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Background and Objective: Pakistan has witnessed a dramatic change in the increasing prevalence and emergence of HIV subtypes for more than two decades. Pakistani population is increasingly engaged in high-risk practices, and the prescribed drugs are potentially causing resistance. There are chances that these resistant strains are beginning to circulate from high-risk to the general population. Methods: The study was conducted at the section of Molecular Pathology Lab of Dow Diagnostic and Research Laboratory, Dow University of Health Sciences (DUHS) Karachi. In this study, we analyzed gene sequences of HIV for drug resistance and molecular epidemiology., along with amino acid sequence variability. Furthermore, we undertook phylogenetic analysis for possible geographic linkages of Pakistani HIV strains. Results: Our results demonstrate that A1 is the leading HIV subtype circulating in the country, whereas other emerging subtypes and recombinant forms, including subtype B, CRF02_AG, CRF10_CD CRF35_AD, and CRF11_cpx were also observed. Our sequences cluster with the Middle East, African, and a few European sequences according to geographical distribution. These sequences showed high-level resistance per drug resistance pattern, with 62.5% of patients exhibiting resistance to NNRTI drugs and 60% mutation at E138A and K103N, respectively, against NNRTI drugs. About 75% sequences showed resistance mutation at M184V against NRTI drugs. The antiretroviral drugs are now causing H-LR to the patients with no effect. Our results also revealed that certain regions of RT exhibited high sequence variability, especially at Amino Acids positions p.119, p.130, p.157, p.164. Conclusion: We hereby report major novel mutations and several minor mutations that may have a drastic change in the drug resistance pattern.

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New-onset diabetes mellitus (NODM) is a common long-term complication after liver transplantation (LT). It is thought to be drug-induced in most cases, no matter the underlying disease that cause liver failure and indicated transplantation. Standard post-transplantation (PT) immunosuppressive regimens include prolonged use of calcineurin inhibitors (CNIs), namely tacrolimus (TAC), alongside corticosteroids to avoid acute and chronic graft rejection. This combination is well known for its diabetogenicity. Significant differences between the applied regimens stand out concerning the duration and dosages to prevent the metabolic side effects of these drugs in the long run without compromising the graft's survival. Studies were collected after an extensive research of PubMed database for this very specific topic using the following MeSH keywords in multiple combinations: "Liver Transplantation," "Diabetes Mellitus," "NODM," "Tacrolimus," "Cyclosporine A," and "Steroids." In addition, we used the same keywords for regular searches in Google Scholar. Only the relevant English human studies between 2010 and 2020 were collected except for review articles. Duplicates were eliminated using Mendeley software. Twelve relevant studies directly related to the targeted topic were collected and discussed, including five retrospective cohorts, four prospective cohorts, one clinical trial, one prospective pilot, and one case report. Their topics included primarily the factors increasing the risk of new-onset diabetes mellitus after liver transplantation (NODALT), TAC-based immunosuppression and its relative blood levels affecting the possible development of NODALT, the role of cyclosporine in substituting TAC regimen, and the effect of different steroids-avoiding protocols on the prevention of NODALT. The reviewed studies suggested that lowering the serum concentration of tacrolimus (cTAC) throughout the PT period and eliminating the corticosteroids regimen as early as possible, among other measures, can significantly impact the rate of emergence of NODM. This traditional review tackles the most recent studies about NODALT to establish a comprehensive view on this issue and guide clinicians and researchers for the safest immunosuppressive regimen to date, while maintaining a balanced metabolic profile.

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BACKGROUND: Quercetin, a well-known naturally occurring polyphenol, has recently been shown by molecular docking, in vitro and in vivo studies to be a possible anti-COVID-19 candidate. Quercetin has strong antioxidant, anti-inflammatory, immunomodulatory, and antiviral properties, and it is characterized by a very high safety profile, exerted in animals and in humans. Like most other polyphenols, quercetin shows a very low rate of oral absorption and its clinical use is considered by most of modest utility. Quercetin in a delivery-food grade system with sunflower phospholipids (Quercetin Phytosome®, QP) increases its oral absorption up to 20-fold. METHODS: In the present prospective, randomized, controlled, and open-label study, a daily dose of 1000 mg of QP was investigated for 30 days in 152 COVID-19 outpatients to disclose its adjuvant effect in treating the early symptoms and in preventing the severe outcomes of the disease. RESULTS: The results revealed a reduction in frequency and length of hospitalization, in need of non-invasive oxygen therapy, in progression to intensive care units and in number of deaths. The results also confirmed the very high safety profile of quercetin and suggested possible anti-fatigue and pro-appetite properties. CONCLUSION: QP is a safe agent and in combination with standard care, when used in early stage of viral infection, could aid in improving the early symptoms and help in preventing the severity of COVID-19 disease. It is suggested that a double-blind, placebo-controlled study should be urgently carried out to confirm the results of our study.

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Late presentation and resistance to chemotherapeutic agents make a deadly combination for ovarian cancer patients. The treatment of these patients is thus challenging. This study explores the possible molecular mechanisms by which tumor cells interact with the extracellular matrix (ECM) constituents, forming metastatic implants and enhancing patients' sensitivity to drugs. For the literature review, PubMed was used as a database. The standard search was done using keywords "collagen, ovarian cancer, extracellular matrix, drug resistance" in different combinations, which finally yielded 32 studies meeting the inclusion/exclusion criteria. The studies included were published in the English language in the past seven years. After analyzing, we found all of them to be histopathological studies. Nine studies also used murine cell lines besides human cell lines and tissue samples from ovarian cancer patients. One study has a retrospective analysis done. Eight studies demonstrate the role of hypoxia and matrix remodeling enzymes in ovarian cancer dissemination. Genetics playing a crucial role in cancer metastasis is demonstrated in eight studies. Ten studies included shows receptors, enzymes, and spheroid organization in disease progression. Six studies address chemotherapeutic resistance. Intraperitoneal dissemination of ovarian cancer and the development of chemotherapeutic resistance depends on certain molecular interactions, and they can be targeted to improve patients' overall survival.

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Attention-deficit hyperactivity disorder (ADHD) is a neuropsychological disorder that causes inattentiveness, hyperactivity, and impulsiveness in patients. Ventral striatal hypo-responsiveness, orbitofrontal cortex, and dopaminergic status in the brain are related to the pathogenesis of ADHD. Reinforcement tasks by monetary incentive delay (MID) was shown to produce more responsiveness in patients. In this study, we reviewed how reinforcement interventions and compensatory mechanisms affect the behavior of ADHD patients. This systematic review was undertaken as per the Preferred Reporting Items for Systematic Review and Meta-Analysis guidelines, and PubMed database was used for literature search. The quality appraisal was completed using the Newcastle-Ottawa scale, and nine case-control studies were included in this systematic review. A total of 976 participants were included, with 493 cases and 330 controls. The studies included discuss reinforcement, attention networks, and compensatory mechanisms. Our review concludes that reinforcement improves responsiveness to gain and loss of rewards in ADHD patients. Reward processing is selectively associated with the salience network. While ADHD, predominantly the inattentive type, is insensitive to stimuli, ADHD combined type and controls showed similar responsiveness. The right visual cortex may also be related to compensatory mechanisms in ADHD. As we only included case-control studies from the last eight years, in the English language, we might have missed some relevant studies related to this research. Because the included studies have a relatively small sample size, we recommend future studies to explore larger cohorts of patients to improve the reliability of findings pertinent to this field.

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Financial, cultural, and managerial hurdles have made biosafety and biosecurity measures difficult in resource-constrained countries like Pakistan. Because of increasing awareness of biorisk management, diagnostic and research laboratories have made major advances in biosafety and biosecurity in the recent decade. As a result, identifying and addressing gaps in biorisk management has never been more critical. The purpose of this study was to assess the current situation of personal protective equipment (PPE), biosafety behavior, waste management, biosafety and biosecurity measures, training and safety, and health services in diagnostic and research laboratories across Pakistan's Khyber Pakhtunkhwa (KP) province. We adapted the WHO Laboratory Assessment tool (2012) and CWA 15793 (Biorisk management guidelines) for conducting a cross-sectional survey, which was distributed among various laboratories in KP. The survey included 30 laboratories, including 11 diagnostic and 19 research laboratories. In comparison to diagnostic laboratories, biorisk management practices in research laboratories were better in terms of PPE, biosafety behavior, waste management, biosafety measures, biosecurity measures, trainings, and safety and health services. KP laboratories' biorisk management practices have improved over time, according to our findings. However, we were able to identify inadequacies that would require considerable improvements to the current setups based on the WHO and CWA 15793 recommendations. Organizations can tailor their biosafety measures and training to address identified gaps using the presented KP snapshot.

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OBJECTIVE: In this pilot study we aimed to evaluate the safety of a single intramuscular methylprednisolone (IM) injection at the time of discharge as a replacement for oral steroid therapy for patients in our population with asthma or chronic obstructive pulmonary disease (COPD). METHODS: This proof-of-concept, open label clinical trial without randomisation was conducted at the Pulmonary Department of Ziauddin Hospital and University, Karachi from January 2018 to March 2018. Patients discharged after in-hospital treatment for exacerbations of either asthma or COPD were recruited for this study. Intramuscular injection of methylprednisolone was administered to these patients who were then followed-up after one week and one month. During that period, information was collected of the patients' self-report of any unscheduled emergency room visit, blood sugar and blood pressure control, symptoms suggestive of thrush, increase gastric acidity and weight gain. For the data analysis, frequency and percentages were calculated with SPSS version 21. RESULTS: A total of 30 patients aged 52.83 ± 14.27 years were recruited for this pilot study. At one month follow-up, no unscheduled emergency room visits were observed in all of the study patients. Symptoms suggestive of oral thrush were recorded in only 2 (6.7%) patients and weight gain was reported by only 5 (16.7%). Controlled blood sugar and blood pressure was reported by all the patients. No incidence of nocturnal symptoms, awakening and dyspepsia were reported. CONCLUSIONS: A single dose of methylprednisolone injection without any obvious side effects over one month among patients with asthma and COPD demonstrated a safe strategy for them.

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Virus-like particle (VLP)-based vaccines have been in the market since decades for preventing viral infection and have proven their usefulness also in other areas of biotechnology. Here, we describe in detail simple small-scale production and purification procedures for the generation of hepatitis B surface antigen (HBsAg) VLPs using Pichia pastoris as expression host. This protocol may also be applicable with variations to other HBsAg-based VLPs additionally carrying antigens of other pathogens.

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Worldwide antiretroviral therapy (ART) has reduced the mortality and morbidity rates in individuals with HIV infection. However, the increasing occurrence of drug resistance is limiting treatment options. In recent years, Pakistan has witnessed a concentrated epidemic of HIV. It is very important to identify geographical linkages and mutations that generate selective pressure and drive resistance of HIV in our population. The aim of this work was to identify genetic diversity and drug resistance patterns of HIV in Pakistan, using available sequences and bioinformatics tools, which may help in selecting effective combination of available drugs. A total of 755 Pakistani HIV gag, pol and env sequences were retrieved from the Los Alamos HIV database. Sequences were aligned with reference sequences of different subtypes. For geographical linkages, sequences of predominant subtypes were aligned with sequences of the same subtypes from different countries. Phylogenetic trees were constructed using the maximum-likelihood method in MEGA 7 software. For drug resistance analysis, sequences were entered into the Stanford University HIV Drug Resistance Database. Phylogenetic trees for studying genetic diversity showed that 82% of the sequences were of subtype A, while the rest of the sequences were of subtypes B (9.5%), K (2%), D (2%) and AE (1%). Moreover, trees that were constructed to examine geographical linkages showed close clustering of strains with those of the neighboring countries Afghanistan and India, as well as some African countries. A search for drug resistance mutations showed that 93% of the sequences had no major or minor mutations. The remaining 7% of the sequences contained a major mutation, Y115F, which causes the virus to exhibit low to intermediate resistance against lamivudine and emtricitabine. Our data indicate that HIV subtype A is the major subtype, while subtypes K, D and AE are also present in our country, suggesting gradual viral evolution and possible entry of different subtypes from neighboring countries. These data suggest that HIV is still sensitive to most of the antiretroviral drugs used in Pakistan.

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Unfortunately, the co-author's name Nouman Mughal was incorrectly published in the original article and the same is corrected here in this erratum.

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National level population-based cancer data have never been published from Pakistan in seven decades since independence (1947). Therefore, generation of high-quality regional data becomes highly relevant. Cancer data for the period of 2010-2015 representing the population from all districts of Karachi (14.6 million) are presented herein. After institutional approval (Ref no. IRB-459/DUHS/-14), a Pathology based cancer registry was established at the largest government-run diagnostic and reference center of Karachi. During 2010-2015, a total of 13,508 cancers (including 686 non-melanoma-skin-cancers (NMSC)) were diagnosed. Of these, 5665 (41.9%) were in males while 7843 (58.1%) were in females. Incidence rates for all cancers (excluding NMSC) were 66.7 per 100,000 (crude) and 105.1 per 100,000 (ASR) for males and 112.0 per 100,000 (crude) and 175.8 per 100,000 (ASR) for females. In males, cancer of lip and oral cavity was the most frequently diagnosed cancer (30.8%, ASR 33.1), followed by NMSC (7.7%, ASR 9.5) and colorectum (7%, ASR 7.3). In females, breast cancer was the most frequently recorded malignancy (49.5%, ASR 87.9), followed by lip and oral cavity (11.2%, ASR 22.0) and oesophagus (5.6%, ASR 10.7). We report that Karachi has the highest incidence of cancers of breast, lip and oral cavity, oesophagus and larynx in females and cancer of lip and oral cavity and larynx (2nd only to Turkey) in males compared to any of the Asian populations. Notably, incidence of tobacco associated cancers is very high in Karachi, demanding urgent attention by relevant authorities to address the un-controlled and drastically high consumption of various forms of tobacco in the city.

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The hepatitis B surface antigen (HBsAg) is a recombinant protein-based vaccine being able to form virus-like particles (VLPs). HBsAg is mainly produced using yeast-based expression systems, however, recent results strongly suggest that VLPs are not formed within the yeast cells during the cultivation but are formed in a gradual manner during the following down-stream procedures. VLPs are also not detectable during the first down-stream steps including mechanical and EDTA/detergent-assisted cell destruction. Moreover, VLPs are not detectable in the cell lysate treated with polyethylene glycol and colloidal silica. The first VLP resembling structures appear after elution of HBsAg from colloidal silica to which it binds through hydrophobic interaction. These first VLP resembling structures are non-symmetrical as well as heterodisperse and exhibit a high tendency toward cluster formation presumably because of surface exposed hydrophobic patches. More symmetrical and monodisperse VLPs appear after the following ion-exchange and size-exclusion chromatography most likely as the result of buffer changes during these purification steps (toward more neutral pH and less salt). Final treatment of the VLPs with the denaturant KSCN at moderate concentrations with following KSCN removal by dialysis does not cause unfolding and VLP disassembly but results in a re- and fine-structuring of the VLP surface topology.

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Following earlier studies on high-level intracellular production of hepatitis B surface antigen (HBsAg) using recombinant Pichia pastoris, we present here in detail an enhanced method for the purification of recombinant HBsAg virus-like particles (VLPs). We have screened various detergents for their ability to promote the solubilization of recombinant intracellular HBsAg. In addition, we have analyzed the effect of cell disruption and extraction regarding their impact on the release of HBsAg. Our results show that introduction of the mild nonionic detergent Tween 20 in the initial process of cell lysis at ∼600bars by high pressure homogenization leads to the best results. The subsequent purification steps involved polyethylene glycol precipitation of host cell contaminants, hydrophobic adsorption of HBsAg to colloidal silica followed by ion-exchange chromatography and either isopycnic density ultracentrifugation or size exclusion chromatography for the recovery of the VLPs. After final KSCN treatment and dialysis, a total yield of ∼3% with a purity of >99% was reached. The pure protein was characterized by electron microscopy, showing the presence of uniform VLPs which are the pre-requisite for immunogenicity. The intramuscular co-administration of HBsAg VLPs, with either alum or a PEGylated-derivative of the toll-like receptor 2/6 agonist MALP-2, to mice resulted in the elicitation of significantly higher HBsAg-specific IgG titers as well as a stronger cellular immune response compared to mice vaccinated with a gold standard vaccine (Engerix™). These results show that P. pastoris derived HBsAg VLPs exhibit a high potential as a superior biosimilar vaccine against hepatitis B.

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