Ankit Gupta

Introduction: Paediatric sepsis remains a formidable challenge in global child health, contributing significantly to morbidity and mortality, especially in resource-constrained settings. Accurate early prognostication of disease trajectory is imperative for effective triage and timely intervention; however, existing biomarkers and clinical scoring systems are often limited by insufficient specificity, operational complexity or limited applicability across diverse clinical settings. One potential avenue for improving early prognostication is investigating novel biomarkers. Cereblon (CRBN), a multifunctional protein implicated in immune modulation, has shown variable associations with outcomes in adult sepsis, while its role in paediatric sepsis remains unexplored. This study aims to assess the potential of CRBN expression as an early prognostic biomarker in paediatric sepsis, thereby informing clinical decision-making and improving patient outcomes. Methods and analysis: This prospective observational paediatric intensive care unit (PICU)-based study will enrol 240 children aged 2 months to 15 years with sepsis using a consecutive non-probability sampling method. Pediatric Sequential Organ Failure Assessment (pSOFA) scores will be recorded at admission and at 24 hours. Blood samples will be collected at admission to assess CRBN mRNA expression by quantitative real-time PCR and protein levels by ELISA. The primary outcome will be the correlation of CRBN with 28-day mortality. Secondary outcomes will assess its correlation with the pSOFA score, C-reactive protein, duration of mechanical ventilation and duration of PICU stay. CRBN levels will be compared between survivors and non-survivors, and correlations with pSOFA score, inflammatory markers and clinical outcomes will be assessed using Pearson’s or Spearman’s tests as appropriate. Prognostic performance will be assessed using Receiver operating characteristics (ROC) curve and area under curve (AUC) value with Youden-derived cut-offs. Multivariable regression will be performed to adjust for key confounders. Ethics and dissemination: Ethical approval was obtained from the Institutional Ethics Committee, All India Institute of Medical Sciences, Raebareli, prior to study initiation (IEC Code-2024-4-EMP-EXP-9). Written informed consent will be obtained from parents or guardians. The study findings will be disseminated through peer-reviewed publications and scientific conferences. The reporting of study results will adhere to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines to ensure transparency and completeness.

The extraction of extracellular RNA from human serum is challenging due to its low abundance, high degree of fragmentation, and susceptibility to rapid degradation. These limitations are further compounded in pediatric research, where low blood sample volume is a major, frequent constraint, necessitating highly sensitive and efficient RNA extraction methods suitable for minimal‐input samples. Existing protocols use TRIzol or commercial kits, which are primarily optimized for the enrichment of small RNAs rather than for the comprehensive recovery of extracellular RNA. Here, we present an optimized protocol for efficient isolation of extracellular RNA from human serum, using a modified TRIzol‐based method following chloroform phase separation. We processed serum samples from pediatric patients using TRI‐Reagent‐based lysis and chloroform phase separation, and then compared the results of subsequent isopropanol precipitation (protocol 1) and silica‐column‐based purification (protocol 2). Protocol 2 significantly improved RNA yield (5‐fold), purity ( A ₂₆₀ / A ₂₈₀ and A ₂₆₀ / A ₂₃₀ ratios), and integrity compared to protocol 1, as confirmed by agarose gel electrophoresis. The extracted RNA was successfully utilized for cDNA synthesis and qRT‐PCR amplification of the human CRBN gene, demonstrating its suitability for downstream applications. Protocol 2 provides a rapid, cost‐effective, and robust approach for isolating extracellular RNA from serum, validated primarily for RT‐PCR‐based applications, yielding RNA of improved quantity and purity suitable for biomarker analysis and gene expression studies from minimal‐input clinical samples. © 2026 Wiley Periodicals LLC. Basic Protocol : Optimized isolation of extracellular RNA from human serum using TRIzol‐based lysis and chloroform phase separation Support Protocol 1 : Isopropanol‐precipitation‐based recovery of extracellular RNA from aqueous phase Support Protocol 2 : Silica‐column‐based purification of extracellular RNA for enhanced yield and purity Support Protocol 3 : cDNA synthesis and qRT‐PCR analysis of extracellular RNA.

Insulin resistance arises from impaired insulin signaling, leading to reduced glucose uptake and the progression of type 2 diabetes mellitus. Experimental evidence indicates that lithium modulates insulin signaling pathways; however, its role in the development of insulin resistance remains poorly defined. Anthropometric, biochemical, and lifestyle data were collected. IR was assessed via HOMA-IR, and insulin processing/clearance was evaluated by insulin to c-peptide ratio (ICPR). Univariate and multivariate logistic regression identified independent predictors of IR. ROC analysis assessed the diagnostic performance of Li. Linear regression examined associations between biochemical parameters and IR. A cross-sectional study was conducted among 211 adults with T2D attending AIIMS Raebareli; of these, 172 participants with complete data constituted the final analytical cohort, including 122 (70.9%) with insulin resistance (IR). The IR group exhibited significantly higher fasting and postprandial glucose, insulin, C-peptide, ICPR, total cholesterol, and TyG index, and lower serum lithium levels than the non-IR group. Multivariate logistic regression analyses identified ICPR, TyG index, and serum lithium (OR = 3.69, 95% CI: 1.190-11.437, P = 0.024) as independent predictors of IR. ROC analysis showed that a lithium cutoff of 0.1 mmol/L predicted IR, with 81.9% sensitivity and 56.5% specificity (AUC = 0.863). Lower lithium levels were associated with higher insulin, C-peptide, and ICPR, and linear regression revealed a significant inverse association between lithium and HOMA-IR (β = -0.233, p = 0.007). Lower serum lithium levels independently predict insulin resistance in T2DM, alongside ICPR and TyG index. This association with hyperinsulinemia and impaired insulin processing/clearance suggests a potential mechanistic role for lithium in metabolic dysregulation.

Malaria pathogenesis is driven by extensive remodeling of infected erythrocytes mediated by parasite-exported proteins; however, many components of this export machinery remain uncharacterized. In this study, we investigated an unannotated Plasmodium falciparum exported protein, PfExP (PF3D7_1149100), to determine its localization, membrane association, and interaction network. Sequence and phylogenetic analyses revealed that PfExP is conserved exclusively within the Laverania subgenus and contains a canonical PEXEL motif along with a conserved α-helical N-terminal region. Structural prediction using AlphaFold indicated a bipartite structure, consisting of a structured N-terminal domain and a disordered C-terminal tail. A transgenic parasite line expressing HA-tagged PfExP was generated to experimentally characterize PfExP. Immunofluorescence analysis demonstrated that PfExP is exported beyond the parasite boundary and localizes to punctate structures within the host erythrocyte, consistent with Maurer’s clefts. Membrane fractionation assays revealed that PfExP functions as a peripheral membrane protein, supporting its transient association with host cell membranes. Co-immunoprecipitation coupled with LC–MS/MS identified interactions with key exported and host cell remodeling proteins, including KAHRP, REX2, RESA, and ETRAMP2, as well as the PTEX component HSP101. These findings place PfExP within a network of proteins involved in trafficking and erythrocyte modification. Collectively, our results suggest that PfExP is a Laverania-specific, peripherally associated exported protein that likely functions as a scaffold or adaptor in protein trafficking pathways at Maurer’s clefts. This study provides novel insights into the molecular mechanisms underlying host cell remodeling and highlights PfExP as a promising target for future functional and therapeutic investigations.

Background The amplification of eukaryotic genes, which contain introns for expression purposes, is very tedious and expensive. The only available lab method is through cDNA synthesis, which has a very low success rate in generating a pure gene fragment lacking any introns. Methods The current paucity of any alternative lab method directed us to devise a strategy to amplify a P. falciparum RPL12mito gene (PfRPL12mito) containing an intron through three sequential PCR steps using gDNA and primers with a minimum 15 bp 5′-overhang for the full-length protein expression. Results Sanger sequencing confirmed the absence of an intronic segment and a continuous exonic region in the amplified gene fragment. The gene was successfully expressed as recombinant protein with a 6xHis tag. Conclusions This method provides a novel and cost-effective approach to amplify intron-containing eukaryotic genes, including those from the malaria parasite, directly from genomic DNA for expression purposes.

Malaria parasites increase their host erythrocyte’s permeability to obtain essential nutrients from plasma and facilitate intracellular growth. In the human Plasmodium falciparum pathogen, this increase is mediated by the plasmodial surface anion channel (PSAC) and has been linked to CLAG3, a protein integral to the host erythrocyte membrane and encoded by a member of the conserved clag multigene family. Whether paralogs encoded by other clag genes also insert at the host membrane is unknown; their contributions to PSAC formation and other roles served are also unexplored. Here, we generated transfectant lines carrying epitope-tagged versions of each CLAG. Each paralog is colocalized with CLAG3, with concordant trafficking via merozoite rhoptries to the host erythrocyte membrane of newly invaded erythrocytes. Each also exists within infected cells in at least two forms: an alkaline-extractable soluble form and a form integral to the host membrane. Like CLAG3, CLAG2 has a variant region cleaved by extracellular proteases, but CLAG8 and CLAG9 are protease resistant. Paralog knockout lines, generated through CRISPR/Cas9 transfection, exhibited uncompromised growth in PGIM, a modified medium with higher physiological nutrient levels; this finding is in marked contrast to a recently reported CLAG3 knockout parasite. CLAG2 and CLAG8 knockout lines exhibited compensatory increases in the transcription of the remaining clags and associated rhoph genes, yielding increased PSAC-mediated uptake for specific solutes. We also report on the distinct transport properties of these knockout lines. Similar membrane topologies at the host membrane are consistent with each CLAG paralog contributing to PSAC, but other roles require further examination.

Malaria parasites increase their host erythrocyte’s permeability to obtain essential nutrients from plasma and facilitate intracellular growth. In the human P. falciparum pathogen, this increase is mediated by the plasmodial surface anion channel (PSAC) and has been linked to CLAG3, as encoded by one member of the conserved clag multigene family. Whether paralogs encoded by other clag genes also contribute to PSAC formation or serve unrelated roles remains unknown. Here, we generated transfectant lines carrying epitope-tagged versions of each CLAG. Each paralog colocalized with CLAG3, with concordant trafficking via merozoite rhoptries to the host erythrocyte membrane of newly-invaded erythrocytes. Each also exists within infected cells in at least two forms: an alkaline-extractable soluble form and a form integral to the host membrane. Like CLAG3, CLAG2 has a variant region cleaved by extracellular protease but CLAG8 and CLAG9 are protease-resistant. Paralog knockout lines, generated through CRISPR/Cas9 transfection, exhibited uncompromised growth in PGIM, a modified medium with more physiological nutrient levels; this finding is in marked contrast to a recently reported CLAG3 knockout parasite. CLAG2 and CLAG8 knockout lines exhibited compensatory increases in transcription of remaining clags and associated rhoph genes, yielding increased PSAC-mediated uptake to specific solutes. We also report distinct transport properties of these knockout lines, suggesting partially overlapping roles of CLAG paralogs in PSAC activity. We propose that the nutrient channel pore is formed by a higher order assembly of CLAG proteins at the host membrane.

Chronic exposure to fluoride through drinking water has been linked to insulin resistance and resulting type 2 diabetes mellitus (T2DM). Here, we aim to study the impact of water fluoride levels on blood glucose and vitamin D levels. A hospital-based study was conducted on diabetic patients (n = 303) at All India Institute of Medical Sciences (AIIMS), Raebareli outstation patient department (OPD). The relationship between vitamin D or fasting blood glucose levels (BGLs) with water fluoride levels was estimated through Spearman's rank correlation. We found a significant negative correlation between water fluoride and vitamin D levels [rs = −0.777, p-value < 0.001] and a positive correlation between water fluoride and fasting BGLs [rs = 0.178, p-value 1.5 mg/L) had higher odds of severe vitamin D deficiency (odds ratio: 5.07, 95% CI: 1.9–13.2, p-value = 0.0009). The results demonstrate that vitamin D deficiency and fasting BGLs are significantly associated with water fluoride levels. This study signifies the role of fluoride toxicity in poor glycemic control and derived vitamin D deficiency. Vitamin D supplementation and the application of standard household water purification devices are recommended to tackle vitamin D deficiency in fluoride-endemic areas.

Background: Anemia is a most common health issue in Indian population mainly due to nutritional deficiencies. A number of factors contribute in anemia which is further categorized based on various parameters in hematological indices and microscopic examination. Methods: A retrospective study has been carried out on the patients (n = 1332) in a retrospective study on the patients (n = 1332) in Raebareli district to determine prevalence of anemia and subsequent categorization into normocytic, microcytic and macrocytic based on complete blood count (CBC) profile and blood smear. Results: This study demonstrates 15.54% patients were diagnosed for anemia and more than half of the total anemic patients (55.07%) were found to be suffered from macrocytosis which was found significantly more prevalent among male (65.17%) compare to female (38.38%) patients. Conclusions: Several factors may contribute to macrocytosis among male population including alcoholism in Raebareli region. However, female patients were found almost equally susceptible to all three forms of anemia: normocytic, microcytic and macrocytic. This study provides a key insight into prevalence and possible causes of different types of anemia in this region which may be useful for implementation of government health programs to counter this problem and provide better treatment plans.