High arsenic contamination in the breast milk of mothers inhabiting the Gangetic plains of Bihar: a major health risk to infants.

Groundwater arsenic poisoning has posed serious health hazards in the exposed population. The objective of the study is to evaluate the arsenic ingestion from breastmilk among pediatric population in Bihar. In the present study, the total women selected were n = 513. Out of which n = 378 women after consent provided their breastmilk for the study, n = 58 subjects were non-lactating but had some type of disease in them and n = 77 subjects denied for the breastmilk sample. Hence, they were selected for the women health study. In addition, urine samples from n = 184 infants' urine were collected for human arsenic exposure study. The study reveals that the arsenic content in the exposed women (in 55%) was significantly high in the breast milk against the WHO permissible limit 0.64 µg/L followed by their urine and blood samples as biological marker. Moreover, the child's urine also had arsenic content greater than the permissible limit (< 50 µg/L) in 67% of the studied children from the arsenic exposed regions. Concerningly, the rate at which arsenic is eliminated from an infant's body via urine in real time was only 50%. This arsenic exposure to young infants has caused potential risks and future health implications. Moreover, the arsenic content was also very high in the analyzed staple food samples such as rice, wheat and potato which is the major cause for arsenic contamination in breastmilk. The study advocates for prompt action to address the issue and implement stringent legislative measures in order to mitigate and eradicate this pressing problem that has implications for future generations.

Pathogenesis of Cardiomyopathy Caused by Variants in ALPK3, an Essential Pseudokinase in the Cardiomyocyte Nucleus and Sarcomere.

BACKGROUND: ALPK3 encodes α-kinase 3, a muscle-specific protein of unknown function. ALPK3 loss-of-function variants cause cardiomyopathy with distinctive clinical manifestations in both children and adults, but the molecular functions of ALPK3 remain poorly understood. METHODS: We explored the putative kinase activity of ALPK3 and the consequences of damaging variants using isogenic human induced pluripotent stem cell-derived cardiomyocytes, mice, and human patient tissues. RESULTS: Multiple sequence alignment of all human α-kinase domains and their orthologs revealed 4 conserved residues that were variant only in ALPK3, demonstrating evolutionary divergence of the ALPK3 α-kinase domain sequence. Phosphoproteomic evaluation of both ALPK3 kinase domain inhibition and overexpression failed to detect significant changes in catalytic activity, establishing ALPK3 as a pseudokinase. Investigations into alternative functions revealed that ALPK3 colocalized with myomesin proteins (MYOM1, MYOM2) at both the nuclear envelope and the sarcomere M-band. ALPK3 loss-of-function variants caused myomesin proteins to mislocalize and also dysregulated several additional M-band proteins involved in sarcomere protein turnover, which ultimately impaired cardiomyocyte structure and function. CONCLUSIONS: ALPK3 is an essential cardiac pseudokinase that inserts in the nuclear envelope and the sarcomere M-band. Loss of ALPK3 causes mislocalization of myomesins, critical force-buffering proteins in cardiomyocytes, and also dysregulates M-band proteins necessary for sarcomere protein turnover. We conclude that ALPK3 cardiomyopathy induces ventricular dilatation caused by insufficient myomesin-mediated force buffering and hypertrophy by impairment of sarcomere proteostasis.

Filamin C Cardiomyopathy Variants Cause Protein and Lysosome Accumulation.

[Figure: see text].

Forensic characterization of 124 SNPs in the central Indian population using precision ID Identity Panel through next-generation sequencing.

With the advent of next-generation sequencing technology, SNP markers are being explored as a useful alternative to conventional capillary electrophoresis-based STR typing. Low mutation rate and short-sized amplicons are added advantages of SNP markers over the STRs. However, to achieve a sufficient level of discrimination among individuals, a higher number of SNPs need to be characterized simultaneously. Hence, the NGS technique is highly useful to analyze a sufficiently higher number of SNPs simultaneously. Though the technique is in its nascent stage, an attempt has been made to assess its usability in the central Indian population by analyzing 124 SNPs (90 autosomal and 34 Y-chromosome) in 95 individuals. Various quality parameters such as locus balance, locus strand balance, heterozygosity balance, and noise level showed a good quality sequence obtained from the Ion GeneStudio S5 instrument. Obtained frequency of SNP alleles ranged from 0.001 to 0.377 in autosomal SNPs. rs9951171 was found to be the most informative SNP in the studied population with the highest PD and lowest MP value. The cumulative MP of 90 SNPs was found to be 4.76698 × 10-37. Analysis of 34 Y-chromosome SNPs reveals 11 unique haplogroups in 54 male samples with R1a1 as the most frequent haplogroup found in 22.22% of samples. Interpopulation comparison by FST analysis, PCA plot, and STRUCTURE analysis showed genetic stratification of the studied population suggesting the utility of SNP markers present in the Precision ID Identity Panel for forensic demands of the Indian population.

Myosin Sequestration Regulates Sarcomere Function, Cardiomyocyte Energetics, and Metabolism, Informing the Pathogenesis of Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is caused by pathogenic variants in sarcomere protein genes that evoke hypercontractility, poor relaxation, and increased energy consumption by the heart and increased patient risks for arrhythmias and heart failure. Recent studies show that pathogenic missense variants in myosin, the molecular motor of the sarcomere, are clustered in residues that participate in dynamic conformational states of sarcomere proteins. We hypothesized that these conformations are essential to adapt contractile output for energy conservation and that pathophysiology of HCM results from destabilization of these conformations. METHODS: We assayed myosin ATP binding to define the proportion of myosins in the super relaxed state (SRX) conformation or the disordered relaxed state (DRX) conformation in healthy rodent and human hearts, at baseline and in response to reduced hemodynamic demands of hibernation or pathogenic HCM variants. To determine the relationships between myosin conformations, sarcomere function, and cell biology, we assessed contractility, relaxation, and cardiomyocyte morphology and metabolism, with and without an allosteric modulator of myosin ATPase activity. We then tested whether the positions of myosin variants of unknown clinical significance that were identified in patients with HCM, predicted functional consequences and associations with heart failure and arrhythmias. RESULTS: Myosins undergo physiological shifts between the SRX conformation that maximizes energy conservation and the DRX conformation that enables cross-bridge formation with greater ATP consumption. Systemic hemodynamic requirements, pharmacological modulators of myosin, and pathogenic myosin missense mutations influenced the proportions of these conformations. Hibernation increased the proportion of myosins in the SRX conformation, whereas pathogenic variants destabilized these and increased the proportion of myosins in the DRX conformation, which enhanced cardiomyocyte contractility, but impaired relaxation and evoked hypertrophic remodeling with increased energetic stress. Using structural locations to stratify variants of unknown clinical significance, we showed that the variants that destabilized myosin conformations were associated with higher rates of heart failure and arrhythmias in patients with HCM. CONCLUSIONS: Myosin conformations establish work-energy equipoise that is essential for life-long cellular homeostasis and heart function. Destabilization of myosin energy-conserving states promotes contractile abnormalities, morphological and metabolic remodeling, and adverse clinical outcomes in patients with HCM. Therapeutic restabilization corrects cellular contractile and metabolic phenotypes and may limit these adverse clinical outcomes in patients with HCM.

SarcTrack.

RATIONALE: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in combination with CRISPR/Cas9 genome editing provide unparalleled opportunities to study cardiac biology and disease. However, sarcomeres, the fundamental units of myocyte contraction, are immature and nonlinear in hiPSC-CMs, which technically challenge accurate functional interrogation of contractile parameters in beating cells. Furthermore, existing analysis methods are relatively low-throughput, indirectly assess contractility, or only assess well-aligned sarcomeres found in mature cardiac tissues. OBJECTIVE: We aimed to develop an analysis platform that directly, rapidly, and automatically tracks sarcomeres in beating cardiomyocytes. The platform should assess sarcomere content, contraction and relaxation parameters, and beat rate. METHODS AND RESULTS: We developed SarcTrack, a MatLab software that monitors fluorescently tagged sarcomeres in hiPSC-CMs. The algorithm determines sarcomere content, sarcomere length, and returns rates of sarcomere contraction and relaxation. By rapid measurement of hundreds of sarcomeres in each hiPSC-CM, SarcTrack provides large data sets for robust statistical analyses of multiple contractile parameters. We validated SarcTrack by analyzing drug-treated hiPSC-CMs, confirming the contractility effects of compounds that directly activate (CK-1827452) or inhibit (MYK-461) myosin molecules or indirectly alter contractility (verapamil and propranolol). SarcTrack analysis of hiPSC-CMs carrying a heterozygous truncation variant in the myosin-binding protein C ( MYBPC3) gene, which causes hypertrophic cardiomyopathy, recapitulated seminal disease phenotypes including cardiac hypercontractility and diminished relaxation, abnormalities that normalized with MYK-461 treatment. CONCLUSIONS: SarcTrack provides a direct and efficient method to quantitatively assess sarcomere function. By improving existing contractility analysis methods and overcoming technical challenges associated with functional evaluation of hiPSC-CMs, SarcTrack enhances translational prospects for sarcomere-regulating therapeutics and accelerates interrogation of human cardiac genetic variants.

Evaluation of diallelic STR markers with inter-population allelic database for their usefulness in paternity trios in the Central Indian population.

BACKGROUND: Most of the forensic DNA laboratories have migrated to new generation STR kits of 6 dye chemistry with more than 20 autosomal STRs. The population-specific databases of such STR markers are lacking in many regions. AIM: To evaluate the effect of the inter-population database in 100 paternity trios with no inconsistencies at 23 STRs. SUBJECTS AND METHODS: 100 paternity trios were evaluated considering inter-population allelic frequency values for calculation of Combined Paternity Index (CPI) and Probability of Paternity (POP). RESULTS: No significant variation (p < 0.05) among the allele frequencies at the interpopulation level was observed. The number of obligate alleles and the likelihood of transferring obligate alleles from the putative father showed a positive correlation (p < 0.005) with Power of Discrimination (PD), Polymorphic Information Content (PIC), Power of Exclusion (PE), Paternity Index (PI), Observed and Expected Heterozygosity (Ho and He), and a statistically significant negative correlation (p < 0.005) with Matching Probability (Pm). The average Combined Paternity Index (CPI) and Probability of Paternity (POP) did not show any statistically significant difference (p < 0.05) at the interpopulation level. CONCLUSION: The allelic database showed no effect on the CPI and POP in 100 paternity trios. This suggests no urgent need for using population-specific databases for statistical evaluation of paternity trios without inconsistencies.

Expanding the clinical and genetic spectrum of ALPK3 variants: Phenotypes identified in pediatric cardiomyopathy patients and adults with heterozygous variants.

INTRODUCTION: Biallelic damaging variants in ALPK3, encoding alpha-protein kinase 3, cause pediatric-onset cardiomyopathy with manifestations that are incompletely defined. METHODS AND RESULTS: We analyzed clinical manifestations of damaging biallelic ALPK3 variants in 19 pediatric patients, including nine previously published cases. Among these, 11 loss-of-function (LoF) variants, seven compound LoF and deleterious missense variants, and one homozygous deleterious missense variant were identified. Among 18 live-born patients, 8 exhibited neonatal dilated cardiomyopathy (44.4%; 95% CI: 21.5%-69.2%) that subsequently transitioned into ventricular hypertrophy. The majority of patients had extracardiac phenotypes, including contractures, scoliosis, cleft palate, and facial dysmorphisms. We observed no association between variant type or location, disease severity, and/or extracardiac manifestations. Myocardial histopathology showed focal cardiomyocyte hypertrophy, subendocardial fibroelastosis in patients under 4 years of age, and myofibrillar disarray in adults. Rare heterozygous ALPK3 variants were also assessed in adult-onset cardiomyopathy patients. Among 1548 Dutch patients referred for initial genetic analyses, we identified 39 individuals with rare heterozygous ALPK3 variants (2.5%; 95% CI: 1.8%-3.4%), including 26 missense and 10 LoF variants. Among 149 U.S. patients without pathogenic variants in 83 cardiomyopathy-related genes, we identified six missense and nine LoF ALPK3 variants (10.1%; 95% CI: 5.7%-16.1%). LoF ALPK3 variants were increased in comparison to matched controls (Dutch cohort, P = 1.6×10-5; U.S. cohort, P = 2.2×10-13). CONCLUSION: Biallelic damaging ALPK3 variants cause pediatric cardiomyopathy manifested by DCM transitioning to hypertrophy, often with poor contractile function. Additional extracardiac features occur in most patients, including musculoskeletal abnormalities and cleft palate. Heterozygous LoF ALPK3 variants are enriched in adults with cardiomyopathy and may contribute to their cardiomyopathy. Adults with ALPK3 LoF variants therefore warrant evaluations for cardiomyopathy.

Feedback regulation of Caulobacter crescentus holdfast synthesis by flagellum assembly via the holdfast inhibitor HfiA.

To permanently attach to surfaces, Caulobacter crescentusproduces a strong adhesive, the holdfast. The timing of holdfast synthesis is developmentally regulated by cell cycle cues. When C. crescentusis grown in a complex medium, holdfast synthesis can also be stimulated by surface sensing, in which swarmer cells rapidly synthesize holdfast in direct response to surface contact. In contrast to growth in complex medium, here we show that when cells are grown in a defined medium, surface contact does not trigger holdfast synthesis. Moreover, we show that in a defined medium, flagellum synthesis and regulation of holdfast production are linked. In these conditions, mutants lacking a flagellum attach to surfaces over time more efficiently than either wild-type strains or strains harboring a paralyzed flagellum. Enhanced adhesion in mutants lacking flagellar components is due to premature holdfast synthesis during the cell cycle and is regulated by the holdfast synthesis inhibitor HfiA. hfiA transcription is reduced in flagellar mutants and this reduction is modulated by the diguanylate cyclase developmental regulator PleD. We also show that, in contrast to previous predictions, flagella are not necessarily required for C. crescentus surface sensing in the absence of flow, and that arrest of flagellar rotation does not stimulate holdfast synthesis. Rather, our data support a model in which flagellum assembly feeds back to control holdfast synthesis via HfiA expression in a c-di-GMP-dependent manner under defined nutrient conditions.