Safety considerations for dietary supplement manufacturers in the United States.

Due to significant dietary supplement use in the US, product manufacturers must understand the importance of implementing a robust approach to establishing safety for all ingredients, including dietary ingredients, components, and finished dietary supplement products. Different regulatory pathways exist by which the safety of dietary ingredients can be established, and thus allowed to be marketed in a dietary supplement. For individual dietary ingredients, safety information may come from a variety of sources including history of safe use, presence of the ingredient in foods, and/or non-clinical and clinical data. On occasion safety data gaps are identified for a specific ingredient, particularly those of botanical origin. Modern toxicological methods and models can prove helpful in satisfying data gaps and are presented in this review. For finished dietary supplement products, issues potentially impacting safety to consider include claims, product labeling, overages, contaminants, residual solvents, heavy metals, packaging, and product stability. In addition, a safety assessment does not end once a product is marketed. It is important that manufacturers actively monitor and record the occurrence of adverse events reported in association with the use of their products, in accordance with the law. Herein, we provide a comprehensive overview of considerations for assessing dietary supplement safety.

A method for estimating coherence of molecular mechanisms in major human disease and traits.

BACKGROUND: Phenotypes such as height and intelligence, are thought to be a product of the collective effects of multiple phenotype-associated genes and interactions among their protein products. High/low degree of interactions is suggestive of coherent/random molecular mechanisms, respectively. Comparing the degree of interactions may help to better understand the coherence of phenotype-specific molecular mechanisms and the potential for therapeutic intervention. However, direct comparison of the degree of interactions is difficult due to different sizes and configurations of phenotype-associated gene networks. METHODS: We introduce a metric for measuring coherence of molecular-interaction networks as a slope of internal versus external distributions of the degree of interactions. The internal degree distribution is defined by interaction counts within a phenotype-specific gene network, while the external degree distribution counts interactions with other genes in the whole protein-protein interaction (PPI) network. We present a novel method for normalizing the coherence estimates, making them directly comparable. RESULTS: Using STRING and BioGrid PPI databases, we compared the coherence of 116 phenotype-associated gene sets from GWAScatalog against size-matched KEGG pathways (the reference for high coherence) and random networks (the lower limit of coherence). We observed a range of coherence estimates for each category of phenotypes. Metabolic traits and diseases were the most coherent, while psychiatric disorders and intelligence-related traits were the least coherent. We demonstrate that coherence and modularity measures capture distinct network properties. CONCLUSIONS: We present a general-purpose method for estimating and comparing the coherence of molecular-interaction gene networks that accounts for the network size and shape differences. Our results highlight gaps in our current knowledge of genetics and molecular mechanisms of complex phenotypes and suggest priorities for future GWASs.

Transcriptomic and genetic analyses reveal potential causal drivers for intractable partial epilepsy.

Mesial temporal lobe epilepsy with hippocampal sclerosis represents the most common epilepsy syndrome in adult patients with medically intractable partial epilepsy. Mesial temporal lobe epilepsy is usually regarded as a polygenic and complex disorder, still poorly understood but probably caused and perpetuated by dysregulation of numerous biological networks and cellular functions. The study of gene expression changes by single nucleotide polymorphisms in regulatory elements (expression quantitative trait loci, eQTLs) has been shown to be a powerful complementary approach to the detection and understanding of risk loci by genome-wide association studies. We performed a whole (gene and exon-level) transcriptome analysis on cortical tissue samples (Brodmann areas 20 and 21) from 86 patients with mesial temporal lobe epilepsy with hippocampal sclerosis and 75 neurologically healthy controls. Genome-wide genotyping data from the same individuals (patients and controls) were analysed and paired with the transcriptome data. We report potential epilepsy-risk eQTLs, some of which are specific to tissue from patients with mesial temporal lobe epilepsy with hippocampal sclerosis. We also found large transcriptional and splicing deregulation in mesial temporal lobe epilepsy with hippocampal sclerosis tissue as well as gene networks involving neuronal and glial mechanisms that provide new insights into the cause and maintenance of the seizures. These data (available via the 'Seizubraineac' web-tool resource, www.seizubraineac.org) will facilitate the identification of new therapeutic targets and biomarkers as well as genetic risk variants that could influence epilepsy and pharmacoresistance.

The Coherence Problem: Finding Meaning in GWAS Complexity.

Genome wide association studies (GWAS) for behavioral traits and psychiatric disorders have inspired both confident optimism and withering criticism. Although many recent findings from well powered GWAS have been replicated in independent data sets, the genes identified have pinned down few if any underlying causal mechanisms. Therefore, a key issue is whether or not the genes implicated by GWAS form a coherent story on their own and thus could in principle lead to insight into the biological mechanisms underlying the trait or disorder. We sketch here four scenarios for how genes may contribute to traits and disorders; genetic studies may help elucidate mechanisms under only two of our scenarios. We also describe here an approach to characterize, in an unbiased fashion, the molecular coherence of the gene sets implicated by GWAS of various behavioral and psychiatric phenotypes and we sketch how the four scenarios may be reflected in our molecular coherence measure.

Spatio-temporal transcriptome of the human brain.

Brain development and function depend on the precise regulation of gene expression. However, our understanding of the complexity and dynamics of the transcriptome of the human brain is incomplete. Here we report the generation and analysis of exon-level transcriptome and associated genotyping data, representing males and females of different ethnicities, from multiple brain regions and neocortical areas of developing and adult post-mortem human brains. We found that 86 per cent of the genes analysed were expressed, and that 90 per cent of these were differentially regulated at the whole-transcript or exon level across brain regions and/or time. The majority of these spatio-temporal differences were detected before birth, with subsequent increases in the similarity among regional transcriptomes. The transcriptome is organized into distinct co-expression networks, and shows sex-biased gene expression and exon usage. We also profiled trajectories of genes associated with neurobiological categories and diseases, and identified associations between single nucleotide polymorphisms and gene expression. This study provides a comprehensive data set on the human brain transcriptome and insights into the transcriptional foundations of human neurodevelopment.

Transcriptome sequencing and genome-wide association analyses reveal lysosomal function and actin cytoskeleton remodeling in schizophrenia and bipolar disorder.

Schizophrenia (SCZ) and bipolar disorder (BPD) are severe mental disorders with high heritability. Clinicians have long noticed the similarities of clinic symptoms between these disorders. In recent years, accumulating evidence indicates some shared genetic liabilities. However, what is shared remains elusive. In this study, we conducted whole transcriptome analysis of post-mortem brain tissues (cingulate cortex) from SCZ, BPD and control subjects, and identified differentially expressed genes in these disorders. We found 105 and 153 genes differentially expressed in SCZ and BPD, respectively. By comparing the t-test scores, we found that many of the genes differentially expressed in SCZ and BPD are concordant in their expression level (q⩽0.01, 53 genes; q⩽0.05, 213 genes; q⩽0.1, 885 genes). Using genome-wide association data from the Psychiatric Genomics Consortium, we found that these differentially and concordantly expressed genes were enriched in association signals for both SCZ (P<10(-7)) and BPD (P=0.029). To our knowledge, this is the first time that a substantially large number of genes show concordant expression and association for both SCZ and BPD. Pathway analyses of these genes indicated that they are involved in the lysosome, Fc gamma receptor-mediated phagocytosis, regulation of actin cytoskeleton pathways, along with several cancer pathways. Functional analyses of these genes revealed an interconnected pathway network centered on lysosomal function and the regulation of actin cytoskeleton. These pathways and their interacting network were principally confirmed by an independent transcriptome sequencing data set of the hippocampus. Dysregulation of lysosomal function and cytoskeleton remodeling has direct impacts on endocytosis, phagocytosis, exocytosis, vesicle trafficking, neuronal maturation and migration, neurite outgrowth and synaptic density and plasticity, and different aspects of these processes have been implicated in SCZ and BPD.

Genome-wide depletion of replication initiation events in highly transcribed regions.

This report investigates the mechanisms by which mammalian cells coordinate DNA replication with transcription and chromatin assembly. In yeast, DNA replication initiates within nucleosome-free regions, but studies in mammalian cells have not revealed a similar relationship. Here, we have used genome-wide massively parallel sequencing to map replication initiation events, thereby creating a database of all replication initiation sites within nonrepetitive DNA in two human cell lines. Mining this database revealed that genomic regions transcribed at moderate levels were generally associated with high replication initiation frequency. In genomic regions with high rates of transcription, very few replication initiation events were detected. High-resolution mapping of replication initiation sites showed that replication initiation events were absent from transcription start sites but were highly enriched in adjacent, downstream sequences. Methylation of CpG sequences strongly affected the location of replication initiation events, whereas histone modifications had minimal effects. These observations suggest that high levels of transcription interfere with formation of pre-replication protein complexes. Data presented here identify replication initiation sites throughout the genome, providing a foundation for further analyses of DNA-replication dynamics and cell-cycle progression.

Human resilience depends on distinctively human brain circuitry and development.

Most studies of psychological resilience in the past century have focused on either biological or social psychological correlates of resilience or depression. This article argues that the two approaches need to be integrated because of uniquely human processes of cortical development during early childhood. The article concludes with some suggestions for integrative research agendas.

Functional classes of SNPs related to psychiatric disorders and behavioral traits contrast with those related to neurological disorders.

We investigated the functional classes of genomic regions containing SNPS contributing most to the SNP-heritability of important psychiatric and neurological disorders and behavioral traits, as determined from recent genome-wide association studies. We employed linkage-disequilibrium score regression with several brain-specific genomic annotations not previously utilized. The classes of genomic annotations conferring substantial SNP-heritability for the psychiatric disorders and behavioral traits differed systematically from the classes associated with neurological disorders, and both differed from the classes enriched for height, a biometric trait used here as a control outgroup. The SNPs implicated in these psychiatric disorders and behavioral traits were highly enriched in CTCF binding sites, in conserved regions likely to be enhancers, and in brain-specific promoters, regulatory sites likely to affect responses to experience. The SNPs relevant for neurological disorders were highly enriched in constitutive coding regions and splice regulatory sites.

HPV+ head and neck cancer-derived small extracellular vesicles communicate with TRPV1+ neurons to mediate cancer pain.

ABSTRACT: Severe pain is often experienced by patients with head and neck cancer and is associated with a poor prognosis. Despite its frequency and severity, current treatments fail to adequately control cancer-associated pain because of our lack of mechanistic understanding. Although recent works have shed some light of the biology underlying pain in HPV-negative oral cancers, the mechanisms mediating pain in HPV+ cancers remain unknown. Cancer-derived small extracellular vesicles (cancer-sEVs) are well positioned to function as mediators of communication between cancer cells and neurons. Inhibition of cancer-sEV release attenuated pain in tumor-bearing mice. Injection of purified cancer-sEVs is sufficient to induce pain hypersensitivity in naive mice that is prevented by QX-314 treatment and in Trpv1-/- mice. Cancer-sEVs triggered calcium influx in nociceptors, and inhibition or ablation of nociceptors protects against cancer pain. Interrogation of published sequencing data of human sensory neurons exposed to human cancer-sEVs suggested a stimulation of protein translation in neurons. Induction of translation by cancer-sEVs was validated in our mouse model, and its inhibition alleviated cancer pain in mice. In summary, our work reveals that HPV+ head and neck squamous cell carcinoma-derived sEVs alter TRPV1+ neurons by promoting nascent translation to mediate cancer pain and identified several promising therapeutic targets to interfere with this pathway.

DNA methyltransferase 1 and DNA methylation patterning contribute to germinal center B-cell differentiation.

The phenotype of germinal center (GC) B cells includes the unique ability to tolerate rapid proliferation and the mutagenic actions of activation induced cytosine deaminase (AICDA). Given the importance of epigenetic patterning in determining cellular phenotypes, we examined DNA methylation and the role of DNA methyltransferases in the formation of GCs. DNA methylation profiling revealed a marked shift in DNA methylation patterning in GC B cells versus resting/naive B cells. This shift included significant differential methylation of 235 genes, with concordant inverse changes in gene expression affecting most notably genes of the NFkB and MAP kinase signaling pathways. GC B cells were predominantly hypomethylated compared with naive B cells and AICDA binding sites were highly overrepresented among hypomethylated loci. GC B cells also exhibited greater DNA methylation heterogeneity than naive B cells. Among DNA methyltransferases (DNMTs), only DNMT1 was significantly up-regulated in GC B cells. Dnmt1 hypomorphic mice displayed deficient GC formation and treatment of mice with the DNA methyltransferase inhibitor decitabine resulted in failure to form GCs after immune stimulation. Notably, the GC B cells of Dnmt1 hypomorphic animals showed evidence of increased DNA damage, suggesting dual roles for DNMT1 in DNA methylation and double strand DNA break repair.

Stem cell and hepatocyte proliferation in hepatitis C cirrhosis and hepatocellular carcinoma: transplant implications.

BACKGROUND. The liver possesses two distinct mechanisms for healing. Wound healing via hepatic stem cells recapitulates early development (hepatoblast proliferation), while liver regeneration resembles late embryonic growth (hepatocyte proliferation). Loss of control over both of these processes have been proposed as mechanisms that may contribute to poor outcomes in HCC. MATERIAL AND METHODS. We used microarray gene expression profiles to examine the involvement of hepatic stem cell and hepatocyte proliferation markers and regulators in HCV-induced cirrhosis and HCC. We compared 30 cirrhosis and 49 HCC samples to 12 disease-free control livers. RESULTS. Cirrhosis and HCC expressed markers of stem cell. Inhibitors of hepatocyte proliferation (HP) were highly expressed in cirrhosis. Loss of these HP inhibitors in HCC patients was associated poor prognosis (94 vs. 38% 2-year recurrence- free survival, p = 0.0003). Principal Components Analysis discriminated cirrhotic and HCC tissues, and HCC patients with poor (< 2 year) vs. good (> 2 year) recurrence-free survival. Loss of CDH1 expression correlated with up-regulation of hepatocyte proliferation promoters MET and YAP1. CDH1, MET, and YAP1 were independent predictors of recurrence-free survival by Cox regression when corrected for tumor stage (p < 0.0001). CONCLUSION. HCV-cirrhosis is characterized by proliferation of liver stem cells and inhibition of hepatocyte proliferation. HCC tumors in which this pattern persists have superior outcomes to those which acquire a hepatocyte proliferation signature. Genes in this signature should be studied further for potential as tissue or serum biomarkers for patient risk stratification. CDH1 and MET are candidates for personalized therapies with targeted pharmaceutical agents.

Phytochemical drug discovery for COVID-19 using high-resolution computational docking and machine learning assisted binder prediction.

The COVID-19 pandemic has resulted in millions of deaths around the world. Multiple vaccines are in use, but there are many underserved locations that do not have adequate access to them. Variants may emerge that are highly resistant to existing vaccines, and therefore cheap and readily obtainable therapeutics are needed. Phytochemicals, or plant chemicals, can possibly be such therapeutics. Phytochemicals can be used in a polypharmacological approach, where multiple viral proteins are inhibited and escape mutations are made less likely. Finding the right phytochemicals for viral protein inhibition is challenging, but in-silico screening methods can make this a more tractable problem. In this study, we screen a wide range of natural drug products against a comprehensive set of SARS-CoV-2 proteins using a high-resolution computational workflow. This workflow consists of a structure-based virtual screening (SBVS), where an initial phytochemical library was docked against all selected protein structures. Subsequently, ligand-based virtual screening (LBVS) was employed, where chemical features of 34 lead compounds obtained from the SBVS were used to predict 53 lead compounds from a larger phytochemical library via supervised learning. A computational docking validation of the 53 predicted leads obtained from LBVS revealed that 28 of them elicit strong binding interactions with SARS-CoV-2 proteins. Thus, the inclusion of LBVS resulted in a 4-fold increase in the lead discovery rate. Of the total 62 leads, 18 showed promising pharmacokinetic properties in a computational ADME screening. Collectively, this study demonstrates the advantage of incorporating machine learning elements into a virtual screening workflow.Communicated by Ramaswamy H. Sarma.

Factor Structure of the Postpartum Bonding Questionnaire in a US-Based Cohort of Mothers.

As research efforts in the field of pediatrics are oriented toward a better understanding of the synergistic relationship between different facets of early relational health (ERH) and child development and wellbeing, it is essential to focus on the quality of research instruments available for measuring different components of ERH. This study investigates the measurement characteristics of a widely used parent/caregiver-reported measure of bonding, the Postpartum Bonding Questionnaire (PBQ), in a US-based sample (n=610) of English-speaking biological mothers who completed the PBQ at 4 months postpartum. To evaluate the factor structure of the PBQ, confirmatory and exploratory statistical techniques were employed. The current study failed to replicate the PBQ's original 4-factor structure. Exploratory factor analysis results supported the creation of a 14-item abbreviated measure, the PBQ-14. The PBQ-14 showed evidence of good psychometric properties, including high internal consistency (ω=.87) and correlation with depression (r=.44, p<.001) assessed using the Patient Health Questionnaire (PHQ-9), as would be expected. The new unidimensional PBQ-14 is suitable for use in the US as a measure of general postnatal parent/caregiver-to-infant bonding.

Genomic sequence analysis and characterization of Sneathia amnii sp. nov.

BACKGROUND: Bacteria of the genus Sneathia are emerging as potential pathogens of the female reproductive tract. Species of Sneathia, which were formerly grouped with Leptotrichia, can be part of the normal microbiota of the genitourinary tracts of men and women, but they are also associated with a variety of clinical conditions including bacterial vaginosis, preeclampsia, preterm labor, spontaneous abortion, post-partum bacteremia and other invasive infections. Sneathia species also exhibit a significant correlation with sexually transmitted diseases and cervical cancer. Because Sneathia species are fastidious and rarely cultured successfully in vitro; and the genomes of members of the genus had until now not been characterized, very little is known about the physiology or the virulence of these organisms. RESULTS: Here, we describe a novel species, Sneathia amnii sp. nov, which closely resembles bacteria previously designated "Leptotrichia amnionii". As part of the Vaginal Human Microbiome Project at VCU, a vaginal isolate of S. amnii sp. nov. was identified, successfully cultured and bacteriologically cloned. The biochemical characteristics and virulence properties of the organism were examined in vitro, and the genome of the organism was sequenced, annotated and analyzed. The analysis revealed a reduced circular genome of ~1.34 Mbp, containing ~1,282 protein-coding genes. Metabolic reconstruction of the bacterium reflected its biochemical phenotype, and several genes potentially associated with pathogenicity were identified. CONCLUSIONS: Bacteria with complex growth requirements frequently remain poorly characterized and, as a consequence, their roles in health and disease are unclear. Elucidation of the physiology and identification of genes putatively involved in the metabolism and virulence of S. amnii may lead to a better understanding of the role of this potential pathogen in bacterial vaginosis, preterm birth, and other issues associated with vaginal and reproductive health.

Predicting drug sensitivity and resistance: profiling ABC transporter genes in cancer cells.

For analysis of multidrug resistance, a major barrier to effective cancer chemotherapy, we profiled mRNA expression of the 48 known human ABC transporters in 60 diverse cancer cell lines (the NCI-60) used by the National Cancer Institute to screen for anticancer activity. The use of real-time RT-PCR avoided artifacts commonly encountered with microarray technologies. By correlating the results with the growth inhibitory profiles of 1,429 candidate anticancer drugs tested against the cells, we identified which transporters are more likely than others to confer resistance to which agents. Unexpectedly, we also found and validated compounds whose activity is potentiated, rather than antagonized, by the MDR1 multidrug transporter. Such compounds may serve as leads for development.

Differential Co-Expression Analysis of RNA-Seq Data Reveals Novel Potential Biomarkers of Device-Tissue Interaction.

The biological response to electrodes implanted in the brain has been a long-standing barrier to achieving a stable tissue device-interface. Understanding the mechanisms underlying this response could explain phenomena including recording instability and loss, shifting stimulation thresholds, off-target effects of neuromodulation, and stimulation-induced depression of neural excitability. Our prior work detected differential expression in hundreds of genes following device implantation. Here, we extend upon that work by providing new analyses using differential co-expression analysis, which identifies changes in the correlation structure between groups of genes detected at the interface in comparison to control tissues. We used an "eigengene" approach to identify hub genes associated with each module. Our work adds to a growing body of literature which applies new techniques in molecular biology and computational analysis to long-standing issues surrounding electrode integration with the brain.

Allelic heterogeneity in genetic association meta-analysis: an application to DTNBP1 and schizophrenia.

BACKGROUND/AIMS: Meta-analysis of genetic association studies is a useful approach when individual investigations do not yield studywise significant results but the evidence across studies is modest and homogeneous. Current meta-analysis methods account for heterogeneity by down-weighting studies as a function of between-study variance. We contend that current approaches may obscure interesting phenomena in genetic association data. However, an appropriate approach to examining heterogeneity across studies is lacking. METHODS: We develop a novel approach, based on the EM algorithm, to detect allelic heterogeneity, identify subpopulations and assign studies to those subpopulations. We then apply these methods to the association between DTNBP1 and schizophrenia (Scz), one of the most studied relationships in complex disease genetics. We examined 32 published and unpublished population and family-based association studies containing up to 14 SNPs spanning the DTNBP1 locus. RESULTS: We explored heterogeneity in several ways including meta-regression and approaches aimed at exploring the mixture of heterogeneous studies at a particular SNP. We found significant evidence for a mixture of association distributions at multiple loci. CONCLUSION: We propose a novel approach that is broadly applicable and may be useful in large scale genetic association meta-analyses to detect significant allelic heterogeneity.

Swimming direction of the glass catfish is responsive to magnetic stimulation.

Several marine species have developed a magnetic perception that is essential for navigation and detection of prey and predators. One of these species is the transparent glass catfish that contains an ampullary organ dedicated to sense magnetic fields. Here we examine the behavior of the glass catfish in response to static magnetic fields which will provide valuable insight on function of this magnetic response. By utilizing state of the art animal tracking software and artificial intelligence approaches, we quantified the effects of magnetic fields on the swimming direction of glass catfish. The results demonstrate that glass catfish placed in a radial arm maze, consistently swim away from magnetic fields over 20 µT and show adaptability to changing magnetic field direction and location.

Non-invasive neuromodulation using rTMS and the electromagnetic-perceptive gene (EPG) facilitates plasticity after nerve injury.

BACKGROUND: Twenty million Americans suffer from peripheral nerve injury. These patients often develop chronic pain and sensory dysfunctions. In the past decade, neuroimaging studies showed that these changes are associated with altered cortical excitation-inhibition balance and maladaptive plasticity. We tested if neuromodulation of the deprived sensory cortex could restore the cortical balance, and whether it would be effective in alleviating sensory complications. OBJECTIVE: We tested if non-invasive repetitive transcranial magnetic stimulation (rTMS) which induces neuronal excitability, and cell-specific magnetic activation via the Electromagnetic-perceptive gene (EPG) which is a novel gene that was identified and cloned from glass catfish and demonstrated to evoke neural responses when magnetically stimulated, can restore cortical excitability. METHODS: A rat model of forepaw denervation was used. rTMS was delivered every other day for 30 days, starting at the acute or at the chronic post-injury phase. A minimally-invasive neuromodulation via EPG was performed every day for 30 days starting at the chronic phase. A battery of behavioral tests was performed in the days and weeks following limb denervation in EPG-treated rats, and behavioral tests, fMRI and immunochemistry were performed in rTMS-treated rats. RESULTS: The results demonstrate that neuromodulation significantly improved long-term mobility, decreased anxiety and enhanced neuroplasticity. The results identify that both acute and delayed rTMS intervention facilitated rehabilitation. Moreover, the results implicate EPG as an effective cell-specific neuromodulation approach. CONCLUSION: Together, these results reinforce the growing amount of evidence from human and animal studies that are establishing neuromodulation as an effective strategy to promote plasticity and rehabilitation.