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Background: Little is known regarding factors associated with calcitriol and a relative measure of calcitriol, the calcitriol-24,25-dihydroxyvitamin D3-calcifediol proportion ratio (C24CPR)., Methods: Using a cross-sectional study design, healthy young adults of African and European descent, matched (1:1) on age (±5 years) provided a blood sample in non-summer months (N = 376). Vitamin D metabolites were measured in plasma with HPLC/MS-MS. West African genetic ancestry proportion (WGA) was estimated using STRUCTURE modeling of genetic ancestry-informative markers. Multivariable regression models were used to estimate the association of WGA and vitamin D-pathway gene variants with calcitriol and C24CPR, controlling for days from summer solstice, age, sex, blood pressure, body mass index, dietary vitamin D intake, oral contraceptive/medroxyprogesterone acetate use, smoking, tanning bed use, and time of day., Results: Calcitriol and C24CPR were not highly correlated (rho = 0.14), although both were significantly, positively, and monotonically associated with WGA (Ptrend 0.025 and <0.001, respectively). In fully adjusted models, genetic factors explained a greater proportion of variability in C24CPR (R2 = 0.121 and 0.310, respectively). Variants in genes with associated with calcitriol (CALB1, CYP27B1, GC, and PPARGC1A) differed from those associated with C24CPR (CYP3A43, FGF23, KL, and VDR)., Conclusions: Both absolute and relative measures of calcitriol were significantly higher among African Americans. Otherwise, these biomarkers appear to be genetically distinct., Impact: C24CPR may be better suited to personalized medicine, due to a higher proportion of population variability explained by genetic variation and a less skewed distribution., (©2023 American Association for Cancer Research.)

We previously reported that the environmental pollutant and tobacco smoke constituent dibenzo[ def,p ]chrysene (DBP) induced DNA damage, altered DNA methylation and induced oral squamous cell carcinoma (OSCC) in mice. In the present study, we showed that 5% dietary black raspberry (BRB) significantly reduced ( P < 0.05) the levels of DBP-DNA adducts in the mouse oral cavity with comparable effect to those of its constitutes. Thus, only BRB was selected to examine if aberrant DNA methylation induced by DBP can be altered by BRB. Using comparative genome-wide DNA methylation analysis, we identified 479 hypermethylated and 481 hypomethylated sites ( q < 0.01, methylation difference >25%) between the oral tissues of mice treated with DBP and fed control diet or diet containing BRB. Among the 30 differential methylated sites (DMS) induced by DBP, we found DMS mapped to Fgf3, Qrich2, Rmdn2 , and Cbarp were hypermethylated by BRB whereas hypomethylated by DBP at either the exact position or proximal sites; DMS mapped to Vamp3, Ppp1rB1, Pkm , and Zfp316 were hypomethylated by BRB but hypermethylated by DBP at proximal sites. In addition to Fgf3 , 2 DMS mapped to Fgf4 and Fgf13 were hypermethylated by BRB; these fibroblast growth factors are involved in regulation of the epithelial-mesenchymal transition (EMT) pathway as identified by IPA. Moreover, BRB significantly reduced ( P < 0.05) the tumor incidence from 70% to 46.7%. Taken together, the inhibitory effects of BRB on DNA damage combined with its effects on epigenetic alterations may account for BRB inhibition of oral tumorigenesis induced by DBP. SIGNIFICANCE: We provided mechanistic insights that can account for the inhibition of oral tumors by BRB, which could serve as the framework for future chemopreventive trials for addicted smokers as well as non- or former smokers who are exposed to environmental carcinogens., (©2020 American Association for Cancer Research.)

The homeostatic iron regulator protein HFE is involved in regulation of iron acquisition for cells. The prevalence of two common HFE gene variants (H63D, C282Y) has been studied in many cancer types; however, the impact of HFE variants, sex and HFE gene expression in lung cancer has not been studied. We determined the prevalence of HFE variants and their impact on cancer phenotypes in lung cancer cell lines, in lung cancer patient specimens, and using The Cancer Genome Atlas (TCGA) database. We found that seven out of ten human lung cancer cell lines carry the H63D or C282Y HFE variant. Analysis of lung cancer specimens from our institute (Penn State Hershey Medical Center) revealed a sex and genotype interaction risk for metastasis in lung adenocarcinoma (LUAD) patients; H63D HFE is associated with less metastasis in males compared to wild type (WT) HFE; however, females with the H63D HFE variant tend to develop more metastatic tumors than WT female patients. In the TCGA LUAD dataset, the H63D HFE variant was associated with poorer survival in females compared to females with WT HFE. The frequency of C282Y HFE is higher in female lung squamous cell carcinoma (LUSC) patients of TCGA than males, however the C282Y HFE variant did not impact the survival of LUSC patients. In the TCGA LUSC dataset, C282Y HFE patients (especially females) had poorer survival than WT HFE patients. HFE expression level was not affected by HFE genotype status and did not impact patient's survival, regardless of sex. In summary, these data suggest that there is a sexually dimorphic effect of HFE polymorphisms in the survival and metastatic disease in lung cancer., Competing Interests: The authors have declared that no competing interests exist.

Epidemiological data confirm a much higher incidence of high-risk human papillomavirus 16 (HPV16)-mediated carcinogenesis of the cervical epithelium than for other target sites. In order to elucidate tissue-specific responses to virus infection, we compared gene expression changes induced by productive HPV16 infection of cervical, foreskin, and tonsil organotypic rafts. These rafts closely mimic persistent HPV16 infection, long before carcinogenesis sets in. The total number of gene expression changes varied considerably across the tissue types, with only 32 genes being regulated in common. Among them, we confirmed the Kelch-like family protein KLHL35 and the laminin-5 complex to be upregulated and downregulated, respectively, in all the three tissues. HPV16 infection induces upregulation of genes involved in cell cycle control, cell division, mitosis, DNA replication, and DNA damage repair in all the three tissues, indicative of a hyperproliferative environment. In the cervical and tonsil epithelium, we observe significant downregulation of genes involved in epidermis development, keratinocyte differentiation, and extracellular matrix organization. On the other hand, in HPV16-positive foreskin (HPV16 foreskin) tissue, several genes involved in interferon-mediated innate immunity, cytokine signaling, and cellular defenses were downregulated. Furthermore, pathway analysis and experimental validations identified important cellular pathways like STAT1 and transforming growth factor β (TGF-β) to be differentially regulated among the three tissue types. The differential modulation of important cellular pathways like TGF-β1 and STAT1 can explain the sensitivity of tissues to HPV cancer progression. IMPORTANCE Although the high-risk human papillomavirus 16 infects anogenital and oropharyngeal sites, the cervical epithelium has a unique vulnerability to progression of cancer. Host responses during persistent infection and preneoplastic stages can shape the outcome of cancer progression in a tissue-dependent manner. Our study for the first time reports differential regulation of critical cellular functions and signaling pathways during productive HPV16 infection of cervical, foreskin, and tonsil tissues. While the virus induces hyperproliferation in infected cells, it downregulates epithelial differentiation, epidermal development, and innate immune responses, according to the tissue type. Modulation of these biological functions can determine virus fitness and pathogenesis and illuminate key cellular mechanisms that the virus employs to establish persistence and finally initiate disease progression., (Copyright © 2019 American Society for Microbiology.)

Diabetes promotes the posttranslational modification of proteins by O- linked addition of GlcNAc ( O- GlcNAcylation) to Ser/Thr residues of proteins and thereby contributes to diabetic complications. In the retina of diabetic mice, the repressor of mRNA translation, eIF4E-binding protein 1 (4E-BP1), is O- GlcNAcylated, and sequestration of the cap-binding protein eukaryotic translation initiation factor (eIF4E) is enhanced. O- GlcNAcylation has also been detected on several eukaryotic translation initiation factors and ribosomal proteins. However, the functional consequence of this modification is unknown. Here, using ribosome profiling, we evaluated the effect of enhanced O- GlcNAcylation on retinal gene expression. Mice receiving thiamet G (TMG), an inhibitor of the O- GlcNAc hydrolase O- GlcNAcase, exhibited enhanced retinal protein O- GlcNAcylation. The principal effect of TMG on retinal gene expression was observed in ribosome-associated mRNAs ( i.e. mRNAs undergoing translation), as less than 1% of mRNAs exhibited changes in abundance. Remarkably, ∼19% of the transcriptome exhibited TMG-induced changes in ribosome occupancy, with 1912 mRNAs having reduced and 1683 mRNAs having increased translational rates. In the retina, the effect of O- GlcNAcase inhibition on translation of specific mitochondrial proteins, including superoxide dismutase 2 (SOD2), depended on 4E-BP1/2. O- GlcNAcylation enhanced cellular respiration and promoted mitochondrial superoxide levels in WT cells, and 4E-BP1/2 deletion prevented O- GlcNAcylation-induced mitochondrial superoxide in cells in culture and in the retina. The retina of diabetic WT mice exhibited increased reactive oxygen species levels, an effect not observed in diabetic 4E-BP1/2-deficient mice. These findings provide evidence for a mechanism whereby diabetes-induced O- GlcNAcylation promotes oxidative stress in the retina by altering the selection of mRNAs for translation., (© 2019 Dierschke et al.)

Human papillomavirus (HPV) infection is the world's most common sexually transmitted infection and is responsible for most cases of cervical cancer. Previous studies of global gene expression changes induced by HPV infection have focused on the cancerous stages of infection, and therefore, not much is known about global gene expression changes at early preneoplastic stages of infection. We show for the first time the global gene expression changes during early-stage HPV16 infection in cervical tissue using 3-dimensional organotypic raft cultures, which produce high levels of progeny virions. cDNA microarray analysis showed that a total of 594 genes were upregulated and 651 genes were downregulated at least 1.5-fold with HPV16 infection. Gene ontology analysis showed that biological processes including cell cycle progression and DNA metabolism were upregulated, while skin development, immune response, and cell death were downregulated with HPV16 infection in cervical keratinocytes. Individual genes were selected for validation at the transcriptional and translational levels, including UBC , which was central to the protein association network of immune response genes, and top downregulated genes RPTN , SERPINB4 , KRT23 , and KLK8 In particular, KLK8 and SERPINB4 were shown to be upregulated in cancer, which contrasts with the gene regulation during the productive replication stage. Organotypic raft cultures, which allow full progression of the HPV life cycle, allowed us to identify novel gene modulations and potential therapeutic targets of early-stage HPV infection in cervical tissue. Additionally, our results suggest that early-stage productive infection and cancerous stages of infection are distinct disease states expressing different host transcriptomes. IMPORTANCE Persistent HPV infection is responsible for most cases of cervical cancer. The transition from precancerous to cancerous stages of HPV infection is marked by a significant reduction in virus production. Most global gene expression studies of HPV infection have focused on the cancerous stages. Therefore, little is known about global gene expression changes at precancerous stages. For the first time, we measured global gene expression changes at the precancerous stages of HPV16 infection in human cervical tissue producing high levels of virus. We identified a group of genes that are typically overexpressed in cancerous stages to be significantly downregulated at the precancerous stage. Moreover, we identified significantly modulated genes that have not yet been studied in the context of HPV infection. Studying the role of these genes in HPV infection will help us understand what drives the transition from precancerous to cancerous stages and may lead to the development of new therapeutic targets., (Copyright © 2018 American Society for Microbiology.)

Homeostatic synaptic downscaling reduces neuronal excitability by modulating the number of postsynaptic receptors. Histone modifications and the subsequent chromatin remodeling play critical roles in activity-dependent gene expression. Histone modification codes are recognized by chromatin readers that affect gene expression by altering chromatin structure. We show that L3mbtl1 (lethal 3 malignant brain tumor-like 1), a polycomb chromatin reader, is downregulated by neuronal activity and is essential for synaptic response and downscaling. Genome-scale mapping of L3mbtl1 occupancies identified Ctnnb1 as a key gene downstream of L3mbtl1. Importantly, the occupancy of L3mbtl1 on the Ctnnb1 gene was regulated by neuronal activity. L3mbtl1 knockout neurons exhibited reduced Ctnnb1 expression. Partial knockdown of Ctnnb1 in wild-type neurons reduced excitatory synaptic transmission and abolished homeostatic downscaling, and transfecting Ctnnb1 in L3mbtl1 knockout neurons enhanced synaptic transmission and restored homeostatic downscaling. These results highlight a role for L3mbtl1 in regulating homeostasis of synaptic efficacy., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

We investigated the association between genetic polymorphisms in cytochrome P450 (CYP2R1, CYP24A1, and the CYP3A family) with nonsummer plasma concentrations of vitamin D metabolites (25-hydroxyvitamin D3 (25(OH)D3) and proportion 24,25-dihydroxyvitamin D3 (24,25(OH)2D3)) among healthy individuals of sub-Saharan African and European ancestry, matched on age (within 5 years; n = 188 in each ancestral group), in central suburban Pennsylvania (2006-2009). Vitamin D metabolites were measured using high-performance liquid chromatography with tandem mass spectrometry. Paired multiple regression and adjusted least-squares mean analyses were used to test for associations between genotype and log-transformed metabolite concentrations, adjusted for age, sex, proportion of West-African genetic ancestry, body mass index, oral contraceptive (OC) use, tanning bed use, vitamin D intake, days from summer solstice, time of day of blood draw, and isoforms of the vitamin D receptor (VDR) and vitamin D binding protein. Polymorphisms in CYP2R1, CYP3A43, vitamin D binding protein, and genetic ancestry proportion remained associated with plasma 25(OH)D3 after adjustment. Only CYP3A43 and VDR polymorphisms were associated with proportion 24,25(OH)2D3. Magnitudes of association with 25(OH)D3 were similar for CYP3A43, tanning bed use, and OC use. Significant least-squares mean interactions (CYP2R1/OC use (P = 0.030) and CYP3A43/VDR (P = 0.013)) were identified. A CYP3A43 genotype, previously implicated in cancer, is strongly associated with biomarkers of vitamin D metabolism. Interactive associations should be further investigated.

Genetic and epigenetic alterations observed at end stage OSCC formation could be considered as a consequence of cancer development and thus changes in normal or premalignant tissues which had been exposed to oral carcinogens such as Dibenzo[def,p]chrysene (DBP) may better serve as predictive biomarkers of disease development. Many types of DNA damage can induce epigenetic changes which can occur early and in the absence of evident morphological abnormalities. Therefore we used ERRBS to generate genome-scale, single-base resolution DNA methylomes from histologically normal oral tissues of mice treated with DBP under experimental conditions known to induce maximum DNA damage which is essential for the development of OSCC induced by DBP in mice. After genome-wide correction, 30 and 48 differentially methylated sites (DMS) were identified between vehicle control and DBP treated mice using 25% and 10% differences in methylation, respectively. RT-PCR was further performed to examine the expressions of nine selected genes. Among them, Fgf3, a gene frequently amplified in head and neck cancer, showed most prominent and significant gene expression change (2.4× increases), despite the hypomethylation of Fgf3 was identified at >10kb upstream of transcription start site. No difference was observed in protein expression between normal oral tissues treated with DBP or vehicle as examined by immunohistochemistry. Collectively, our results indicate that Fgf3 hypomethylation and gene overexpression, but not protein expression, occurred in the early stage of oral carcinogenesis induced by DBP. Thus, Fgf3 hypomethylation may serve as a potential biomarker for early detection of OSCC.

Excessive alcohol consumption results in significant changes in gene expression and isoforms due to altered mRNA splicing. As such, an intriguing possibility is that disturbances in alternative splicing are involved in key pathological pathways triggered by alcohol exposure. However, no resources have been available to systematically analyze this possibility at a genome-wide scale. Here, we performed RNA sequencing of human fetal cortical slices that were obtained at the late first trimester and exposed to ethanol or control medium. We report 382 events that were identified as changes affecting the ratio of splicing isoforms in the ethanol-exposed fetal human cortex. Additionally, previously unreported novel isoforms of several genes were also identified. These results provide a broad perspective on the post-transcriptional regulatory network underlying ethanol-induced pathogenesis in the developing human cortex., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Transcriptome complexity is substantially increased by the use of multiple transcription start sites for a given gene. By utilizing a rod photoreceptor-specific chromatin signature, and the RefSeq database of established transcription start sites, we have identified essentially all known rod photoreceptor genes as well as a group of novel genes that have a high probability of being expressed in rod photoreceptors. Approximately half of these novel rod genes are transcribed into multiple mRNA and/or protein isoforms through alternative transcriptional start sites (ATSS), only one of which has a rod-specific epigenetic signature and gives rise to a rod transcript. This suggests that, during retina development, some genes use ATSS to regulate cell type and temporal specificity, effectively generating a rod transcript from otherwise ubiquitously expressed genes. Biological confirmation of the relationship between epigenetic signatures and gene expression, as well as comparison of our genome-wide chromatin signature maps with available data sets for retina, namely a ChIP-on-Chip study of Polymerase-II (Pol-II) binding sites, ChIP-Seq studies for NRL- and CRX- binding sites and DHS (University of Washington data, available on UCSC mouse Genome Browser as a part of ENCODE project) fully support our hypothesis and together accurately identify and predict an array of new rod transcripts. The same approach was used to identify a number of TSS that are not currently in RefSeq. Biological conformation of the use of some of these TSS suggests that this method will be valuable for exploring the range of transcriptional complexity in many tissues. Comparison of mouse and human genome-wide data indicates that most of these alternate TSS appear to be present in both species, indicating that our approach can be useful for identification of regulatory regions that might play a role in human retinal disease.

Repetitive prenatal exposure to identical or similar doses of harmful agents results in highly variable and unpredictable negative effects on fetal brain development ranging in severity from high to little or none. However, the molecular and cellular basis of this variability is not well understood. This study reports that exposure of mouse and human embryonic brain tissues to equal doses of harmful chemicals, such as ethanol, activates the primary stress response transcription factor heat shock factor 1 (Hsf1) in a highly variable and stochastic manner. While Hsf1 is essential for protecting the embryonic brain from environmental stress, excessive activation impairs critical developmental events such as neuronal migration. Our results suggest that mosaic activation of Hsf1 within the embryonic brain in response to prenatal environmental stress exposure may contribute to the resulting generation of phenotypic variations observed in complex congenital brain disorders.

Previous studies have linked increased frequency of glycosylphosphatidylinositol-anchor protein (GPI-AP) deficiency with genomic instability and the risk of carcinogenesis. However, the underlying mechanism is still not clear. A randomForest analysis of the gene expression array data from 55 MDS patients (GSE4619) demonstrated a significant (p = 0.0007) correlation (Pearson r =-0.4068) between GPI-anchor biosynthesis gene expression and genomic instability, in which PIGN, a gene participating in GPI-AP biosynthesis, was ranked as the third most important in predicting risk of MDS progression. Furthermore, we observed that PIGN gene expression aberrations (increased transcriptional activity but diminished to no protein production) were associated with increased frequency of GPI-AP deficiency in leukemic cells during leukemic transformation/progression. PIGN gene expression aberrations were attributed to partial intron retentions between exons 14 and 15 resulting in frameshifts and premature termination which were confirmed by examining the RNA-seq data from a group of AML patients (phs001027.v1.p1). PIGN gene expression aberration correlated with the elevation of genomic instability marker expression that was independent of the TP53 regulatory pathway. Suppression/elimination of PIGN protein expression caused a similar pattern of genomic instability that was rescued by PIGN restoration. Finally, we found that PIGN bound to the spindle assembly checkpoint protein, MAD1, and regulated its expression during the cell cycle. In conclusion, PIGN gene is crucial in regulating mitotic integrity to maintain chromosomal stability and prevents leukemic transformation/progression.

Human hemochromatosis protein (HFE) is involved in iron metabolism. Two major HFE polymorphisms, H63D and C282Y, have been associated with an increased risk of cancers. Previously, we reported decreased gender effects in overall survival based on H63D or C282Y HFE polymorphisms patients with glioblastoma multiforme (GBM). However, the effect of other single nucleotide variation (SNV) in the HFE gene on the cancer development and progression has not been systematically studied. To expand our finding in a larger sample, and to identify other HFE SNV, we analyzed the frequency of somatic SNV in HFE gene and its relationship to survival in GBM patients using The Cancer Genome Atlas (TCGA) GBM (Caucasian only) database. We found 9 SNVs with increased frequency in blood normal of TCGA GBM patients compared to the 1000Genome. Among 9 SNVs, 7 SNVs were located in the intron and 2 SNVs (i.e., H63D, C282Y) in the exon of HFE gene. The statistical analysis demonstrated that blood normal samples of TCGA GBM have more H63D (p = 0.0002, 95% Confidence interval (CI): 0.2119-0.3223) or C282Y (p = 0.0129, 95% CI: 0.0474-0.1159) HFE polymorphisms than 1000Genome. The Kaplan-Meier survival curve for the 264 GBM samples revealed no difference between wild type (WT) HFE and H63D, and WT HFE and C282Y GBM patients. In addition, there was no difference in the survival of male/female GBM patients based on HFE genotype. There was no correlation between HFE expression and survival. In conclusion, the current results suggest that somatic HFE polymorphisms do not impact GBM patients' survival in the TCGA data set of GBM.

A facultative heterochromatin mark, histone H3 lysine 9 dimethylation (H3K9me2), which is mediated by histone methyltransferases G9a/GLP (EHMT2/1), undergoes dramatic rearrangements during myeloid cell differentiation as observed by chromatin imaging. To determine whether these structural transitions also involve genomic repositioning of H3K9me2, we used ChIP-sequencing to map genome-wide topography of H3K9me2 in normal human granulocytes, normal CD34+ hematopoietic progenitors, primary myeloblasts from acute myeloid leukemia (AML) patients, and a model leukemia cell line K562. We observe that H3K9me2 naturally repositions from the previously designated "repressed" chromatin state in hematopoietic progenitors to predominant association with heterochromatin regions in granulocytes. In contrast, AML cells accumulate H3K9me2 on previously undefined large (> 100 Kb) genomic blocks that are enriched with AML-specific single nucleotide variants, sites of chromosomal translocations, and genes downregulated in AML. Specifically, the AML-specific H3K9me2 blocks are enriched with genes regulated by the proto-oncogene ERG that promotes stem cell characteristics. The AML-enriched H3K9me2 blocks (in contrast to the heterochromatin-associated H3K9me2 blocks enriched in granulocytes) are reduced by pharmacological inhibition of the histone methyltransferase G9a/GLP in K562 cells concomitantly with transcriptional activation of ERG and ETS1 oncogenes. Our data suggest that G9a/GLP mediate formation of transient H3K9me2 blocks that are preserved in AML myeloblasts and may lead to an increased rate of AML-specific mutagenesis and chromosomal translocations.

Background: Diabetic nephropathy (DN) is a major complication of type1 and type 2 diabetes. Understanding how diabetes regulate transcriptome dynamics in DN is important for understanding the biology of the disease and for guiding development of new treatments., Results: We analyzed the kidney transcriptome of a DN mouse model, D2.B6-Ins2 Akita /MatbJ, before/after treatment with P78-PEDF. Age, weight, and gender-matched mice and wild-type (wt) littermates were treated at 6 weeks (early treatment) or 12 weeks (late treatment) of age for the duration of 6 weeks. Animals were implanted with an osmotic mini pump delivering 0.3 ug/g/day P78-PEDF or vehicle. Using RNA-seq, we identified14,316 transcripts (12,328 coding;1,988 non-coding) that were significant and reliably expressed (FPKM > =1) in diabetic kidneys. Expression of 1,129 (7.9%) including 901 coding genes was altered by diabetes with log2 fold changes (FC) between -86.2 and +86.0 (q < 0.05) compared to wt. Of these, 164 (14.5%) showed increased and 965 (85.5%) decreased expression with FC > 1.5. Coding genes with highest FC in diabetic kidneys include Nhej1 (32.04), Ept1 (8.6), Srd5a2 (-6.55), Aif1 (-6.05), and Angptl7 (-4.71). Early and late stage diabetic groups receiving continuous infusion of P78 showed altered expression of 316/14,316 (2.2%) transcripts, including 121 coding genes compared to non-treated diabetic controls. Of these, 183 were upregulated and 133 downregulated with FC +50.9--93.3 (q < 0.05). P78 reversed diabetes-induced changes in 138/1129 (12.2%) transcripts, including 49/901 (5.44%) coding genes. Nhej1 (-37.94), Tceanc2 (5.76), Ept1 (-4.45), Ugt1a2 (3.03), and Tmsb15l (-3.0) showed the highest FC with treatment. The DNA repair gene, Nhej1 with the greatest FC in diabetic kidneys was completely restored to control levels by both early and late P78 treatments. Expression of other coding genes regulated by diabetes with FC > =(+/-) 1.5 and completely reversed by P78 include Mamdc4, Kdm4b, Tmem252, Selm, and Hpd. RT and QRT-PCR validated expression of gene with FC > (+/-)2.0. Transcriptome changes were also observed between early and late-stage treatments. Precursor non-coding miRNAs showed the highest fold changes in expression in the diabetic and P78 treatment groups. Several diabetic-induced changes were reversed in direction of expression by treatment including Gm24083, GM25953, miR1905, Gm25535, Gm27903, and miR196a1 with FC > =(+/-)20. From Ingenuity pathway analysis (IPA), mitochondrial dysfunction, Nrf-2- mediated oxidative stress and renal injury pathways emerged as key mechanisms in DN. DN-enriching genes in these pathways were reduced in number or regulated in the opposite direction by treatment., Conclusions: Unique biomarkers and canonical pathways identified in this study may hold the key to understanding mechanisms of DN pathobiology with value for clinical translation. Our data suggest that mitochondrial dysfunction, genotoxicity and oxidative stress are principal events in DN and that P78-PEDF holds promise for its management.

Epigenetic modifiers can work in concert with transcription factors to control the transition of cells from proliferating progenitors into quiescent terminally differentiated cells. This transition involves changes in histone methylation and one of the key regulators of this is the H3K4me2/1 histone demethylase LSD1. Here, we show that the highest expression of LSD1 occurs in postmitotic retinal cells during the peak period of rod photoreceptor differentiation. Pharmacological inhibition of LSD1 in retinal explants cultured from PN1 to PN8 had three major effects. It prevented the normal decrease in expression of genes associated with progenitor function, it blocked rod photoreceptor development, and it increased expression of genes associated with other retinal cell types. The maintained expression of progenitor genes was associated with a maintained level of H3K4me2 over the gene and its promoter. Among the genes whose expression was maintained was Hes1, a repressor known to block rod photoreceptor development. The inhibition of rod photoreceptor gene expression occurred in spite of the normal expression of transcription factors CRX and NRL, and the normal accumulation of H3K4me2 marks over the promoter and gene body. We suggest that LSD1 acts in concert with a series of nuclear receptors to modify chromatin structure and repress progenitor genes as well as to inhibit ectopic patterns of gene expression in the differentiating postmitotic retinal cells.

Transmission of olfactory information to higher brain regions is mediated by olfactory bulb (OB) projection neurons, the mitral and tufted cells. Although mitral/tufted cells are often characterized as the OB counterpart of cortical projection neurons (also known as pyramidal neurons), they possess several unique morphological characteristics and project specifically to the olfactory cortices. Moreover, the molecular networks contributing to the generation of mitral/tufted cells during development are largely unknown. To understand the developmental patterns of gene expression in mitral/tufted cells in the OB, we performed transcriptome analyses targeting purified OB projection neurons at different developmental time points with next-generation RNA sequencing (RNA-seq). Through these analyses, we found 1202 protein-coding genes that are temporally differentially-regulated in developing projection neurons. Among them, 388 genes temporally changed their expression level only in projection neurons. The data provide useful resource to study the molecular mechanisms regulating development of mitral/tufted cells. We further compared the gene expression profiles of developing mitral/tufted cells with those of three cortical projection neuron subtypes, subcerebral projection neurons, corticothalamic projection neurons, and callosal projection neurons, and found that the molecular signature of developing olfactory projection neuron bears resemblance to that of subcerebral neurons. We also identified 3422 events that change the ratio of splicing isoforms in mitral/tufted cells during maturation. Interestingly, several genes expressed a novel isoform not previously reported. These results provide us with a broad perspective of the molecular networks underlying the development of OB projection neurons., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Consumption of a protein-containing meal by a fasted animal promotes protein accretion in skeletal muscle, in part through leucine stimulation of protein synthesis and indirectly through repression of protein degradation mediated by its metabolite, α-ketoisocaproate. Mice lacking the mitochondrial branched-chain aminotransferase (BCATm/Bcat2), which interconverts leucine and α-ketoisocaproate, exhibit elevated protein turnover. Here, the transcriptomes of gastrocnemius muscle from BCATm knockout (KO) and wild-type mice were compared by next-generation RNA sequencing (RNA-Seq) to identify potential adaptations associated with their persistently altered nutrient signaling. Statistically significant changes in the abundance of 1,486/∼39,010 genes were identified. Bioinformatics analysis of the RNA-Seq data indicated that pathways involved in protein synthesis [eukaryotic initiation factor (eIF)-2, mammalian target of rapamycin, eIF4, and p70S6K pathways including 40S and 60S ribosomal proteins], protein breakdown (e.g., ubiquitin mediated), and muscle degeneration (apoptosis, atrophy, myopathy, and cell death) were upregulated. Also in agreement with our previous observations, the abundance of mRNAs associated with reduced body size, glycemia, plasma insulin, and lipid signaling pathways was altered in BCATm KO mice. Consistently, genes encoding anaerobic and/or oxidative metabolism of carbohydrate, fatty acids, and branched chain amino acids were modestly but systematically reduced. Although there was no indication that muscle fiber type was different between KO and wild-type mice, a difference in the abundance of mRNAs associated with a muscular dystrophy phenotype was observed, consistent with the published exercise intolerance of these mice. The results suggest transcriptional adaptations occur in BCATm KO mice that along with altered nutrient signaling may contribute to their previously reported protein turnover, metabolic and exercise phenotypes., (Copyright © 2015 the American Physiological Society.)

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease resulting from the destruction of insulin-producing pancreatic beta cells and is fatal unless treated with insulin. During the last four decades, multiple insulin-dependent diabetes (Idd) susceptibility/resistance loci that regulate T1D development have been identified in humans and non-obese diabetic (NOD) mice, an established animal model for T1D. However, the exact mechanisms by which these loci confer diabetes risk and the identity of the causative genes remain largely elusive. To identify genes and molecular mechanisms that control the function of diabetogenic T cells, we conducted DNA microarray analysis in islet-specific CD4 + T cells from BDC2.5 TCR transgenic NOD mice that contain the Idd9 locus from T1D-susceptible NOD mice or T1D-resistant C57BL/10 mice. Here we describe in detail the contents and analyses for these gene expression data associated with our previous study [1]. Gene expression data are available at the Gene Expression Omnibus (GEO) repository from the National Center for Biotechnology Information (accession number GSE64674).

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology., (© The Author 2015. Published by Oxford University Press.)

Environmental contributions to cancer development are widely accepted, but only a fraction of all pertinent exposures have probably been identified. Traditional toxicological approaches to the problem have largely focused on the effects of individual agents at singular endpoints. As such, they have incompletely addressed both the pro-carcinogenic contributions of environmentally relevant low-dose chemical mixtures and the fact that exposures can influence multiple cancer-associated endpoints over varying timescales. Of these endpoints, dysregulated metabolism is one of the most common and recognizable features of cancer, but its specific roles in exposure-associated cancer development remain poorly understood. Most studies have focused on discrete aspects of cancer metabolism and have incompletely considered both its dynamic integrated nature and the complex controlling influences of substrate availability, external trophic signals and environmental conditions. Emerging high throughput approaches to environmental risk assessment also do not directly address the metabolic causes or consequences of changes in gene expression. As such, there is a compelling need to establish common or complementary frameworks for further exploration that experimentally and conceptually consider the gestalt of cancer metabolism and its causal relationships to both carcinogenesis and the development of other cancer hallmarks. A literature review to identify environmentally relevant exposures unambiguously linked to both cancer development and dysregulated metabolism suggests major gaps in our understanding of exposure-associated carcinogenesis and metabolic reprogramming. Although limited evidence exists to support primary causal roles for metabolism in carcinogenesis, the universality of altered cancer metabolism underscores its fundamental biological importance, and multiple pleiomorphic, even dichotomous, roles for metabolism in promoting, antagonizing or otherwise enabling the development and selection of cancer are suggested., (Published by Oxford University Press 2015.)

Recent studies suggest that low vitamin D-binding protein (VDBP aka group-specific complement or Gc) concentrations may be linked with inflammatory-mediated conditions, including asthma, chronic obstructive pulmonary disease, and cancer. However, these studies may be confounded by substantial racial and ethnic or genetic differences. The purpose of this study was to test the hypothesis that circulating VDBP concentrations are significantly associated with genetic ancestry. We used a validated high-performance liquid chromatography tandem mass spectrometry assay of 25-hydroxyvitamin D3 and its downstream metabolite 24,25-dihydroxyvitamin D3. VDBP concentrations (milligrams per liter) were measured in duplicate using a commercial enzyme-linked immunosorbent assay among healthy African American (n = 56) and Caucasian American (n = 60) participants. Ancestry informative markers across the genome were used to estimate individual genetic ancestry proportions, designed to robustly distinguish between West African and European ancestry. Genotype-defined Gc isoforms were defined using rs7041 and rs4588 combination groups. VDBP concentration was correlated with both Gc isoform (r = 0.93, P < 0.001) and West African genetic ancestry (r = -0.66, P < 0.001). In the final model, Gc isoform, the catabolic ratio of serum vitamin D, oral contraceptive use, and body mass index remained significantly associated with VDBP concentration, after adjustment for genetic ancestry. Failure to adjust for Gc isoform may lead to spurious associations in studies of VDBP concentration and disease risk, particularly when the condition of interest may also be associated with genetic ancestry. The higher circulating VDBP concentrations and higher vitamin D catabolic rate among Caucasian Americans observed here appear to be consistent with lower bone mineral density and racial and ethnic differences in vitamin D-inducing cytokines., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Second generation antipsychotics (SGAs), like olanzapine, exhibit acute metabolic side effects leading to metabolic inflexibility, hyperglycemia, adiposity and diabetes. Understanding how SGAs affect the skeletal muscle transcriptome could elucidate approaches for mitigating these side effects. Male Sprague-Dawley rats were infused intravenously with vehicle or olanzapine for 24h using a dose leading to a mild hyperglycemia. RNA-Seq was performed on gastrocnemius muscle, followed by alignment of the data with the Rat Genome Assembly 5.0. Olanzapine altered expression of 1347 out of 26407 genes. Genes encoding skeletal muscle fiber-type specific sarcomeric, ion channel, glycolytic, O2- and Ca2+-handling, TCA cycle, vascularization and lipid oxidation proteins and pathways, along with NADH shuttles and LDH isoforms were affected. Bioinformatics analyses indicate that olanzapine decreased the expression of slower and more oxidative fiber type genes (e.g., type 1), while up regulating those for the most glycolytic and least metabolically flexible, fast twitch fiber type, IIb. Protein turnover genes, necessary to bring about transition, were also up regulated. Potential upstream regulators were also identified. Olanzapine appears to be rapidly affecting the muscle transcriptome to bring about a change to a fast-glycolytic fiber type. Such fiber types are more susceptible than slow muscle to atrophy, and such transitions are observed in chronic metabolic diseases. Thus these effects could contribute to the altered body composition and metabolic disease olanzapine causes. A potential interventional strategy is implicated because aerobic exercise, in contrast to resistance exercise, can oppose such slow to fast fiber transitions.

Type 1 diabetes (T1D) is a polygenic disease with multiple insulin-dependent diabetes (Idd) loci predisposing humans and NOD mice to disease. NOD.B10 Idd9 congenic mice, in which the NOD Idd9 chromosomal region is replaced by the Idd9 from T1D-resistant C57BL/10 mice, are significantly protected from T1D development. However, the genes and pathways conferring T1D development or protection by Idd9 remain to be fully elucidated. We have developed novel NOD.B10-Idd9 (line 905) congenic mice that predominantly harbor islet-reactive CD4(+) T cells expressing the BDC2.5 TCR (BDC-Idd9.905 mice). To establish functional links between the Idd9 genotype and its phenotype, we used microarray analyses to investigate the gene expression profiles of ex vivo and Ag-activated CD4(+) T cells from these mice and BDC2.5 (BDC) NOD controls. Among the differentially expressed genes, those located within the Idd9 region were greatly enriched in islet-specific CD4(+) T cells. Bioinformatics analyses of differentially expressed genes between BDC-Idd9.905 and BDC CD4(+) T cells identified Eno1, Rbbp4, and Mtor, all of which are encoded by Idd9 and part of gene networks involved in cellular growth and development. As predicted, proliferation and Th1/Th17 responses of islet-specific CD4(+) T cells from BDC-Idd9.905 mice following Ag stimulation in vitro were reduced compared with BDC mice. Furthermore, proliferative responses to endogenous autoantigen and diabetogenic function were impaired in BDC-Idd9.905 CD4(+) T cells. These findings suggest that differential expression of the identified Idd9 genes contributed to Idd9-dependent T1D susceptibility by controlling the diabetogenic function of islet-specific CD4(+) T cells., (Copyright © 2015 by The American Association of Immunologists, Inc.)

The UDP-glucuronosyltransferase (UGT) 1A enzymes are involved in the phase II metabolism of many important endogenous and exogenous compounds. The nine UGT1A isoforms exhibit high interindividual differences in expression, but their epigenetic regulation is not well understood. The purpose of the present study was to examine microRNA (miRNA) regulation of hepatic UGT1A enzymes and determine whether or not that regulation impacts enzymatic activity. In silico analysis identified miRNA 491-3p (miR-491-3p) as a potential regulator of the UGT1A gene family via binding to the shared UGT1A 3'-untranslated region common to all UGT1A enzymes. Transfection of miR-491-3p mimic into HuH-7 cells significantly repressed UGT1A1 (P < 0.001), UGT1A3 (P < 0.05), and UGT1A6 (P < 0.05) mRNA levels. For UGT1A1, this repression correlated with significantly reduced metabolism of raloxifene into raloxifene-6-glucuronide (ral-6-gluc; P < 0.01) and raloxifene-4'-glucuronide (ral-4'-gluc; P < 0.01). In HuH-7 cells with repressed miR-491-3p expression, there was a significant increase (~80%; P < 0.01) in UGT1A1 mRNA and a corresponding increase in glucuronidation of raloxifene into ral-6-gluc (50%; P < 0.05) and ral-4'-gluc (22%; P < 0.01). Knockdown of endogenous miR-491-3p in HepG2 cells did not significantly alter UGT1A1 mRNA levels but did increase the formation of ral-6-gluc (50%; P < 0.05) and ral-4'-gluc (34%; P < 0.001). A significant inverse correlation between miR-491-3p expression and both UGT1A3 (P < 0.05) and UGT1A6 (P < 0.01) mRNA levels was observed in a panel of normal human liver specimens, with a significant (P < 0.05) increase in UGT1A3 and UGT1A6 mRNA levels observed in miR-491-3p nonexpressing versus expressing liver specimens. These results suggest that miR-491-3p is an important factor in regulating the expression of UGT1A enzymes in vivo.

The epigenetic contribution to neurogenesis is largely unknown. There is, however, growing evidence that posttranslational modification of histones is a dynamic process that shows many correlations with gene expression. Here we have followed the genome-wide distribution of two important histone H3 modifications, H3K4me2 and H3K27me3 during late mouse retina development. The retina provides an ideal model for these studies because of its well-characterized structure and development and also the extensive studies of the retinal transcriptome and its development. We found that a group of genes expressed only in mature rod photoreceptors have a unique signature consisting of de-novo accumulation of H3K4me2, both at the transcription start site (TSS) and over the whole gene, that correlates with the increase in transcription, but no accumulation of H3K27me3 at any stage. By in silico analysis of this unique signature we have identified a larger group of genes that may be selectively expressed in mature rod photoreceptors. We also found that the distribution of H3K4me2 and H3K27me3 on the genes widely expressed is not always associated with their transcriptional levels. Different histone signatures for retinal genes with the same gene expression pattern suggest the diversities of epigenetic regulation. Genes without H3K4me2 and H3K27me3 accumulation at any stage represent a large group of transcripts never expressed in retina. The epigenetic signatures defined by H3K4me2 and H3K27me3 can distinguish cell-type specific genes from widespread transcripts and may be reflective of cell specificity during retina maturation. In addition to the developmental patterns seen in wild type retina, the dramatic changes of histone modification in the retinas of mutant animals lacking rod photoreceptors provide a tool to study the epigenetic changes in other cell types and thus describe a broad range of epigenetic events in a solid tissue in vivo.

Thyroid transcription factor 1 (TTF-1 or NKX2-1) is an essential fetal lung developmental factor, which can be recurrently activated by gene amplification in adult lung cancer. We have discovered the first microRNA (i.e., miR-365) that directly regulates TTF-1 by interacting with its 3'-untranslated region. By gene expression profiling, we identified other putative targets of miR-365 and miR-365*. In line with the microRNA/target relationship, the expression patterns of miR-365 and TTF-1 were in an inverse relationship in human lung cancer. Exploration of human lung cancer genomics data uncovered that TTF-1 gene amplification was significantly associated with DNA copy number loss at one of the two genomic loci encoding the precursor RNA of mature miR-365 (i.e., mir-365-1). This implies the existence of genetic selection pressure to lose the repressive miR-365 that would otherwise suppress amplified TTF-1. We detected a signaling loop between transforming growth factor beta (TGFb) and miR-365 and this loop reinforced suppression of TTF-1 via miR-365. Mir-365 also targeted an epithelial mesenchymal transition (EMT)-promoting gene HMGA2. In summary, these data connect the lung transcriptional program to the microRNA network.

Background: Biochemical diagnostics of ethanol intake would improve alcohol abuse treatment and have applications in clinical trial and public safety settings. Self-reporting of alcohol use has clinical utility but lacks the desired reliability. Previously, proposed single-analyte biochemical tests of alcohol intake suffer from low sensitivity and specificity or examine only acute drinking and have therefore seen limited clinical use., Methods: To address this unmet need, plasma protein biomarker discovery and validation were performed with an alcohol self-administering nonhuman primate model system to develop a diagnostic that accurately classifies subjects into nondrinking, nonabusive drinking, and abusive drinking categories., Results: A 17-plasma protein panel was determined that correctly classifies abusive drinking with 100% sensitivity and also differentiates any level of drinking from alcohol abstinence with 88% accuracy., Conclusions: The biomarker panel reflects changes in multiple organ systems and suggests robust changes in the plasma proteome with drinking that might serve as a sensitive and specific diagnostic test. The specific plasma proteins altered with alcohol self-administration might represent indicators of alcohol-induced stress on a variety of organ systems., (Copyright 2010 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Lead generation is a major hurdle in small-molecule drug discovery, with an estimated 60% of projects failing from lack of lead matter or difficulty in optimizing leads for drug-like properties. It would be valuable to identify these less-druggable targets before incurring substantial expenditure and effort. Here we show that a model-based approach using basic biophysical principles yields good prediction of druggability based solely on the crystal structure of the target binding site. We quantitatively estimate the maximal affinity achievable by a drug-like molecule, and we show that these calculated values correlate with drug discovery outcomes. We experimentally test two predictions using high-throughput screening of a diverse compound collection. The collective results highlight the utility of our approach as well as strategies for tackling difficult targets.

We separately have shown that the maximal druglike affinity of a given binding site on a protein can be calculated on the basis of the binding-site structure alone by using a desolvation-based free energy model along with the notion that druglike ligands fall into certain physiochemical property ranges. Here, we present an approach where we reformulate the calculated druggability affinity as an additive free energy to facilitate the searching of whole protein surfaces for druglike binding sites. The highest-scoring patches in many cases represent known ligand-binding sites for druggable targets, but not for difficult targets. This approach differs from other approaches in that it does not simply identify pockets with the greatest volume but instead identifies pockets that are likely to be amenable to druglike small-molecule binding. Combining the method with a functional residue prediction method called SCA (statistical coupling analysis) results in the prediction of potentially druggable allosteric binding sites on p38alpha kinase.