 2, 11.1%; 95% CI: 3.1-32.8) and G (n = 2, 11.1%; 95% CI: 3.1-32.8). Although we screened 18 samples for drug-resistance-associated mutations, except for an accessory mutation (M46K) in the protease (PR) region in one subject, we found a lack of drug-resistance-associated substitutions in the PR region. On the other hand, we found two subjects (2/18) carrying a resistance-associated mutation (V106I) conferring a low level of resistance against non-nucleoside reverse transcriptase inhibitors. The present study shows that multiple subtypes of HIV-1 are present in the affected population. Continuous disease surveillance coupled with evaluation of drug resistance at higher resolution should be done in future studies.

Molecular Insights into Inhibition of the Methylated Histone-Plant Homeodomain Complexes by Calixarenes.

Plant homeodomain (PHD) finger-containing proteins are implicated in fundamental biological processes, including transcriptional activation and repression, DNA damage repair, cell differentiation, and survival. The PHD finger functions as an epigenetic reader that binds to posttranslationally modified or unmodified histone H3 tails, recruiting catalytic writers and erasers and other components of the epigenetic machinery to chromatin. Despite the critical role of the histone-PHD interaction in normal and pathological processes, selective inhibitors of this association have not been well developed. Here we demonstrate that macrocyclic calixarenes can disrupt binding of PHD fingers to methylated lysine 4 of histone H3 in vitro and in vivo. The inhibitory activity relies on differences in binding affinities of the PHD fingers for H3K4me and the methylation state of the histone ligand, whereas the composition of the aromatic H3K4me-binding site of the PHD fingers appears to have no effect. Our approach provides a novel tool for studying the biological roles of methyllysine readers in epigenetic signaling.

Dihydropyrimidine based hydrazine dihydrochloride derivatives as potent urease inhibitors.

Four series of heterocyclic compounds 4-dihydropyrimidine-2-thiones 7-12 (series A), N,S-dimethyl-dihydropyrimidines 13-18 (series B), hydrazine derivatives of dihydropyrimidine 19-24 (series C), and tetrazolo dihydropyrimidine derivatives 25-30 (series D), were synthesized and evaluated for in vitro urease inhibitory activity. The series B-D were first time examined for urease inhibition. Series A and C were found to be significantly active with IC50 values between 34.7-42.9 and 15.0-26.0 µM, respectively. The structure-activity relationship showed that the free S atom and hydrazine moiety are the key pharmacophores against urease enzyme. The kinetic studies of the active series A (7-12) and C (19-24) were carried out to determine their modes of inhibition and dissociation constants Ki. Compounds of series A (7-12) and series C (19-24) showed a mixed-type of inhibition with Ki values ranging between 15.76-25.66 and 14.63-29.42 µM, respectively. The molecular docking results showed that all the active compounds of both series have significant binding interactions with the active sites specially Ni-ion of the urease enzyme. Cytotoxicity of all series A-D was also evaluated against mammalian mouse fibroblast 3T3 cell lines, and no toxicity was observed in cellular model.

Molecular basis for chromatin binding and regulation of MLL5.

The human mixed-lineage leukemia 5 (MLL5) protein mediates hematopoietic cell homeostasis, cell cycle, and survival; however, the molecular basis underlying MLL5 activities remains unknown. Here, we show that MLL5 is recruited to gene-rich euchromatic regions via the interaction of its plant homeodomain finger with the histone mark H3K4me3. The 1.48-Å resolution crystal structure of MLL5 plant homeodomain in complex with the H3K4me3 peptide reveals a noncanonical binding mechanism, whereby K4me3 is recognized through a single aromatic residue and an aspartate. The binding induces a unique His-Asp swapping rearrangement mediated by a C-terminal α-helix. Phosphorylation of H3T3 and H3T6 abrogates the association with H3K4me3 in vitro and in vivo, releasing MLL5 from chromatin in mitosis. This regulatory switch is conserved in the Drosophila ortholog of MLL5, UpSET, and suggests the developmental control for targeting of H3K4me3. Together, our findings provide first insights into the molecular basis for the recruitment, exclusion, and regulation of MLL5 at chromatin.

Diverse functions of PHD fingers of the MLL/KMT2 subfamily.

Five members of the KMT2 family of lysine methyltransferases, originally named the mixed lineage leukemia (MLL1-5) proteins, regulate gene expression during embryogenesis and development. Each KMT2A-E contains a catalytic SET domain that methylates lysine 4 of histone H3, and one or several PHD fingers. Over the past few years a growing number of studies have uncovered diverse biological roles of the KMT2A-E PHD fingers, implicating them in binding to methylated histones and other nuclear proteins, and in mediating the E3 ligase activity and dimerization. Mutations in the PHD fingers or deletion of these modules are linked to human diseases including cancer and Kabuki syndrome. In this work, we summarize recently identified biological functions of the KMT2A-E PHD fingers, discuss mechanisms of their action, and examine preference of these domains for histone and non-histone ligands.

Low-cost design and fabrication of an anthropomorphic robotic hand.

Human hand signifies a magnificent and challenging example for scientists and engineers trying to replicate its complex structure and functionality. This paper proposes a bio-mechatronic approach for the design of an anthropomorphic artificial hand capable of performing basic human hand motions with fundamental gripping functionality. The dexterity of the artificial hand is exhibited by imitating the natural motion of the human fingers. Imitation is produced according to the data acquired from the flex sensors attached to the human fingers. In order to have proper gripping, closed-loop control is implemented using the tactile sensors. Feedback for the closed-loop control is provided by force sensing resistors (FSRs), attached on the fingertips of the robotic hand. These sensors also enable handling of fragile objects. The mathematical model is derived using forward kinematics and also simulated on MATLAB to ascertain the position of robotic fingers in 3D space.

Performance predication of a solar assisted desiccant air conditioning system using radial basis function neural network: An integrated machine learning approach.

In this Paper solar desiccant air conditioning system integrated with cross flow Maisotsenko cycle (M-cycle) indirect evaporative cooler is used to investigate the performance of whole system in different range of parameters. Solar evacuated tube electric heater is used to supply the regeneration temperature to the desiccant wheel, whereas, Desiccant Wheel (DW) and M-cycle is used to handle latent load and sensible load separately. Major contribution of this research is to predict system level performance parameters of a Solar Assisted Desiccant Air Conditioning (Sol-DAC) system using Radial Basis Function Neural Network (RBF-NN) under real transient experimental inlet conditions. Nine parameters are mainly considered as input parameters to train the RBF-NN model, which are, supply Air temperature at the process side of desiccant wheel, supply air humidity ratio at process side of the desiccant wheel, outlet temperature from the desiccant wheel at process side, outlet humidity ratio from the desiccant wheel at process side, regeneration temperature at regeneration side of the DW, outlet temperature from the heat recovery wheel at process side, outlet humidity ratio out from the Heat Recovery Wheel (HRW) at process side, temperature before heat recovery wheel regeneration side of the system, humidity ratio before heat recovery wheel regeneration side of the system. Four parameters are considered as the output of the RBF-NN model, namely: output temperature, output humidity, Cooling Capacity (CC), and Coefficient of Performance (COP). The results of the RBF-NN model shows that the best Mean Squared Error (MSE) and Regression coefficient (R) for outlet temperature prediction are 0.00998279 and 0.99832 when regeneration temperature is 70 °C and inlet humidity at 18 g/kg. Best MSE and R for predication of outlet humidity are 0.0102932 and 0.99485 when the regeneration temperature is 70 °C and inlet humidity at 16 g/kg. Best MSE and R for predication of COP are 0.0106691 and 0.9981 when the regeneration temperature is 70 °C and inlet humidity 12 g/kg. Best MSE and R for predication of CC are 0.0144943 and 0.99711 when the regeneration temperature is 70 °C and inlet humidity 14 g/kg. Experimental and predicted performance parameters were in close agreement and showed minimal deviation. Investigations of predicted results revealed that trained RBF-NN model was capable of predicting the trend of output result under the varying input condition.

Mapping Genetic Markers Associated with Antigenicity and Host Range in H9N2 Influenza A Viruses Infecting Poultry in Pakistan.

The aim of the current study was to map the genetic diversity in the haemagglutinin (HA) glycoprotein of influenza A viruses (IAVs) of the H9N2 subtype. Twenty-five H9N2 IAVs were isolated from broiler chickens from March to July 2019. The HA gene was amplified, and phylogenetic analysis was performed to determine the evolutionary relationship. Important antigenic amino acid residues of HA attributed to immune escape and zoonotic potential were compared among H9N2 IAVs. Phylogenetic analysis revealed that sublineage B2 under the G1 lineage in Pakistan was found to be diversified, and newly sequenced H9N2 isolates were nested into two clades (A and B). Mutations linked to the antigenic variation and potential immune escape were observed as G72E (1/25, 4%), A180T (3/25, 12%), and A180V (1/25, 4%). A twofold significant reduction (P < 0.01) in log2 hemagglutination inhibition titers was observed with H9N2 IAV naturally harboring amino acid V180 instead of A180 in HA protein. Moreover, in the last 20 years, complete substitution at residues (T127D, D135N, and L150N) and partial substitution at residues (72, 74, 131, 148, 180, 183, 188, 216, 217, and 249, mature H9 HA numbering) associated with changes in antigenicity were observed. The presence of L216 in all H9N2 IAV isolates and T/V180 in four isolates in the receptor-binding site reveals the potential of these viruses to cross the species barrier to infect human or mammals. The current study observed the circulation of antigenically diverse H9N2 IAV variants that possess potential mutations that can escape the host immune system.

Nota de investigación- Mapeo de marcadores genéticos asociados con la antigenicidad y el rango de huéspedes en los virus de la influenza tipo A subtipo H9N2 que infectan a la avicultura en Pakistán. El objetivo del presente estudio fue mapear la diversidad genética en la glicoproteína hemaglutinina (HA) de los virus de la influenza A (IAV) del subtipo H9N2. Se aislaron veinticinco virus de influenza H9N2 de pollos de engorde de marzo a julio del 2019. Se amplificó el gene HA y se realizó un análisis filogenético para determinar la relación evolutiva. Se compararon importantes residuos de aminoácidos antigénicos de la hemaglutinina atribuidos al escape inmunológico y al potencial zoonótico entre los virus de la influenza aviar H9N2. El análisis filogenético reveló que el sublinaje B2 bajo el linaje G1 en Pakistán estaba diversificado, y los aislados de H9N2 recién secuenciados se agruparon en dos clados (A y B). Se observaron mutaciones relacionadas con la variación antigénica y el posible escape inmunológico como los residuos de aminoácidos G72E (1/25, 4%), A180T (3/25, 12%) y A180V (1/25, 4%). Se observó una reducción significativa al doble (P < 0.01) en los títulos de inhibición de la hemaglutinación log2 cuando el virus de la influenza aviar H9N2 albergaba naturalmente el aminoácido V180 en lugar del A180 en la proteína HA. Además, en los últimos 20 años, sustitución completa en los residuos (T127D, D135N y L150N) y sustitución parcial en los residuos (72, 74, 131, 148, 180, 183, 188, 216, 217 y 249, de acuerdo con la numeración de la HA subtipo madura) asociados con cambios en la antigenicidad. La presencia del residuo L216 en todos los aislados de influenza aviar H9N2 y T/V180 en cuatro aislados en el sitio de unión al receptor revela el potencial de estos virus para cruzar la barrera de las especies para infectar a humanos o mamíferos. El estudio actual observó la circulación de variantes antigénicamente diversas del virus de influenza aviar H9N2 que poseen mutaciones potenciales que pueden escapar del sistema inmunológico del huésped.

The Practicality of the Robotic Total Hip Arthroplasty for the Treatment of Complex Bilateral Adult Hip Dysplasia. Technology Makes It Easy.

Adult hip dysplasia provides many challenges for joint surgeons. Due to the abnormal bone morphology and altered biomechanics of the hip, surgeons must ensure accurate implant positioning to avoid postoperative complications. We present a 56-year-old female with a history of bilateral Legg-Calve-Perthes disease and subsequent dysplasia who underwent bilateral total hip arthroplasty using robotic navigation. We highlight the utility of robotic navigation in adult hip dysplasia to improve implant positioning and ensure optimal patient outcomes.

Early Superior Clinical Outcomes in Robotic-Assisted TKA Compared to Conventional TKA in the Same Patient: A Comparative Analysis.

Robotic-assisted total knee arthroplasty (RA-TKA) has demonstrated improved alignment and outcome scores when compared with manual total knee arthroplasty (M-TKA); however, few studies compare differences in the same patient. This study is a retrospective review that assesses clinical outcomes of 36 patients who underwent a primary RA-TKA and had undergone a prior contralateral M-TKA. All surgeries were performed by a single surgeon at the same institution. Patients were assessed for differences in hospital length of stay, improvement in pre- versus postoperative range of motion, Knee Injury and Osteoarthritis Outcome Score (KOOS), and Western Ontario and McMaster Universities Arthritis Index (WOMAC) scores. Student's t-test and Fisher's exact test were utilized to detect significant differences. Patient demographics showed a mean age of 64.5, 24 females (67%), and mean body mass index of 35.1 ± 5.7. The average follow-up time was 2.9 years for M-TKA and 1.3 years for RA-TKA. Hospital length of stay was decreased by 5.5 hours for RA-TKA (p = 0.03). Total postoperative WOMAC score was not statistically different between RA-TKA and M-TKA (p = 0.061); however, pain and stiffness components were statistically improved in RA-TKA (p = 0.041 and p = 0.007), respectively. KOOS was higher in RA-TKA, which approached statistical significance (p = 0.005). Pre- versus postoperative knee flexion improved significantly in both cohorts. There was a significant difference in pre- versus postoperative range of motion at 3, 6, and 12 months follow-up after RA-TKA in comparison to M-TKA (p < 0.05). There were no postoperative complications. Patients who underwent RA-TKA demonstrated early improvement at 1-year follow-up in pain, stiffness, and knee flexion when compared with their prior contralateral M-TKA. There was a significant decrease in postoperative length of stay by 5.5 hours in the RA-TKA group. Limitations include a small sample size and differences in follow-up times between RA-TKA and M-TKA.

Molecular Characterization and Selection of Indigenous SARS-CoV-2 Delta Variant for the Development of the First Inactivated SARS-CoV-2 Vaccine of Pakistan.

Vaccines are one of the efficient means available so far for preventing and controlling the infection rate of COVID-19. Several researchers have focused on the whole virus's (SARS-CoV-2) inactivated vaccines which are economically efficient to produce. In Pakistan, multiple variants of SARS-CoV-2 have been reported since the start of the pandemic in February 2020. Due to the continuous evolution of the virus and economic recessions, the present study was designed to develop an indigenous inactivated SARS-CoV-2 vaccine that might help not only to prevent the COVID-19 in Pakistan, it will also save the country's economic resources. The SARS-CoV-2 were isolated and characterized using the Vero-E6 cell culture system. The seed selection was carried out using cross-neutralization assay and phylogenetic analysis. The selected isolate of SARS-CoV-2 (hCoV-19/Pakistan/UHSPK3-UVAS268/2021) was inactivated using beta-propiolactone followed by vaccine formulation using Alum adjuvant, keeping the S protein concentration as 5 µg/dose. The vaccine efficacy was evaluated by in vivo immunogenicity testing in laboratory animals and in in vitro microneutralization test. The phylogenetic analysis revealed that all the SARS-CoV-2 isolates reported from Pakistan nested into different clades, representing multiple introductions of the virus into Pakistan. The antisera raised against various isolates from different waves in Pakistan showed a varied level of neutralization titers. However, the antisera produced against a variant (hCoV-19/Pakistan/UHSPK3-UVAS268/2021; fourth wave) efficiently neutralized (1:64-1:512) all the tested SARS-CoV-2 isolates. The inactivated whole virus vaccine of SARS-CoV-2 was safe and it also elicited a protective immune response in rabbits and rhesus macaques on the 35th-day post-vaccination. The activity of neutralizing antibodies of vaccinated animals was found at 1:256-1:1024 at 35 days post-vaccination, indicating the effectiveness of the double-dose regime of the indigenous SARS-CoV-2 vaccine.