Crosstalk of ubiquitin system and non-coding RNA in fibrosis.

Chronic tissue injury triggers changes in the cell type and microenvironment at the site of injury and eventually fibrosis develops. Current research suggests that fibrosis is a highly dynamic and reversible process, which means that human intervention after fibrosis has occurred has the potential to slow down or cure fibrosis. The ubiquitin system regulates the biological functions of specific proteins involved in the development of fibrosis, and researchers have designed small molecule drugs to treat fibrotic diseases on this basis, but their therapeutic effects are still limited. With the development of molecular biology technology, researchers have found that non-coding RNA (ncRNA) can interact with the ubiquitin system to jointly regulate the development of fibrosis. More in-depth explorations of the interaction between ncRNA and ubiquitin system will provide new ideas for the clinical treatment of fibrotic diseases.

Epigenetic orchestration of the DNA damage response: Insights into the regulatory mechanisms.

The DNA damage response (DDR) is a critical cellular mechanism that safeguards genome integrity and prevents the accumulation of harmful DNA lesions. Increasing evidence highlights the intersection between DDR signaling and epigenetic regulation, offering profound insights into various aspects of cellular function including oncogenesis. This comprehensive review explores the intricate relationship between the epigenetic modifications and DDR activation, with a specific focus on the impact of viral infections. Oncogenic viruses, such as human papillomavirus, hepatitis virus (HBV or HCV), and Epstein-Barr virus have been shown to activate the DDR. Consequently, these DNA damage events trigger a cascade of epigenetic alterations, including changes in DNA methylation patterns, histone modifications and the expression of noncoding RNAs. These epigenetic changes exert profound effects on chromatin structure, gene expression, and maintenance of genome stability. Importantly, elucidation of the viral-induced epigenetic alterations in the context of DDR holds significant implications for comprehending the complexity of cancer and provides potential targets for therapeutic interventions.

Exploring the pathogenesis of pulmonary vascular disease.

Pulmonary hypertension (PH) is a complex cardiopulmonary disorder impacting the lung vasculature, resulting in increased pulmonary vascular resistance that leads to right ventricular dysfunction. Pulmonary hypertension comprises of 5 groups (PH group 1 to 5) where group 1 pulmonary arterial hypertension (PAH), results from alterations that directly affect the pulmonary arteries. Although PAH has a complex pathophysiology that is not completely understood, it is known to be a multifactorial disease that results from a combination of genetic, epigenetic and environmental factors, leading to a varied range of symptoms in PAH patients. PAH does not have a cure, its incidence and prevalence continue to increase every year, resulting in higher morbidity and mortality rates. In this review, we discuss the different pathologic mechanisms with a focus on epigenetic modifications and their roles in the development and progression of PAH. These modifications include DNA methylation, histone modifications, and microRNA dysregulation. Understanding these epigenetic modifications will improve our understanding of PAH and unveil novel therapeutic targets, thus steering research toward innovative treatment strategies.

Abscisic acid-mediated autoregulation of the MYB41-BRAHMA module enhances drought tolerance in Arabidopsis.

Drought stress poses a substantial challenge to plant growth and agricultural productivity worldwide. Upon water depletion, plants activate an abscisic acid (ABA) signaling pathway, leading to stomatal closure to reduce water loss. The MYB family of transcription factors plays diverse roles in growth, development, stress responses and biosynthesis, yet their involvement in stomatal regulation remains unclear. Here, we demonstrate that ABA significantly upregulates the expression of MYB41, MYB74, and MYB102, with MYB41 serving as a key regulator that induces the expression of both MYB74 and MYB102. Through luciferase assays, chromatin immunoprecipitation (ChIP) assays and electrophoretic mobility shift assays (EMSA), we reveal that MYB41 engages in positive feedback regulation by binding to its own promoter, thus amplifying its transcription in Arabidopsis (Arabidopsis thaliana). Furthermore, our investigation showed that MYB41 recruits BRAHMA (BRM), the core ATPase subunit of the SWI/SNF complex, to the MYB41 promoter, facilitating the binding of HISTONE DEACETYLASE 6 (HDA6). This recruitment triggers epigenetic modifications, resulting in reduced MYB41 expression characterized by elevated H3K27me3 levels and concurrent decreases in H3ac, H3K27ac, and H3K14ac levels in wild-type plants compared to brm knockout mutant plants. Our genetic and molecular analyses show that ABA mediates autoregulation of the MYB41-BRM module, which intricately modulates stomatal movement in A. thaliana. This discovery sheds light on a drought response mechanism with the potential to greatly enhance agricultural productivity.

Histone (de)acetylation in epigenetic regulation of Phytophthora pathobiology.

Phytophthora species are oomycetes that have evolved a broad spectrum of biological processes and improved strategies to cope with host and environmental challenges. A growing body of evidence indicates that the high pathogen plasticity is based on epigenetic regulation of gene expression linked to Phytophthora's rapid adjustment to endogenous cues and various stresses. As 5mC DNA methylation has not yet been identified in Phytophthora, the reversible processes of acetylation/deacetylation of histone proteins seem to play a pivotal role in the epigenetic control of gene expression in oomycetes. To explore this issue, we review the structure, diversity, and phylogeny of histone acetyltransferases (HATs) and histone deacetylases (HDACs) in six plant-damaging Phytophthora species: P. capsici, P. cinnamomi, P. infestans, P. parasitica, P. ramorum, and P. sojae. To further integrate and improve our understanding of the phylogenetic classification, evolutionary relationship, and functional characteristics, we supplement this review with a comprehensive view of HATs and HDACs using recent genome- and proteome-level databases. Finally, the potential functional role of transcriptional reprogramming mediated by epigenetic changes during Phytophthora species saprophytic and parasitic phases under nitro-oxidative stress is also briefly discussed.

Exploring regulatory mechanisms on miRNAs and their implications in inflammation-related diseases.

This comprehensive exploration delves into the pivotal role of microRNAs (miRNAs) within the intricate tapestry of cellular regulation. As potent orchestrators of gene expression, miRNAs exhibit diverse functions in cellular processes, extending their influence from the nucleus to the cytoplasm. The complex journey of miRNA biogenesis, involving transcription, processing, and integration into the RNA-induced silencing complex, showcases their versatility. In the cytoplasm, mature miRNAs finely tune cellular functions by modulating target mRNA expression, while their reach extends into the nucleus, influencing transcriptional regulation and epigenetic modifications. Dysregulation of miRNAs becomes apparent in various pathologies, such as cancer, autoimmune diseases, and inflammatory conditions. The adaptability of miRNAs to environmental signals, interactions with transcription factors, and involvement in intricate regulatory networks underscore their significance. DNA methylation and histone modifications adds depth to understanding the dynamic regulation of miRNAs. Mechanisms like competition with RNA-binding proteins, sponging, and the control of miRNA levels through degradation and editing contribute to this complex regulation process. In this review, we mainly focus on how dysregulation of miRNA expression can be related with skin-related autoimmune and autoinflammatory diseases, arthritis, cardiovascular diseases, inflammatory bowel disease, autoimmune and autoinflammatory diseases, and neurodegenerative disorders. We also emphasize the multifaceted roles of miRNAs, urging continued research to unravel their complexities. The mechanisms governing miRNA functions promise advancements in therapeutic interventions and enhanced insights into cellular dynamics in health and disease.

Update: the role of epigenetics in the metabolic memory of diabetic complications.

Diabetes, a chronic disease characterized by hyperglycemia, is associated with significantly accelerated complications, including diabetic kidney disease (DKD), which increases morbidity and mortality. Hyperglycemia and other diabetes-related environmental factors such as overnutrition, sedentary lifestyles, and hyperlipidemia can induce epigenetic changes. Working alone or with genetic factors, these epigenetic changes that occur without alterations in the underlying DNA sequence, can alter the expression of pathophysiological genes and impair functions of associated target cells/organs, leading to diabetic complications like DKD. Notably, some hyperglycemia-induced epigenetic changes persist in target cells/tissues even after glucose normalization, leading to sustained complications despite glycemic control, so-called metabolic memory. Emerging evidence from in vitro and in vivo animal models and clinical trials with subjects with diabetes identified clear associations between metabolic memory and epigenetic changes including DNA methylation, histone modifications, chromatin structure, and noncoding RNAs at key loci. Targeting such persistent epigenetic changes and/or molecules regulated by them can serve as valuable opportunities to attenuate, or erase metabolic memory, which is crucial to prevent complication progression. Here, we review these cell/tissue-specific epigenetic changes identified to-date as related to diabetic complications, especially DKD, and the current status on targeting epigenetics to tackle metabolic memory. We also discuss limitations in current studies, including the need for more (epi)genome-wide studies, integrative analysis using multiple epigenetic marks and Omics datasets, and mechanistic evaluation of metabolic memory. Considering the tremendous technological advances in epigenomics, genetics, sequencing, and availability of genomic datasets from clinical cohorts, this field is likely to see considerable progress in the upcoming years.

ORC1 enhances repressive epigenetic modifications on HIV-1 LTR to promote HIV-1 latency.

The human immunodeficiency virus type 1 (HIV-1) reservoir consists of latently infected cells which present a major obstacle to achieving a functional cure for HIV-1. The formation and maintenance of HIV-1 latency have been extensively studied, and latency-reversing agents (LRAs) that can reactivate latent HIV-1 by targeting the involved host factors are developed; however, their clinical efficacies remain unsatisfactory. Therefore, it is imperative to identify novel targets for more potential candidates or better combinations for LRAs. In this study, we utilized CRISPR affinity purification in situ of regulatory elements system to screen for host factors associated with the HIV-1 long terminal repeat region that could potentially be involved in HIV-1 latency. We successfully identified that origin recognition complex 1 (ORC1), the largest subunit of the origin recognition complex, contributes to HIV-1 latency in addition to its function in DNA replication initiation. Notably, ORC1 is enriched on the HIV-1 promoter and recruits a series of repressive epigenetic elements, including DNMT1 and HDAC1/2, and histone modifiers, such as H3K9me3 and H3K27me3, thereby facilitating the establishment and maintenance of HIV-1 latency. Moreover, the reactivation of latent HIV-1 through ORC1 depletion has been confirmed across various latency cell models and primary CD4+ T cells from people living with HIV-1. Additionally, we comprehensively validated the properties of liquid-liquid phase separation (LLPS) of ORC1 from multiple perspectives and identified the key regions that promote the formation of LLPS. This property is important for the recruitment of ORC1 to the HIV-1 promoter. Collectively, these findings highlight ORC1 as a potential novel target implicated in HIV-1 latency and position it as a promising candidate for the development of novel LRAs. IMPORTANCE: Identifying host factors involved in maintaining human immunodeficiency virus type 1 (HIV-1) latency and understanding their mechanisms prepares the groundwork to discover novel targets for HIV-1 latent infection and provides further options for the selection of latency-reversing agents in the "shock" strategy. In this study, we identified a novel role of the DNA replication factor origin recognition complex 1 (ORC1) in maintaining repressive chromatin structures surrounding the HIV-1 promoter region, thereby contributing to HIV-1 latency. This discovery expands our understanding of the non-replicative functions of the ORC complex and provides a potential therapeutic strategy for HIV-1 cure.

Targeted bisulfite sequencing of Scots pine adaptation-related genes.

During environmental changes, epigenetic processes can enable adaptive responses faster than natural selection. In plants, very little is known about the role of DNA methylation during long-term adaptation. Scots pine is a widely distributed coniferous species which must adapt to different environmental conditions throughout its long lifespan. Thus, epigenetic modifications may contribute towards this direction. We provide bisulfite next-generation sequencing data from the putative promoters and exons of eight adaptation-related genes (A3IP2, CCA1, COL1, COL2, FTL2, MFT1, PHYO, and ZTL) in three Scots pine populations located in northern and southern parts of Finland. DNA methylation levels were studied in the two seed tissues: the maternal megagametophyte which contributes to embryo viability, and the biparental embryo which represents the next generation. In most genes, differentially methylated cytosines (DMCs) were in line with our previously demonstrated gene expression differences found in the same Scots pine populations. In addition, we found a strong correlation of total methylation levels between the embryo and megagametophyte tissues of a given individual tree, which indicates that DNA methylation can be inherited from the maternal parent. In conclusion, our results imply that DNA methylation differences may contribute to the adaptation of Scots pine populations in different climatic conditions.

Mechanisms of Embryonic Stem Cell Pluripotency Maintenance and Their Application in Livestock and Poultry Breeding.

Embryonic stem cells (ESCs) are remarkably undifferentiated cells that originate from the inner cell mass of the blastocyst. They possess the ability to self-renew and differentiate into multiple cell types, making them invaluable in diverse applications such as disease modeling and the creation of transgenic animals. In recent years, as agricultural practices have evolved from traditional to biological breeding, it has become clear that pluripotent stem cells (PSCs), either ESCs or induced pluripotent stem cells (iPSCs), are optimal for continually screening suitable cellular materials. However, the technologies for long-term in vitro culture or establishment of cell lines for PSCs in livestock are still immature, and research progress is uneven, which poses challenges for the application of PSCs in various fields. The establishment of a robust in vitro system for these cells is critically dependent on understanding their pluripotency maintenance mechanisms. It is believed that the combined effects of pluripotent transcription factors, pivotal signaling pathways, and epigenetic regulation contribute to maintaining their pluripotent state, forming a comprehensive regulatory network. This article will delve into the primary mechanisms underlying the maintenance of pluripotency in PSCs and elaborate on the applications of PSCs in the field of livestock.

Replenishment of TCA cycle intermediates and long-noncoding RNAs regulation in breast cancer.

The tricarboxylic acid (TCA) cycle is an essential interface that coordinates cellular metabolism and is as a primary route determining the fate of a variety of fuel sources, including glucose, fatty acid and glutamate. The crosstalk of nutrients replenished TCA cycle regulates breast cancer (BC) progression by changing substrate levels-induced epigenetic alterations, especially the methylation, acetylation, succinylation and lactylation. Long non-coding RNAs (lncRNA) have dual roles in inhibiting or promoting energy reprogramming, and so altering the metabolic flux of fuel sources to the TCA cycle, which may regulate epigenetic modifications at the cellular level of BC. This narrative review discussed the central role of the TCA cycle in interconnecting numerous fuels and the induced epigenetic modifications, and the underlying regulatory mechanisms of lncRNAs in BC.

LL37/self-DNA complexes mediate monocyte reprogramming.

LL37 alone and in complex with self-DNA triggers inflammatory responses in myeloid cells and plays a crucial role in the development of systemic autoimmune diseases, like psoriasis and systemic lupus erythematosus. We demonstrated that LL37/self-DNA complexes induce long-term metabolic and epigenetic changes in monocytes, enhancing their responsiveness to subsequent stimuli. Monocytes trained with LL37/self-DNA complexes and those derived from psoriatic patients exhibited heightened glycolytic and oxidative phosphorylation rates, elevated release of proinflammatory cytokines, and affected naïve CD4+ T cells. Additionally, KDM6A/B, a demethylase of lysine 27 on histone 3, was upregulated in psoriatic monocytes and monocytes treated with LL37/self-DNA complexes. Inhibition of KDM6A/B reversed the trained immune phenotype by reducing proinflammatory cytokine production, metabolic activity, and the induction of IL-17-producing T cells by LL37/self-DNA-treated monocytes. Our findings highlight the role of LL37/self-DNA-induced innate immune memory in psoriasis pathogenesis, uncovering its impact on monocyte and T cell dynamics.

Unraveling the Progression of Colon Cancer Pathogenesis Through Epigenetic Alterations and Genetic Pathways.

In the modern age, colon cancer has attained a widespread status, affecting a considerable number of people. It develops due to the progressive accumulation of genetic and epigenetic alterations. While genetic mutations have been extensively studied in the context of colon cancer, emerging evidence highlights the pivotal role of epigenetic alterations in its pathogenesis. These alterations ultimately result in the transformation of normal colonic epithelium into colon adenocarcinoma. Key mechanisms of epigenetic modifications include DNA methylation, histone modification, and nucleosome positioning. Research findings have linked these modifications to the development, progression, or metastasis of tumors. Through the assessment of the colon cancer epigenome, it has been discovered that practically all colorectal cancers (CRCs) display gene methylation abnormalities and changes in miRNA expression. Advancements in this area indicate that epigenetic modifications will likely be commonly used in the near future to direct the prevention and treatment of CRC. The maintenance of genome stability is essential for preserving cellular integrity. The development of CRC is primarily influenced by the loss of genomic stability, which allows for the emergence of new mutations contributing to tumor characteristics. Although genetic mutations have been extensively researched in the realm of colon cancer, recent evidence underscores the pivotal role of epigenetic changes in its pathogenesis. The following types of genomic instability will be discussed: chromosomal instability, microsatellite instability, CpG island methylation phenotype, and aberrant DNA methylation.

Machine learning and related approaches in transcriptomics.

Data acquisition for transcriptomic studies used to be the bottleneck in the transcriptomic analytical pipeline. However, recent developments in transcriptome profiling technologies have increased researchers' ability to obtain data, resulting in a shift in focus to data analysis. Incorporating machine learning to traditional analytical methods allows the possibility of handling larger volumes of complex data more efficiently. Many bioinformaticians, especially those unfamiliar with ML in the study of human transcriptomics and complex biological systems, face a significant barrier stemming from their limited awareness of the current landscape of ML utilisation in this field. To address this gap, this review endeavours to introduce those individuals to the general types of ML, followed by a comprehensive range of more specific techniques, demonstrated through examples of their incorporation into analytical pipelines for human transcriptome investigations. Important computational aspects such as data pre-processing, task formulation, results (performance of ML models), and validation methods are encompassed. In hope of better practical relevance, there is a strong focus on studies published within the last five years, almost exclusively examining human transcriptomes, with outcomes compared with standard non-ML tools.

Genome-wide mapping of DNase I hypersensitive sites revealed differential chromatin accessibility and regulatory DNA elements under drought stress in rice cultivars.

Drought stress (DS) is one of the major constraints limiting yield in crop plants including rice. Gene regulation under DS is largely governed by accessibility of the transcription factors (TFs) to their cognate cis-regulatory elements (CREs). In this study, we used DNase I hypersensitive assays followed by sequencing to identify the accessible chromatin regions under DS in a drought-sensitive (IR64) and a drought-tolerant (N22) rice cultivar. Our results indicated that DNase I hypersensitive sites (DHSs) were highly enriched at transcription start sites (TSSs) and numerous DHSs were detected in the promoter regions. DHSs were concurrent with epigenetic marks and the genes harboring DHSs in their TSS and promoter regions were highly expressed. In addition, DS induced changes in DHSs (∆DHSs) in TSS and promoter regions were positively correlated with upregulation of several genes involved in drought/abiotic stress response, those encoding TFs and located within drought-associated quantitative trait loci, much preferentially in the drought-tolerant cultivar. The CREs representing the binding sites of TFs involved in DS response were detected within the ∆DHSs, suggesting differential accessibility of TFs to their cognate sites under DS in different rice cultivars, which may be further deployed for enhancing drought tolerance in rice.

N6-methyladenosine dynamics in placental development and trophoblast functions, and its potential role in placental diseases.

N6-methyladenosine (m6A) is the most abundant modification controlling RNA metabolism and cellular functions, but its roles in placental development are still poorly understood. Here, we characterized the synchronization of m6A modifications and placental functions by mapping the m6A methylome in human placentas (n = 3, each trimester), revealing that the dynamic patterns of m6A were associated with gene expression homeostasis and different biological pathways in placental development. Then, we generated trophoblast-specific knockout mice of Wtap, a critical component of methyltransferase complex, and demonstrated that Wtap was essential for trophoblast proliferation, placentation and perinatal growth. Further in vitro experiments which includes cell viability assays and series molecular binding assays demonstrated that WTAP-m6A-IGF2BP3 axis regulated the RNA stability and translation of Anillin (ANLN) and VEGFA, promoting trophoblast proliferation and secretion. Dysregulation of this regulatory axis was observed in placentas from pregnancies with fetal growth restriction (FGR) or preeclampsia, revealing the pathogenic effects of imbalanced m6A modifications. Therefore, our findings provide novel insights into the functions and regulatory mechanisms of m6A modifications in placental development and placental-related gestational diseases.

CCAAT Promoter element regulates transgenerational expression of the MHC class I gene.

Transgenerational gene expression depends on both underlying DNA sequences and epigenetic modifications. The latter, which can result in transmission of variegated gene expression patterns across multiple generations without DNA alterations, has been termed epigenetic inheritance and has been documented in plants, worms, flies and mammals. Whereas transcription factors binding to cognate DNA sequence elements regulate gene expression, the molecular basis for epigenetic inheritance has been linked to histone and DNA modifications and non-coding RNA. Here we report that mutation of the CCAAT box promoter element abrogates NF-Y binding and disrupts the stable transgenerational expression of an MHC class I transgene. Transgenic mice with a mutated CCAAT box in the MHC class I transgene display variegated expression of the transgene among littermates and progeny in multiple independently derived transgenic lines. After 4 generations, CCAAT mutant transgenic lines derived from a single founder stably displayed distinct patterns of expression. Histone modifications and RNA polymerase II binding correlate with expression of CCAAT mutant transgenic lines, whereas DNA methylation and nucleosome occupancy do not. Mutation of the CCAAT box also results in changes to CTCF binding and DNA looping patterns across the transgene that correlate with expression status. These studies identify the CCAAT promoter element as a regulator of stable transgenerational gene expression such that mutation of the CCAAT box results in variegated transgenerational inheritance. Considering that the CCAAT box is present in 30% of eukaryotic promoters, this study provides insights into how fidelity of gene expression patterns is maintained through multiple generations.

Mitochondrial metabolism regulation and epigenetics in hypoxia.

The complex and dynamic interaction between cellular energy control and gene expression modulation is shown by the intersection between mitochondrial metabolism and epigenetics in hypoxic environments. Poor oxygen delivery to tissues, or hypoxia, is a basic physiological stressor that sets off a series of reactions in cells to adapt and endure oxygen-starved environments. Often called the "powerhouse of the cell," mitochondria are essential to cellular metabolism, especially regarding producing energy through oxidative phosphorylation. The cellular response to hypoxia entails a change in mitochondrial metabolism to improve survival, including epigenetic modifications that control gene expression without altering the underlying genome. By altering the expression of genes involved in angiogenesis, cell survival, and metabolism, these epigenetic modifications help cells adapt to hypoxia. The sophisticated interplay between mitochondrial metabolism and epigenetics in hypoxia is highlighted by several important points, which have been summarized in the current article. Deciphering the relationship between mitochondrial metabolism and epigenetics during hypoxia is essential to understanding the molecular processes that regulate cellular adaptation to reduced oxygen concentrations.

Heavy metals toxicity on epigenetic modifications in the pathogenesis of Alzheimer's disease (AD).

Alzheimer's disease (AD) is a progressive decline in cognitive ability and behavior which eventually disrupts daily activities. AD has no cure and the progression rate varies unlikely. Among various causative factors, heavy metals are reported to be a significant hazard in AD pathogenesis. Metal-induced neurodegeneration has been focused globally with thorough research to unravel the mechanistic insights in AD. Recently, heavy metals suggested to play an important role in epigenetic alterations which might provide evidential results on AD pathology. Epigenetic modifications are known to play towards novel therapeutic approaches in treating AD. Though many studies focus on epigenetics and heavy metal implications in AD, there is a lack of research on heavy metal influence on epigenetic toxicity in neurological disorders. The current review aims to elucidate the plausible role of cadmium (Cd), iron (Fe), arsenic (As), copper (Cu), and lithium (Li) metals on epigenetic factors and the increase in amyloid beta and tau phosphorylation in AD. Also, the review discusses the common methods of heavy metal detection to implicate in AD pathogenesis. Hence, from this review, we can extend the need for future research on identifying the mechanistic behavior of heavy metals on epigenetic toxicity and to develop diagnostic and therapeutic markers in AD.

Mitochondrial DNA: Inherent Complexities Relevant to Genetic Analyses.

Mitochondrial DNA (mtDNA) exhibits distinct characteristics distinguishing it from the nuclear genome, necessitating specific analytical methods in genetic studies. This comprehensive review explores the complex role of mtDNA in a variety of genetic studies, including genome-wide, epigenome-wide, and phenome-wide association studies, with a focus on its implications for human traits and diseases. Here, we discuss the structure and gene-encoding properties of mtDNA, along with the influence of environmental factors and epigenetic modifications on its function and variability. Particularly significant are the challenges posed by mtDNA's high mutation rate, heteroplasmy, and copy number variations, and their impact on disease susceptibility and population genetic analyses. The review also highlights recent advances in methodological approaches that enhance our understanding of mtDNA associations, advocating for refined genetic research techniques that accommodate its complexities. By providing a comprehensive overview of the intricacies of mtDNA, this paper underscores the need for an integrated approach to genetic studies that considers the unique properties of mitochondrial genetics. Our findings aim to inform future research and encourage the development of innovative methodologies to better interpret the broad implications of mtDNA in human health and disease.