Acute Impacts of Ionizing Radiation Exposure on the Gastrointestinal Tract and Gut Microbiome in Mice.

Radiation therapy for abdominopelvic malignancies often results in damage to the gastrointestinal tract (GIT) and permanent changes in bowel function. An overlooked component of the pathophysiology of radiation-induced bowel injury is the role of the gut microbiome. The goal of this research was to identify the impacts of acute radiation exposure on the GIT and gut microbiome. C57BL/6 mice exposed to whole-body X-rays (0.1-3 Gy) were assessed for histological and microbiome changes 48 h post-radiation exposure. Within the ileum, a dose of 3 Gy significantly decreased crypt depth as well as the number of goblet cells, but increased overall goblet cell size. Overall, radiation altered the microbial distribution within each of the main phyla in a dose- and tissue-dependent manner. Within the Firmicutes phylum, high dose irradiation resulted in significant alterations in bacteria from the class Bacilli within the small bowels, and from the class Clostridia in the large bowels. The 3 Gy radiation also significantly increased the abundance of bacterial families from the Bacteroidetes phylum in the colon and feces. Overall, we identified various alterations in microbiome composition following acute radiation exposure, which could potentially lead to novel biomarkers for tracking patient toxicities or could be used as targets for mitigation strategies against radiation damage.

Absence of Depressive and Anxious Behavior with Genetic Dysregulation in Adult C57Bl/6J Mice after Prenatal Exposure to Ionizing Radiation.

The exposure of ionizing radiation during early gestation often leads to deleterious and even lethal effects; however, few extensive studies have been conducted on late gestational exposures. This research examined the behavior al effects of C57Bl/6J mouse offspring exposed to low dose ionizing gamma irradiation during the equivalent third trimester. Pregnant dams were randomly assigned to sham or exposed groups to either low dose or sublethal dose radiation (50, 300, or 1000 mGy) at gestational day 15. Adult offspring underwent a behavioral and genetic analysis after being raised under normal murine housing conditions. Our results indicate very little change in the behavioral tasks measuring general anxiety, social anxiety, and stress-management in animals exposed prenatally across the low dose radiation conditions. Quantitative real-time polymerase chain reactions were conducted on the cerebral cortex, hippocampus, and cerebellum of each animal; results indicate some dysregulation in markers of DNA damage, synaptic activity, reactive oxygen species (ROS) regulation, and methylation pathways in the offspring. Together, our results provide evidence in the C57Bl/6J strain, that exposure to sublethal dose radiation (<1000 mGy) during the last period of gestation leads to no observable changes in behaviour when assessed as adults, although some changes in gene expression were observed for specific brain regions. These results indicate that the level of oxidative stress occurring during late gestation for this mouse strain is not sufficient for a change in the assessed behavioral phenotype, but results in some modest dysregulation of the genetic profile of the brain.

Metabolic adaptation and ATP homeostasis in Pseudomonas fluorescens exposed to phosphate stress.

Phosphate (Pi) is essential for life as it is an integral part of the universal chemical energy adenosine triphosphate (ATP), and macromolecules such as, DNA, RNA proteins and lipids. Despite the core roles and the need of this nutrient in living cells, some bacteria can grow in environments that are poor in Pi. The metabolic mechanisms that enable bacteria to proliferate in a low phosphate environment are not fully understood. In this study, the soil microbe Pseudomonas (P.) fluorescens was cultured in a control and a low Pi (stress) medium in order to delineate how energy homeostasis is maintained. Although there was no significant variation in biomass yield in these cultures, metabolites like isocitrate, oxaloacetate, pyruvate and phosphoenolpyruvate (PEP) were markedly increased in the phosphate-starved condition. Components of the glycolytic, glyoxylate and tricarboxylic acid cycles operated in tandem to generate ATP by substrate level phosphorylation (SLP) as NADH-producing enzymes were impeded. The α-ketoglutarate (KG) produced when glutamine, the sole carbon nutrient was transformed into phosphoenol pyruvate (PEP) and succinyl-CoA (SC), two high energy moieties. The metabolic reprogramming orchestrated by isocitrate lyase (ICL), phosphoenolpyruvate synthase (PEPS), pyruvate phosphate dikinase (PPDK), and succinyl-CoA synthetase fulfilled the ATP budget. Cell free extract experiments confirmed ATP synthesis in the presence of such substrates as PEP, oxaloacetate and isocitrate respectively. Gene expression profiling revealed elevated transcripts associated with numerous enzymes including ICL, PEPS, and succinyl-CoA synthetase (SCS). This microbial adaptation will be critical in promoting biological activity in Pi-poor ecosystems.

Metabolic adaptation and NADPH homeostasis evoked by a sulfur-deficient environment in Pseudomonas fluorescens.

Sulfur is essential for all living organisms due to its ability to mediate a variety of enzymatic reactions, signalling networks, and redox processes. The interplay between sulfhydryl group (SH) and disulfide bond (S-S) is central to the maintenance of intracellular oxidative balance. Although most aerobic organisms succumb to sulfur starvation, the nutritionally versatile soil microbe Pseudomonas fluorescens elaborates an intricate metabolic reprogramming in order to adapt to this challenge. When cultured in a sulfur-deficient medium with glutamine as the sole carbon and nitrogen source, the microbe reconfigures its metabolism aimed at the enhanced synthesis of NADPH, an antioxidant and the limited production of NADH, a pro-oxidant. While oxidative phosphorylation (OXPHOS) and tricarboxylic acid (TCA) cycle, metabolic modules known to generate reactive oxygen species are impeded, the activities NADPH-producing enzymes such as malic enzyme, and glutamate dehydrogenase (GDH) NADP-dependent are increased. The α-ketoglutarate (KG) generated from glutamine rapidly enters the TCA cycle via α-ketoglutarate dehydrogenase (KGDH), an enzyme that was prominent in the control cultures. In the S-deficient media, the severely impeded KGDH coupled with the increased activity of the reversible isocitrate dehydrogenase (ICDH) that fixes KG into isocitrate in the presence of NADH and HCO3- ensures a constant supply of this critical tricarboxylic acid. The up-regulation of ICDH-NADP dependent in the soluble fraction of the cells obtained from the S-deficient media results in enhanced NADPH synthesis, a reaction aided by the concomitant increase in NAD kinase activity. The latter converts NAD into NADP in the presence of ATP. Taken together, the data point to a metabolic network involving isocitrate, α-KG, and ICDH that converts NADH into NADPH in P. fluorescens subjected to a S-deprived environment.

Reactive nitrogen species (RNS)-resistant microbes: adaptation and medical implications.

Nitrosative stress results from an increase in reactive nitrogen species (RNS) within the cell. Though the RNS - nitric oxide (·NO) and peroxynitrite (ONOO-) - play pivotal physiological roles, at elevated concentrations, these moieties can be poisonous to both prokaryotic and eukaryotic cells alike due to their capacity to disrupt a variety of essential biological processes. Numerous microbes are known to adapt to nitrosative stress by elaborating intricate strategies aimed at neutralizing RNS. In this review, we will discuss both the enzymatic systems dedicated to the elimination of RNS as well as the metabolic networks that are tailored to generate RNS-detoxifying metabolites - α-keto-acids. The latter has been demonstrated to nullify RNS via non-enzymatic decarboxylation resulting in the production of a carboxylic acid, many of which are potent signaling molecules. Furthermore, as aerobic energy production is severely impeded during nitrosative stress, alternative ATP-generating modules will be explored. To that end, a holistic understanding of the molecular adaptation to nitrosative stress, reinforces the notion that neutralization of toxicants necessitates significant metabolic reconfiguration to facilitate cell survival. As the alarming rise in antimicrobial resistant pathogens continues unabated, this review will also discuss the potential for developing therapies that target the alternative ATP-generating machinery of bacteria.

Endothelial FoxO proteins impair insulin sensitivity and restrain muscle angiogenesis in response to a high-fat diet.

Skeletal muscle microvascular dysfunction contributes to disease severity in type 2 diabetes. Recent studies indicate a role for Forkhead box O (FoxO) transcription factors in modulating endothelial cell phenotype. We hypothesized that a high-fat (HF) diet generates a dysfunctional vascular niche through an increased expression of endothelial FoxO. FoxO1 protein increased (+130%) in the skeletal muscle capillaries from HF compared to normal chow-fed mice. FoxO1 protein was significantly elevated in cultured endothelial cells exposed to the saturated fatty acid palmitate or the proinflammatory cytokine TNF-α. In HF-fed mice, endothelium-directed depletion of FoxO1/3/4 (FoxO(Δ)) improved insulin sensitivity (+110%) compared to that of the controls (FoxO(L/L)). The number of skeletal muscle capillaries increased significantly in the HF-FoxO(Δ) mice. Transcript profiling of skeletal muscle identified significant increases in genes associated with angiogenesis and lipid metabolism in HF-FoxO(Δ) vs. HF-FoxO(L/L) mice. HF-FoxO(Δ) muscle also was characterized by a decrease in inflammation-related genes and an enriched M2 macrophage signature. We conclude that endothelial FoxO proteins promote insulin resistance in HF diet, which may in part result from FoxO proteins establishing an antiangiogenic and proinflammatory microenvironment within skeletal muscle. These findings provide mechanistic insight into the development of microvascular dysfunction in the progression of type 2 diabetes.-Nwadozi, E., Roudier, E., Rullman, E., Tharmalingam, S., Liu, H.-Y., Gustafsson, T., Haas, T. L. Endothelial FoxO proteins impair insulin sensitivity and restrain muscle angiogenesis in response to a high-fat diet.

Phenotypic and transcriptional changes in lens epithelial cells following acute and fractionated ionizing radiation exposure.

PURPOSE: Exposure to ionizing radiation is one of the known risk factors for the development of lens opacities. It is believed that radiation interactions with lens epithelial cells (LEC) are the underlying cause of cataract development, however, the exact mechanisms have yet to be identified. The aim of this study was to investigate how different radiation dose and fractionation impact normal LEC function. MATERIALS AND METHODS: A human derived LEC cell line (HLE-B3) was exposed to a single acute x-ray dose (0.25 Gy) and 6 fractionated doses (total dose of 0.05, 0.1, 0.25, 0.5, 1, and 2 Gy divided over 5 equal fractions). LEC were examined for proliferation using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and migration using a Boyden chamber assay at various time points (0.25, 0.5, 1, 2, 4, 7, 9, 11, and 14 d) post-irradiation. Transcriptomic analysis through RNA sequencing was also performed to identify differentially expressed genes and regulatory networks in cells following 4 different acute exposures and 1 fractionated exposure. RESULTS: Exposure to an acute dose of 0.25 Gy significantly increased proliferation and migration rates, peaking at 7 d post irradiation (20% and 240% greater than controls, respectively), before returning to baseline levels by day 14. Fractionated exposures had minimal effects up to a dose of 0.5 Gy, but significantly reduced proliferation and migration after 1 and 2 Gy by up to 50%. The largest transcriptional response occurred 12 h after an acute 0.25 Gy dose, with 362 genes up-regulated and 288 genes down-regulated. A unique panel of differentially expressed genes was observed between moderate versus high dose exposures, suggesting a dose-dependent transcriptional response in LEC that is more pronounced at lower doses. Gene ontology and upstream regulator analysis identified multiple biological processes and molecular functions implicated in the radiation response, in particular differentiation, motility, receptor/ligand binding, cell signaling and epithelial-mesenchymal cell transition. CONCLUSIONS: Overall, this research provides novel insights into the dose and fractionation effects on functional changes and transcriptional regulatory networks in LEC, furthering our understanding of the mechanisms behind radiation induced cataracts.

Protracted Exposure to a Sub-background Radiation Environment Negatively Impacts the Anhydrobiotic Recovery of Desiccated Yeast Sentinels.

ABSTRACT: Experiments that examine the impacts of subnatural background radiation exposure provide a unique approach to studying the biological effects of low-dose radiation. These experiments often need to be conducted in deep underground laboratories in order to filter surface-level cosmic radiation. This presents some logistical challenges in experimental design and necessitates a model organism with minimal maintenance. As such, desiccated yeast ( Saccharomyces cerevisiae ) is an ideal model system for these investigations. This study aimed to determine the impact of prolonged sub-background radiation exposure in anhydrobiotic (desiccated) yeast at SNOLAB in Sudbury, Ontario, Canada. Two yeast strains were used: a normal wild type and an isogenic recombinational repair-deficient rad51 knockout strain ( rad51 Δ). Desiccated yeast samples were stored in the normal background surface control laboratory (68.0 nGy h -1 ) and in the sub-background environment within SNOLAB (10.1 nGy h -1 ) for up to 48 wk. Post-rehydration survival, growth rate, and metabolic activity were assessed at multiple time points. Survival in the sub-background environment was significantly reduced by a factor of 1.39 and 2.67 in the wild type and rad51 ∆ strains, respectively. Post-rehydration metabolic activity measured via alamarBlue reduction remained unchanged in the wild type strain but was 26% lower in the sub-background rad51 ∆ strain. These results demonstrate that removing natural background radiation negatively impacts the survival and metabolism of desiccated yeast, highlighting the potential importance of natural radiation exposure in maintaining homeostasis of living organisms.

The Effects of Oral Probiotics on Type 2 Diabetes Mellitus (T2DM): A Clinical Trial Systematic Literature Review.

Type 2 diabetes mellitus (T2DM) remains a global health concern. Emerging clinical trial (CT) evidence suggests that probiotic intervention may promote a healthy gut microbiome in individuals with T2DM, thereby improving management of the disease. This systematic literature review summarizes thirty-three CTs investigating the use of oral probiotics for the management of T2DM. Here, twenty-one studies (64%) demonstrated an improvement in at least one glycemic parameter, while fifteen studies (45%) showed an improvement in at least one lipid parameter. However, no article in this review was able to establish a uniform decrease in glycemic, lipid, or blood pressure profiles. The lack of consistency across the studies may be attributed to differences in probiotic composition, duration of probiotic consumption, and probiotic dose. An interesting finding of this literature review was the beneficial trend of metformin and probiotic co-administration. Here, patients with T2DM taking metformin demonstrated enhanced glycemic control via the co-administration of probiotics. Taken together, the overall positive findings reported across the studies in combination with minimal adverse effects constitute ground for further quality CTs. This review provides recommendations for future CTs that may address the shortcomings of the current studies and help to extract useful data from future investigations of the use of probiotics in T2DM management.

The REPAIR Project, a Deep-Underground Radiobiology Experiment Investigating the Biological Effects of Natural Background Radiation: The First 6 Years.

In 2017, a special edition of Radiation Research was published [Oct; Vol. 188 4.2 (https://bioone.org/journals/radiation-research/volume-188/issue-4.2)] which focused on a recently established radiobiology project within SNOLAB, a unique deep-underground research facility. This special edition included original articles, reviews and commentaries relevant to the research goals of this new project which was titled Researching the Effects of the Presence and Absence of Ionizing Radiation (REPAIR). These research goals were founded in understanding the biological effects of terrestrial and cosmic natural background radiation (NBR). Since 2017, REPAIR has evolved into a sub-NBR radiobiology research program which investigates these effects using multiple model systems and various biological endpoints. This paper summarizes the evolution of the REPAIR project over the first 6-years including its experimental scope and capabilities as well as research accomplishments.

Low-Dose Hemibody Radiation, a Treatment Option for Recurrent Prostate Cancer: A Phase 2 Single-Arm Trial.

Purpose: Nontargeted low-dose ionizing radiation has been proposed as a cancer therapeutic for several decades; however, questions remain about the duration of hematological changes and optimal dosing regimen. Early studies delivering fractionated low doses of radiation to patients with cancer used varying doses and schedules, which make it difficult to standardize a successful dose and scheduling system for widespread use. The aim of this phase 2 two-stage trial was to determine whether low-dose radiation therapy (LD-RT) reduced prostate-specific antigen (PSA) in patients with recurrent prostate cancer in efforts to delay initiation of conventional therapies that are known to decrease quality of life. The primary study outcome was reduction in PSA levels by at least 50%. Methods and Materials: Sixteen patients with recurrent prostate cancer were recruited and received 2 doses of 150 mGy of nontargeted radiation per week, for 5 consecutive weeks, with 15 participants completing the study. Results: A maximal response of 40.5% decrease in PSA at 3 months was observed. A total of 8 participants remained off any additional interventions, of whom 3 had minor fluctuations in PSA for at least 1 year after treatment. The most common adverse event reported was mild fatigue during active treatment (n = 4), which did not persist in the follow-up period. No participants withdrew due to safety concerns or hematological abnormalities (ie, platelet ≤50 × 109/L, leukocyte ≤3 × 109/L, granulocyte ≤2 × 109/L). Conclusions: Our study did not meet the primary objective; however, LD-RT may be a potential therapy for some patients with recurrent prostate cancer by stalling rising PSA. This study also demonstrates that low-dose radiation is well tolerated by participants with minimal toxicities and no change in quality of life.

Molecular Mechanisms Associated with Antifungal Resistance in Pathogenic Candida Species.

Candidiasis is a highly pervasive infection posing major health risks, especially for immunocompromised populations. Pathogenic Candida species have evolved intrinsic and acquired resistance to a variety of antifungal medications. The primary goal of this literature review is to summarize the molecular mechanisms associated with antifungal resistance in Candida species. Resistance can be conferred via gain-of-function mutations in target pathway genes or their transcriptional regulators. Therefore, an overview of the known gene mutations is presented for the following antifungals: azoles (fluconazole, voriconazole, posaconazole and itraconazole), echinocandins (caspofungin, anidulafungin and micafungin), polyenes (amphotericin B and nystatin) and 5-fluorocytosine (5-FC). The following mutation hot spots were identified: (1) ergosterol biosynthesis pathway mutations (ERG11 and UPC2), resulting in azole resistance; (2) overexpression of the efflux pumps, promoting azole resistance (transcription factor genes: tac1 and mrr1; transporter genes: CDR1, CDR2, MDR1, PDR16 and SNQ2); (3) cell wall biosynthesis mutations (FKS1, FKS2 and PDR1), conferring resistance to echinocandins; (4) mutations of nucleic acid synthesis/repair genes (FCY1, FCY2 and FUR1), resulting in 5-FC resistance; and (5) biofilm production, promoting general antifungal resistance. This review also provides a summary of standardized inhibitory breakpoints obtained from international guidelines for prominent Candida species. Notably, N. glabrata, P. kudriavzevii and C. auris demonstrate fluconazole resistance.

Genomic Loss and Epigenetic Silencing of the FOSL1 Tumor Suppressor Gene in Radiation-induced Neoplastic Transformation of Human CGL1 Cells Alters the Tumorigenic Phenotype In Vitro and In Vivo.

The CGL1 human hybrid cell system has been utilized for many decades as an excellent cellular tool for investigating neoplastic transformation. Substantial work has been done previously implicating genetic factors related to chromosome 11 to the alteration of tumorigenic phenotype in CGL1 cells. This includes candidate tumor suppressor gene FOSL1, a member of the AP-1 transcription factor complex which encodes for protein FRA1. Here we present novel evidence supporting the role of FOSL1 in the suppression of tumorigenicity in segregants of the CGL1 system. Gamma-induced mutant (GIM) and control (CON) cells were isolated from 7 Gy gamma-irradiated CGL1s. Western, Southern and Northern blot analysis were utilized to assess FOSL1/FRA1 expression as well as methylation studies. GIMs were transfected to re-express FRA1 and in vivo tumorigenicity studies were conducted. Global transcriptomic microarray and RT-qPCR analysis were used to further characterize these unique cell segregants. GIMs were found to be tumorigenic in vivo when injected into nude mice whereas CON cells were not. GIMs show loss of Fosl/FRA1 expression as confirmed by Western blot. Southern and Northern blot analysis further reveals that FRA1 reduction in tumorigenic CGL1 segregants is likely due to transcriptional suppression. Results suggest that radiation-induced neoplastic transformation of CGL1 is in part due to silencing of the FOSL1 tumor suppressor gene promoter by methylation. The radiation-induced tumorigenic GIMs transfected to re-express FRA1 resulted in suppression of subcutaneous tumor growth in nude mice in vivo. Global microarray analysis and RT-qPCR validation elucidated several hundred differentially expressed genes. Downstream analysis reveals a significant number of altered pathways and enriched Gene Ontology terms genes related to cellular adhesion, proliferation, and migration. Together these findings provide strong evidence that FRA1 is a tumor suppressor gene deleted and epigenetically silenced after ionizing radiation-induced neoplastic transformation in the CGL1 human hybrid cell system.

Overexpression of FRA1 (FOSL1) Leads to Global Transcriptional Perturbations, Reduced Cellular Adhesion and Altered Cell Cycle Progression.

FRA1 (FOSL1) is a transcription factor and a member of the activator protein-1 superfamily. FRA1 is expressed in most tissues at low levels, and its expression is robustly induced in response to extracellular signals, leading to downstream cellular processes. However, abnormal FRA1 overexpression has been reported in various pathological states, including tumor progression and inflammation. To date, the molecular effects of FRA1 overexpression are still not understood. Therefore, the aim of this study was to investigate the transcriptional and functional effects of FRA1 overexpression using the CGL1 human hybrid cell line. FRA1-overexpressing CGL1 cells were generated using stably integrated CRISPR-mediated transcriptional activation, resulting in a 2-3 fold increase in FRA1 mRNA and protein levels. RNA-sequencing identified 298 differentially expressed genes with FRA1 overexpression. Gene ontology analysis showed numerous molecular networks enriched with FRA1 overexpression, including transcription-factor binding, regulation of the extracellular matrix and adhesion, and a variety of signaling processes, including protein kinase activity and chemokine signaling. In addition, cell functional assays demonstrated reduced cell adherence to fibronectin and collagen with FRA1 overexpression and altered cell cycle progression. Taken together, this study unravels the transcriptional response mediated by FRA1 overexpression and establishes the role of FRA1 in adhesion and cell cycle progression.

Calcium-sensing receptor modulates cell adhesion and migration via integrins.

The calcium-sensing receptor (CaSR) is a family C G protein-coupled receptor that is activated by elevated levels of extracellular divalent cations. The CaSR couples to members of the G(q) family of G proteins, and in the endocrine system this receptor is instrumental in regulating the release of parathyroid hormone from the parathyroid gland and calcitonin from thyroid cells. Here, we demonstrate that in medullary thyroid carcinoma cells, the CaSR promotes cellular adhesion and migration via coupling to members of the integrin family of extracellular matrix-binding proteins. Immunopurification and mass spectrometry, co-immunoprecipitation, and co-localization studies showed that the CaSR and ß1-containing integrins are components of a macromolecular protein complex. In fibronectin-based cell adhesion and migration assays, the CaSR-positive allosteric modulator NPS R-568 induced a concentration-dependent increase in cell adhesion and migration; both of these effects were blocked by a specific CaSR-negative allosteric modulator. These effects were mediated by integrins because they were blocked by a peptide inhibitor of integrin binding to fibronectin and ß1 knockdown experiments. An analysis of intracellular signaling pathways revealed a key role for CaSR-induced phospholipase C activation and the release of intracellular calcium. These results demonstrate for the first time that an ion-sensing G protein-coupled receptor functionally couples to the integrins and, in conjunction with intracellular calcium release, promotes cellular adhesion and migration in tumor cells. The significance of this interaction is further highlighted by studies implicating the CaSR in cancer metastasis, axonal growth, and stem cell attachment, functions that rely on integrin-mediated cell adhesion.

A Metabolic Network Mediating the Cycling of Succinate, a Product of ROS Detoxification into α-Ketoglutarate, an Antioxidant.

Sulfur is an essential element for life. However, the soil microbe Pseudomonas (P.) fluorescens can survive in a low sulfur environment. When cultured in a sulfur-deficient medium, the bacterium reprograms its metabolic pathways to produce α-ketoglutarate (KG) and regenerate this keto-acid from succinate, a by-product of ROS detoxification. Succinate semialdehyde dehydrogenase (SSADH) and KG decarboxylase (KGDC) work in partnership to synthesize KG. This process is further aided by the increased activity of the enzymes glutamate decarboxylase (GDC) and γ-amino-butyrate transaminase (GABAT). The pool of succinate semialdehyde (SSA) generated is further channeled towards the formation of the antioxidant. Spectrophotometric analyses, HPLC experiments and electrophoretic studies with intact cells and cell-free extracts (CFE) pointed to the metabolites (succinate, SSA, GABA) and enzymes (SSADH, GDC, KGDC) contributing to this KG-forming metabolic machinery. Real-time polymerase chain reaction (RT-qPCR) revealed significant increase in transcripts of such enzymes as SSADH, GDC and KGDC. The findings of this study highlight a novel pathway involving keto-acids in ROS scavenging. The cycling of succinate into KG provides an efficient means of combatting an oxidative environment. Considering the central role of KG in biological processes, this metabolic network may be operative in other living systems.

Effect of Prenatal Glucocorticoid Exposure on Circadian Rhythm Gene Expression in the Brains of Adult Rat Offspring.

Circadian clocks control many vital aspects of physiology from the sleep-wake cycle to metabolism. The circadian clock operates through transcriptional-translational feedback loops. The normal circadian signaling relies on a 'master clock', located in the suprachiasmatic nucleus (SCN), which synchronizes peripheral oscillators. Glucocorticoid receptor (GR) signaling has the ability to reset the phase of peripheral clocks. It has been shown that maternal exposure to glucocorticoids (GCs) can lead to modification of hypothalamic-pituitary-adrenal (HPA) function, impact stress-related behaviors, and result in a hypertensive state via GR activation. We previously demonstrated altered circadian rhythm signaling in the adrenal glands of offspring exposed to the synthetic GC, dexamethasone (Dex). Results from the current study show that prenatal exposure to Dex affects circadian rhythm gene expression in a brain region-specific and a sex-specific manner within molecular oscillators of the amygdala, hippocampus, paraventricular nucleus, and prefrontal cortex, as well as the main oscillator in the SCN. Results also show that spontaneously hypertensive rats (SHR) exhibited dysregulated circadian rhythm gene expression in these same brain regions compared with normotensive Wistar-Kyoto rats (WKY), although the pattern of dysregulation was markedly different from that seen in adult offspring prenatally exposed to GCs.

Radiation-Induced Alterations in Proliferation, Migration, and Adhesion in Lens Epithelial Cells and Implications for Cataract Development.

The lens of the eye is one of the most radiosensitive tissues. Although the exact mechanism of radiation-induced cataract development remains unknown, altered proliferation, migration, and adhesion have been proposed as factors. Lens epithelial cells were exposed to X-rays (0.1-2 Gy) and radiation effects were examined after 12 h and 7 day. Proliferation was quantified using an MTT assay, migration was measured using a Boyden chamber and wound-healing assay, and adhesion was assessed on three extracellular matrices. Transcriptional changes were also examined using RT-qPCR for a panel of genes related to these processes. In general, a nonlinear radiation response was observed, with the greatest effects occurring at a dose of 0.25 Gy. At this dose, a reduction in proliferation occurred 12 h post irradiation (82.06 ± 2.66%), followed by an increase at 7 day (116.16 ± 3.64%). Cell migration was increased at 0.25 Gy, with rates 121.66 ± 6.49% and 232.78 ± 22.22% greater than controls at 12 h and 7 day respectively. Cell adhesion was consistently reduced above doses of 0.25 Gy. Transcriptional alterations were identified at these same doses in multiple genes related to proliferation, migration, and adhesion. Overall, this research began to elucidate the functional changes that occur in lens cells following radiation exposure, thereby providing a better mechanistic understanding of radiation-induced cataract development.

Identification of Radiation-Induced miRNA Biomarkers Using the CGL1 Cell Model System.

MicroRNAs (miRNAs) have emerged as a potential class of biomolecules for diagnostic biomarker applications. miRNAs are small non-coding RNA molecules, produced and released by cells in response to various stimuli, that demonstrate remarkable stability in a wide range of biological fluids, in extreme pH fluctuations, and after multiple freeze-thaw cycles. Given these advantages, identification of miRNA-based biomarkers for radiation exposures can contribute to the development of reliable biological dosimetry methods, especially for low-dose radiation (LDR) exposures. In this study, an miRNAome next-generation sequencing (NGS) approach was utilized to identify novel radiation-induced miRNA gene changes within the CGL1 human cell line. Here, irradiations of 10, 100, and 1000 mGy were performed and the samples were collected 1, 6, and 24 h post-irradiation. Corroboration of the miRNAome results with RT-qPCR verification confirmed the identification of numerous radiation-induced miRNA expression changes at all doses assessed. Further evaluation of select radiation-induced miRNAs, including miR-1228-3p and miR-758-5p, as well as their downstream mRNA targets, Ube2d2, Ppp2r2d, and Id2, demonstrated significantly dysregulated reciprocal expression patterns. Further evaluation is needed to determine whether the candidate miRNA biomarkers identified in this study can serve as suitable targets for radiation biodosimetry applications.

Subchronic administration and combination metabotropic glutamate and GABAB receptor drug therapy in fragile X syndrome.

The most common cause of inherited mental retardation, fragile X syndrome, results from a triplet repeat expansion in the FMR1 gene and loss of the mRNA binding protein, fragile X mental retardation protein (FMRP). In the absence of FMRP, signaling through group I metabotropic glutamate receptors (mGluRs) is enhanced. We previously proposed a mechanism whereby the audiogenic seizures exhibited by FMR1 null mice result from an imbalance in excitatory mGluR and inhibitory GABA(B) receptor (GABA(B)R) signaling (Mol Pharmacol 76:18-24, 2009). Here, we tested the mGluR5-positive allosteric modulator 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB), the mGluR5 inverse agonist 2-methyl-6-(phenylethynyl)pyridine (MPEP), and GABA(B) receptor agonists, alone and in combination on receptor protein expression and audiogenic seizures in FMR1 mice. Single doses of MPEP (30 mg/kg), the GABA(B)R orthosteric agonist R-baclofen (1 mg/kg), or the GABA(B)R-positive allosteric modulator N,N'-dicyclopentyl-2-(methylthio)-5-nitro-4,6-pyrimidine diamine (GS-39783) (30 mg/kg), reduced the incidence of seizures. However, when administered subchronically (daily injections for 6 days), MPEP retained its anticonvulsant activity, whereas R-baclofen and GS-39783 did not. When administered at lower doses that had no effect when given alone, a single injection of MPEP plus R-baclofen also reduced seizures, but the effect was lost after subchronic administration. We were surprised to find that subchronic treatment with R-baclofen also induced tolerance to a single high dose of MPEP. These data demonstrate that tolerance develops rapidly to the antiseizure properties of R-baclofen alone and R-baclofen coadministered with MPEP, but not with MPEP alone. Our findings suggest that cross-talk between the G-protein signaling pathways of these receptors affects drug efficacy after repeated treatment.