Affects Gene Expression Profiles Research Articles

Comparison of Minced Cartilage Implantation with Autologous Chondrocyte Transplantation in an In Vitro Inflammation Model.

The current gold standard to treat large cartilage defects is autologous chondrocyte transplantation (ACT). As a new surgical method of cartilage regeneration, minced cartilage implantation (MCI) is increasingly coming into focus. The aim of this study is to investigate the influence of chondrogenesis between isolated and cultured chondrocytes compared to cartilage chips in a standardized inflammation model with the proinflammatory cytokine TNFα. Articular chondrocytes from bovine cartilage were cultured according to the ACT method to passage 3 and transferred to spheroid culture. At the same time, cartilage was fragmented (<1 mm3) to produce cartilage chips. TNFα (20 ng/mL) was supplemented to simulate an inflammatory process. TNFα had a stronger influence on the passaged chondrocytes compared to the non-passaged ones, affecting gene expression profiles differently between isolated chondrocytes and cartilage chips. MCI showed less susceptibility to TNFα, with reduced IL-6 release and less impact on inflammation markers. Biochemical and histological analyses supported these findings, showing a greater negative influence of TNFα on the passaged pellet cultures compared to the unpassaged cells and MCI constructs. This study demonstrated the negative influence of TNFα on chondrogenesis in a chondrocyte spheroid culture and cartilage fragment model. Passaged chondrocytes are more sensitive to cytokine influences compared to non-passaged cells and chondrons. This suggests that MCI may have superior regeneration potential in osteoarthritic conditions compared to ACT. Further investigations are necessary for the translation of these findings into clinical practice.

DNA Methylation in the Hypothalamic Feeding Center and Obesity.

Obesity rates have been increasing worldwide for decades, mainly due to environmental factors, such as diet, nutrition, and exercise. However, the molecular mechanisms through which environmental factors induce obesity remain unclear. Several mechanisms underlie the body's response to environmental factors, and one of the main mechanisms involves epigenetic modifications, such as DNA methylation. The pattern of DNA methylation is influenced by environmental factors, and altered DNA methylation patterns can affect gene expression profiles and phenotypes. DNA methylation may mediate the development of obesity caused by environmental factors. Similar to the factors governing obesity, DNA methylation is influenced by nutrients and metabolites. Notably, DNA methylation is associated with body size and weight programming. The DNA methylation levels of proopiomelanocortin (Pomc) and neuropeptide Y (Npy) in the hypothalamic feeding center, a key region controlling systemic energy balance, are affected by diet. Conditional knockout mouse studies of epigenetic enzymes have shown that DNA methylation in the hypothalamic feeding center plays an indispensable role in energy homeostasis. In this review, we discuss the role of DNA methylation in the hypothalamic feeding center as a potential mechanism underlying the development of obesity induced by environmental factors.

The Nutriepigenome.

Unlike genetic changes, epigenetics modulates gene expression without stable modification of the genome. Even though all cells, including sperm and egg, have an epigenome pattern, most of these modifications occur during lifetime and interestingly, some of them, are reversible. Lifestyle and especially nutrients as well as diet regimens are presently gaining importance due to their ability to affect the epigenome. On the other hand, since the epigenome profoundly affects gene expression profile it can be speculated that the epigenome could modulate individual response to nutrients. Recent years have thus seen growing interest on nutrients, macronutrients ratio and diet regimens capable to affect the epigenetic pattern. In fact, while genetic alterations are mostly detrimental at the individual level, reshaping the epigenome may be a feasible strategy to positively counteract the detrimental effect of aging. Here, I review nutrient consumption and diet regimens as a possible strategy to counteract aging-driven epigenome derangement.

The effects of short-term glyphosate-based herbicide exposure on insect gene expression profiles

Glyphosate-based herbicides (GBHs) are the most frequently used herbicides worldwide. The use of GBHs is intended to tackle weeds, but GBHs have been shown to affect the life-history traits and antioxidant defense system of invertebrates found in agroecosystems. Thus far, the effects of GBHs on detoxification pathways among invertebrates have not been sufficiently investigated. We performed two different experiments—1) the direct pure glyphosate and GBH treatment, and 2) the indirect GBH experiment via food—to examine the possible effects of environmentally relevant GBH levels on the survival of the Colorado potato beetle (Leptinotarsa decemlineata) and the expression profiles of their detoxification genes. As candidate genes, we selected four cytochrome P450 (CYP), three glutathione-S-transferase (GST), and two acetylcholinesterase (AChE) genes that are known to be related to metabolic or target-site resistances in insects. We showed that environmentally relevant levels of pure glyphosate and GBH increased the probability for higher mortality in the Colorado potato beetle larvae in the direct experiment, but not in the indirect experiment. The GBHs or glyphosate did not affect the expression profiles of the studied CYP, GST, or AChE genes; however, we found a large family-level variation in expression profiles in both the direct and indirect treatment experiments. These results suggest that the genes selected for this study may not be the ones expressed in response to glyphosate or GBHs. It is also possible that the relatively short exposure time did not affect gene expression profiles, or the response may have already occurred at a shorter exposure time. Our results show that glyphosate products may affect the survival of the herbivorous insect already at lower levels, depending on their sensitivity to pesticides.

Non-alcoholic fatty liver disease: an epigenetic view of pathogenesis and a new treatment options

Nonalcoholic fatty liver disease (NAFLD) is the most common reason of chronic liver disease. NAFLD causes a wide array of liver conditions ranging from simple steatosis - to nonalcoholic steatohepatitis (NASH) and advanced hepatic fibrosis. Numerous studies show that epigenetic processes are also involved in the pathogenesis of NAFLD. Shifts in the regularity of genomic DNA methylation can cause aberrant gene expression in NAFLD. Pathogenesis of NAFLD is not entirely understood, but it is well-known that obesity, diabetes and metabolic abnormalities played a significant role in the disease development and progression. Epigenetics is known as an inheritable phenomenon which influences the expression of gene without altering the DNA sequence, offers a new view on the pathogenesis of NAFLD. Moreover, epigenetic mechanisms including DNA methylation, posttranslational histone modifications and non-coding RNAs seem to orchestrate various aspects of NAFLD. Histone acetylation affects gene expression profiles in NAFLD. Abnormal histone changes induce insulin resistance, progression of type 2 diabetes mellitus, and subsequent development of NAFLD. This review reflects new advances in the study of epigenetic mechanisms for the development of NAFLD and the formation of innovative therapeutic targets and the long-awaited diagnostic and prognostic tools based on them.

Quercetin positively affects gene expression profiles and metabolic pathway of antibiotic-treated mouse gut microbiota.

Quercetin has a wide range of biological properties that can be used to prevent or decrease particular inflammatory diseases. In this study, we aimed to investigate the gene expression profile and metabolic pathway of the gut microbiota of an antibiotic-treated mouse model administered quercetin. Blood, feces, and intestinal tissue samples were collected and metagenomic sequencing, enzyme-linked immunosorbent assay, and western blot analysis were used to detect variations. The results showed that the quercetin-treated group exhibited increased levels of health beneficial bacterial species, including Faecalibaculum rodentium (103.13%), Enterorhabdus caecimuris (4.13%), Eggerthella lenta (4%), Roseburia hominis (1.33%), and Enterorhabdus mucosicola (1.79%), compared with the model group. These bacterial species were positively related to butyrate, propionate, and intestinal tight junction proteins (zonula occludens-1 and occludin) expression, but negatively related to serum lipopolysaccharide and tumor necrosis factor-α level. In addition, the metabolic pathway analysis showed that dietary quercetin significantly enhanced spliceosomes (111.11%), tight junctions (62.96%), the citrate cycle (10.41%), pyruvate metabolism (6.95%), and lysine biosynthesis (5.06%), but decreasing fatty acid biosynthesis (23.91%) and N-glycan (7.37%) biosynthesis. Furthermore, these metabolic pathway changes were related to relative changes in the abundance of 10 Kyoto Encyclopedia of Genes and Genomes genes (K00244, K00341, K02946, K03737, K01885, k10352, k11717, k10532, K02078, K01191). In conclusion, dietary quercetin increased butyrate-producing bacterial species, and the acetyl-CoA-mediated increased butyrate accelerated carbohydrate, energy metabolism, reduced cell motility and endotoxemia, and increased the gut barrier function, thereby leading to healthy colonic conditions for the host.

Repeated exposure of fluazinam fungicides affects gene expression profiles yet carries no costs on a nontarget pest.

Fungicides are used to control pathogenic fungi of crop species, but they have also been shown to alter behavioral, life history and fitness related traits of nontarget insects. Here, we tested the fungicide effects on feeding behavior, survival and physiology of the nontarget pest insect, the Colorado potato beetle (CPB) (Leptinotarsa decemlineata). Feeding behavior was studied by a choice test of adult beetles, which were allowed to choose between a control and a fungicide (fluazinam) treated potato leaf. Larval survival was recorded after 24 and 72h exposure to control and fungicide-treated leaves with 2 different concentrations. The adults did not show fungicide avoidance behavior. Similarly, survival of the larvae was not affected by the exposure to fungicides. Finally, to understand the effects of fungicides at the physiological level (gene expression), we tested whether the larval exposure to fungicide alter the expression of 5 metabolic pathway and stress associated genes. Highest concentration and 72-h exposure caused upregulation of 1 cytochrome P450 (CYP9Z14v2) and 1 insecticide resistance gene (Ldace1), whereas metabolic detoxification gene (Ugt1) was downregulated. At 24-h exposure, highest concentration caused downregulation of another common detoxification gene (Gs), while both exposure times to lowest concentration caused upregulation of the Hsp70 stress tolerance gene. Despite these overall effects, there was a considerable amount of variation among different families in the gene expression levels. Even though the behavioral effects of the fungicide treatments were minor, the expression level differences of the studied genes indicate changes on the metabolic detoxifications and stress-related pathways.

Timing of Diapause Initiation and Overwintering Conditions Alter Gene Expression Profiles in Megachile rotundata.

Within the United States and Canada, the primary pollinator of alfalfa is the alfalfa leafcutting bee (ALCB), Megachile rotundata. Our previous findings showed that overwintering conditions impacted gene expression profile in ALCB prepupae that entered diapause early in the season. However, ALCB are a bivoltine species, which begs the question of whether bees entering diapause later in the season also show this trend. To better understand the effects of the timing of diapause initiation, we analyzed mRNA copy number of genes known to be involved in diapause regulation in early and late season diapausing ALCB that were overwintered in field conditions or using current agricultural management conditions. We hypothesized that overwintering conditions for late diapausing bees also affects gene expression profiles. Our results showed that expression profiles were altered by both overwintering condition and timing of diapause initiation, with bees that entered diapause earlier in the season showing different expression patterns than those that entered diapause later in the season. This trend was seen in expression of members of the cyclin family and several targets of the insulin signaling pathway, including forkhead box protein O (FOXO), which is known to be important for diapause regulation and stress responses. But, of the genes screened, the proto-oncogene, Myc, was the most impacted by the timing of diapause initiation. Under field conditions, there were significant differences in Myc expression between the early and late season samples in all months except for November and February. This same general trend in Myc expression was also seen in the laboratory-maintained bees with significant difference in expression in all months except for November, February, and May. These results support previous conclusions from our research showing that the molecular regulation of diapause development in ALCB is not a simple singular cascade of gene expression but a highly plastic response that varies between bees depending upon their environmental history.

Abstract P5-12-13: Exploring the impact of the tumor microenvironment on nuclear morphometry: Lessons learned for sensitivity to cytotoxic treatment

Abstract Globally, breast cancer remains the leading cause of death in females. While systemic therapy for breast cancer may work effectively in the early phases, for more than 10% of primary and 50% of metastatic cases, the disease eventually progresses, resisting treatments. To overcome this issue, recognizing markers of resistance as early as possible is critical. Microenvironmental factors influence tumors, and increased stiffness of the extracellular matrix (ECM) is associated with cancer progression. Additionally, neighboring cells such as non-neoplastic epithelial cells(that may constitute an important portion of the tumor microenvironment -TME) are suspected to affect tumors, but details on their impact are lacking. Here, we asked whether the response to increased ECM stiffness differed depending on the degree of aggressiveness of triple negative breast cancers (TNBC) and the presence of the non-neoplastic epithelium. We investigated two morphometric parameters, nuclear circularity and area (used in cancer pathology), and tumors’ sensitivity to cisplatin to assess tumor aggressiveness- with TNBC cultured at normal (770 Pa) and increased (2000 and 3300 Pa) collagen I stiffness. Reduced nuclear circularity and increased heterogeneity for this parameter in the cancer cell population resulted from higher matrix stiffness and were associated with decreased sensitivity to the cytotoxic drug, cisplatin, notably in the more invasive cancer type. Interestingly, the impact of stiffness increase was reduced by the conditioned medium from the non-neoplastic cells. These findings warrant further investigations to identify whether changes in nuclear circularity that illustrate a global nuclear reorganization affect gene expression profiles linked to cell survival. Citation Format: Apekshya Chhetri. Exploring the impact of the tumor microenvironment on nuclear morphometry: Lessons learned for sensitivity to cytotoxic treatment [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-12-13.

C-Abl regulates a synaptic plasticity-related transcriptional program involved in memory and learning.

Memory consolidation requires activation of a gene expression program that allows de novo protein synthesis. But the molecular mechanisms that favour or restrict that program are poorly understood. The kinase c-Abl can modulate gene expression through transcription factors and chromatin modifiers. Here, we show that c-Abl ablation in the brain improves learning acquisition and memory consolidation in mice. Its absence also affects gene expression profiles in the mouse hippocampus. We found that genes involved in synaptic plasticity and actin cytoskeleton dynamics, such as Arp2 and Thorase, are up-regulated at the mRNA and protein levels in trained c-Abl KO mice and by a chemical-LTP stimulus. Trained c-Abl KO mice also show that dendritic spines are larger than in wild-type mice and present at a higher density. These results indicate that c-Abl kinase is an important part of the mechanism that limits or restricts signalling of relevant gene programs involved in morphological and functional spine changes upon neuronal stimulation.

Role of epigenetic modifications in the development of crops essential traits.

Epigenetic modification refers to the chemical modifications of chromosomal DNA and histones, mainly including DNA methylation, histone modifications and non-coding RNAs. Without altering the DNA sequence, these heritable modifications can affect gene expression profiles by changing the chromatin state and play an important role in regulating the growth and development of plants. When the specific epigenetic modifications are changed, crops can obtain excellent phenotypes and stronger environmental adaptability. Therefore, artificially changing the epigenetic modifications are expected to obtain high-quality germplasm resources more suitable for agricultural production. In this review, we summarize the main types of plant epigenetic modifications, highlight the research progresses of functional plant epigenetic modifications on the important traits and responses to environmental stress, and identify the main problems that need be solved in the application of epigenetics in crop improvement, thereby providing new insights for the functional epigenetic modifications on crop breeding and improvement.

High promoter sequence variation in subgroup 6 members of R2R3-MYB genes is involved in different floral anthocyanin color patterns in Lilium spp.

The spatially and temporally distinct expression of R2R3-MYB positive regulators is among the major mechanisms that create various anthocyanin color patterns in many flowers. However, we do not know how these positive regulators have gained different expression profiles. In the Asiatic hybrid lily 'Lollypop' (derived from the crosses of species belonging to Sinomartagon/Daurolirion section), MYB12 and MYB19S regulate the pigmentation at whole tepals and raised tepal spots, respectively. In the Oriental hybrid lily 'Sorbonne' (derived from the crosses of species belonging to the Archelirion section), MYB12 regulates both whole tepal and raised spot pigmentation. The genes have similar amino acid sequences with similar protein functions but exhibit different expression profiles in lily flowers. As promoters are among the most significant factors affecting gene expression profiles, their promoter sequences were determined in this study. The three genes had very different promoter sequences, and putative cis-regulatory elements were not conserved in numbers or order. To further confirm the promoter functions, tobacco plants were transformed with native promoter-driven MYB12 or MYB19S genes of 'Lollypop.' Expression levels of MYB12 were higher in corolla tubes than in lobes, while those of MYB19S were higher in corolla lobes than in tubes. Thus, the diverse promoter functions were likely to be the leading causes of their different expression profiles and generation of unique color patterns. Finally, the history of R2R3-MYB gene establishment during lily evolution was estimated using sequence data.

56 Nobiletin affects gene expression profiles of the ERK1/2 pathway in bovine embryos produced invitro

During embryo development the embryonic genome activation (EGA) is one of the most important events and in bovine embryos it occurs at the 8- to 16-cell stage. Invitro embryo production increases the levels of reactive oxygen species (ROS), which leads to the low quality of the produced blastocysts, possibly by affecting EGA. Nobiletin is an antioxidant that affects cell cycle regulation (Huang et al. 2016 Evid. Based. Complement. Alternat. Med. 2016, 2918796, https://doi.org/10.1155/2016/2918796). Therefore, we aimed to evaluate the effect of nobiletin supplementation, in two key periods of early embryo development, on blastocyst yield and expression of selected genes of the ERK1/2 pathway and oxidative stress on produced embryos. Invitro zygotes were cultured in synthetic oviductal fluid (SOF) with 5% fetal calf serum (control, C); C with 5 or 10µM nobiletin (MedChemExpress) (N5, N10); or C with 0.03% dimethyl sulfoxide (CDMSO; vehicle for nobiletin dilution) during the minor (21–54h post-insemination (hpi): 2- to 8-cell; MNEGA; 12 replicates) or major (54–96 hpi: 8- to 16-cell; MJEGA; 10 replicates) phase of EGA. The speed of development was considered and embryos that reached ≥8 cells at 54 hpi from MNEGA phase and ≥16 cells at 96 hpi from MJEGA phase, were selected and further cultured in control medium until Day 7. Embryos at ≥8 cell (MNEGA), ≥16 cell (MJEGA) stage, and Day 7 blastocysts from both periods were snap-frozen in liquid N2 for gene expression analysis (3 pools of 10 embryos/treatment). The expression of genes related to ERK1/2 pathway (H3–3B, H3–3A, NFE2L2) and oxidative stress (GPX1) were measured by quantitative PCR; H2AFZ and ACTB were used as housekeeping genes. Statistical analysis was assessed by one-way ANOVA. At 54 hpi, irrespective of nobiletin supplementation, no differences were found in the proportion of embryos that reached the 8-cell stage between groups in both phases (≈60%). At 96 hpi, nobiletin during MJEGA showed a higher proportion of embryos reaching the 16-cell stage than control groups (≈70% vs. ≈60%, respectively; P&amp;lt;0.001). Blastocyst yield for MNEGA and MJEGA was higher (P&amp;lt;0.001) for N5 (40.0±0.8% and 46.7±0.8%) and N10 (41.0±0.9% and 54.5±1.1%) compared with C (32.0±0.6% and 38.4±1.1%) and CDMSO (31.2±0.4% and 35.8±1.0%) groups, while N10 was higher (P&amp;lt;0.05) compared to N5 group in MJEGA. The expression of H3–3B and H3–3A were higher (P&amp;lt;0.05) in 8-cell embryos from N5 and N10 groups during MNEGA; while in 16-cell embryos, H3–3B and NFE2L2 were higher (P&amp;lt;0.05) only in the N10 group compared with both controls during MJEGA. GPX1 was upregulated in nobiletin-supplemented groups from both phases (8- and 16-cell embryos and blastocysts) compared with controls (P&amp;lt;0.05). In conclusion, nobiletin supplementation during minor or major EGA has a positive effect in pre-implantation embryo development and modifies the transcription of cell cycle and oxidative stress genes in early embryos. These benefits can be attributed to its bioactivity and indicate that it might be a tool to overcome EGA and ROS disorders in bovine invitro-produced embryos.This research was funded by MINECO-Spain AGL2015-70140-R, PID2019-111641RB-I00, RTI2018-093548-B-I00; SENESCYT-Ecuador; FAPESP-Brazil 2017/20339-3, CNPq-Brazil 304276/2018-9.

The effects of postmortem delay on mouse and human microglia gene expression.

Microglia are specialized macrophages of the central nervous system (CNS) and first to react to pathogens or injury. Over the last decade, transcriptional profiling of microglia significantly contributed to our understanding of their functions. In the case of human CNS samples, either potential CNS pathology in the case of surgery samples, or a postmortem delay (PMD) due to the time needed for tissue access and collection, are potential factors that affect gene expression profiles. To determine the effect of PMD on the microglia transcriptome, we first analyzed mouse microglia, where genotype, antemortem conditions and PMD can be controlled. Microglia were isolated from mice after different PMDs (0, 4, 6, 12, and 24 hr) using fluorescence‐activated cell sorting (FACS). The number of viable microglia significantly decreased with increasing PMD, but even after a 12 hr PMD, high‐quality RNA could be obtained. PMD had very limited effect on mouse microglia gene expression, only 50 genes were differentially expressed between different PMDs. These genes were related to mitochondrial, ribosomal, and protein binding functions. In human microglia transcriptomes we previously generated, 31 of the 50 PMD‐associated mouse genes had human homologs, and their relative expression was also affected by PMD. This study provides a set of genes that shows relative expression changes in relation to PMD, both in mouse and human microglia. Although the gene expression changes detected are subtle, these genes need to be accounted for when analyzing microglia transcriptomes generated from samples with variable PMDs.

Genome-Wide Gene Expression Analyses of BRCA1- and BRCA2-Associated Breast and Ovarian Tumours.

Simple SummaryVariants in the breast cancer susceptibility genes BRCA1 and BRCA2 increase the risk of developing breast and ovarian cancers. Over the past two decades researchers have aimed to identify gene expression changes associated with high-risk BRCA1 and BRCA2 variants. In this review we explore the replicability of BRCA1- and BRCA2-associated gene expression profiles in diseased and normal tissue. We highlight the impact of experimental factors and study designs on the comparability and utility of gene expression profiles associated with high-risk BRCA1 and BRCA2 variants. Additionally, we emphasise the importance of controlling for confounding molecular features that may influence the design of study cohort groups.Germline pathogenic variants in BRCA1 and BRCA2 increase cumulative lifetime risk up to 75% for breast cancer and 76% for ovarian cancer. Genetic testing for BRCA1 and BRCA2 pathogenic variants has become an important part of clinical practice for cancer risk assessment and for reducing individual risk of developing cancer. Genetic testing can produce three outcomes: positive (a pathogenic variant), uninformative (no pathogenic variant) and uncertain significance (a variant of unknown clinical significance). More than one third of BRCA1 and BRCA2 variants identified have been classified as variants of uncertain significance, presenting a challenge for clinicians. To address this important clinical challenge, a number of studies have been undertaken to establish a gene expression phenotype for pathogenic BRCA1 and BRCA2 variant carriers in several diseased and normal tissues. However, the consistency of gene expression phenotypes described in studies has been poor. To determine if gene expression analysis has been a successful approach for variant classification, we describe the design and comparability of 23 published gene expression studies that have profiled cells from BRCA1 and BRCA2 pathogenic variant carriers. We show the impact of advancements in expression-based technologies, the importance of developing larger study cohorts and the necessity to better understand variables affecting gene expression profiles across different tissue types.

Hyperandrogenic Milieu Dysregulates the Expression of Insulin Signaling Factors and Glucose Transporters in the Endometrium of Patients With Polycystic Ovary Syndrome

Purpose: Subfertility associated with polycystic ovary syndrome (PCOS) mainly originates from oligoovulation/anovulation. Although insulin resistance and androgen excess are known to cause PCOS-associated implantation failure, the consequences of PCOS on endometrial homeostasis and pathophysiology have not been comprehensively understood. In this study, we examined whether the pathophysiologic milieu of PCOS intrinsically affects expression profiles of genes related to insulin signaling and facilitative glucose transporters (GLUTs) in the human endometrium and/or during in vitro decidualization. Study Design: Seven healthy women with regular menstrual cycles and 13 patients with PCOS were recruited for this study. To mimic the hyperandrogenic or hyperinsulinemic milieu in the endometrium of patient with PCOS (PCOSE) in vitro, human endometrial stromal cells (hESCs) were treated with dihydrotestosterone (DHT) or insulin, respectively. Results: In PCOSE, messenger RNA (mRNA) levels of insulin receptor (IR), IR substrate (IRS) 1, and IRS2 were significantly increased. Furthermore, GLUT1 and GLUT12 were aberrantly increased. Chronic exposure to insulin or DHT aberrantly increased IRS1/IRS2 phosphorylation and protein levels of GLUT1 and GLUT12 in hESCs, suggesting that not only hyperinsulinemic but also hyperandrogenic conditions affect insulin signaling and glucose metabolism. The mRNA microarrays demonstrated that DHT dysregulates various gene sets, including cell cycle and glucose metabolism, in hESCs. Furthermore, DHT suppressed the expression of GLUT1 and GLUT12 as well as decidualization markers, IGFBP1 and prolactin, during in vitro decidualization. Conclusions: The hyperandrogenic milieu affects gene expression profiles, including gene sets associated with insulin signaling, cell cycle, glucose metabolism, and/or glucose transport, in human endometrium and during in vitro decidualization.

Glucocorticoids counteract hypertrophic effects of myostatin inhibition in dystrophic muscle.

Duchenne muscular dystrophy (DMD) is a devastating genetic muscle disease resulting in progressive muscle degeneration and wasting. Glucocorticoids, specifically prednisone/prednisolone and deflazacort, are commonly used by DMD patients. Emerging DMD therapeutics include those targeting the muscle-wasting factor, myostatin (Mstn). The aim of this study was to investigate how chronic glucocorticoid treatment impacts the efficacy of Mstn inhibition in the D2.mdx mouse model of DMD. We report that chronic treatment of dystrophic mice with prednisolone (Pred) causes significant muscle wasting, entailing both activation of the ubiquitin-proteasome degradation pathway and inhibition of muscle protein synthesis. Combining Pred with Mstn inhibition, using a modified Mstn propeptide (dnMstn), completely abrogates the muscle hypertrophic effects of Mstn inhibition independently of Mstn expression or SMAD3 activation. Transcriptomic analysis identified that combining Pred with dnMstn treatment affects gene expression profiles associated with inflammation, metabolism, and fibrosis. Additionally, we demonstrate that Pred-induced muscle atrophy is not prevented by Mstn ablation. Therefore, glucocorticoids interfere with potential muscle mass benefits associated with targeting Mstn, and the ramifications of glucocorticoid use should be a consideration during clinical trial design for DMD therapeutics. These results have significant implications for past and future Mstn inhibition trials in DMD.

Comprehensive analysis of whole genome methylation in mouse blastocysts cultured with four different constituents following in vitro fertilization

BackgroundWith the development of assisted reproductive technology (ART), diseases believed to be caused by ART have begun to be identified as imprinted disease. However, no conclusion has been reached. So we sought to determine whether ART procedures disturb gene methylation and whether imprinted genes alone are selectively disturbed. To examine whether the constituents of the culture medium contribute to the changes in methylation, we used a mouse model to conduct IVF and comprehensively analyzed 5′–C–phosphate–G–3′ (CpG) by reduced representation bisulfite sequencing (RRBS) using a second-generation sequencer to determine changes in methylation using four types of culture media with different amino acid constituents.ResultsWe cultured ova to the blastocyst stage in a mouse model in culture media with four different amino acid constituents. Each culture medium included (1) KSOM culture medium (NoAA), (2) KSOM media + essential amino acids (EAAs), (3) KSOM medium + non-essential amino acids (NEAAs), or (4) KSOM medium + EAAs + NEAAs (AllAA) analyzed by reduced representation bisulfite sequencing. The results showed that (1) there were many regions that maintained hypermethylation with NEAAs, (2) there was little effect of demethylation on reprogramming in the 5′UTR and promoter regions, and (3) specific changes were observed in imprinted genes such as Nnat and Nespas.ConclusionsCompared with EAAs, NEAAs could protect genes from demethylation caused by reprogramming. On the imprinted genes, methylation of the promoter region of H19 was decreased by NEAAs, suggesting that specific genes were prone to changes in methylation. It was suggested that these changes could provide similar results in humans. Further studies are needed to understand how changes in methylation may affect gene expression profiles.

Transcriptomic changes triggered by ouabain in rat cerebellum granule cells: Role of α3- and α1-Na+,K+-ATPase-mediated signaling.

It was shown previously that inhibition of the ubiquitous α1 isoform of Na+,K+-ATPase by ouabain sharply affects gene expression profile via elevation of intracellular [Na+]i/[K+]i ratio. Unlike other cells, neurons are abundant in the α3 isoform of Na+,K+-ATPase, whose affinity in rodents to ouabain is 104-fold higher compared to the α1 isoform. With these sharp differences in mind, we compared transcriptomic changes in rat cerebellum granule cells triggered by inhibition of α1- and α3-Na+,K+-ATPase isoforms. Inhibition of α1- and α3-Na+,K+-ATPase isoforms by 1 mM ouabain resulted in dissipation of transmembrane Na+ and K+ gradients and differential expression of 994 transcripts, whereas selective inhibition of α3-Na+,K+-ATPase isoform by 100 nM ouabain affected expression of 144 transcripts without any impact on the [Na+]i/[K+]i ratio. The list of genes whose expression was affected by 1 mM ouabain by more than 2-fold was abundant in intermediates of intracellular signaling and transcription regulators, including augmented content of Npas4, Fos, Junb, Atf3, and Klf4 mRNAs, whose upregulated expression was demonstrated in neurons subjected to electrical and glutamatergic stimulation. The role [Na+]i/[K+]i-mediated signaling in transcriptomic changes involved in memory formation and storage should be examined further.

Hippocampal transcriptomic responses to enzyme-mediated cellular dissociation.

Single-neuron gene expression studies may be especially important for understanding nervous system structure and function because of the neuron-specific functionality and plasticity that defines functional neural circuits. Cellular dissociation is a prerequisite technical manipulation for single-cell and single cell-population studies, but the extent to which the cellular dissociation process affects neural gene expression has not been determined. This information is necessary for interpreting the results of experimental manipulations that affect neural function such as learning and memory. The goal of this research was to determine the impact of cellular dissociation on brain transcriptomes. We compared gene expression of microdissected samples from the dentate gyrus (DG), CA3, and CA1 subfields of the mouse hippocampus either prepared by a standard tissue homogenization protocol or subjected to enzymatic digestion used to dissociate cells within tissues. We report that compared to homogenization, enzymatic dissociation alters about 350 genes or 2% of the hippocampal transcriptome. While only a few genes canonically implicated in long-term potentiation and fear memory change expression levels in response to the dissociation procedure, these data indicate that sample preparation can affect gene expression profiles, which might confound interpretation of results depending on the research question. This study is important for the investigation of any complex tissues as research effort moves from subfield level analysis to single cell analysis of gene expression.