Search

Objective: Low plasma levels of carotenoids are associated with mortality and chronic disease states. Genetic studies in animals revealed that the tissue accumulation of these dietary pigments is associated with the genes encoding β-carotene oxygenase 2 (BCO2) and the scavenger receptor class B type 1 (SR-B1). Here we examined in mice how BCO2 and SR-B1 affect the metabolism of the model carotenoid zeaxanthin that serves as a macular pigment in the human retina. Methods: We used mice with a lacZ reporter gene knock-in to determine Bco2 expression patterns in the small intestine. By genetic dissection, we studied the contribution of BCO2 and SR-B1 to zeaxanthin uptake homeostasis and tissue accumulation under different supply conditions (50 mg/kg and 250 mg/kg). We determined the metabolic profiles of zeaxanthin and its metabolites in different tissues by LC-MS using standard and chiral columns. An albino Isx−/−/Bco2−/− mouse homozygous for Tyrc−2J was generated to study the effect of light on ocular zeaxanthin metabolites. Results: We demonstrate that BCO2 is highly expressed in enterocytes of the small intestine. Genetic deletion of Bco2 led to enhanced accumulation of zeaxanthin, indicating that the enzyme serves as a gatekeeper of zeaxanthin bioavailability. Relaxing the regulation of SR-B1 expression in enterocytes by genetic deletion of the transcription factor ISX further enhanced zeaxanthin accumulation in tissues. We observed that the absorption of zeaxanthin was dose-dependent and identified the jejunum as the major zeaxanthin-absorbing intestinal region. We further showed that zeaxanthin underwent oxidation to ε,ε-3,3′-carotene-dione in mouse tissues. We detected all three enantiomers of the zeaxanthin oxidation product whereas the parent zeaxanthin only existed as (3R, 3′R)-enantiomer in the diet. The ratio of oxidized to parent zeaxanthin varied between tissues and was dependent on the supplementation dose. We further showed in an albino Isx−/−/Bco2−/− mouse that supra-physiological supplementation doses (250 mg/kg) with zeaxanthin rapidly induced hypercarotenemia with a golden skin phenotype and that light stress increased the concentration of oxidized zeaxanthin in the eyes. Conclusions: We established the biochemical basis of zeaxanthin metabolism in mice and showed that tissue factors and abiotic stress affect the metabolism and homeostasis of this dietary lipid.

The scavenger receptor class B type 1 (SR-B1) facilitates uptake of cholesterol and carotenoids into the plasma membrane (PM) of mammalian cells. Downstream of SR-B1, ASTER-B protein mediates the nonvesicular transport of cholesterol to mitochondria for steroidogenesis. Mitochondria also are the place for the processing of carotenoids into diapocarotenoids by β-carotene oxygenase-2. However, the role of these lipid transport proteins in carotenoid metabolism has not yet been established. Herein, we showed that the recombinant StART-like lipid-binding domain of ASTER-A and B preferentially binds oxygenated carotenoids such as zeaxanthin. We established a novel carotenoid uptake assay and demonstrated that ASTER-B expressing A549 cells transport zeaxanthin to mitochondria. In contrast, the pure hydrocarbon β-carotene is not transported to the organelles, consistent with its metabolic processing to vitamin A in the cytosol by β-carotene oxygenase-1. Depletion of the PM from cholesterol by methyl-β-cyclodextrin treatment enhanced zeaxanthin but not β-carotene transport to mitochondria. Loss-of-function assays by siRNA in A549 cells and the absence of zeaxanthin accumulation in mitochondria of ARPE19 cells confirmed the pivotal role of ASTER-B in this process. Together, our study in human cell lines established ASTER-B protein as key player in nonvesicular transport of zeaxanthin to mitochondria and elucidated the molecular basis of compartmentalization of the metabolism of nonprovitamin A and provitamin A carotenoids in mammalian cells.

Vitamin A is distributed within the body to support chromophore synthesis in the eyes and retinoid signaling in most other tissues. Two pathways exist for the delivery of vitamin A: the extrinsic pathway transports dietary vitamin A in lipoproteins from intestinal enterocytes to tissues, while the intrinsic pathway distributes vitamin A from hepatic stores bound to serum retinol binding protein (RBP). Previously, the intestine-specific homeodomain transcription factor (ISX) and the RBP receptor STRA6 were identified as gatekeepers of these pathways; however, it is not clear how mutations in the corresponding genes affect retinoid homeostasis. Here, we used a genetic dissection approach in mice to examine the contributions of these proteins in select tissues. We observed that ISX deficiency increased utilization of both preformed and provitamin A. We found that increased storage of retinoids in peripheral tissues of ISX-deficient mice was dependent on STRA6 and induced by retinoid signaling. In addition, double-mutant mice exhibited a partial rescue of the Stra6 mutant ocular phenotype. This rescue came at the expense of a massive accumulation of vitamin A in other tissues, demonstrating that vitamin A is randomly distributed when present in excessive amounts. Remarkably, provitamin A supplementation of mutant mice induced the expression of the RBP receptor 2 in the liver and was accompanied by increased hepatic retinyl ester stores. Taken together, these findings indicate dynamic crosstalk between the delivery pathways for this essential nutrient and suggest that hepatic reuptake of vitamin A takes place when excessive amounts circulate in the blood.

The RLBP1 gene encodes the 36 kDa cellular retinaldehyde-binding protein, CRALBP, a soluble retinoid carrier, in the visual cycle of the eyes. Mutations in RLBP1 are associated with recessively inherited clinical phenotypes, including Bothnia dystrophy, retinitis pigmentosa, retinitis punctata albescens, fundus albipunctatus, and Newfoundland rod–cone dystrophy. However, the etiology of these retinal disorders is not well understood. Here, we generated homologous zebrafish models to bridge this knowledge gap. Duplication of the rlbp1 gene in zebrafish and cell-specific expression of the paralogs rlbp1a in the retinal pigment epithelium and rlbp1b in Müller glial cells allowed us to create intrinsically cell type-specific knockout fish lines. Using rlbp1a and rlbp1b single and double mutants, we investigated the pathological effects on visual function. Our analyses revealed that rlbp1a was essential for cone photoreceptor function and chromophore metabolism in the fish eyes. rlbp1a-mutant fish displayed reduced chromophore levels and attenuated cone photoreceptor responses to light stimuli. They accumulated 11-cis and all-trans-retinyl esters which displayed as enlarged lipid droplets in the RPE reminiscent of the subretinal yellow-white lesions in patients with RLBP1 mutations. During aging, these fish developed retinal thinning and cone and rod photoreceptor dystrophy. In contrast, rlbp1b mutants did not display impaired vision. The double mutant essentially replicated the phenotype of the rlbp1a single mutant. Together, our study showed that the rlbp1a zebrafish mutant recapitulated many features of human blinding diseases caused by RLBP1 mutations and provided novel insights into the pathways for chromophore regeneration of cone photoreceptors.

Abstract: There is increasing recognition that dietary lipids can affect the expression of genes encoding their metabolizing enzymes, transporters, and binding proteins. This mechanism plays a pivotal role in controlling tissue homeostasis of these compounds and avoiding diseases. The regulation of retinoid biosynthesis from β-carotene (BC) is a classic example for such an interaction. The intestine-specific homeodomain transcription factor (ISX) controls the activity of the vitamin A-forming enzyme β-carotene oxygenase-1 in intestinal enterocytes in response to increasing concentration of the vitamin A metabolite retinoic acid. However, it is unclear how cells control the concentration of the signaling molecule in this negative-feedback loop. We demonstrate in mice that the sequestration of retinyl esters by the enzyme lecithin:retinol acyltransferase (LRAT) is central for this process. Using genetic and pharmacological approaches in mice, we observed that in LRAT deficiency, the transcription factor ISX became hypersensitive to dietary vitamin A and suppressed retinoid biosynthesis. The dysregulation of the pathway resulted in BC accumulation and vitamin A deficiency of extrahepatic tissues. Pharmacological inhibition of retinoid signaling and genetic depletion of the Isx gene restored retinoid biosynthesis in enterocytes. We provide evidence that the catalytic activity of LRAT coordinates the negative-feedback regulation of intestinal retinoid biosynthesis and maintains optimal retinoid levels in the body.

Emerging evidence indicates that diabetes disturbs photoreceptor function and vitamin A homeostasis. However, the biochemical basis of this phenotype is not well established. Here, we compared the effects of streptozotocin-induced diabetes in wild-type (WT) mice and Stra6-/- mice, a mouse model for ocular vitamin A deficiency. After 8 weeks, diabetes increased serum retinyl esters in mice of both genotypes. The eyes of diabetic WT mice displayed increased superoxide levels but no changes in retinoid concentrations. Diabetic Stra6-/- mice showed increased ocular retinoid concentrations, but superoxide levels remained unchanged. After 30 weeks, significant alterations in liver and fat retinoid concentrations were observed in diabetic mice. Diabetic WT mice exhibited a decreased expression of visual cycle proteins and a thinning of the photoreceptor layer. Stra6-/- mice displayed significantly lower ocular retinoid concentration than WT mice. An altered retinal morphology and a reduced expression of photoreceptor marker genes paralleled these biochemical changes and were more pronounced in the diabetic animals. Taken together, we observed that diabetes altered vitamin A homeostasis in several organ systems and aggravated photoreceptor pathologies in the vitamin-deficient mouse eyes.

The health of poultry flock is crucial in sustainable farming. Recent advances in machine learning and speech analysis have opened up opportunities for real-time monitoring of the behavior and health of flock. However, there has been little research on using Tiny Machine Learning (Tiny ML) for continuous vocalization monitoring in poultry. This study addresses this gap by developing and deploying Tiny ML models on low-power edge devices to monitor chicken vocalizations. The focus is on overcoming challenges such as memory limitations, processing power, and battery life to ensure practical implementation in agricultural settings. In collaboration with avian researchers, a diverse dataset of poultry vocalizations representing a range of health and environmental conditions was created to train and validate the algorithms. Digital Signal Processing (DSP) blocks of the Edge Impulse platform were used to generate spectral features for studying fowl vocalization. A one-dimensional Convolutional Neural Network (CNN) model was employed for classification. The study emphasizes accurately identifying and categorizing different chicken noises associated with emotional states such as discomfort, hunger, and satisfaction. To improve accuracy and reduce background noise, noise-robust Tiny ML algorithms were developed. Before the removal of background noise, our average accuracy and F1 scores were 91.6% and 0.92, respectively. After the removal, they improved to 96.6% and 0.95., Competing Interests: No Competing interests., (Copyright: © 2025 Srinivasagan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Perinatal nutrition exerts a profound influence on adult metabolic health. This study aimed to investigate whether increased maternal vitamin A (VA) supply can lead to beneficial metabolic phenotypes in the offspring. The researchers utilized mice deficient in the intestine-specific homeobox (ISX) transcription factor, which exhibits increased intestinal VA retinoid production from dietary β-carotene (BC). ISX-deficient dams were fed a VA-sufficient or a BC-enriched diet during the last week of gestation and the whole lactation period. Total retinol levels in milk and weanling livers were 2- to 2.5-fold higher in the offspring of BC-fed dams (BC offspring), indicating increased VA supplies during late gestation and lactation. The corresponding VA-sufficient and BC offspring (males and females) were compared at weaning and adulthood after being fed either a standard or high-fat diet (HFD) with regular VA content for 13 weeks from weaning. HFD-induced increases in adiposity metrics, such as fat depot mass and adipocyte diameter, were more pronounced in males than females and were attenuated or suppressed in the BC offspring. Notably, the BC offspring were protected from HFD-induced increases in circulating triacylglycerol levels and hepatic steatosis. These protective effects were associated with reduced food efficiency, enhanced capacity for thermogenesis and mitochondrial oxidative metabolism in adipose tissues, and increased adipocyte hyperplasia rather than hypertrophy in the BC offspring. In conclusion, maternal VA nutrition influenced by genetics may confer metabolic benefits to the offspring, with mild increases in late gestation and lactation protecting against obesity and metabolic dysregulation in adulthood. NEW & NOTEWORTHY A genetic mouse model, deficient in intestine-specific homeobox (ISX) transcription factor, is used to show that a mildly increased maternal vitamin A supply from β-carotene feeding during late gestation and lactation programs energy and lipid metabolism in tissues and protects the offspring from diet-induced hypertrophic obesity and hepatic steatosis. This knowledge may have implications for human populations where polymorphisms in ISX and ISX target genes involved in vitamin A homeostasis are prevalent.

A sharp drop in lenticular glutathione (GSH) plays a pivotal role in age-related cataract (ARC) formation. Despite recognizing GSH's importance in lens defense for decades, its decline with age remains puzzling. Our recent study revealed an age-related truncation affecting the essential GSH biosynthesis enzyme, the γ-glutamylcysteine ligase catalytic subunit (GCLC), at aspartate residue 499. Intriguingly, these truncated GCLC fragments compete with full-length GCLC in forming a heterocomplex with the modifier subunit (GCLM) but exhibit markedly reduced enzymatic activity. Crucially, using an aspartate-to-glutamate mutation knock-in (D499E-KI) mouse model that blocks GCLC truncation, we observed a notable delay in ARC formation compared to WT mice: Nearly 50% of D499E-KI mice remained cataract-free versus ~20% of the WT mice at their age of 20 months. Our findings concerning age-related GCLC truncation might be the key to understanding the profound reduction in lens GSH with age. By halting GCLC truncation, we can rejuvenate lens GSH levels and considerably postpone cataract onset.

Fresh dates have a limited shelf life and are susceptible to spoilage, which can lead to economic losses for producers and suppliers. The problem of accurate shelf life estimation for fresh dates is essential for various stakeholders involved in the production, supply, and consumption of dates. Modified atmosphere packaging (MAP) is one of the essential methods that improves the quality and increases the shelf life of fresh dates by reducing the rate of ripening. Therefore, this study aims to apply fast and cost-effective non-destructive techniques based on machine learning (ML) to predict and estimate the shelf life of stored fresh date fruits under different conditions. Predicting and estimating the shelf life of stored date fruits is essential for scheduling them for consumption at the right time in the supply chain to benefit from the nutritional advantages of fresh dates. The study observed the physicochemical attributes of fresh date fruits, including moisture content, total soluble solids, sugar content, tannin content, pH, and firmness, during storage in a vacuum and MAP at 5 and 24 ∘C every 7 days to determine the shelf life using a non-destructive approach. TinyML-compatible regression models were employed to predict the stages of fruit development during the storage period. The decrease in the shelf life of the fruits begins when they transition from the Khalal stage to the Rutab stage, and the shelf life ends when they start to spoil or ripen to the Tamr stage. Low-cost Visible-Near-Infrared (VisNIR) spectral sensors (AS7265x-multi-spectral) were used to capture the internal physicochemical attributes of the fresh fruit. Regression models were employed for shelf life estimation. The findings indicated that vacuum and modified atmosphere packaging with 20% CO 2 and N balance efficiently increased the shelf life of the stored fresh fruit to 53 days and 44 days, respectively, when maintained at 5 ∘C. However, the shelf life decreased to 44 and 23 days when the vacuum and modified atmosphere packaging with 20% CO 2 and N balance were maintained at room temperature (24 ∘C). Edge Impulse supports the training and deployment of models on low-cost microcontrollers, which can be used to predict real-time estimations of the shelf life of fresh dates using TinyML sensors.

The Internet of Things (IoT) plays a critical role in remotely monitoring a wide variety of different application sectors, including agriculture, building, and energy. The wind turbine energy generator (WTEG) is a real-world application that can take advantage of IoT technologies, such as a low-cost weather station, where human activities can be significantly affected by enhancing the production of clean energy based on the known direction of the wind. Meanwhile, common weather stations are neither affordable nor customizable for specific applications. Moreover, due to weather forecast changes over time and location within the same city, it is not efficient to rely on a limited number of weather stations that may be located far away from a recipient's location. Therefore, in this paper, we focus on presenting a low-cost weather station that relies on an artificial intelligence (AI) algorithm that can be distributed across a WTEG area with minimal cost. The proposed study measures multiple weather parameters, such as wind direction, wind velocity (WV), temperature, pressure, mean sea level, and relative humidity to provide current measurements to recipients and AI-based forecasts. In addition, the proposed study consists of several heterogeneous nodes and a controller for each station in a target area. The collected data can be transmitted through Bluetooth low energy (BLE). The experimental results reveal that the proposed study matches the standard of the National Meteorological Center (NMC), with a nowcast measurement of 95% accuracy for WV and 92% for wind direction (WD).

Purpose: Ultraviolet B (UVB) has been well documented to induce capsular cataracts; however, the mechanism of the lens epithelial cell-mediated repair process after UVB irradiation is not fully understood. The purpose of this study was to better understand lens epithelial cell repair after UVB-induced epithelium damage., Method: C57BL/6J mice were irradiated by various doses of UVB. Lens morphology and lens capsule opacity were monitored by slit lamp, darkfield microscopy, and phase-contrast microscopy. Lens epithelial cell mitotic activation and cell apoptosis were measured by immunohistochemistry. Lens epithelial ultrastructure was analyzed by transmission electron microscopy., Results: UVB irradiation above a dose of 2.87 kJ/m2 triggered lens epithelial cell apoptosis and subcapsular cataract formation, with a ring-shaped structure composed of multilayered epithelial cell clusters manifesting a dense ring-shaped capsular cataract. The epithelial cells immediately outside the edge of the ring-shaped aggregates transitioned to mitotically active cells and performed wound healing through the epithelialization process. However, repairs ceased when lens epithelial cells made direct contact, and scar-like tissue in the center of the anterior capsule remained even by 6 months after UVB irradiation., Conclusions: Our present study demonstrates that normally quiescent lens epithelial cells can be reactivated for epithelialization repair in response to UV-induced damage.

Age-related cataracts (ARC) are the primary cause of blindness worldwide, and oxidative stress is considered the central pathogenesis of age-related cataractogenesis. Interestingly, ample evidence suggests that there is no remarkable apoptosis present in aged and cataractous human lenses despite the profound disruption of redox homeostasis, raising an essential question regarding the existence of other cell death mechanisms. Here we sought to explore the lens epithelial cell's (LEC) susceptibility to ferroptosis after documentation has concluded that aged and cataractous human lenses manifest with increased reactive oxygen species (ROS) formation, elevated lipid peroxidation, and accumulative intracellular redox-active iron, constituting the three hallmarks of ferroptosis during aging and cataractogenesis. Here we show that very low concentrations of system X c - inhibitor Erastin (0.5 μM) and glutathione peroxidase 4 (GPX4) inhibitor RSL3 (0.1 μM) can drastically induce human LEC (FHL124) ferroptosis in vitro and mouse lens epithelium ferroptosis ex vivo. Depletion of intracellular glutathione (GSH) in human LECs and mouse lens epithelium significantly sensitizes ferroptosis, particularly under RSL3 challenge. Intriguingly, both human LECs and the mouse lens epithelium demonstrate an age-related sensitization of ferroptosis. Transcriptome analysis indicates that clusters of genes are up-or down-regulated in aged LECs, impacting cellular redox and iron homeostases, such as downregulation of both cystine/glutamate antiporter subunits SLC7A11 and SLC3A2 and iron exporter ferroportin (SLC40A1). Here, for the first time, we are suggesting that LECs are highly susceptible to ferroptosis. Moreover, aged and cataractous human lenses may possess more pro-ferroptotic criteria than any other organ in the human body., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The enzyme β-carotene oxygenase 2 (BCO2) converts carotenoids into more polar metabolites. Studies in mammals, fish, and birds revealed that BCO2 controls carotenoid homeostasis and is involved in the pathway for vitamin A production. However, it is controversial whether BCO2 function is conserved in humans, because of a 4-amino acid long insertion caused by a splice acceptor site polymorphism. We here show that human BCO2 splice variants, BCO2a and BCO2b, are expressed as pre-proteins with mitochondrial targeting sequence (MTS). The MTS of BCO2a directed a green fluorescent reporter protein to the mitochondria when expressed in ARPE-19 cells. Removal of the MTS increased solubility of BCO2a when expressed in Escherichia coli and rendered the recombinant protein enzymatically active. The expression of the enzymatically active recombinant human BCO2a was further improved by codon optimization and its fusion with maltose-binding protein. Introduction of the 4-amino acid insertion into mouse Bco2 did not impede the chimeric enzyme's catalytic proficiency. We further showed that the chimeric BCO2 displayed broad substrate specificity and converted carotenoids into two ionones and a central C14-apocarotendial by oxidative cleavage reactions at C9,C10 and C9',C10'. Thus, our study demonstrates that human BCO2 is a catalytically competent enzyme. Consequently, information on BCO2 becomes broadly applicable in human biology with important implications for the physiology of the eyes and other tissues., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Background: Astaxanthin is a red lipophilic carotenoid that is often undetectable in human plasma due to the limited supply in typical Western diets. Despite its presence at lower than detectable concentrations, previous clinical feeding studies have reported that astaxanthin exhibits potent antioxidant properties., Objective: We examined astaxanthin accumulation and its effects on gut microbiota, inflammation, and whole-body metabolic homeostasis in wild-type C57BL/6 J (WT) and β-carotene oxygenase 2 (BCO2) knockout (KO) mice., Methods: Six-wk-old male and female BCO2 KO and WT mice were provided with either nonpurified AIN93M (e.g., control diet) or the control diet supplemented with 0.04% astaxanthin (wt/wt) ad libitum for 8 wk. Whole-body energy expenditure was measured by indirect calorimetry. Feces were collected from individual mice for short-chain fatty acid assessment. Hepatic astaxanthin concentrations and liver metabolic markers, cecal gut microbiota profiling, inflammation markers in colonic lamina propria, and plasma samples were assessed. Data were analyzed by 3-way ANOVA followed by Tukey's post hoc analysis., Results: BCO2 KO but not WT mice fed astaxanthin had ∼10-fold more of this compound in liver than controls (P < 0.05). In terms of the microbiota composition, deletion of BCO2 was associated with a significantly increased abundance of Mucispirillum schaedleri in mice regardless of gender. In addition to more liver astaxanthin in male KO compared with WT mice fed astaxanthin, the abundance of gut Akkermansia muciniphila was 385% greater, plasma glucagon-like peptide 1 was 27% greater, plasma glucagon and IL-1β were 53% and 30% lower, respectively, and colon NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome activation was 23% lower (all P < 0.05) in male KO mice than the WT mice., Conclusions: Astaxanthin affects the gut microbiota composition in both genders, but the association with reductions in local and systemic inflammation, oxidative stress, and improvement of metabolic homeostasis only occurs in male mice., (Copyright © The Author(s) on behalf of the American Society for Nutrition 2020.)

The epithelial-mesenchymal transition (EMT) process plays a pivotal role in the pathogenesis of posterior capsular opacification because of remnant lens epithelial cell proliferation, migration, and transformation after cataract surgery. The latter, we hypothesize, may result in posterior capsule wrinkling and opacification because of a profound change in the lens growth environment via a 1000-fold reduction of extracellular glutathione (GSH) levels. To test this hypothesis, we investigated the EMT process in cell culture and GSH biosynthesis deficiency mouse models. Our data indicate a dramatic increase of pro-EMT markers, such as type I collagen, α-smooth muscle actin, vimentin, and fibronectin, under conditions of lens GSH depletion. Further study suggests that decreased GSH triggers the Wnt/β-catenin signal transduction pathway, independent of transforming growth factor-β. Equally important, the antioxidants N-acetyl cysteine and GSH ethyl ester could significantly attenuate the EMT signaling stimulated by decreased GSH levels. These findings were further confirmed by mock cataract surgery in both gamma glutamyl-cysteine ligase, catalytic subunit, and gamma glutamyl-cysteine ligase, modifier subunit, knockout mouse models. Remarkably, increased EMT marker expression, β-catenin activation, and translocation into the nucleus were found in both knockout mice compared with the wild type, and such increased expression could be significantly attenuated by N-acetyl cysteine or GSH ethyl ester treatment. This study, for the first time we believe, links oxidative stress to lens fibrosis and posterior capsular opacification formation via EMT-mediated mechanisms., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Linguistic and genetic studies on Roma populations inhabited in Europe have unequivocally traced these populations to the Indian subcontinent. However, the exact parental population group and time of the out-of-India dispersal have remained disputed. In the absence of archaeological records and with only scanty historical documentation of the Roma, comparative linguistic studies were the first to identify their Indian origin. Recently, molecular studies on the basis of disease-causing mutations and haploid DNA markers (i.e. mtDNA and Y-chromosome) supported the linguistic view. The presence of Indian-specific Y-chromosome haplogroup H1a1a-M82 and mtDNA haplogroups M5a1, M18 and M35b among Roma has corroborated that their South Asian origins and later admixture with Near Eastern and European populations. However, previous studies have left unanswered questions about the exact parental population groups in South Asia. Here we present a detailed phylogeographical study of Y-chromosomal haplogroup H1a1a-M82 in a data set of more than 10,000 global samples to discern a more precise ancestral source of European Romani populations. The phylogeographical patterns and diversity estimates indicate an early origin of this haplogroup in the Indian subcontinent and its further expansion to other regions. Tellingly, the short tandem repeat (STR) based network of H1a1a-M82 lineages displayed the closest connection of Romani haplotypes with the traditional scheduled caste and scheduled tribe population groups of northwestern India.