Heterotypic electrostatic interactions control complex phase separation of tau and prion into multiphasic condensates and co-aggregates.

Biomolecular condensates formed via phase separation of proteins and nucleic acids are thought to perform a wide range of critical cellular functions by maintaining spatiotemporal regulation and organizing intracellular biochemistry. However, aberrant phase transitions are implicated in a multitude of human diseases. Here, we demonstrate that two neuronal proteins, namely tau and prion, undergo complex coacervation driven by domain-specific electrostatic interactions to yield highly dynamic, mesoscopic liquid-like droplets. The acidic N-terminal segment of tau interacts electrostatically with the polybasic N-terminal intrinsically disordered segment of the prion protein (PrP). We employed a unique combination of time-resolved tools that encompass several orders of magnitude of timescales ranging from nanoseconds to seconds. These studies unveil an intriguing symphony of molecular events associated with the formation of heterotypic condensates comprising ephemeral, domain-specific, short-range electrostatic nanoclusters. Our results reveal that these heterotypic condensates can be tuned by RNA in a stoichiometry-dependent manner resulting in reversible, multiphasic, immiscible, and ternary condensates of different morphologies ranging from core-shell to nested droplets. This ternary system exhibits a typical three-regime phase behavior reminiscent of other membraneless organelles including nucleolar condensates. We also show that upon aging, tau:PrP droplets gradually convert into solid-like co-assemblies by sequestration of persistent intermolecular interactions. Our vibrational Raman results in conjunction with atomic force microscopy and multi-color fluorescence imaging reveal the presence of amorphous and amyloid-like co-aggregates upon maturation. Our findings provide mechanistic underpinnings of overlapping neuropathology involving tau and PrP and highlight a broader biological role of complex phase transitions in physiology and disease.

An intrinsically disordered pathological prion variant Y145Stop converts into self-seeding amyloids via liquid-liquid phase separation.

Biomolecular condensation via liquid-liquid phase separation of intrinsically disordered proteins/regions (IDPs/IDRs) along with other biomolecules is proposed to control critical cellular functions, whereas aberrant phase transitions are associated with a range of neurodegenerative diseases. Here, we show that a disease-associated stop codon mutation of the prion protein (PrP) at tyrosine 145 (Y145Stop), resulting in a truncated, highly disordered, N-terminal IDR, spontaneously phase-separates into dynamic liquid-like droplets. Phase separation of this highly positively charged N-terminal segment is promoted by the electrostatic screening and a multitude of weak, transient, multivalent, intermolecular interactions. Single-droplet Raman measurements, in conjunction with an array of bioinformatic, spectroscopic, microscopic, and mutagenesis studies, revealed a highly mobile internal organization within the liquid-like condensates. The phase behavior of Y145Stop is modulated by RNA. Lower RNA:protein ratios promote condensation at a low micromolar protein concentration under physiological conditions. At higher concentrations of RNA, phase separation is abolished. Upon aging, these highly dynamic liquid-like droplets gradually transform into ordered, ß-rich, amyloid-like aggregates. These aggregates formed via phase transitions display an autocatalytic self-templating characteristic involving the recruitment and binding-induced conformational conversion of monomeric Y145Stop into amyloid fibrils. In contrast to this intrinsically disordered truncated variant, the wild-type full-length PrP exhibits a much lower propensity for both condensation and maturation into amyloids, hinting at a possible protective role of the C-terminal domain. Such an interplay of molecular factors in modulating the protein phase behavior might have much broader implications in cell physiology and disease.

Integrated single-cell transcriptome analysis of CD34 + enriched leukemic stem cells revealed intra- and inter-patient transcriptional heterogeneity in pediatric acute myeloid leukemia.

To gain insights into the idiosyncrasies of CD34 + enriched leukemic stem cells, we investigated the nature and extent of transcriptional heterogeneity by single-cell sequencing in pediatric AML. Whole transcriptome analysis of 28,029 AML single cells was performed using the nanowell cartridge-based barcoding technology. Integrated transcriptional analysis identified unique leukemic stem cell clusters of each patient and intra-patient heterogeneity was revealed by multiple LSC-enriched clusters differing in their cell cycle processes and BCL2 expression. All LSC-enriched clusters exhibited gene expression profile of dormancy and self-renewal. Upregulation of genes involved in non-coding RNA processing and ribonucleoprotein assembly were observed in LSC-enriched clusters relative to HSC. The genes involved in regulation of apoptotic processes, response to cytokine stimulus, and negative regulation of transcription were upregulated in LSC-enriched clusters as compared to the blasts. Validation of top altered genes in LSC-enriched clusters confirmed upregulation of TCF7L2, JUP, ARHGAP25, LPAR6, and PRDX1 genes, and serine/threonine kinases (STK24, STK26). Upregulation of LPAR6 showed trend towards MRD positive status (Odds ratio = 0.126; 95% CI = 0.0144-1.10; p = 0.067) and increased expression of STK26 significantly correlated with higher RFS (HR = 0.231; 95% CI = 0.0506-1.052; p = 0.04). Our findings addressed the inter- and intra-patient diversity within AML LSC and potential signaling and chemoresistance-associated targets that warrant investigation in larger cohort that may guide precision medicine in the near future.

Study of Thyroid Profile in Patients of Type II Diabetes Mellitus.

INTRODUCTION: Thyroid hormones play an indispensable role in various metabolic process in our body. Excess or deficiency of either insulin or thyroid hormones can result in functional abnormalities of one another. Though it has been since long recognised that diabetes and thyroid disorder share a complex interplay of factors, the present study is planned to study the thyroid profile in patients of type 2 diabetes mellitus. MATERIALS: To study the Thyroid profile in patients of Type 2 Diabetes. Observational, single centre, case control study. All patients of either gender, aged 30 to 60 years, with Type 2 diabetes mellitus were included in the study. RESULT: it was found that only 61% of the patients with Diabetes were euthyroid. Amongst the patients with thyroid disorder, hypothyroidism was more prevalent than hyperthyroidism 31% and 10% respectively. On further assessment with respect to primary and subclinical thyroid disorder, it was found that subclinical hypothyroidism (21%) was more common as compared to Primary hypothyroidism (10%) and subclinical hyperthyroidism (5%) was more common than hyperthyroidism (3%) in the patients with diabetes. CONCLUSION: The study concluded that screening for thyroid dysfunction among patients with diabetes mellitus should be routinely performed, to recognize these dysfunctions early. This will ensure timely therapeutic intervention and in turn will improve the management of both diseases leading to better quality of life and decreasing the burden of complications. References American Diabetes Association. 2. Classification and Diagnosis of Diabetes: Standards of Medical Care in Diabetes-2018. Diabetes Care 2018;41(Suppl 1):S13-S27. Akbar DH, Ahmed MM, Al-Mughales J. Thyroid dysfunction and thyroid autoimmunity in Saudi type 2 diabetics. Acta Diabetol 2006;43(1):14-18.

Understanding Childhood Constipation through the Prism of the Caretaker.

Aims: Childhood constipation is presenting with increasing frequency at pediatric surgical clinics. The caregiver's role in prevention and management is pivotal. This study aimed at determining mothers' knowledge, attitudes, and practices with regard to childhood constipation and the association of these with demographic variables. Materials and Methods: This was a survey-based descriptive study conducted at a tertiary care hospital in South India. Randomly selected mothers of children aged 1-10 years consulting for any problem other than constipation were included in the study. Data collection was done by means of a pretested and prevalidated questionnaire. Results: There were 169 mothers with a median age of 30 years. Over half were homemakers and of a rural background. Urban mothers scored better than their rural counterparts in the attitude section (P = 0.034). Mothers with greater knowledge had better attitude (P = 0.001) and practice (P = 0.020) scores. Those with higher attitude scores also fared better in the practice section (P = 0.04). Conclusions: Knowledge, attitude and practice concerning childhood constipation are connected to each other. South Indian mothers are sufficiently aware of the nuances surrounding childhood constipation, but focused large-scale outreach programs and health education are necessary to bridge the gaps.

Endothelial colony forming cells' tetrahydrobiopterin level in coronary artery disease patients and its association with circulating endothelial progenitor cells.

Endothelial colony forming cells (ECFCs) participate in neovascularization. Endothelial nitric oxide synthase (eNOS) derived NO· helps in homing of endothelial progenitor cells (EPCs) at the site of vascular injury. The enzyme cofactor tetrahydrobiopterin (BH4) stabilizes the catalytic active state of eNOS. Association of intracellular ECFCs biopterins and ratio of reduced to oxidized biopterin (BH4:BH2) with circulatory EPCs and ECFCs functionality have not been studied. We investigated ECFCs biopterin levels and its association with circulatory EPCs as well as ECFCs proliferative potential in terms of day of appearance in culture. Circulatory EPCs were enumerated by flowcytometry in 53 coronary artery disease (CAD) patients and 42 controls. ECFCs were cultured, characterized, and biopterin levels assessed by high performance liquid chromatography. Appearance of ECFCs' colony and their number were recorded. Circulatory EPCs were significantly lower in CAD and ECFCs appeared in 56% and 33% of CAD and control subjects, respectively. Intracellular BH4 and BH4:BH2 were significantly reduced in CAD. BH4:BH2 was positively correlated with circulatory EPCs (p = 0.01), and negatively with day of appearance of ECFCs (p = 0.04). Circulatory EPCs negatively correlated with ECFCs appearance (p = 0.02). These findings suggest the role of biopterins in maintaining circulatory EPCs and functional integrity of ECFCs.

The Syringe Technique for Ultrasound-Guided Hydrostatic Intussusception Reduction.

Background: Ultrasound-guided hydrostatic reduction (UGHR) is a well accepted and widely used method of paediatric intussusception reduction, with the saline drip technique being the most commonly employed. Aims and Objectives: In this study we aimed to assess the outcomes of a novel technique of UGHR. Materials and Methods: Data was obtained from a 15 year retrospective chart review of paediatric intussusceptions. Following resuscitation, UGHR was performed for uncomplicated intussusceptions using a 50cc syringe to infuse saline into the colon. It was performed in the ultrasound suite without sedation and time taken was monitored. A maximum of 3 attempts were done to achieve reduction. Results: UGHR was attempted in 66 of 93 intussusceptions. The commonest type of intussusception was ileo-colic(91%) and the commonest symptom was vomiting(70%). Surgery was performed only when there was shock, peritonitis or repeated failed reductions. The median time taken for reduction was 4.9 minutes. The overall success rate was 83% with 89% of these requiring only a single attempt. There were no deaths or procedure related complications. Conclusions: The syringe technique for intussusception reduction is a safe, effective, and time-saving technique. Additionally, it offers the advantages of simplicity and rapidity of reduction and in experienced hands may not require pressure monitoring.

Critical evaluation of the utility of pre- and post-therapy immunophenotypes in assessment of measurable residual disease in B-ALL.

Measurable residual disease (MRD) is an important parameter to predict outcome in B-cell acute lymphoblastic leukemia (B-ALL). Two different approaches have been used for the assessment of MRD by multiparametric flow cytometry that include the "Leukemia Associated Aberrant Immunophenotype (LAIP)" and "Difference from Normal (DFN)" approach. In this retrospective study, we analyzed 539 samples obtained from 281 patients of which 258 were paired samples and the remaining 23 samples were from post-induction time point only, to explore the utility of baseline immunophenotype (IPT) for MRD assessment. Single-tube 10-color panel was used both at diagnosis and MRD time points. Out of 281 patients, 31.67% (n = 89) were positive and 68.32% (n = 192) were negative for MRD. Among 258 paired diagnostic and follow-up samples, baseline IPT was required in only 9.31% (24/258) cases which included cases with hematogone pattern and isolated dim to negative CD10 expression patterns. Comparison of baseline IPT with post-induction MRD positive samples showed a change in expression of at least one antigen in 94.04% cases. Although the immunophenotypic change in expression of various antigens is frequent in post-induction samples of B-ALL, it does not adversely impact the MRD assessment. In conclusion, the baseline IPT is required in less than 10% of B-ALL, specifically those with hematogone pattern and/or dim to negative expression of CD10. Hence, a combination of DFN and LAIP approach is recommended for reliable MRD assessment.

Cystine Urolithiasis in Early Childhood.

Urolithiasis in children is rare with reported incidence of 1.8 per 1000 children. A metabolic cause is identifiable in 40-50% of children with stones and is considered when multiple, recurrent and bilateral. Cystinuria is an important preventable cause of urolithiasis. We present an infant with recurrent gross hematuria due to cystine urolithiasis for its rarity.

Immunophenotypic analysis of T-acute lymphoblastic leukemia. A CD5-based ETP-ALL perspective of non-ETP T-ALL.

T-cell antigens [CD5,CD1a,CD8] define early T-cell precursor acute lymphoblastic leukemia (ETP-ALL). To understand immature T-ALL of which ETP-ALL is part, we used these antigens to subcategorize non-ETP T-ALL for examining expression of myeloid/stem cell antigens (M/S) and clinical features. Using CD5 (+/-) to start categorization, we studied 69 routinely immunophenotyped patients with T-ALL. CD5(-) was a homogenous (CD8,CD1a)(-) M/S(+) ETP-ALL group (n = 9). CD5(+) cases were (CD8,CD1a)(-) pre-T-ALL (n = 22) or (CD8,CD1a)(+) (n = 38) thymic/cortical T-ALL; M/S(+) 20/22 (90.91%) in former and 22/38 (57.89%) in latter (P = 0.007). ETP- and pre-T-ALL together (CD1a(-) ,CD5(-/+) immature T-ALL group) were nearly always M/S(+) (29/31; 93.55%). In multivariate analysis, only ETP-ALL predicted poor overall survival (P = 0.02). We conclude (i) CD5 negativity in T-ALL almost always means ETP-ALL. CD1a and CD8 negativity, as much as CD5, marks immaturity in T-ALL, and the CD5(+/-) /CD1a(-) /CD8(-) immature T-ALL group needs further study to understand the biology of the T-ALL-myeloid interface. (ii) ETP-ALL patients may be pre-T-ALL if CD2(+) ; CD2(+) , conversely, CD5(-) /CD1a(-) /CD8(-) pre-T ALL patients are ETP-ALL. (iii) Immunophenotypic workup of T-ALL must not omit CD1a, CD5, CD8 and CD2, and positivity of antigens should preferably be defined as recommended for ETP-ALL, so that this entity can be better evaluated in future studies of immature T-ALL, a group to which ETP-ALL belongs. (iv) ETP-ALL has poor prognosis.

Obstructing urethral membrane in a girl: a rare cause of female bladder outlet obstruction.

Bladder outlet obstruction is known to produce back pressure changes on the urinary tract with devastating sequelae more often than not. Among the causes, posterior urethral valve, which is the most common, is documented to occur exclusively in males. Female posterior urethral valves have been reported in the past in less than 25 cases in existing literature. We discuss the case of a female toddler who presented with symptoms of straining to void and recurrent urinary tract infections. On evaluation, she was found to have an obstructing urethral membrane causing bladder outlet obstruction, which was endoscopically ablated with success.

Bridging the Gap: A Strategic Approach to Upscale Knowledge Among Diverse Healthcare Providers for Effective Tuberculosis Management in Gujarat, India.

INTRODUCTION: Tuberculosis (TB) remains a global health challenge, particularly in low- and middle-income countries. Knowledge gaps among healthcare providers (HCPs) significantly impact TB management, hindering timely care-seeking and effective interventions. OBJECTIVE: The primary objective was to assess knowledge gaps among 3086 HCPs engaged in the National Tuberculosis Elimination Program (NTEP) implementation in Gujarat, India. The study provided a platform to develop and implement cadre-specific training modules to address identified knowledge deficiencies and enhance TB management. METHODOLOGY: The study was conducted in two phases. Phase one was designed as a cross-sectional assessment to identify the knowledge gaps. Phase two involved the development of cadre-specific training modules based on identified deficiencies in the knowledge, crafted with collaboration from an expert panel. The training impact will be evaluated after completion of the training of all cadres through a comprehensive assessment. RESULTS: Out of 3086 assessed HCPs, 26% scored below the passing benchmark, revealing significant knowledge gaps. The variations were observed among and within the same cadres, with the accredited social health activists (ASHAs) and community health workers showing higher proficiency while pharmacists and medical officers showed lower proficiency. The cadre-specific training modules and training cascade were designed to address these gaps and improve TB-related knowledge and skills. CONCLUSION: The study underscores the critical need for targeted interventions to address knowledge gaps among HCPs involved in TB control. The customized HCP-specific training programs are recommended to enhance knowledge, improve TB management, and contribute to national TB elimination goals.

Predictors for Concurrent Diabetes in Tuberculosis Patients. Perspectives from Two Mining Districts of Eastern Tribal State Jharkhand, in India.

Background: Tuberculosis and diabetes both diseases are present in large numbers in the country and we are major contributors to both globally. With the objective to understand the various traits of patients having both tuberculosis and diabetes and to ascertain various possible predictors for such occurrence based on the public health database we carried out this study. We seek answers to questions like they have any effects? Are they having any additive role to play? Methods: One-year data from the NIKSHAY portal of both districts were analyzed to look for possible associations and other variable traits. Data were analyzed using standard methods to express data in frequency and percentage. Chi-square test was used to establish association, while step-wise approach was used to calculate univariate and multivariate logistic regression analysis for knowing various predictors. P-value of <0.05 was considered statistically significant. Results: Concurrent diabetes in tuberculosis patients was close to 294 (6%) in the 4933 individuals. In total, 65.2% of the study population were male. Diagnosis of tuberculosis was made most of the time by chest X-ray (49.4%) followed by Microscopy ZN staining and cartridge-based nucleic acid amplification test (CBNAAT). Death was more among diabetics (4.4%) as compared to nondiabetics (3.5%). Conclusion: Diabetes is increasing in tuberculosis patients; improvement in data quality is needed. More research is required to reveal various other reasons that make tuberculosis patients more prone to develop diabetes.

Masquerading fungal bowel mass in an adolescent: a clinicopathological challenge.

Abdominal masses clubbed with weight loss in the paediatric age group can raise hairs, especially since malignancy is a differential. We present the case of an early adolescent male who presented with abdominal pain and was found to have a mass mimicking a malignancy. The resected surgical specimen revealed entomophthoromycosis of the jejunum and he made a complete recovery following surgery and adjuvant itraconazole. The diagnosis of a fungal aetiology in these cases requires a high index of suspicion and background knowledge of the risk factors, disease occurrence and mode of presentation. Gastrointestinal entomophthoromycosis has an impressive potential for cure if promptly diagnosed and treated.

Hydrogen-Bonded Network of Water in Phase-Separated Biomolecular Condensates.

Biomolecular condensates formed via phase separation of intrinsically disordered proteins/regions (IDPs/IDRs) and nucleic acids are associated with cell physiology and disease. Water makes up for ∼60-70% of the condensate volume and is thought to influence the complex interplay of chain-chain and chain-solvent interactions, modulating the mesoscale properties of condensates. The behavior of water in condensates and the key roles of protein hydration water in driving the phase separation remain elusive. Here, we employ single-droplet vibrational Raman spectroscopy to illuminate the structural redistribution within protein hydration water during the phase separation of neuronal IDPs. Our Raman measurements reveal the changes in the water hydrogen bonding network during homotypic and heterotypic phase separation governed by various molecular drivers. Such single-droplet water Raman measurements offer a potent generic tool to unmask the intriguing interplay of sequence-encoded chain-chain and chain-solvent interactions governing macromolecular phase separation into membraneless organelles, synthetic condensates, and protocells.

Immunophenotypic characterization of leukemic stem cells in acute myeloid leukemia using single tube 10-colour panel by multiparametric flow cytometry: Deciphering the spectrum, complexity and immunophenotypic heterogeneity.

INTRODUCTION: Despite extensive research, comprehensive characterization of leukaemic stem cells (LSC) and information on their immunophenotypic differences from normal haematopoietic stem cells (HSC) is lacking. Herein, we attempted to unravel the immunophenotypic (IPT) characteristics and heterogeneity of LSC using multiparametric flow cytometry (MFC) and single-cell sequencing. MATERIALS AND METHODS: Bone marrow aspirate samples from patients with acute myeloid leukaemia (AML) were evaluated using MFC at diagnostic and post induction time points using a single tube-10-colour-panel containing LSC-associated antibodies CD123, CD45RA, CD44, CD33 and COMPOSITE (CLL-1, TIM-3, CD25, CD11b, CD22, CD7, CD56) with backbone markers that is, CD45, CD34, CD38, CD117, sCD3. Single-cell sequencing of the whole transcriptome was also done in a bone marrow sample. RESULTS: LSCs and HSCs were identified in 225/255 (88.2%) and 183/255 (71.6%) samples, respectively. Significantly higher expression was noted for COMPOSITE, CD45RA, CD123, CD33, and CD44 in LSCs than HSCs (p < 0.0001). On comparing the LSC specific antigen expressions between CD34+ (n = 184) and CD34- LSCs (n = 41), no difference was observed between the groups. More than one sub-population of LSC was demonstrated in 4.4% of cases, which further revealed high concordance between MFC and single cell transcriptomic analysis in one of the cases displaying three LSC subpopulations by both methods. CONCLUSION: A single tube-10-colour MFC panel is proposed as an easy and reproducible tool to identify and discriminate LSCs from HSCs. LSCs display both inter- and intra-sample heterogeneity in terms of antigen expressions, which opens the facets for single cell molecular analysis to elucidate the role of subpopulations of LSCs in AML progression.

Development and user acceptability testing of healthy heart mobile application - a tool for cardiovascular risk modification among patients with type 2 diabetes mellitus.

OBJECTIVES: Cardiovascular disease (CVD) remains the primary cause of mortality in individuals with type 2 diabetes mellitus. Digital health has quickly emerged as a technology with the ability to bridge the gap in cardiovascular disease self-management and revolutionize the way healthcare has traditionally been delivered. However, there is little data on the application of mobile technologies for cardiovascular risk reduction among diabetic patients. The current study has been constructed with this in mind. METHODS: A framework for the development of a healthy heart mobile application for CVD risk stratification and risk management among Type 2 diabetes mellitus patients was finalized after consultation with diabetologists, nutritionists, and scientists. The mobile app has three user cases: Patient login, doctor login, and admin login. A questionnaire was designed and the feedback of patients and Physicians was taken regarding the design, presentation, content, and user-friendliness of the app based on responses obtained on the questionnaire. RESULTS: The Android version of the healthy heart mobile mobile app was developed for CVD risk stratification and risk management among type 2 diabetes mellitus patients. The dashboard of the mobile app displayed the CVD risk score and category (mild, moderate, high, or very high CVD risk; which was colored coded), health tracker to monitor medication adherence, lipid profile, diabetes control, CVD risk profile and compliance with the WHO recommendations regarding diet, physical activity and addictions, User acceptability and experience were tested for the developed healthy heart mobile app among patients and physicians. The majority of the respondents graded the design, presentation, content, and user-friendliness of the app as either excellent or good. CONCLUSIONS: The mobile app for self-management and CVD risk reduction among diabetic patients was successfully developed. The paper and mobile-based CVD risk calculation and stratification were found to be a match for all the participants. The app was updated based on suggestions from the pilot study and was well-accepted by both patients and physicians.

Utility of CD229 as novel marker in measurable residual disease assessment in multiple myeloma-An evidence-based approach.

INTRODUCTION: CD229 has been found to be a useful plasma cell (PC) gating marker in multiple myeloma (MM). This study analyses the expression profile of CD229 on various bone marrow compartments namely, PC, non-PC and hematogones (HGs) using Multiparameter flow cytometry (MFC). Furthermore, it evaluates the ability of CD229 to delineate normal PC (NPC) from aberrant PC (APC) in measurable residual disease assessment (MRD) in MM. METHODS: Bone marrow aspirates from patients diagnosed with MM (per standard IMWG criteria) were collected in EDTA and processed for MFC using a single tube 14-color antibody panel as per standard operating procedure. RESULTS: A total of 74 patients with a diagnosis of MM (26 treatment naïve and 48 on therapy) were evaluated. The expression of CD229 was homogenous on both the PC and HG compartments as compared to CD138 and CD38. On comparing the expression of individual markers, it was found to be statistically significant between PC, HGs and non-PC for all three markers (p < 0.001). APC showed lower median expression of CD38 and higher median expression of CD138 and CD229 as compared to NPC and was found to be statistically significant for all markers (p < 0.001). In terms of differential expression on NPC and APC; CD38 was found to be the most aberrantly expressed (70%; 52/74) followed by CD229 (7%; 5/74) and CD138 (5%; 4/74). CONCLUSIONS: CD229 can be used for the identification of PC and due to relatively homogenous expression; it can be used as a suitable marker for targeted therapies. However, precise discrimination of NPC from APC cannot be reliably achieved with CD229, limiting its utility as a useful marker of diagnostic relevance and MRD assessment in MM.