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BackgroundMicrorchidia proteins (MORCs) are involved in epigenetic gene silencing in a variety of eukaryotic organisms. Deletion of MORCs result in several developmental abnormalities and their dysregulation has been implicated in developmental disease and multiple cancers. Specifically, mammalian MORC3 mutations are associated with immune system defects and human cancers such as bladder, uterine, stomach, lung, and diffuse large B cell lymphomas. While previous studies have shown that MORC3 binds to H3K4me3 in vitro and overlaps with H3K4me3 ChIP-seq peaks in mouse embryonic stem cells, the mechanism by which MORC3 regulates gene expression is unknown.ResultsIn this study, we identified that mutation in Morc3 results in a suppressor of variegation phenotype in a Modifiers of murine metastable epialleles Dominant (MommeD) screen. We also find that MORC3 functions as an epigenetic silencer of transposable elements (TEs) in mouse embryonic stem cells (mESCs). Loss of Morc3 results in upregulation of TEs, specifically those belonging to the LTR class of retrotransposons also referred to as endogenous retroviruses (ERVs). Using ChIP-seq we found that MORC3, in addition to its known localization at H3K4me3 sites, also binds to ERVs, suggesting a direct role in regulating their expression. Previous studies have shown that these ERVs are marked by the repressive histone mark H3K9me3 which plays a key role in their silencing. However, we found that levels of H3K9me3 showed only minor losses in Morc3 mutant mES cells. Instead, we found that loss of Morc3 resulted in increased chromatin accessibility at ERVs as measured by ATAC-seq.ConclusionsOur results reveal MORC3 as a novel regulator of ERV silencing in mouse embryonic stem cells. The relatively minor changes of H3K9me3 in the Morc3 mutant suggests that MORC3 acts mainly downstream of, or in a parallel pathway with, the TRIM28/SETDB1 complex that deposits H3K9me3 at these loci. The increased chromatin accessibility of ERVs in the Morc3 mutant suggests that MORC3 may act at the level of chromatin compaction to effect TE silencing.

The movement of ribosomes on mRNA is often interrupted by secondary structures that present mechanical barriers and play a central role in translation regulation. We investigate how ribosomes couple their internal conformational changes with the activity of translocation factor EF-G to unwind mRNA secondary structures using high-resolution optical tweezers with single-molecule fluorescence capability. We find that hairpin opening occurs during EF-G-catalyzed translocation and is driven by the forward rotation of the small subunit head. Modulating the magnitude of the hairpin barrier by force shows that ribosomes respond to strong barriers by shifting their operation to an alternative 7-fold-slower kinetic pathway prior to translocation. Shifting into a slow gear results from an allosteric switch in the ribosome that may allow it to exploit thermal fluctuations to overcome mechanical barriers. Finally, we observe that ribosomes occasionally open the hairpin in two successive sub-codon steps, revealing a previously unobserved translocation intermediate.

This study investigated the psychometric properties of the modified checklist for autism in toddlers (M-CHAT) in a diverse rural American low-socioeconomic status (SES) sample. Four hundred and forty-seven English (n = 335) and Spanish (n = 112) speaking caregivers completed the M-CHAT during their toddler's 18- or 24-month well visit in a Southwest Virginia pediatric clinic. The M-CHAT did not show acceptable internal consistency in groups with low maternal education or minority status. Caregivers reporting low maternal education and minority status were more likely to endorse items suggestive of autism. These results indicate that the M-CHAT may require modifications to be more internally consistent and accurate across ethnic and educational groups in rural areas with low levels of SES. Recommendations to increase the utility of the M-CHAT are discussed.

Preclinical and clinical findings suggest sexual dimorphism in cardiotoxicity induced by a chemotherapeutic drug, doxorubicin (DOX). However, molecular alterations leading to sex‐related differential vulnerability of heart to DOX toxicity are not fully explored. In the present study, RNA sequencing in hearts of B6C3F1 mice indicated more differentially expressed genes in males than females (224 vs. 19; ≥1.5‐fold, False Discovery Rate [FDR] < 0.05) at 1 week after receiving 24 mg/kg total cumulative DOX dose that induced cardiac lesions only in males. Pathway analysis further revealed probable inactivation of cardiac apelin fibroblast signaling pathway (p = 0.00004) only in DOX‐treated male mice that showed ≥1.25‐fold downregulation in the transcript and protein levels of the apelin receptor, APJ. In hearts of DOX‐treated females, the transcript levels of apelin (1.24‐fold) and APJ (1.47‐fold) were significantly (p < 0.05) increased compared to saline‐treated controls. Sex‐related differential DOX effect was also observed on molecular targets downstream of the apelin‐APJ pathway in cardiac fibroblasts and cardiomyocytes. In cardiac fibroblasts, upregulation of Tgf‐β2, Ctgf, Sphk1, Serpine1, and Timp1 (fibrosis; FDR < 0.05) in DOX‐treated males and upregulation of only Tgf‐β2 and Timp1 (p < 0.05) in females suggested a greater DOX toxicity in hearts of males than females. Additionally, Ryr2 and Serca2 (calcium handling; FDR < 0.05) were downregulated in conjunction with 1.35‐fold upregulation of Casp12 (sarcoplasmic reticulum‐mediated apoptosis; FDR < 0.05) in DOX‐treated male mice. Drug effect on the transcript level of these genes was less severe in female hearts. Collectively, these data suggest a likely role of the apelin‐APJ axis in sex‐related differential DOX‐induced cardiotoxicity in our mouse model. RNA sequencing in mouse hearts 1‐week after 24 mg/kg cumulative doxorubicin dose suggested a potential role of the apelin‐APJ pathway in sex‐related differential DOX‐induced cardiotoxicity. In males, the apelin receptor, APJ, was downregulated, whereas apelin and APJ were upregulated in females compared to saline‐treated counterparts. Downstream alterations were also seen, including significantly altered expression of Tgf‐β2, Sphk1, Serpine1, Ctgf, and Timp1 (fibrosis) in cardiac fibroblasts and Ryr2 and Serca2 (Ca2+ homeostasis) in cardiomyocytes with greater effects in males than females. [ABSTRACT FROM AUTHOR]

This study investigated the psychometric properties of the modified checklist for autism in toddlers (M-CHAT) in a diverse rural American low-socioeconomic status (SES) sample. Four hundred and forty-seven English (n = 335) and Spanish (n = 112) speaking caregivers completed the M-CHAT during their toddler's 18- or 24-month well visit in a Southwest Virginia pediatric clinic. The M-CHAT did not show acceptable internal consistency in groups with low maternal education or minority status. Caregivers reporting low maternal education and minority status were more likely to endorse items suggestive of autism. These results indicate that the M-CHAT may require modifications to be more internally consistent and accurate across ethnic and educational groups in rural areas with low levels of SES. Recommendations to increase the utility of the M-CHAT are discussed. Keywords M-CHAT * Autism screening * Reliability * Diverse populations, Introduction Autism spectrum disorder (ASD) as defined by diagnostic criteria (American Psychiatric Association [APA] 2000) and first described by Kanner (1943) is considered the fastest growing developmental disability in the [...]

Subclinical cardiotoxicity at low total cumulative doxorubicin (DOX) doses can manifest into cardiomyopathy in long‐term cancer survivors. However, the underlying mechanisms are poorly understood. In male B6C3F1 mice, assessment of cardiac function by echocardiography was performed at 1, 4, 10, 17, and 24 weeks after exposure to 6, 9, 12, and 24 mg/kg total cumulative DOX doses or saline (SAL) to monitor development of delayed‐onset cardiotoxicity. The 6‐ or 9‐mg/kg total cumulative doses resulted in a significant time‐dependent decline in systolic function (left ventricular ejection fraction (LVEF) and fractional shortening (FS)) during the 24‐week recovery although there was not a significant alteration in % LVEF or % FS at any specific time point during the recovery. A significant decline in systolic function was elicited by the cardiotoxic cumulative DOX dose (24 mg/kg) during the 4‐ to 24‐week period after treatment compared to SAL‐treated counterparts. At 24 weeks after DOX treatment, a significant dose‐related decrease in the expression of genes and proteins involved in sarcoplasmic reticulum (SR) calcium homeostasis (Ryr2 and Serca2) was associated with a dose‐related increase in the transcript level of Casp12 (SR‐specific apoptosis) in hearts. These mice also showed enhanced apoptotic activity in hearts indicated by a significant dose‐related elevation in the number of apoptotic cardiomyocytes compared to SAL‐treated counterparts. These findings collectively suggest that a steady decline in SR calcium handling and apoptosis might be involved in the development of subclinical cardiotoxicity that can evolve into irreversible cardiomyopathy later in life. Subclinical cardiotoxicity elicited by low total cumulative doxorubicin (DOX) doses can occur years after treatment in cancer patients. In male B6C3F1 mice, a progressive deterioration in heart function measured by echocardiography was evidenced by a significant time‐dependent decline in systolic function during 24 weeks after treatment with 6‐ or 9‐mg/kg total cumulative DOX doses. A significant dose‐related downregulation of Ryr2 and Serca2 and increased number of apoptotic cardiomyocytes at 24 weeks after treatment may underlie delayed‐onset subclinical cardiac dysfunction. [ABSTRACT FROM AUTHOR]

Abstract In studies that use DNA arrays to assess changes in gene expression, it is preferable to measure the significance of treatment effects on a group of genes from a pathway or functional category such as gene ontology terms (GO terms, http://www.geneontology.org) because this facilitates the interpretation of effects and may markedly increase significance. A modified meta-analysis method to combine p-values was developed to measure the significance of an overall treatment effect on such functionally-defined groups of genes, taking into account the correlation structure among genes. For hypothesis testing that allows gene expression to change in both directions, p-values are calculated under the null distribution generated by a Monte Carlo method. As a test of this procedure, we attempted to distinguish altered pathways in microarray studies performed with Mitochips, oligonucleotide microarrays specific to mitochondrial DNA-encoded transcripts. We found that our analytic method improves the specificity of selection for altered pathways, due to incorporation of the inter-gene correlation structure in each pathway. It is thus a practical method to measure treatment effects on GO groups. In many actual applications, microarray experiments measure treatment effects under complicated design structures and with small sample sizes. For such applications to real data of limited statistical power, and also in computer simulations, we demonstrate that our method gives reasonable test results.

Background. As assessed by flow cytometry, the increase in hydrogen peroxide in individual neutrophils from old volunteers was significantly greater than in neutrophils from young volunteers. To explain the discrepancy in previous reports that showed reduced superoxide generation with age and our finding, we measured the kinetics of antioxidative enzymes. Methods. Neutrophils were obtained from young (ages 21-34) and old (ages over 65) volunteers. The increase in hydrogen peroxide following stimulation with formyl peptide in individual neutrophils was assessed by flow cytometry by using dihydrorhodamine 123. The enzyme kinetics was determined from the best fit curve using Michaelis-Menten equations. Results. Aging was associated with a significant reduction in the [V.sub.max] for glutathione peroxidase. The decreased activity was not due to selenium deficiency as the serum and neutrophil concentrations were identical with age. Following activation, a significant increase in the [K.sub.m] was noted in neutrophils from young but not from old volunteers. Conclusions. These results account for the increased intracellular accumulation of hydrogen peroxide as a function of age in stimulated neutrophils. These results provide evidence in humans of an age-related impairment in antioxidative defense mechanisms that support the free radical theory of aging.

Abstract Background Age- and sex-related susceptibility to adverse drug reactions and disease is a key concern in understanding drug safety and disease progression. We hypothesize that the underlying suite of hepatic genes expressed at various life cycle stages will impact susceptibility to adverse drug reactions. Understanding the basal liver gene expression patterns is a necessary first step in addressing this hypothesis and will inform our assessments of adverse drug reactions as the liver plays a central role in drug metabolism and biotransformation. Untreated male and female F344 rats were sacrificed at 2, 5, 6, 8, 15, 21, 52, 78, and 104 weeks of age. Liver tissues were collected for histology and gene expression analysis. Whole-genome rat microarrays were used to query global expression profiles. Results An initial list of differentially expressed genes was selected using criteria based upon p-value (p < 0.05) and fold-change (+/- 1.5). Three dimensional principal component analyses revealed differences between males and females beginning at 2 weeks with more divergent profiles beginning at 5 weeks. The greatest sex-differences were observed between 8 and 52 weeks before converging again at 104 weeks. K-means clustering identified groups of genes that displayed age-related patterns of expression. Various adult aging-related clusters represented gene pathways related to xenobiotic metabolism, DNA damage repair, and oxidative stress. Conclusions These results suggest an underlying role for genes in specific clusters in potentiating age- and sex-related differences in susceptibility to adverse health effects. Furthermore, such a comprehensive picture of life cycle changes in gene expression deepens our understanding and informs the utility of liver gene expression biomarkers.

Abstract Background Environmental ozone can rapidly degrade cyanine 5 (Cy5), a fluorescent dye commonly used in microarray gene expression studies. Cyanine 3 (Cy3) is much less affected by atmospheric ozone. Degradation of the Cy5 signal relative to the Cy3 signal in 2-color microarrays will adversely reduce the Cy5/Cy3 ratio resulting in unreliable microarray data. Results Ozone in central Arkansas typically ranges between ~22 ppb to ~46 ppb and can be as high as 60–100 ppb depending upon season, meteorological conditions, and time of day. These levels of ozone are common in many areas of the country during the summer. A carbon filter was installed in the laboratory air handling system to reduce ozone levels in the microarray laboratory. In addition, the airflow was balanced to prevent non-filtered air from entering the laboratory. These modifications reduced the ozone within the microarray laboratory to ~2–4 ppb. Data presented here document reductions in Cy5 signal on both in-house produced microarrays and commercial microarrays as a result of exposure to unfiltered air. Comparisons of identically hybridized microarrays exposed to either carbon-filtered or unfiltered air demonstrated the protective effect of carbon-filtration on microarray data as indicated by Cy5 and Cy3 intensities. LOWESS normalization of the data was not able to completely overcome the effect of ozone-induced reduction of Cy5 signal. Experiments were also conducted to examine the effects of high humidity on microarray quality. Modest, but significant, increases in Cy5 and Cy3 signal intensities were observed after 2 or 4 hours at 98–99% humidity compared to 42% humidity. Conclusion Simple installation of carbon filters in the laboratory air handling system resulted in low and consistent ozone levels. This allowed the accurate determination of gene expression by microarray using Cy5 and Cy3 fluorescent dyes.