Humoral and T cell responses to SARS-CoV-2 reveal insights into immunity during the early pandemic period in Pakistan.

BACKGROUND: Protection against SARS-CoV-2 is mediated by humoral and T cell responses. Pakistan faced relatively low morbidity and mortality from COVID-19 through the pandemic. To examine the role of prior immunity in the population, we studied IgG antibody response levels, virus neutralizing activity and T cell reactivity to Spike protein in a healthy control group (HG) as compared with COVID-19 cases and individuals from the pre-pandemic period (PP). METHODS: HG and COVID-19 participants were recruited between October 2020 and May 2021. Pre-pandemic sera was collected before 2018. IgG antibodies against Spike and its Receptor Binding Domain (RBD) were determined by ELISA. Virus neutralization activity was determined using a PCR-based micro-neutralization assay. T cell - IFN-γ activation was assessed by ELISpot. RESULTS: Overall, the magnitude of anti-Spike IgG antibody levels as well as seropositivity was greatest in COVID-19 cases (90%) as compared with HG (39.8%) and PP (12.2%). During the study period, Pakistan experienced three COVID-19 waves. We observed that IgG seropositivity to Spike in HG increased from 10.3 to 83.5% during the study, whilst seropositivity to RBD increased from 7.5 to 33.3%. IgG antibodies to Spike and RBD were correlated positively in all three study groups. Virus neutralizing activity was identified in sera of COVID-19, HG and PP. Spike reactive T cells were present in COVID-19, HG and PP groups. Individuals with reactive T cells included those with and without IgG antibodies to Spike. CONCLUSIONS: Antibody and T cell responses to Spike protein in individuals from the pre-pandemic period suggest prior immunity against SARS-CoV-2, most likely from cross-reactive responses. The rising seroprevalence observed in healthy individuals through the pandemic without known COVID-19 may be due to the activation of adaptive immunity from cross-reactive memory B and T cells. This may explain the more favourable COVID-19 outcomes observed in this population.

Analysis of the Antimicrobial and Anti-Biofilm Activity of Natural Compounds and Their Analogues against Staphylococcus aureus Isolates.

(1) Background: Staphylococcus aureus (S. aureus) is one of the most frequent causes of biofilm-associated infections. With the emergence of antibiotic-resistant, especially methicillin-resistant S. aureus (MRSA), there is an urgent need to discover novel inhibitory compounds against this clinically important pathogen. In this study, we evaluated the antimicrobial and anti-biofilm activity of 11 compounds, including phenyl propenes and phenolic aldehydes, eugenol, ferulic acid, sinapic acid, salicylaldehyde, vanillin, cinnamoyl acid, and aldehydes, against drug-resistant S. aureus isolates. (2) Methods: Thirty-two clinical S. aureus isolates were obtained from Alkhidmat Diagnostic Center and Blood Bank, Karachi, Pakistan, and screened for biofilm-forming potential, and susceptibility/resistance against ciprofloxacin, chloramphenicol, ampicillin, amikacin, cephalothin, clindamycin, streptomycin, and gentamicin using the Kirby-Bauer disk diffusion method. Subsequently, 5 representative clinical isolates were selected and used to test the antimicrobial and anti-biofilm potential of 11 compounds using both qualitative and quantitative assays, followed by qPCR analysis to examine the differences in the expression levels of biofilm-forming genes (ica-A, fnb-B, clf-A and cna) in treated (with natural compounds and their derivatives) and untreated isolates. (3) Results: All isolates were found to be multi-drug resistant and dominant biofilm formers. The individual Minimum Inhibitory Concentration (MIC) of natural compounds and their analogues ranged from 0.75−160 mg/mL. Furthermore, the compounds, Salicylaldehyde (SALI), Vanillin (VAN), α-methyl-trans-cinnamaldehyde (A-MT), and trans-4-nitrocinnamic acid (T4N) exhibited significant (15−92%) biofilm inhibition/reduction percentage capacity at the concentration of 1−10 mg/mL. Gene expression analysis showed that salicylaldehyde, α-methyl-trans-cinnamaldehyde, and α-bromo-trans-cinnamaldehyde resulted in a significant (p < 0.05) downregulation of the expression of ica-A, clf-A, and fnb-A genes compared to the untreated resistant isolate. (4) Conclusions: The natural compounds and their analogues used in this study exhibited significant antimicrobial and anti-biofilm activity against S. aureus. Biofilms persist as the main concern in clinical settings. These compounds may serve as potential candidate drug molecules against biofilm forming S. aureus.

Interactions of Apigenin and Safranal with the 5HT1A and 5HT2A Receptors and Behavioral Effects in Depression and Anxiety: A Molecular Docking, Lipid-Mediated Molecular Dynamics, and In Vivo Analysis.

BACKGROUND: The current study utilizes in silico molecular docking/molecular dynamics to evaluate the binding affinity of apigenin and safranal with 5HT1AR/5HT2AR, followed by assessment of in vivo effects of these compounds on depressive and anxious behavior. METHODS: The docking between apigenin and safranal and the 5HT1A and 5HT2A receptors was performed utilizing AutoDock Vina software, while MD and protein-lipid molecular dynamics simulations were executed by AMBER16 software. For in vivo analysis, healthy control (HC), disease control (DC), fluoxetine-, and apigenin-safranal-treated rats were tested for changes in depression and anxiety using the forced swim test (FST) and the elevated plus-maze test (EPMT), respectively. RESULTS: The binding affinity estimations identified the superior interacting capacity of apigenin over safranal for 5HT1A/5HT2A receptors over 200 ns MD simulations. Both compounds exhibit oral bioavailability and absorbance. In the rodent model, there was a significant increase in the overall mobility time in the FST, while in the EPMT, there was a decrease in latency and an increase in the number of entries for the treated and HC rats compared with the DC rats, suggesting a reduction in depressive/anxiety symptoms after treatment. CONCLUSIONS: Our analyses suggest apigenin and safranal as prospective medication options to treat depression and anxiety.

HBV S antigen evolution in the backdrop of HDV infection affects epitope processing and presentation.

INTRODUCTION: HBV can evolve under selection pressure exerted by drugs and/or host immunity, resulting in accumulation of escape mutations that can affect the drug or the immune activity. Hepatitis delta virus (HDV) coinfection is also known to exert selection pressure on HBV, which leads to selective amplification of certain mutations, especially in genes that are required for HDV pathogenesis, such as HBsAg. However, little is known about the function of these mutations on HBV or HDV life cycle. The purpose of this study is to determine mutations selectively amplified in the backdrop of HDV, and how these mutations affect processing of CD4- and CD8-T cell epitopes. METHODS: HBsAg was successfully amplified from 49/50 HBV mono- and 36/50 coinfected samples. The sequences were used to identify mutations specific to each study group, followed by an in silico analysis to determine the effect of these mutations on (1) proteasomal degradation, (2) MHC-I and MHC-II biding, and (3) processing of T-cell epitopes. RESULTS: HBV-HDV coinfected sequences exhibited certain unique mutations in HBsAg genes. Some of these mutations affected the generation of proteasomal sites, binding of HBsAg epitopes to MHC-I and -II ligands, and subsequent generation of T- cell epitopes. CONCLUSION: These observations suggest that HBV selectively amplifies certain mutations in the backdrop of HDV coinfection. Selective amplification of these mutations at certain strategic locations might not only enable HBV to counteract the inhibitory effects of HDV on HBV replication but also facilitate its survival by escaping the immune response.

Blue Biotechnology: Computational Screening of Sarcophyton Cembranoid Diterpenes for SARS-CoV-2 Main Protease Inhibition.

The coronavirus pandemic has affected more than 150 million people, while over 3.25 million people have died from the coronavirus disease 2019 (COVID-19). As there are no established therapies for COVID-19 treatment, drugs that inhibit viral replication are a promising target; specifically, the main protease (Mpro) that process CoV-encoded polyproteins serves as an Achilles heel for assembly of replication-transcription machinery as well as down-stream viral replication. In the search for potential antiviral drugs that target Mpro, a series of cembranoid diterpenes from the biologically active soft-coral genus Sarcophyton have been examined as SARS-CoV-2 Mpro inhibitors. Over 360 metabolites from the genus were screened using molecular docking calculations. Promising diterpenes were further characterized by molecular dynamics (MD) simulations based on molecular mechanics-generalized Born surface area (MM-GBSA) binding energy calculations. According to in silico calculations, five cembranoid diterpenes manifested adequate binding affinities as Mpro inhibitors with ΔGbinding < -33.0 kcal/mol. Binding energy and structural analyses of the most potent Sarcophyton inhibitor, bislatumlide A (340), was compared to darunavir, an HIV protease inhibitor that has been recently subjected to clinical-trial as an anti-COVID-19 drug. In silico analysis indicates that 340 has a higher binding affinity against Mpro than darunavir with ΔGbinding values of -43.8 and -34.8 kcal/mol, respectively throughout 100 ns MD simulations. Drug-likeness calculations revealed robust bioavailability and protein-protein interactions were identified for 340; biochemical signaling genes included ACE, MAPK14 and ESR1 as identified based on a STRING database. Pathway enrichment analysis combined with reactome mining revealed that 340 has the capability to re-modulate the p38 MAPK pathway hijacked by SARS-CoV-2 and antagonize injurious effects. These findings justify further in vivo and in vitro testing of 340 as an antiviral agent against SARS-CoV-2.

Baseline MMP expression in periapical granuloma and its relationship with periapical wound healing after surgical endodontic treatment.

BACKGROUND: Matrix metalloproteinases (MMPs) catalyzes the degradation of the extracellular matrix components and have a major role in many physiological processes including wound healing. In the current study, we examined the correlation of baseline MMPs 1, 2, 7, and 9 expressions with periapical wound healing after surgical endodontic treatment. METHODS: 27 patients aged between 15 and 57 years presenting with chronic apical periodontitis or chronic apical abscess of an anterior tooth with previously attempted or failed root canal treatment were included in this study. During surgical endodontic treatment, tissue from the periapical lesion sample was collected and used for gross histopathological analysis as well as mRNA expression analysis of MMPs 1, 2, 7, and 9. Patients were recalled for follow-up after 6  months to evaluate the healing status both clinically and radiographically and healing was correlated with baseline MMP expression. RESULTS: Out of 27 patients, healing was observed in 15 patients at the end of 6 months, and in 21 patients after 12 months.. Six patients showed no healing even after 12 months. Analysis of baseline MMP 1, 2, 7, and 9 expression levels with healing status showed the mean relative expression of MMP2 and MMP9 to be considerably increased in the non-healing group as compared to the healing group. CONCLUSION: Overexpression of MMP2 and MMP9 may be considered as a potential prognostic biomarker for periapical wound healing after surgical endodontic treatment. However, further studies are desirable to establish its precise relationship with periapical wound healing.

Probabilistic measures of HIV-1 transmission in different HIV-1 key population groups of Larkana, Pakistan.

OBJECTIVE: To estimate the probability of human immunodeficiency virus (HIV)-1 transmission from different key HIV population groups using probabilistic modelling. METHODS: This study was conducted in December 2020. A probabilistic model was used to estimate the probability of HIV-1 transmission from different key HIV population groups in Larkana. Our model was run on three probabilistic assumptions: 1) each replication gave two conceivable results: 'true' or 'false'; 2) the chance of giving a 'true' result is the same for each replication; and 3) the replications are independent - 'true' in one will not impact the likelihood of 'true' in another. RESULTS: The results estimated the probability of HIV transmission in key HIV population groups in Larkana to range between 0.42-0.54 per trial, where the highest probability of transmission was predicted for men who have sex with men (MSM; 0.54 per trial), followed by transgender (TG; 0.46 per trial) and people who inject drugs (PWID; 0.457 per trial). CONCLUSIONS: Our results suggest that there is a high likelihood of HIV transmission by key population groups in Larkana, such as MSM, TG, and PWID. Mathematic models, such as one proposed in our study can aid the HIV and acquired immunodeficiency syndrome (AIDS) control programmes in evaluating and optimising the strategies in controlling transmission of HIV from the key population groups.

The emergence of an unassigned complex recombinant form in a Pakistani HIV-infected individual.

In Pakistan, the HIV situation has gone from an outbreak to a concentrated epidemic, and the virus has now crossed into the low-risk population. In addition, several new HIV outbreaks have occurred in different parts of the country. HIV-1 subtype A has been the major epidemic subtype in Pakistan; however, as the epidemic has grown, the emergence of several new subtypes and recombinant forms has been observed. Here, we present the first case and genetic analysis of an unassigned, complex recombinant form in a Pakistani HIV-infected individual with virological failure. Genetic analysis of the sequence indicated that this recombinant form is multi-drug resistant, harboring drug resistance mutations against more than one class of antiretroviral drugs.

Analysis of anti-microbial and anti-biofilm activity of hand washes and sanitizers against S. aureus and P. aeruginosa.

OBJECTIVE: To analyse the biofilm-forming potential of clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa, and to assess antimicrobial activity of commonly used sanitizers in hospital and laboratory settings. METHODS: The study was conducted at Aga Khan University Karachi from August 2016 to January 2017. The biofilm-forming potential of Staphylococcus aureus and Pseudomonas aeruginosa clinical isolates were evaluated qualitatively using air-liquid interface tube method, and air-liquid interface cover slip assay. The antimicrobial activity of commonly-used hand-washes and sanitizers were assessed using agar well diffusion method, while the anti-biofilm activity of the hand-washes and sanitizers was qualitatively assessed using air-liquid interface covers lip as s ay. RESULTS: Of the eight hand-washes and sanitizers, 2(25%) showed antimicrobial activity against both Staphylococcus aureus and Pseudomonas aeruginosa, while 2(25%) exhibited antimicrobial activity against either S. aureus or P. aeruginosa. Also, 4 (50%) of them showed no inhibitory activity against S. aureus and P. aeruginosa. CONCLUSIONS: The findings shall have important consequences with regards to infection control in hospital and laboratory settings.

The antibiofilm activity of Acetylsalicylic acid, Mefenamic acid, Acetaminophen against biofilms formed by P. aeruginosa and S. epidermidis.

OBJECTIVE: To evaluate the antibacterial activity of Aspirin, Mefenamic acid and Acetaminophen against Pseudomonas aeruginosa and Staphylococcus epidermidis biofilms. METHODS: The study was conducted AKU Karachi in collaboration with DIHE Karachi from March 2018 to December 2018.Quantitative spectrophotometric method was used to study the reduction and removal of the Pseudomonas aeruginosa and Staphylococcus epidermidis formed biofilms. Statistical tests were performed using Graph Pad Prism software. . RESULTS: Acetaminophen showed maximum biofilm reduction activity against the biofilms formed by Pseudomonas aeruginosa, and Staphylococcus epidermidis. Mefanamic acid showed maximum biofilm removal potential against Pseudomonas aeruginosa, while Aspirin and Mefanamic acid were equally effective in removing biofilms formed by Staphylococcus epidermidis as well. CONCLUSIONS: There is a continuous need to look for non-antibiotic agents for their potential antimicrobial and antibiofilm potential.

Association between a single nucleotide polymorphism in neuregulin-1 and schizophrenia in Pakistani patients.

OBJECTIVE: To determine the association of single-nucleotide polymorphism8nrg433E1006 in the neuregulin-1 gene associated with schizophrenia. METHODS: This case-control study was conducted at the Fountain House, Lahore, and the psychiatric clinics at the Aga Khan University, Karachi, from 2010 to 2013.The total genomic deoxyribonucleic acid was isolated and single-nucleotide polymorphism8nrg433E1006 was screened by nested polymerase chain reaction followed by sequencing. These sequences, from patients and controls, were aligned with the human neuregulin-1-glial growth factor 2 gene sequence, which served as a reference sequence. The single nucleotide polymorphism genetic algorithm was characterised at position 433 in the neuregulin-1 gene by aligning test and control sequences with the neuregulin-1-glial growth factor 2reference sequence using ClustalW algorithm, implemented in the BioEdit software. RESULTS: Of the 630 samples, 321(51%) were of cases and 309(49%)of controls. Moreover, 99(30.8%) cases and 79(25.6%) controls rendered correct neuregulin-1 gene frames. Of them, the single-nucleotide polymorphism8nrg433E1006 was present in 62(62.6%) cases and 24(30.4%) controls. The analysis showed that the odds ratio of having schizophrenia is 3.8 times higher in the presence of this single-nucleotide polymorphism at the 92 bp of neuregulin-1 gene with the 95% confidence interval(p=0.0001). CONCLUSIONS: There was a strong association of single-nucleotide polymorphism8nrg433E1006 in the neuregulin-1 gene with schizophrenia.

Opium trade and the spread of HIV in the Golden Crescent.

The Golden Crescent region of South Asia-comprising Afghanistan, Iran, and Pakistan-is a principal global site for opium production and distribution. Over the past few decades, war, terrorism, and a shifting political landscape have facilitated an active heroin trade throughout the region. Protracted conflict has exacerbated already dire socio-economic conditions and political strife within the region and contributed to a consequent rise in opiate trafficking and addiction among the region's inhabitants. The worsening epidemic of injection drug use has paralleled the rising incidence of HIV and other blood-borne infections in the region and drawn attention to the broader implications of the growing opiate trade in the Golden Crescent. The first step in addressing drug use is to recognize that it is not a character flaw but a form of mental illness, hence warranting humane treatment of drug users. It is also recommended that the governments of the Golden Crescent countries encourage substitution of opium with licit crops and raise awareness among the general public about the perils of opium use.

HIV-1 progression links with viral genetic variability and subtype, and patient's HLA type: analysis of a Nairobi-Kenyan cohort.

In a Nairobi-Kenyan cohort of 50 HIV-1 positive patients, we analysed the prevalence of HIV-1 subtypes and human leucocyte antigen (HLA) alleles. From this cohort, 33 patients were selected for the analysis of HIV-1 infection progression markers (i.e. CD4 cell counts and viral loads) and their association with HIV-1 genetic variability and subtype, and patient's HLA type. HIV-1 gag genetic variability, analysed using bioinformatics tools, showed an inverse relationship with CD4 cell count whereas with viral load that relationship was direct. Certain HLA types and viral subtypes were also found to associate with patients' viral load. Associations between disease parameters and the genetic makeup of the host and virus may be crucial in determining the outcome of HIV-1 infection.

Ensemble Clustering to Generate Phenotypes of Kidney Transplant Donors and Recipients.

In this paper we investigate the generation of phenotypes for kidney transplant donors and recipients to assist with decision making around organ allocation. We present an ensemble clustering approach for multi-type data (numerical and categorical) using two different clustering approaches-i.e., model based and vector quantization based clustering. These clustering approaches were applied to a large, US national deceased donor kidney transplant recipient database to characterize members of each cluster (in an unsupervised fashion) and to determine whether the subsequent risk of graft failure differed for each cluster. We generated three distinct clusters of recipients, which were subsequently used to generate phenotypes. Each cluster phenotype had recipients with varying clinical features, and the risk of kidney transplant graft failure and mortality differed across clusters. Importantly, the clustering results by both approaches demonstrated a significant overlap. Utilization of two distinct clustering approaches may be a novel way to validate unsupervised clustering techniques and clustering can be used for organ allocation decision making on the basis of differential outcomes.

Evaluation of the Training Program to Train HIV Treatment Center Staff in Pakistan.

Introduction In Pakistan, HIV training programs, especially for health professionals working in HIV treatment centers, are limited. Consequently, there is little data about HIV awareness among physicians and allied health workers and how it may affect their care for people living with HIV (PLWH). Recently, the Global Fund to Fight AIDS, Tuberculosis, and Malaria (GFATM) grant Principal Recipient UNDP engaged an NGO experienced in HIV/AIDS training, on a competitive basis, to develop a training manual and conduct training of all categories of HIV treatment centers staff. The goal of this study was to assess the training program's influence on trainees' (both physicians and allied health staff) knowledge and abilities and describe its major lessons. Methodology This was a one-group pre-post test study, carried out between January 17 and February 22, 2023. The study was carried out in three phases. In the first phase, a team of experts developed an antiretroviral treatment (ART) training manual. In the second phase, 9- and three-day training workshops were conducted in six different cities of Pakistan, which were attended by physicians and allied health staff working in different HIV treatment centers across Pakistan. The workshops had plenary lectures, discussions, role plays, video cases, and case studies. In the third phase, a quiz, comprising multiple/best choice questions (MCQs/BCQs) and true and false questions, was administered before (pre) and after the workshop (post) to assess the impact of these training sessions in enhancing the level of HIV knowledge, especially related to ART. The workshop was attended by a total of 256 health workers from different cities in Pakistan. The participants had backgrounds in medical science, psychology, laboratory science, nursing, and computer science. Pre-and post-test responses were statistically analyzed to determine the impact of the training program on participant's knowledge. For this, the Shapiro-Wilk test was applied to test data normality, followed by the application of paired t-test or Wilcoxon Signed Rank Test for normally and non-normally distributed data, respectively. Finally, a chi-square test was applied to examine the significant (p<0.05) association between training workshops and improvement in the participant's level of understanding of HIV. In all statistical tests, p<0.05 was considered significant.  Results The results from our study showed that before the training session, both physicians and allied staff possessed limited knowledge about HIV-related domains. After the workshops, participants from all cities demonstrated a uniform enhancement of knowledge related to different HIV-related domains, evident from the improvement in post-test scores compared to pre-test scores (p<0.0001). The chi-square test showed a significant association between training workshops and improvement in the participant's level of understanding about HIV (p-values for BCQ, MCQ, and true and false: 0.001, 0.0047, and 0.0024, respectively). Conclusions Pre- and post-test evaluation provides an objective, data-driven method for measuring the impact of educational interventions in improving healthcare workers' awareness about HIV. The results emphasize the role of continuous workshops and training programs in enhancing the knowledge and understanding of healthcare and allied health workers regarding HIV.

Differential analysis of histopathological and genetic markers of cancer aggressiveness, and survival difference in EBV-positive and EBV-negative prostate carcinoma.

Several studies have shown an association between prostate carcinoma (PCa) and Epstein-Barr virus (EBV); however, none of the studies so far have identified the histopathological and genetic markers of cancer aggressiveness associated with EBV in PCa tissues. In this study, we used previously characterized EBV-PCR-positive (n = 39) and EBV-negative (n = 60) PCa tissues to perform an IHC-based assessment of key histopathological and molecular markers of PCa aggressiveness (EMT markers, AR expression, perineural invasion, and lymphocytic infiltration characterization). Additionally, we investigated the differential expression of key oncogenes, EMT-associated genes, and PCa-specific oncomiRs, in EBV-positive and -negative tissues, using the qPCR array. Finally, survival benefit analysis was also performed in EBV-positive and EBV-negative PCa patients. The EBV-positive PCa exhibited a higher percentage (80%) of perineural invasion (PNI) compared to EBV-negative PCa (67.3%) samples. Similarly, a higher lymphocytic infiltration was observed in EBV-LMP1-positive PCa samples. The subset characterization of T and B cell lymphocytic infiltration showed a trend of higher intratumoral and tumor stromal lymphocytic infiltration in EBV-negative tissues compared with EBV-positive tissues. The logistic regression analysis showed that EBV-positive status was associated with decreased odds (OR = 0.07; p-value < 0.019) of CD3 intratumoral lymphocytic infiltration in PCa tissues. The analysis of IHC-based expression patterns of EMT markers showed comparable expression of all EMT markers, except vimentin, which showed higher expression in EBV-positive PCa tissues compared to EBV-negative PCa tissues. Furthermore, gene expression analysis showed a statistically significant difference (p < 0.05) in the expression of CDH1, AR, CHEK-2, CDKN-1B, and CDC-20 and oncomiRs miR-126, miR-152-3p, miR-452, miR-145-3p, miR-196a, miR-183-3p, and miR-146b in EBV-positive PCa tissues compared to EBV-negative PCa tissues. Overall, the survival proportion was comparable in both groups. The presence of EBV in the PCa tissues results in an increased expression of certain oncogenes, oncomiRs, and EMT marker (vimentin) and a decrease in CD3 ITL, which may be associated with the aggressive forms of PCa.

Candidate protein biomarkers in chronic kidney disease: a proteomics study.

Proteinuria poses a substantial risk for the progression of chronic kidney disease (CKD) and its related complications. Kidneys excrete hundreds of individual proteins, some with a potential impact on CKD progression or as a marker of the disease. However, the available data on specific urinary proteins and their relationship with CKD severity remain limited. Therefore, we aimed to investigate the urinary proteome and its association with kidney function in CKD patients and healthy controls. The proteomic analysis of urine samples showed CKD stage-specific differences in the number of detected proteins and the exponentially modified protein abundance index for total protein (p = 0.007). Notably, specific urinary proteins such as B2MG, FETUA, VTDB, and AMBP exhibited robust negative associations with kidney function in CKD patients compared to controls. Also, A1AG2, CD44, CD59, CERU, KNG1, LV39, OSTP, RNAS1, SH3L3, and UROM proteins showed positive associations with kidney function in the entire cohort, while LV39, A1BG, and CERU consistently displayed positive associations in patients compared to controls. This study suggests that specific urinary proteins, which were found to be negatively or positively associated with the kidney function of CKD patients, can serve as markers of dysfunctional or functional kidneys, respectively.

Identification of a novel first-generation HIV-1 circulating recombinant form (CRF152_DG) among people living with HIV in Karachi, Pakistan.

The objective of this study was to characterize a novel circulating recombinant form of human immunodeficiency virus type 1 (HIV-1) among people living with HIV in Karachi, Pakistan. We conducted near-full-length genome (NFLG) sequencing on eight samples exhibiting D/G recombination signals in the pol gene region. We successfully obtained NFLG sequences (790-9,614; with reference to the HXB2 genome) from four of the eight samples and then conducted phylogenetic and recombination analyses on them. The four NFLG sequences from our study and one DG unique recombinant form previously identified in the United Kingdom (GenBank accession: MF109700) formed a distinct monophyletic cluster with an Shimodaira-Hasegawa approximate likelihood ratio test node support value of 100%. Bootscan analyses of the five NFLG sequences of DG recombinants showed that all five NFLGs shared the same unique mosaic pattern of recombination breakpoints between D and G clades, with two D fragments in the pol and vif regions inserted into a G backbone. Subregion phylogenetic analyses confirmed these sequences to be a novel circulating recombinant form (CRF) composed of subtypes D and G. The DG recombinant sequences were eventually designated as CRF152_DG by the Los Alamos HIV Sequence Database staff. IMPORTANCE: In Pakistan, the genetic diversity of human immunodeficiency virus type 1 (HIV-1) is becoming increasingly complex, compared to the early years of the epidemic that started after the detection of the first cases of HIV-1 in 1987 in Karachi. Based on the available molecular studies, two dominant HIV-1 clades, sub-subtype A1 and CRF02_AG, have been found to co-circulate with other clades, namely B, C, D, G, CRF01_AE, CRF35_A1D, and CRF56_cpx, in various urban areas of Pakistan. Several novel recombinant forms have also been detected. This first report of CRF152_DG highlights the complex nature of the HIV epidemic in Pakistan and emphasizes the importance of continual molecular surveillance (ideally based on whole-genome sequences) of HIV.

Human Immunodeficiency Virus Type-1 Genetic Diversity and Drugs Resistance Mutations among People Living with HIV in Karachi, Pakistan.

The human immunodeficiency virus type-1 epidemic in Pakistan has significantly increased over the last two decades. In Karachi, Pakistan, there is a lack of updated information on the complexity of HIV-1 genetic diversity and the burden of drug resistance mutations (DRMs) that can contribute to ART failure and poor treatment outcomes. This study aimed to determine HIV-1 genetic diversity and identify drug-resistance mutations among people living with HIV in Karachi. A total of 364 HIV-positive individuals, with a median age of 36 years, were enrolled in the study. The HIV-1 partial pol gene was successfully sequenced from 268 individuals. The sequences were used to generate phylogenetic trees to determine clade diversity and also to assess the burden of DRMs. Based on the partial pol sequences, 13 distinct HIV-1 subtypes and recombinant forms were identified. Subtype A1 was the most common clade (40%), followed by CRF02_AG (33.2%). Acquired DRMs were found in 30.6% of the ART-experienced patients, of whom 70.7%, 20.7%, and 8.5% were associated with resistance to NNRTIs, NRTIs, and PIs, respectively. Transmitted DRMs were found in 5.6% of the ART-naïve patients, of whom 93% were associated with resistance against NNRTIs and 7% to PIs. The high prevalence of DRMs in ART-experienced patients poses significant challenges to the long-term benefits and sustainability of the ART program. This study emphasizes the importance of continuous HIV molecular epidemiology and drug resistance surveillance to support evidence-based HIV prevention, precise ART, and targeted AIDS care.

Autophagy-associated biomarkers ULK2, UVRAG, and miRNAs miR-21, miR-126, and miR-374: Prognostic significance in glioma patients.

As the pioneering study from Pakistan, our research distinctly focuses on validating the roles of autophagy-associated genes and MicroRNAs (miRs) in the unique context of our population for glioma prognosis. The study delves into the nuanced interplay of autophagy within a miR-modulated environment, prompting an exploration of its potential impact on glioma development and survival. Employing real-time PCR (qPCR), we meticulously assessed the expression profiles of autophagy genes and miRs in glioma tissues, complemented by immunohistochemistry on Formalin-fixed paraffin-embedded tissues from the same patients. Our comprehensive statistical analyses, including the data normality hypothesis Shapiro-Wilk test, the Mann-Whitney U-test, Spearman correlation test, and Kaplan-Meier survival analysis, were tailored to unravel the intricate associations specific to low- and high-grade glioma within our population. Clinicopathological analysis revealed a predominance of male patients (66%) with a median age of 35 years. Glioblastoma (32%) and Astrocytoma (36%) were the most prevalent histopathological subtypes. Molecular analysis showed significant correlations between prognostic markers (Ki-67, IDH-1, p53) and clinicopathological factors, including age, histological type, radiotherapy, and chemotherapy. In high-grade glioma, increased expression of AKT and miR-21, coupled with reduced ULK2 and LC3 expression was distinctly observed. While correlation analysis identified a strong positive correlation between ULK2 and UVRAG, PTEN, miR-7, and miR-100 in low-grade glioma, unveiling distinctive molecular signatures unique to our study. Furthermore, a moderate positive correlation emerged between ULK2 and mTOR, miR-7, miR-30, miR-100, miR-204, and miR-374, also between miR-21 and miR-126. Similarly, a positive correlation appeared between ULK2 and AKT, LC3, PI3K, PTEN, ULK1, VPS34, mTOR, Beclin1, UVRAG, miR-7 and miR-374. AKT positively correlated with LC3, PI3K, PTEN, ULK1, VPS34, mTOR, Beclin1, UVRAG, miR-7, miR-30, miR-204, miR-374, miR-126 and miR-21 weakly correlated with AKT and miR-30 in high-grade glioma, providing further insights into the autophagy pathway within our population. The enrichment analysis for miR-21, miR-126, and miR-374 showed MAPK pathway as a common pathway along with Ras, PI3K, and mTOR pathway. The low ULK2, UVRAG, and miR-374 expression group exhibited significantly poor overall survival in glioma, while miR-21 over-expression indicated a poor prognosis in glioma patients, validating it in our population. This study provides comprehensive insights into the molecular landscape of gliomas, highlighting the dysregulation of autophagy genes ULK2, and UVRAG and the associated miR-21, miR-126 and miR-374 as potential prognostic biomarkers and emphasizing their unique significance in shaping survival outcomes in gliomas within the specific context of the Pakistani population.