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ABSTRACTBile acids (BA) are among the most abundant metabolites produced by the gut microbiome. Primary BAs produced in the liver are converted by gut bacterial 7-α-dehydroxylation into secondary BAs, which can differentially regulate host health via signaling based on their varying affinity for BA receptors. Despite the importance of secondary BAs in host health, the regulation of 7-α-dehydroxylation and the role of diet in modulating this process is incompletely defined. Understanding this process could lead to dietary guidelines that beneficially shift BA metabolism. Dietary fiber regulates gut microbial composition and metabolite production. We tested the hypothesis that feeding mice a diet rich in a fermentable dietary fiber, resistant starch (RS), would alter gut bacterial BA metabolism. Male and female wild-type mice were fed a diet supplemented with RS or an isocaloric control diet (IC). Metabolic parameters were similar between groups. RS supplementation increased gut luminal deoxycholic acid (DCA) abundance. However, gut luminal cholic acid (CA) abundance, the substrate for 7-α-dehydroxylation in DCA production, was unaltered by RS. Further, RS supplementation did not change the mRNA expression of hepatic BA producing enzymes or ileal BA transporters. Metagenomic assessment of gut bacterial composition revealed no change in the relative abundance of bacteria known to perform 7-α-dehydroxylation. P. ginsenosidimutans and P. multiformis were positively correlated with gut luminal DCA abundance and increased in response to RS supplementation. These data demonstrate that RS supplementation enriches gut luminal DCA abundance without increasing the relative abundance of bacteria known to perform 7-α-dehydroxylation.

Background: Fragile X syndrome, with an approximate incidence rate of 1 in 4000 males to 1 in 8000 females, is the most prevalent genetic cause of heritable intellectual disability and the most common monogenic cause of autism spectrum disorder. The full mutation of the Fragile X Messenger Ribonucleoprotein-1 gene, characterized by an expansion of CGG trinucleotide repeats (>200 CGG repeats), leads to fragile X syndrome. Currently, there are no targeted treatments available for fragile X syndrome. In a recent large multi-site trial, FXLEARN, the effects of the mGluR5 negative allosteric modulator, AFQ056 (mavoglurant), were investigated, but did not show a significant impact of AFQ056 on language development in children with fragile X syndrome aged 3–6 years. Objectives: The current analyses from biospecimens collected in the FXLEARN study aimed to determine whether AFQ056 affects the level of potential biomarkers associated with Akt/mTOR and matrix metalloproteinase 9 signaling in young individuals with fragile X syndrome. Previous research has indicated that these biomarkers play crucial roles in the pathophysiology of fragile X syndrome. Design: A double-blind placebo-controlled parallel-group flexible-dose forced titration design. Methods: Blood samples for biomarkers were collected during the FXLEARN at baseline and subsequent visits (1- and 8-month visits). Biomarker analyses included fragile X messenger ribonucleoprotein-1 genotyping by Southern blot and PCR approaches, fragile X messenger ribonucleoprotein-1 mRNA levels determined by PCR, matrix metalloproteinase 9 levels’ detection using a magnetic bead panel, and targets of the Akt/mTOR signaling pathway with their phosphorylation levels detected. Results: This research revealed that administering AFQ056 does not affect the expression levels of the investigated blood biomarkers in young children with fragile X syndrome. Conclusion: Our findings of the lack of association between clinical improvement and biomarkers’ levels in the treatment group are in line with the lack of benefit observed in the FXLEARN study. These findings indicate that AFQ056 does not provide benefits as assessed by primary or secondary endpoints. Registration: ClincalTrials.gov NCT02920892.

Abstract Mesenchymal stem cells (MSCs) are novel therapeutics for the treatment of Crohn’s disease. However, their mechanism of action is unclear, especially in disease-relevant chronic models of inflammation. Thus, we used SAMP-1/YitFc (SAMP), a chronic and spontaneous murine model of small intestinal inflammation, to study the therapeutic effects and mechanism of action of human bone marrow-derived MSCs (hMSC). hMSC dose-dependently inhibited naïve T lymphocyte proliferation via prostaglandin E2 (PGE2) secretion and reprogrammed macrophages to an anti-inflammatory phenotype. We found that the hMSCs promoted mucosal healing and immunologic response early after administration in SAMP when live hMSCs are present (until day 9) and resulted in a complete response characterized by mucosal, histological, immunologic, and radiological healing by day 28 when no live hMSCs are present. hMSCs mediate their effect via modulation of T cells and macrophages in the mesentery and mesenteric lymph nodes (mLN). Sc-RNAseq confirmed the anti-inflammatory phenotype of macrophages and identified macrophage efferocytosis of apoptotic hMSCs as a mechanism that explains their long-term efficacy. Taken together, our findings show that hMSCs result in healing and tissue regeneration in a chronic model of small intestinal inflammation and despite being short-lived, exert long-term effects via sustained anti-inflammatory programming of macrophages via efferocytosis.

Abstract Carriers of a premutation allele (PM) in the FMR1 gene are at risk of developing a number of Fragile X premutation asssociated disorders (FXPAC), including Fragile X-associated Tremor/Ataxia Syndrome (FXTAS), Fragile X-associated Primary Ovarian Insufficiency (FXPOI), and Fragile X-associated neuropsychiatric disorders (FXAND). We have recently reported somatic CGG allele expansion in female PM; however, its clinical significance remains unclear. The aim of this study was to examine the potential clinical association between somatic FMR1 allele instability and PM associated disorders. Participants comprised of 424 female PM carriers age 0.3– 90 years. FMR1 molecular measures and clinical information on the presence of medical conditions, were determined for all subjects for primary analysis. Two sub-groups of participants (age ≥ 25, N = 377 and age ≥ 50, N = 134) were used in the analysis related to presence of FXPOI and FXTAS, respectively. Among all participants (N = 424), the degree of instability (expansion) was significantly higher (median 2.5 vs 2.0, P = 0.026) in participants with a diagnosis of attention deficit hyperactivity disorder (ADHD) compared to those without. FMR1 mRNA expression was significantly higher in subjects with any psychiatric disorder diagnosis (P = 0.0017); specifically, in those with ADHD (P = 0.009), and with depression (P = 0.025). Somatic FMR1 expansion was associated with the presence of ADHD in female PM and FMR1 mRNA levels were associated with the presence of mental health disorders. The findings of our research are innovative as they suggest a potential role of the CGG expansion in the clinical phenotype of PM and may potentially guide clinical prognosis and management.

IntroductionIrrigation management dramatically alters soil water availability and distribution and could impact soil microbial communities and carbon (C) and nitrogen (N) cycling to an even greater degree than observed in rainfed systems. Adoption of subsurface drip irrigation (SDI) in California’s Mediterranean agroecosystems provides agronomic benefits but wets only a portion of the soil volume near the root zone, leaving the rest dry throughout the growing season. In contrast, traditional furrow irrigation (FI) has periodic wetting events with more homogenous moisture distribution. With conversion to precision irrigation methods, how will the microbiome respond to changes moisture availability, and how is their response influenced by soil C and N resource levels?MethodsIn a field experiment in California, we compared SDI and FI’s effects on microbial communities and evaluated how long-term organic and conventional management systems impact outcomes. Throughout the growing season, soil samples were collected at two depths (0-15, 15-30 cm) and three distances from bed center (10, 25, 45 cm) where the drip tape is located.ResultsAt harvest, soils irrigated using SDI had lower microbial biomass C (MBC) than under FI at the surface and showed a build-up of soluble C and N relative to MBC at the bed edge, indicating reduced microbial uptake. Community composition at the bed edge also diverged between SDI and FI, favoring Actinobacteria in the former and Acidobacteria and Gemmatimonadetes in the latter. Regardless of irrigation type, dry areas of the bed had the highest alpha diversity indices. Response to SDI was similar in organic and conventional systems, though organic had higher MBC, DOC, and relative abundance of Proteobacteria and fungal lipids, regardless of irrigation.DiscussionProlonged dry conditions in SDI appeared to limit microbial access to resources and changed community composition. As seen in non-agricultural systems, the severity and frequency of moisture changes, adaptation of the communities, and resource availability affect microbial response. Decoupling of C and N pools in dry surface soils under SDI may increase the potential for losses of DOC and nitrate with the first winter rains in this Mediterranean climate.

Purpose: The goals of this cross-sectional community-based survey study were to assess the impact of the Covid-19 pandemic on actionable factors which are known to contribute to worse cancer outcomes, and to determine whether race and ethnicity-based differences exist. Methods: A survey study which captured demographic information and changes in cancer outcomes-related factors since the start of the Covid-19 pandemic, was conducted at a public Covid-19 vaccination clinic over a period of 10 days during March 2021. Surveys were administered in multiple languages. Chi-square tests and ANOVA followed by post-hoc Dunnett testing assessed for race and ethnicity-based differences. Results: A total of 949 people participated (61.6% participation rate). Ninety-three surveys were removed based on inclusion criteria giving a final participant number of 856. Many participants reported postponing cancer screenings (17.8%) and cancellation of medical appointments (22.8% and 25.8% reported cancelled appointments by providers or themselves, respectively) due to the pandemic. Participants also reported decreased physical activity (44.7%) and increased tobacco and/or marijuana usage (7.0%). Conversely, participants reported consuming more fruits and vegetables (21.4%) and decreasing alcohol consumption (21.4%). Several race-related differences but no ethnicity-related differences were observed. Conclusion: Our data can be used to help guide pharmacist-led targeted outreach in our community which will help mitigate Covid-19 pandemic-driven changes in behaviors associated with worse cancer outcomes and exacerbation of cancer health disparities. To our knowledge, this is the first cancer outcomes-related study to be conducted at a public Covid-19 vaccination site and is the first pharmacist-led study in this area.

Using a mouse model for Rett syndrome (RTT), Neier et al analyze gut microbiome and metabolome profiles during disease progression. They show that pathophysiology and progression of RTT is different in females than in males and suggest that changes in the microbiome and metabolism of the gastrointestinal tract influence disease progression.

The course of pathophysiological mechanisms involved in fragile X-associated tremor/ataxia syndrome (FXTAS) remains largely unknown. Previous proteomics and metabolomics studies conducted in blood samples collected from FMR1 premutation carriers with FXTAS reported abnormalities in energy metabolism, and precursors of gluconeogenesis showed significant changes in plasma expression levels in FMR1 premutation carriers who developed FXTAS. We conducted an analysis of postmortem human brain tissues from 44 donors, 25 brains with FXTAS, and 19 matched controls. We quantified the metabolite relative abundance in the inferior temporal gyrus and the cerebellum using untargeted mass spectrometry (MS)-based metabolomics. We investigated how the metabolite type and abundance relate to the number of cytosine-guanine-guanine (CGG) repeats, to markers of neurodegeneration, and to the symptoms of FXTAS. A metabolomic analysis identified 191 primary metabolites, the data were log-transformed and normalized prior to the analysis, and the relative abundance was compared between the groups. The changes in the relative abundance of a set of metabolites were region-specific with some overlapping results; 22 metabolites showed alterations in the inferior temporal gyrus, while 21 showed differences in the cerebellum. The relative abundance of cytidine was decreased in the inferior temporal gyrus, and a lower abundance was found in the cases with larger CGG expansions; oleamide was significantly decreased in the cerebellum. The abundance of 11 metabolites was influenced by changes in the CGG repeat number. A histological evaluation found an association between the presence of microhemorrhages in the inferior temporal gyrus and a lower abundance of 2,5-dihydroxypyrazine. Our study identified alterations in the metabolites involved in the oxidative-stress response and bioenergetics in the brains of individuals with FXTAS. Significant changes in the abundance of cytidine and oleamide suggest their potential as biomarkers and therapeutic targets for FXTAS.

Abstract Over the last decade oxylipins have become more recognized for their involvement in several diseases. Non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen are known to inhibit cyclooxygenase (COX) enzymes, but how NSAIDs affect oxylipins, in addition to COX products, in animal tissues is not well understood. Oxylipins in livers from male and female mice treated with 100 mg/kg/day of ibuprofen for 7 days were investigated. The results showed that ibuprofen treated male livers contained 7 times more altered oxylipins than ibuprofen treated female livers. In male and female livers some prostaglandins were altered, while diols, hydroxy fatty acids and epoxides were significantly altered in male livers. Some soluble epoxide hydrolase (sEH) products, such as 9,10-DiHODE were found to be decreased, while sEH substrates (such as 9(10)-EpODE and 5(6)-EpETrE) were found to be increased in male livers treated with ibuprofen, but not in ibuprofen treated female livers. The enzymatic activities of sEH and microsomal epoxide hydrolase (mEH) were elevated by ibuprofen in both males and females. Analyzing the influence of sex on the effect of ibuprofen on oxylipins and COX products showed that approximately 27% of oxylipins detected were influenced by sex. The results reveal that ibuprofen disturbs not only the COX pathway, but also the CYP450 and lipoxygenase pathways in male mice, suggesting that ibuprofen is likely to generate sex related differences in biologically active oxylipins. Increased sEH activity after ibuprofen treatment is likely to be one of the mechanisms by which the liver reduces the higher levels of EpODEs and EpETrEs.

Abstract Fragile X associated tremor/ataxia syndrome (FXTAS) is a late adult-onset neurodegenerative disorder that affects movement and cognition in male and female carriers of a premutation allele of 55–200 CGG repeats in the Fragile X mental retardation (FMR1) gene. It is currently unknown if and when an individual carrier of a premutation allele will develop FXTAS, as clinical assessment fails to identify carriers at risk before significant neurological symptoms are evident. The primary objective of this study was to investigate the alternative splicing landscape at the FMR1 locus in conjunction with brain measures in male individuals with a premutation allele enrolled in a very first longitudinal study, compared to age-matched healthy male controls, with the purpose of identifying biomarkers for early diagnosis, disease prediction and, a progression of FXTAS. Our findings indicate that increased expression of FMR1 mRNA isoforms, including Iso4/4b, Iso10/10b, as well as of the ASFMR1 mRNAs Iso131bp, are present in premutation carriers as compared to non-carrier healthy controls. More specifically, we observed a higher expression of Iso4/4b and Iso10/10b, which encode for truncated proteins, only in those premutation carriers who developed symptoms of FXTAS over time as compared to non-carrier healthy controls, suggesting a potential role in the development of the disorder. In addition, we found a significant association of these molecular changes with various measurements of brain morphology, including the middle cerebellar peduncle (MCP), superior cerebellar peduncle (SCP), pons, and midbrain, indicating their potential contribution to the pathogenesis of FXTAS. Interestingly, the high expression levels of Iso4/4b observed both at visit 1 and visit 2 and found to be associated with a decrease in mean MCP width only in those individuals who developed FXTAS over time, suggests their role as potential biomarkers for early diagnosis of FXTAS.

ABSTRACT Antimicrobial resistance (AMR) represents a significant source of morbidity and mortality worldwide, with expectations that AMR-associated consequences will continue to worsen throughout the coming decades. Since resistance to antibiotics is encoded in the microbiome, interventions aimed at altering the taxonomic composition of the gut might allow us to prophylactically engineer microbiomes that harbor fewer antibiotic resistant genes (ARGs). Diet is one method of intervention, and yet little is known about the association between diet and antimicrobial resistance. To address this knowledge gap, we examined diet using the food frequency questionnaire (FFQ; habitual diet) and 24-h dietary recalls (Automated Self-Administered 24-h [ASA24®] tool) coupled with an analysis of the microbiome using shotgun metagenome sequencing in 290 healthy adult participants of the United States Department of Agriculture (USDA) Nutritional Phenotyping Study. We found that aminoglycosides were the most abundant and prevalent mechanism of AMR in these healthy adults and that aminoglycoside-O-phosphotransferases (aph3-dprime) correlated negatively with total calories and soluble fiber intake. Individuals in the lowest quartile of ARGs (low-ARG) consumed significantly more fiber in their diets than medium- and high-ARG individuals, which was concomitant with increased abundances of obligate anaerobes, especially from the family Clostridiaceae, in their gut microbiota. Finally, we applied machine learning to examine 387 dietary, physiological, and lifestyle features for associations with antimicrobial resistance, finding that increased phylogenetic diversity of diet was associated with low-ARG individuals. These data suggest diet may be a potential method for reducing the burden of AMR. IMPORTANCE Antimicrobial resistance (AMR) represents a considerable burden to health care systems, with the public health community largely in consensus that AMR will be a major cause of death worldwide in the coming decades. Humans carry antibiotic resistance in the microbes that live in and on us, collectively known as the human microbiome. Diet is a powerful method for shaping the human gut microbiome and may be a tractable method for lessening antibiotic resistance, and yet little is known about the relationship between diet and AMR. We examined this relationship in healthy individuals who contained various abundances of antibiotic resistance genes and found that individuals who consumed diverse diets that were high in fiber and low in animal protein had fewer antibiotic resistance genes. Dietary interventions may be useful for lessening the burden of antimicrobial resistance and might ultimately motivate dietary guidelines which will consider how nutrition can reduce the impact of infectious disease.

Carriers of the FMR1 premutation (PM) allele are at risk of one or more clinical conditions referred to as FX premutation-associated conditions (FXPAC). Since the FMR1 gene is on the X chromosome, the activation ratio (AR) may impact the risk, age of onset, progression, and severity of these conditions. The aim of this study was to evaluate the reliability of AR measured using different approaches and to investigate potential correlations with clinical outcomes. Molecular and clinical assessments were obtained for 30 PM female participants, and AR was assessed using both Southern blot analysis (AR-Sb) and methylation PCR (AR-mPCR). Higher ARs were associated with lower FMR1 transcript levels for any given repeat length. The higher AR-Sb was significantly associated with performance, verbal, and full-scale IQ scores, confirming previous reports. However, the AR-mPCR was not significantly associated (p > 0.05) with these measures. Similarly, the odds of depression and the number of medical conditions were correlated with higher AR-Sb but not correlated with a higher AR-mPCR. This study suggests that AR-Sb may be a more reliable measure of the AR in female carriers of PM alleles. However, further studies are warranted in a larger sample size to fully evaluate the methylation status in these participants and how it may affect the clinical phenotype.

Summary: Ninety percent of Americans consume less than the estimated average requirements of dietary vitamin E (vitE). Severe vitE deficiency due to genetic mutations in the tocopherol transfer protein (TTPA) in humans results in ataxia with vitE deficiency (AVED), with proprioceptive deficits and somatosensory degeneration arising from dorsal root ganglia neurons (DRGNs). Single-cell RNA-sequencing of DRGNs was performed in Ttpa−/− mice, an established model of AVED. In stark contrast to expected changes in proprioceptive neurons, Ttpa−/− DRGNs showed marked upregulation of voltage-gated Ca2+ and K+ channels in mechanosensitive, tyrosine-hydroxylase positive (TH+) DRGNs. The ensuing significant conductance changes resulted in reduced excitability in mechanosensitive Ttpa−/− DRGNs. A highly supplemented vitE diet (600 mg dl-α-tocopheryl acetate/kg diet) prevented the cellular and molecular alterations and improved mechanosensation. VitE deficiency profoundly alters the molecular signature and functional properties of mechanosensitive TH+ DRGN, representing an intriguing shift of the prevailing paradigm from proprioception to mechanical sensation. : Neuroscience; Molecular Neuroscience; Transcriptomics Subject Areas: Neuroscience, Molecular Neuroscience, Transcriptomics

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late adult-onset neurodegenerative disorder that affects movement and cognition in male and female carriers of a premutation allele (55–200 CGG repeats; PM) in the fragile X mental retardation (FMR1) gene. It is currently unknown how the observed brain changes are associated with metabolic signatures in individuals who develop the disorder over time. The primary objective of this study was to investigate the correlation between longitudinal changes in the brain (area of the pons, midbrain, and MCP width) and the changes in the expression level of metabolic biomarkers of early diagnosis and progression of FXTAS in PM who, as part of an ongoing longitudinal study, emerged into two distinct categories. These included those who developed symptoms of FXTAS (converters, CON) at subsequent visits and those who did not meet the criteria of diagnosis (non-converters, NCON) and were compared to age-matched healthy controls (HC). We assessed CGG repeat allele size by Southern Blot and PCR analysis. Magnetic Resonance Imaging (MRIs) acquisition was obtained on a 3T Siemens Trio scanner and metabolomic profile was obtained by ultra-performance liquid chromatography, accurate mass spectrometer, and an Orbitrap mass analyzer. Our findings indicate that differential metabolite levels are linked with the area of the pons between healthy control and premutation groups. More specifically, we observed a significant association of ceramides and mannonate metabolites with a decreased area of the pons, both at visit 1 (V1) and visit 2 (V2) only in the CON as compared to the NCON group suggesting their potential role in the development of the disorder. In addition, we found a significant correlation of these metabolic signatures with the FXTAS stage at V2 indicating their contribution to the progression and pathogenesis of FXTAS. Interestingly, these metabolites, as part of lipid and sphingolipid lipids pathways, provide evidence of the role that their dysregulation plays in the development of FXTAS and inform us as potential targets for personalized therapeutic development.

Sertraline is one among several selective serotonin reuptake inhibitors (SSRIs) that exhibited improvement of language development in Autism Spectrum Disorder (ASD); however, the molecular mechanism has not been elucidated. A double blind, randomized, 6-month, placebo-controlled, clinical trial of low-dose sertraline in children ages (3–6 years) with ASD was conducted at the UC Davis MIND Institute. It aimed at evaluating the efficacy and benefit with respect to early expressive language development and global clinical improvement. This study aimed to identify molecular biomarkers that might be key players in the serotonin pathway and might be predictive of a clinical response to sertraline. Fifty eight subjects with the diagnosis of ASD were randomized to sertraline or placebo. Eight subjects from the sertraline arm and five from the placebo arm discontinued from the study. Furthermore, four subjects did not have a successful blood draw. Hence, genotypes for 41 subjects (20 on placebo and 21 on sertraline) were determined for several genes involved in the serotonin pathway including the serotonin transporter-linked polymorphic region (5-HTTLPR), the tryptophan hydroxylase 2 (TPH2), and the Brain-Derived Neurotrophic Factor (BDNF). In addition, plasma levels of BDNF, Matrix metallopeptidase 9 (MMP-9) and a selected panel of cytokines were determined at baseline and post-treatment. Intent-to-treat analysis revealed several primary significant correlations between molecular changes and the Mullen Scales of Early Learning (MSEL) and Clinical Global Impression Scale – Improvement (CGI-I) of treatment and control groups but they were not significant after adjustment for multiple testing. Thus, sertraline showed no benefit for treatment of young children with ASD in language development or changes in molecular markers in this study. These results indicate that sertraline may not be beneficial for the treatment of children with ASD; however, further investigation of larger groups as well as longer term follow-up studies are warranted.

Genomic loops and their temporal dynamics play an important role in gene expression of DNA viruses, but are incompletely understood. Here, the authors use capture Hi-C analyses and identify genomic architectural dynamics of KSHV that are regulated by the viral transactivator K-Rta.

The prevalence of the fragile X premutation (55–200 CGG repeats) among the general population is relatively high, but there remains a lack of clear understanding of the links between molecular biomarkers and clinical outcomes. In this study we investigated the correlations between molecular measures (CGG repeat size, FMR1 mRNA, FMRP expression levels, and methylation status at the promoter region and in FREE2 site) and clinical phenotypes (anxiety, obsessive compulsive symptoms, depression and executive function deficits) in 36 adult premutation female carriers and compared to 24 normal control subjects. Premutation carriers reported higher levels of obsessive compulsive symptoms, depression, and anxiety, but demonstrated no significant deficits in global cognitive functions or executive function compared to the control group. Increased age in carriers was significantly associated with increased anxiety levels. As expected, FMR1 mRNA expression was significantly correlated with CGG repeat number. However, no significant correlations were observed between molecular (including epigenetic) measures and clinical phenotypes in this sample. Our study, albeit limited by the sample size, establishes the complexity of the mechanisms that link the FMR1 locus to the clinical phenotypes commonly observed in female carriers suggesting that other factors, including environment or additional genetic changes, may have an impact on the clinical phenotypes. However, it continues to emphasize the need for assessment and treatment of psychiatric problems in female premutation carriers.

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability. FXS is an X-linked, neurodevelopmental disorder caused by a CGG trinucleotide repeat expansion in the 5′ untranslated region (UTR) of the Fragile X Mental Retardation gene, FMR1. Greater than 200 CGG repeats results in epigenetic silencing of the gene leading to the deficiency or absence of Fragile X mental retardation protein (FMRP). The loss of FMRP is considered the root cause of FXS. The relationship between neurological function and FMRP expression in peripheral blood mononuclear cells (PBMCs) has not been well established. Assays to detect and measure FMR1 and FMRP have been described; however, none are sufficiently sensitive, precise, or quantitative to properly characterize the relationships between cognitive ability and CGG repeat number, FMR1 mRNA expression, or FMRP expression measured in PBMCs. To address these limitations, two novel immunoassays were developed and optimized, an electro-chemiluminescence immunoassay and a multiparameter flow cytometry assay. Both assays were performed on PMBCs isolated from 27 study participants with FMR1 CGG repeats ranging from normal to full mutation. After correcting for methylation, a significant positive correlation between CGG repeat number and FMR1 mRNA expression levels and a significant negative correlation between FMRP levels and CGG repeat expansion was observed. Importantly, a high positive correlation was observed between intellectual quotient (IQ) and FMRP expression measured in PBMCs.

Recent advances in neurobiology have provided several molecular entrees for targeted treatments for Fragile X syndrome (FXS). However, the efficacy of these treatments has been demonstrated mainly in animal models and has not been consistently predictive of targeted drugs’ response in the preponderance of human clinical trials. Because of the heterogeneity of FXS at various levels, including the molecular level, phenotypic manifestation, and drug response, it is critically important to identify biomarkers that can help in patient stratification and prediction of therapeutic efficacy. The primary objective of this study was to assess the ability of molecular biomarkers to predict phenotypic subgroups, symptom severity, and treatment response to metformin in clinically treated patients with FXS. We specifically tested a triplex protein array comprising of hexokinase 1 (HK1), RAS (all isoforms), and Matrix Metalloproteinase 9 (MMP9) that we previously demonstrated were dysregulated in the FXS mouse model and in blood samples from patient with FXS. Seventeen participants with FXS, 12 males and 5 females, treated clinically with metformin were included in this study. The disruption in expression abundance of these proteins was normalized and associated with significant self-reported improvement in clinical phenotypes (CGI-I in addition to BMI) in a subset of participants with FXS. Our preliminary findings suggest that these proteins are of strong molecular relevance to the FXS pathology that could make them useful molecular biomarkers for this syndrome.

Friedreich's ataxia (FA) is a neurodegenerative disease with no approved therapy that is the result of frataxin deficiency. The identification of human FA blood biomarkers related to disease severity and neuro-pathomechanism could support clinical trials of drug efficacy. To try to identify human biomarkers of neuro-pathomechanistic relevance, we compared the overlapping gene expression changes of primary blood and skin cells of FA patients with changes in the Dorsal Root Ganglion (DRG) of the KIKO FA mouse model. As DRG is the primary site of neurodegeneration in FA, our goal was to identify which changes in blood and skin of FA patients provide a 'window' into the FA neuropathomechanism inside the nervous system. In addition, gene expression in frataxin-deficient neuroglial cells and FA mouse hearts were compared for a total of 5 data sets. The overlap of these changes strongly supports mitochondrial changes, apoptosis and alterations of selenium metabolism. Consistent biomarkers were observed, including three genes of mitochondrial stress (MTIF2, ENO2), apoptosis (DDIT3/CHOP), oxidative stress (PREX1), and selenometabolism (SEPW1). These results prompted our investigation of the GPX1 activity as a marker of selenium and oxidative stress, in which we observed a significant change in FA patients. We believe these lead biomarkers that could be assayed in FA patient blood as indicators of disease severity and progression, and also support the involvement of mitochondria, apoptosis and selenium in the neurodegenerative process.

Not available

Approximately 30–40% of male and 8–16% of female carriers of the Fragile X premutation will develop a neurodegenerative movement disorder characterized by intentional tremor, gait ataxia, autonomic dysfunction, cognitive decline, and Parkinsonism during their lifetime. At the molecular level, premutation carriers have increased expression levels of the FMR1 and the antisense FMR1 (ASFMR1) mRNAs. Both genes undergo alternative splicing giving rise to a number of different transcripts. Alteration in the alternative splicing process might be associated with FXTAS. In this study, we have investigated the correlation between objective measures of movement (balance and tremor using the CATSYS battery) and the expression of both the FMR1 and the ASFMR1 genes. In addition, we investigated whether their expression level and that of the ASFMR1 131 bp splice isoform could distinguish between premutation carriers with FXTAS and non-FXTAS premutation carriers. Confirming previous findings, the expression levels of transcripts at the FMR1 locus positively correlated with the CGG repeat number and significantly differentiated the premutation carriers from the control groups. Furthermore, premutation carriers with and without FXTAS, showed a significant difference in the expression level of the ASFMR1 131 bp splice isoform when compared to age and gender matched controls. However, there was no significant difference in the ASFMR1 131 bp splice isoform expression level when comparing premutation carriers with and without FXTAS. Finally, our results indicate significant group differences in CATSYS dominant hand reaction time and postural sway with eyes closed in premutation carriers without FXTAS compared to controls. In addition, a significant inverse association between the tremor intensity and the expression level of ASFMR1 131 bp splice isoform in premutation carriers compared to controls, was observed, suggesting a potential role in the pathogenesis of FXTAS.

We used the Affymetrix® Genome-Wide Human SNP Array 6.0 to identify heterospecific markers and compare copy number and structural genomic variation between humans and rhesus macaques. Over 200,000 human copy number variation (CNV) probes were mapped to a Chinese and an Indian rhesus macaque sample. Observed genomic rearrangements and synteny were in agreement with the results of a previously published genomic comparison between humans and rhesus macaques. Comparisons between each of the two rhesus macaques and humans yielded 206 regions with copy numbers that differed by at least two fold in the Indian rhesus macaque and human, 32 in the Chinese rhesus macaque and human, and 147 in both rhesus macaques. The detailed genomic map and preliminary CNV data are useful for better understanding genetic variation in rhesus macaques, identifying derived changes in human CNVs that may have evolved by selection, and determining the suitability of rhesus macaques as human models for particular biomedical studies.

Osteoporosis has been associated with pulmonary silicosis in California horses exposed to soils rich in cytotoxic silica dioxide crystals, a syndrome termed silicate associated osteoporosis (SAO). The causal mechanism for the development of osteoporosis is unknown. Osteoporotic lesions are primarily located in bone marrow-rich sites such as ribs, scapula and pelvis. Gene transcription patterns within bone marrow and pulmonary lymph nodes of affected horses may offer clues to disease pathobiology. Bone marrow core and tracheobronchial lymph node tissue samples harvested postmortem from affected and unaffected horses were examined histologically and subjected to RNA sequencing (RNA-seq). Sequenced data were analyzed for differential gene expression and gene ontology. Metatranscriptomic and metagenomic assays evaluated samples for infectious agents. Thirteen of 17 differentially expressed transcripts in bone marrow were linked to bone and cartilage formation such as integrin binding bone sialoprotein (log2FC = 3.39, PFDR = 0.013) and chondroadherin (log2FC = 4.48, PFDR = 0.031). Equus caballus solute carrier family 9, subfamily A2 (log2FC = 3.77, PFDR = 0.0034) was one of the four differentially expressed transcripts linked to osteoclast activity. Osteoblasts were hyperplastic and hypertrophic in bone marrow from affected horses. Biological pathways associated with skeletal morphogenesis were significantly enriched in affected horses. The 30 differentially expressed genes in affected lymph nodes were associated with inflammatory responses. Evidence of infectious agents was not found. The SAO affected bone marrow molecular signature demonstrated increased transcription and heightened activation of osteoblasts. Increased osteoblastic activity could be part of the pathological mechanism for osteoporosis or a compensatory response to the accelerated osteolysis. Transcriptome data offer gene targets for inquiries into the role of osteocytes and osteoblasts in SAO pathogenesis. Viral or bacterial infectious etiology in SAO is less likely based on metatranscriptomic and metagenomic data but cannot be completely ruled out.

Aim: Recent studies raise concerns for higher incidence of hepatocellular carcinoma (HCC) after direct-acting antiviral therapy for hepatitis C virus (HCV). Methods: In this study, using analysis of liver imaging pre- and post-DAA treatment, we queried new occurrence or ‘de novo’ of HCC in patients with HCV-cirrhosis treated with DAAs. Of 150 patients who met study criteria, 7 (4.7%; 95% CI: 2.1–9.5%) patients developed de novo HCC which did not differ from historical rates of 3% (p = 0.22). Results: Notably, patients with decompensated cirrhosis had significantly higher rate of de novo HCC (9.3%; 95% CI: 3.12–22.2%; p = 0.04). Conclusion: Our data support the need for continued surveillance for HCC in HCV cirrhotics even after successful therapy.

Introduction: Peyronie's disease (PD) and Dupuytren contractures (DC) are often comorbid and are believed to have a similar underlying pathophysiologic mechanism. Aim: To investigate the prevalence of PD-like symptoms (PDLS) in men with DC. Methods: From October 2013 to December 2016, men who were seen and evaluated for DC were offered the opportunity to participate in an anonymous survey. The survey assessed several basic demographic and sexual health factors and included items from the International Index of Erectile Function and the Erection Hardness Scale. Men who reported PDLS were asked a series of questions derived from the Peyronie's Disease Questionnaire and for their opinions on theoretical treatment modalities for sexual problems and penile deformity. Main Outcome Measure: Prevalence of PDLS in men with DC. Results: One hundred forty men with DC were invited to participate; 85 surveys were returned (response rate = 61%). Twenty-two respondents (26%, 95% confidence interval = 17–35) reported PDLS. Approximately one fourth of all respondents had an Erection Hardness Scale score lower than 3. The most common specific PDLS concerns were penile curvature (91%), length loss (55%), narrowing (36%), and hinging (32%). In men with PDLS, 73% felt at least a little bothered by the symptoms when attempting sexual activity and 40% reported having sex less frequently because of the symptoms. Just 27% of men with PDLS had ever used a treatment for a sexual concern. In terms of treatments for penile deformities, 64% of men with PDLS expressed an interest in treatment administered in the form of an in-office procedure; 41% were potentially amenable to a surgical procedure. Conclusion: The prevalence of PDLS in men with DC is similar to the prevalence of DC in men diagnosed with PD. A substantial number of these men have distress and would consider standard-of-care treatments for PD. Shindel AW, Sweet G, Thieu W, et al. Prevalence of Peyronie's Disease-Like Symptoms in Men Presenting With Dupuytren Contractures. Sex Med 2017;5:e135–e141.

Background: Little is known about the effect of a minimum number of slides required in generating image datasets used to build generalizable machine-learning (ML) models. In addition, the assumption within deep learning is that the increased number of training images will always enhance accuracy and that the initial validation accuracy of the models correlates well with their generalizability. In this pilot study, we have been able to test the above assumptions to gain a better understanding of such platforms, especially when data resources are limited. Methods: Using 10 colon histology slides (5 carcinoma and 5 benign), we were able to acquire 1000 partially overlapping images (Dataset A) that were then trained and tested on three convolutional neural networks (CNNs), ResNet50, AlexNet, and SqueezeNet, to build a large number of unique models for a simple task of classifying colon histopathology into benign and malignant. Different quantities of images (10–1000) from Dataset A were used to construct >200 unique CNN models whose performances were individually assessed. The performance of these models was initially assessed using 20% of Dataset A's images (not included in the training phase) to acquire their initial validation accuracy (internal accuracy) followed by their generalization accuracy on Dataset B (a very distinct secondary test set acquired from public domain online sources).Results: All CNNs showed similar peak internal accuracies (>97%) from the Dataset A test set. Peak accuracies for the external novel test set (Dataset B), an assessment of the ability to generalize, showed marked variation (ResNet50: 98%; AlexNet: 92%; and SqueezeNet: 80%). The models with the highest accuracy were not generated using the largest training sets. Further, a model's internal accuracy did not always correlate with its generalization accuracy. The results were obtained using an optimized number of cases and controls. Conclusions: Increasing the number of images in a training set does not always improve model accuracy, and significant numbers of cases may not always be needed for generalization, especially for simple tasks. Different CNNs reach peak accuracy with different training set sizes. Further studies are required to evaluate the above findings in more complex ML models prior to using such ancillary tools in clinical settings.

Deletion of the 1.5-3 Mb region of chromosome 22 at locus 11.2 gives rise to the chromosome 22q11.2 deletion syndrome (22q11DS), also known as DiGeorge and Velocardiofacial Syndromes. It is the most common micro-deletion disorder in humans and one of the most common multiple malformation syndromes. The syndrome is characterized by a broad phenotype, whose characterization has expanded considerably within the last decade and includes many associated findings such as craniofacial anomalies (40%), conotruncal defects of the heart (CHD; 70-80%), hypocalcemia (20-60%), and a range of neurocognitive anomalies with high risk of schizophrenia, all with a broad phenotypic variability. These phenotypic features are believed to be the result of a change in the copy number or dosage of the genes located in the deleted region. Despite this relatively clear genetic etiology, very little is known about which genes modulate phenotypic variations in humans or if they are due to combinatorial effects of reduced dosage of multiple genes acting in concert. Here, we report on decreased expression levels of genes within the deletion region of chromosome 22, including DGCR8, in peripheral leukocytes derived from individuals with 22q11DS compared to healthy controls. Furthermore, we found dysregulated miRNA expression in individuals with 22q11DS, including miR-150, miR-194 and miR-185. We postulate this to be related to DGCR8 haploinsufficiency as DGCR8 regulates miRNA biogenesis. Importantly we demonstrate that the level of some miRNAs correlates with brain measures, CHD and thyroid abnormalities, suggesting that the dysregulated miRNAs may contribute to these phenotypes and/or represent relevant blood biomarkers of the disease in individuals with 22q11DS.

Sexual reproduction of Toxoplasma gondii occurs exclusively within enterocytes of the definitive felid host. The resulting immature oocysts are excreted into the environment during defecation, where in the days following, they undergo a complex developmental process. Within each oocyst, this culminates in the generation of two sporocysts, each containing 4 sporozoites. A single felid host is capable of shedding millions of oocysts, which can survive for years in the environment, are resistant to most methods of microbial inactivation during water-treatment and are capable of producing infection in warm-blooded hosts at doses as low as 1-10 ingested oocysts. Despite its extremely interesting developmental biology and crucial role in initiating an infection, almost nothing is known about the oocyst stage beyond morphological descriptions. Here, we present a complete transcriptomic analysis of the oocyst from beginning to end of its development. In addition, and to identify genes whose expression is unique to this developmental form, we compared the transcriptomes of developing oocysts with those of in vitro-derived tachyzoites and in vivo-derived bradyzoites. Our results reveal many genes whose expression is specifically up- or down-regulated in different developmental stages, including many genes that are likely critical to oocyst development, wall formation, resistance to environmental destruction and sporozoite infectivity. Of special note is the up-regulation of genes that appear "off" in tachyzoites and bradyzoites but that encode homologues of proteins known to serve key functions in those asexual stages, including a novel pairing of sporozoite-specific paralogues of AMA1 and RON2, two proteins that have recently been shown to form a crucial bridge during tachyzoite invasion of host cells. This work provides the first in-depth insight into the development and functioning of one of the most important but least studied stages in the Toxoplasma life cycle.

Fragile X associated disorders are caused by a premutation allele in the fragile X mental retardation 1 gene (FMR1) and are hypothesized to result from the toxic effect of elevated levels of expanded FMR1 transcripts. Increased levels of FMR1 mRNA have indeed been reported in premutation carriers; however the mechanism by which expanded alleles lead to elevated levels of FMR1 mRNA in premutation carriers is unknown. Within the CGG repeat tract AGG interruptions are found, generally 1-3 present in normal/intermediate alleles (6-54 CGG repeats) and usually 0-1 in premutation alleles (55-200 CGG repeats). They are present at specific locations, generally occurring after 9 or 10 uninterrupted CGG repeats [(CGG)(9)AGG(CGG)(9)AGG(CGG)(n)]. We evaluated both the number of AGG interruptions and the resulting length of the uninterrupted 3' CGG repeat pure tract in premutation alleles derived from two large cohorts of male and female carriers to determine whether the presence of AGG interruptions or the length of a pure stretch of CGG repeats influence the levels of FMR1 mRNA in blood. Our findings indicate that neither the number of AGG interruptions, nor their position along the CGG tract have a significant affect on mRNA levels in premutation carriers. We also, as expected based on previous findings, observed a highly significant correlation between CGG repeat number (as both total length and length of pure CGG stretch) and FMR1 mRNA expression levels, in both males and females. Importantly, we did not observe any significant difference in FMR1 mRNA levels in premutation carriers based on age.