Muhammad Asghar

Background: Malaria is a severe and fatal disease in the non-immune, but severity lessens with increasing exposure. Children in endemic areas are at a particularly high-risk for malaria, often experiencing multiple sequential clinical and asymptomatic P. falciparum infections annually. Whether the associated persistent cellular activation has additional hidden costs, as reflected by cellular aging, has not been studied. Methods: We measured correlations between telomere length (TL) in cross-sectional blood samples (2007, 2010 and 2013) and cumulative malaria and P. falciparum infection episodes in children from two longitudinal cohorts under active weekly surveillance for febrile malaria. TL was quantified by qPCR in 218 children from Junju, an area of moderate transmission, and 90 age-matched controls from Ngerenya, where P. falciparum transmission is no longer endemic. Results: Although TL declined with age (independently of cumulative malaria exposure; linear effects model, p>0.05) in both cohorts, the decline was more pronounced in those with longer TL at baseline. Asymptomatic P. falciparum parasitaemia, at the time of the cross-sectional survey, was associated with a positive change in TL of 0.22 kb (95% confidence interval 0.02-0.42, p=0.03). Conclusion: Overall, repeated asymptomatic and symptomatic P. falciparum malaria episodes were not associated with TL shortening in the children in our cohorts. The apparent increase in length with asymptomatic malaria was of marginal statistical significance and could have been a chance finding. Alternatively, the suppression of cellular activation and proliferation, recently reported in asymptomatic Plasmodium spp infections, preserves TLs from degrading.

Background: Access to safe and timely blood transfusion is a cornerstone of modern healthcare but depends on a stable supply of voluntary donations and rigorous hemovigilance systems. O-negative red blood cells are universally compatible and essential for emergency transfusions; however, their scarcity, particularly in low-resource regions, poses significant challenges. To address this challenge, a compact trispecific triabody was designed to block A, B, and Rh(D) antigens on RBCs. Results: In this study, two triabody configurations differing in the placement of the anti-Rh(D) variable domain were generated, producing closed (C1) and open (O1) formats. The selected triabody-C1 was expressed in Escherichia coli BL21(DE3) and purified into two fractions, AE3-B1 and AE3-B2. Hemagglutination assays demonstrated that AE3-B2 did not induce hemagglutination, whereas AE3-B1 showed mixed-field hemagglutination under standard conditions and complete hemagglutination under potentiator-enhanced hemagglutination conditions. ELISA-based binding assays indicated that the triabody’s monomers functioned independently with free antigens, while RBC-bound antigen assays revealed altered binding behavior upon sequential antigen engagement. Blood incompatibility related hemagglutination assays using monoclonal antibodies and incompatible O-negative blood plasma demonstrated complete prevention of hemagglutination by AE3-B2 triabody-coated RBCs, confirming effective antigen blocking. Conclusions: The trispecific triabody efficiently prevents hemagglutination by blocking A, B, and Rh(D) antigens on RBCs, enabling them to exhibit enhanced compatibility and hemagglutinating patterns similar to O-negative cells. These findings provide a promising strategy to increase the pool of compatible blood for transfusion, particularly in emergency and resource-limited settings, while emphasizing that future in vivo investigations are needed to confirm efficacy and safety.

Background Access to safe and timely blood transfusion is a cornerstone of modern healthcare but depends on a stable supply of voluntary donations and rigorous hemovigilance systems. O-negative red blood cells (RBCs) are universally compatible and essential for emergency transfusions; however, their scarcity, particularly in low-resource regions, poses significant challenges. To help overcome this challenge, a compact trispecific triabody was designed to block A, B, and Rh(D) antigens on RBCs, thereby conferring universal compatibility similar to O-negative RBCs. Results In this study, two combinations based on the placement of the anti-Rh(D) variable chain at the C- or N-terminus were generated, and fusion proteins from the first combination produced the closed (C1) and open (O1) triabodies. Intramolecular cooperative binding affinities of the selected triabody-C1 were predicted computationally using blood group antigens A, B, and Rh(D), with no significant changes observed in binding free energies. The triabody-C1 was expressed in Escherichia coli BL21(DE3) using a two-plasmid system and purified through a three-step process, yielding two fractions, AE3-B1 and AE3-B2. The hemagglutination potential of the triabody was evaluated both visually and microscopically through immunocytochemistry. Visually, no hemagglutination was observed, while microscopically, mixed-field hemagglutination occurred when AB+ and AB − RBCs were incubated with the triabody in fraction AE3-B1, but not when A+ or B+ RBCs were tested. No hemagglutination of AB+ RBCs was detected with the triabody in fraction AE3-B2. ELISA-based cooperative binding assays using free antigens showed that the triabody’s monomers functioned independently, with no changes in binding parameters K _D or B _max . In contrast, assays with RBC-bound antigens revealed increased K _D and decreased B _max across successive binding events, particularly when Rh(D) antigens were engaged first. Hemagglutination assays confirmed that triabody-coated RBCs exhibited a complete absence of hemagglutination with anti-A, anti-B, and anti-Rh(D) IgM antibodies, confirming effective antigen blocking. Conclusions The trispecific triabody effectively blocks A, B, and Rh(D) antigens, rendering RBCs with O-negative like universal compatibility and offering a promising strategy to expand the supply of universally transfusable blood, particularly in emergency and resource-limited settings.

Persistence of malaria parasites in asymptomatic hosts is crucial in areas of seasonally-interrupted transmission, where P. falciparum bridges wet seasons months apart. During the dry season, infected erythrocytes exhibit extended circulation with reduced cytoadherence, increasing the risk of splenic clearance of infected cells and hindering parasitaemia increase. However, what determines parasite persistence for long periods of time remains unknown. Here, we investigated whether seasonality affects plasma composition so that P. falciparum can detect and adjust to changing serological cues; or if alternatively, parasite infection length dictates clinical presentation and persistency. Data from Malian children exposed to alternating ~6-month wet and dry seasons show that plasma composition is unrelated to time of year in non-infected children, and that carrying P. falciparum only minimally affects plasma constitution in asymptomatic hosts. Parasites persisting in the blood of asymptomatic children from the dry into the ensuing wet season rarely if ever appeared to cause malaria in their hosts as seasons changed. In vitro culture in the presence of plasma collected in the dry or the wet seasons did not affect parasite development, replication or host-cell remodelling. The absence of a parasite-encoded sensing mechanism was further supported by the observation of similar features in P. falciparum persisting asymptomatically in the dry season and parasites in age- and sex-matched asymptomatic children in the wet season. Conversely, we show that P. falciparum clones transmitted early in the wet season had lower chance of surviving until the end of the following dry season, contrasting with a higher likelihood of survival of clones transmitted towards the end of the wet season, allowing for the re-initiation of transmission. We propose that the decreased virulence observed in persisting parasites during the dry season is not due to the parasites sensing ability, nor is it linked to a decreased capacity for parasite replication but rather a consequence decreased cytoadhesion associated with infection length.

Glioblastoma multiforme (GBM) IDH-wildtype is the most prevalent brain malignancy in adults. However, molecular mechanisms, which leads to GBM have not been completely elucidated. Granulocyte colony-stimulating factor (GCSF), Granulocyte colony-stimulating factor receptor GCSFR, and Signal transducers and activators of transcription 3 (STAT3) have been involved in the occurrence and development of various cancers, but their role in GBM is little known. Herein, we have investigated the gene and protein expression of GCSF, GCSFR, and STAT3 in 21 tissue biopsy samples and also in tumor associated normal tissue (TANT) samples derived from glioblastoma patients, which revealed significantly differential expression of these genes. To validate our findings, we performed a comprehensive integrated analysis of transcriptomic and proteomic profiling of respective genes by retrieving GBM RNA-sequence data from Genome Atlas Databases. GO and KEGG analysis revealed enrichment in disease-related pathways, such as JAK/STAT pathway activation, which were associated with GBM progression. We further performed computational docking analysis of potential drug candidate Nisin against GCSF, and the results were validated in vitro through cytotoxic activity assay using a human glioblastoma cell line SF-767 in a dose-dependent manner. Our comprehensive analysis reveals that GCSF augments glioma progression, and its blockade with anticancer bacteriocin peptide Nisin can potentially inhibit the growth and metastasis of GBM.

Glioblastoma multiforme (GBM) is the most prevalent brain malignancy in adults. However, molecular mechanisms, which leads to GBM have not been completely elucidated. Granulocyte colony-stimulating factor (GCSF), Granulocyte colony-stimulating factor receptor GCSFR, and Signal transducers and activators of transcription 3 (STAT3) have been involved in the occurrence and development of various cancers, but their role in GBM is little known. Herein, we have investigated the gene and protein expression of GCSF, GCSFR, and STAT3 in 21 tissue biopsy samples and also in tumor associated normal tissue (TANT) samples derived from glioblastoma patients, which revealed significantly differential expression of these genes. To validate our findings, we performed a comprehensive integrated analysis of transcriptomic and proteomic profiling of respective genes by retrieving Glioblastoma multiforme RNA-sequence data from Genome Atlas Databases. GO and KEGG analysis revealed enrichment in disease-related pathways, such as JAK/STAT pathway activation, which were associated with GBM progression. We further performed computational docking analysis of potential drug candidate Nisin against GCSF, and the results were validated in vitro through cytotoxic activity assay using a human glioblastoma cell line SF-767 in a dose-dependent manner. Our comprehensive analysis reveal that GCSF augments glioma progression, and its blockade with anticancer bacteriocin peptide Nisin can potentially inhibit the growth and metastasis of glioblastoma.

Mpox (previously named Monkeypox) is one of the neglected viral infectious diseases that remained silent for a long period before finally emerging as a threat to the healthcare system in endemic regions of the world in recent years. It has been mostly centered in African countries but has now been reported in other non-endemic regions as well. While keeping a strict eye on COVID pandemic handling, there is a need to remain concerned and alert about viral threats such as Mpox infections in the future. This situation has altered the healthcare system of endemic regions, including Pakistan, to stay vigilant against the expected Mpox outbreaks in the coming months. Though no specific cases have been reported in Pakistan, the healthcare system needs to take mitigation measures to tackle an expected threat before it arrives. This is important in order to avoid another major shock to the health care system of Pakistan. Moreover, since no specific treatment is available for Mpox, we can only rely upon mitigation measures, involving preventive and treatment strategies devised around some already in-use antiviral agents against Mpox viruses. Moreover, there is an imperative need to proactively prepare the healthcare system against Mpox outbreaks, spread awareness, and involve the public in a participatory approach to stay well prepared against any such infection. Moreover, there is a need to utilize financial sources, aids, and funds wisely, to create awareness in the public about such expected healthcare outbreaks in the future.

Progressive age is the single major risk factor for neurodegenerative diseases. Cellular aging markers during Parkinson’s disease (PD) have been implicated in previous studies, however the majority of studies have investigated the association of individual cellular aging hallmarks with PD but not jointly. Here, we have studied the association of PD with three aging hallmarks (telomere attrition, mitochondrial dysfunction, and cellular senescence) in blood and the brain tissue. Our results show that PD patients had 20% lower mitochondrial DNA copies but 26% longer telomeres in blood compared to controls. Moreover, telomere length in blood was positively correlated with medication (Levodopa Equivalent Daily Dose, LEDD) and disease duration. Similar results were found in brain tissue, where patients with Parkinson’s disease (PD), Parkinson’s disease dementia (PDD) and Dementia with Lewy Bodies (DLB) showed (46–95%) depleted mtDNA copies, but (7–9%) longer telomeres compared to controls. In addition, patients had lower mitochondrial biogenesis (PGC-1α and PGC-1β) and higher load of a cellular senescence marker in postmortem prefrontal cortex tissue, with DLB showing the highest effect among the patient groups. Our results suggest that mitochondrial dysfunction (copy number and biogenesis) in blood might be a valuable marker to assess the risk of PD. However, further studies with larger sample size are needed to evaluate these findings.