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Carrageenans (CGNs) are widely used in foods and pharmaceuticals although their safety remains controversial. To investigate the effects of CGNs and CGN-degrading bacteria in the human colon, we screened for CGN degradation by human fecal microbiota, and for inflammatory response to CGNs and/or CGN-degrading bacteria in germ free mice. Thin-layer chromatography indicated that high molecular weight (MW) CGNs (≥100 kDa) remained undegraded in the presence of human fecal microbiota, whereas low MW CGNs, i.e., κ-carrageenan oligosaccharides (KCO, ~4.5 kDa) were degraded when exposed to seven of eight human fecal samples, although sulfate groups were not removed during degradation. Bacteroides xylanisolvens and Escherichia coli isolates from fecal samples apparently degraded KCO synergistically, with B. xylanisolvens serving as the primary degrader. Combined treatment of KCO with KCO-degrading bacteria led to greater pro-inflammatory effects in the colon and rectum of germ-free mice than either KCO or bacteria alone. Similarly, p-p38-, CD3-, and CD79a-positive immune cells were more abundant in combined treatment group mice than in either single treatment group. Our study shows that KCO-degrading bacteria and the low MW products of KCO can promote proinflammatory effects in mice, and represent two key markers for evaluating CGN safety in foods or medicines.

Previous population health studies examining adults with acute myeloid leukemia (AML); however many of these, such as the Cancer Genome Atlas, are derived from databases collected by large urban centers. Due to its unique industry and environmental exposures, we hypothesized the West Virginia Appalachian population may have different mutational trends and clinical outcomes.To address the concern of under-representation of rural minorities in cancer genomic databases, we performed exploratory whole exome sequencing in patients with newly diagnosed AML in rural Appalachia.Correlations between genetic variants and clinical outcome variables were examined via retrospective chart review. A total of 26 patients were identified and whole exome sequencing was performed. Median age was 68 years old. Twenty-one patients had de novo AML (84%). As per European LeukemiaNet (ELN) criteria, 8 patients were favorable (32%), 12 were intermediate (48%), and 5 were adverse risk (20%). Eight patients proceeded to transplant. The median progression-free survival and overall survival were 16.5 months and 26.6 months, respectively. We noted an increased tumor mutation burden and a higher frequency of specific known driver mutations when compared to The Cancer Genome Atlas database; we also found novel mutations in MUC3A, MUC5AC, HCAR3, ORT2B, and PABPC. Survival outcomes were slightly lower than national average and BCOR mutation correlated with inferior outcomes.Our findings provide novel insight into detrimental mutations in AML in a rural, underrepresented population. We discovered several novel mutations and higher frequency of some known driver mutations, which will help us identify therapeutic targets to improve patient outcomes.

Background Prior work demonstrated that female rats (but not their male littermates) exposed to methamphetamine become hypersensitive to myocardial ischemic injury. Importantly, this sex-dependent effect persists following 30 days of subsequent abstinence from the drug, suggesting that it may be mediated by long term changes in gene expression that are not rapidly reversed following discontinuation of methamphetamine use. The goal of the present study was to determine whether methamphetamine induces sex-dependent changes in myocardial gene expression and whether these changes persist following subsequent abstinence from methamphetamine. Results Methamphetamine induced changes in the myocardial transcriptome were significantly greater in female hearts than male hearts both in terms of the number of genes affected and the magnitude of the changes. The largest changes in female hearts involved genes that regulate the circadian clock (Dbp, Per3, Per2, BMal1, and Npas2) which are known to impact myocardial ischemic injury. These genes were unaffected by methamphetamine in male hearts. All changes in gene expression identified at day 11 returned to baseline by day 30. Conclusions These data demonstrate that female rats are more sensitive than males to methamphetamine-induced changes in the myocardial transcriptome and that methamphetamine does not induce changes in myocardial transcription that persist long term after exposure to the drug has been discontinued.

Introduction The prototype DNA hypomethylating agents 5-azacytidine (5AC) and decitabine (DAC) are currently FDA-approved for treatment of blood and bone marrow disorders like myelodysplastic syndrome. 5AC and DAC are considered similar drugs and were shown to induce histone modifications that modulate gene expression. The aim of this study is to compare the effect of both drugs on histone acetylation and methylation at multiple histone amino acids residues. Methods Mass spectrometry was used to compare the effect of both drugs on 95 different histone posttranslational modifications (PTMs) in leukemia cells. ChIP-Seq analysis was used to compare the impact of both drugs on the genome-wide acetylation of the H3K9 mark using primary leukemia cells from six de-identified AML patients. Results Both DAC and 5AC induced histone PTMs in different histone isoforms like H1.4, H2A, H3, H3.1, and H4. Changes in both histone methylation and acetylation were observed with both drugs; however, there were distinct differences in the histone modifications induced by the two drugs. Since both drugs were shown to increase the activity of the HDAC SIRT6 previously, we tested the effect of 5AC on the acetylation of H3K9, the physiological substrate SIRT6, using ChIP-Seq analysis and compared it to the previously published DAC-induced changes. Significant H3K9 acetylation changes ( P< .05) were detected at 925 genes after 5AC treatment vs only 182 genes after DAC treatment. Nevertheless, the gene set modified by 5AC was different from that modified by DAC with only ten similar genes modulated by both drugs. Conclusion Despite similarity in chemical structure and DNA hypomethylating activity, 5AC and DAC induced widely different histone PTMs and considering them interchangeable should be carefully evaluated. The mechanism of these histone PTM changes is not clear and may involve modulation of the activity or the expression of the enzymes inducing histone PTMs.

The phenoxazine dye resazurin exhibits bactericidal activity against the Gram-negative pathogens Francisella tularensis and Neisseria gonorrhoeae. One resazurin derivative, resorufin pentyl ether, significantly reduces vaginal colonization by Neisseria gonorrhoeae in a mouse model of infection. The narrow spectrum of bacteria susceptible to resazurin and its derivatives suggests these compounds have a novel mode of action. To identify potential targets of resazurin and mechanisms of resistance, we isolated mutants of F. tularensis subsp. holarctica live vaccine strain (LVS) exhibiting reduced susceptibility to resazurin and performed whole genome sequencing. The genes pilD (FTL_0959) and dipA (FTL_1306) were mutated in half of the 46 resazurin-resistant (RZR) strains sequenced. Complementation of select RZR LVS isolates with wild-type dipA or pilD partially restored sensitivity to resazurin. To further characterize the role of dipA and pilD in resazurin susceptibility, a dipA deletion mutant, ΔdipA, and pilD disruption mutant, FTL_0959d, were generated. Both mutants were less sensitive to killing by resazurin compared to wild-type LVS with phenotypes similar to the spontaneous resazurin-resistant mutants. This study identified a novel role for two genes dipA and pilD in F. tularensis susceptibility to resazurin.

Consumption of walnuts has slowed breast cancer growth and/or reduced the risk of mammary cancer in mice. The benefit against cancer was associated with altered expression of genes for cancer growth and survival. We hypothesized that walnut consumption would alter gene expression in pathologically confirmed breast cancers of women in a direction that would be expected to decrease breast cancer growth and survival, as was seen in mice. The study was a nonplacebo, 2-arm, clinical trial. Women with breast lumps large enough for research and pathology biopsies were recruited and randomized to walnut consuming or control groups. Immediately after biopsy collection, women in the walnut group began to consume 2 oz of walnuts per day until follow-up surgery. Pathological studies confirmed that lumps were breast cancer in all women who remained in the trial. At surgery, about 2 weeks after biopsy, additional specimens were taken from the breast cancers. Changes in gene expression in the surgical specimen compared to baseline were determined in each individual woman in walnut-consuming (n = 5) and control (n = 5) groups. RNA sequencing expression profiling revealed that expression of 456 identified genes was significantly changed in the tumor due to walnut consumption. Ingenuity Pathway Analysis showed activation of pathways that promote apoptosis and cell adhesion, and inhibition of pathways that promote cell proliferation and migration. These results support the hypothesis that, in humans, walnut consumption could suppress growth and survival of breast cancers.

The FDA-approved DNA hypomethylating agents (DHAs) like 5-azacytidine (5AC) and decitabine (DAC) demonstrate efficacy in the treatment of hematologic malignancies. Despite previous reports that showed histone acetylation changes upon using these agents, the exact mechanism underpinning these changes is unknown. In this study, we investigated the relative potency of the nucleoside analogs and non-nucleoside analogs DHAs on DNA methylation reversal using DNA pyrosequencing. Additionally, we screened their effect on the enzymatic activity of the histone deacetylase sirtuin family (SIRT1, SIRT2, SIRT3, SIRT5 and SIRT6) using both recombinant enzymes and nuclear lysates from leukemia cells. The nucleoside analogs (DAC, 5AC and zebularine) were the most potent DHAs and increased the enzymatic activity of SIRT6 without showing any significant increase in other sirtuin isoforms. ChIP-Seq analysis of bone marrow cells derived from six acute myeloid leukemia (AML) patients and treated with the nucleoside analog DAC induced genome-wide acetylation changes in H3K9, the physiological substrate for SIRT6. Data pooling from the six patients showed significant acetylation changes in 187 gene loci at different chromosomal regions including promoters, coding exons, introns and distal intergenic regions. Signaling pathway analysis showed that H3K9 acetylation changes are linked to AML-relevant signaling pathways like EGF/EGFR and Wnt/Hedgehog/Notch. To our knowledge, this is the first report to identify the nucleoside analogs DHAs as activators of SIRT6. Our findings provide a rationale against the combination of the nucleoside analogs DHAs with SIRT6 inhibitors or chemotherapeutic agents in AML due to the role of SIRT6 in maintaining genome integrity and DNA repair.

Background: Carrageenans (CGNs) are widely used in food and pharmaceutical industries. However, the safety of CGNs is still under debate, because degraded CGNs have been reported to promote an intestinal inflammatory response in animal models. Here, we studied the relationship among CGNs, human gut microbiota, and the host inflammatory response.Methods: TLC was selected for detecting the degradation of KCPs by human gut microbiota in vitro batch fermentation system. PCR-DGGE and real time PCR were used for studying bacterial community. ESI-MS was used for KCPs structure analysis. Hematoxylin-eosin staining (HE), immunohistochemistry (IHC) and RNA-seq were used to evaluated the KCPs on host inflammation response in germ-free mice.Results: Thin-layer chromatography (TLC) data showed that CGNs with a molecular weight (Mw) higher than 100 kDa are not degraded by human fecal microbiota, but low Mw CGNs with an Mw around ~4.5 kDa (KCOs) could be degraded by seven of eight human fecal microbiota samples. KCO degrading B. xylanisolvens was isolated from fecal samples, and PCR-DGGE profiling with band sequencing suggested that B. xylanisolvens was the key KCO degrader in the human gut. Two putative κ-carrageenase genes were identified in the genome sequence of B. xylanisolvens. However, their function on KCO degrading was not verified in vitro. And the sulfate group from KCO is not removed after in vitro degradation by human fecal microbiota, as shown by ESI-MS analysis. The effects of KCO and KCO degrading bacteria on the inflammatory response were investigated in germ-free mice. Increased numbers of P-P38-, CD3a-, and CD79a-positive cells were found in the colon and rectum in mice fed with KCO plus KCO degrading bacteria than in mice fed with only KCO or only B. xylanisolvens and E. coli, as shown by RNA-Seq analysis, HE staining, and IHC. Conclusion: Our data suggested that the presence of KCO degrading bacteria promote the pro-inflammatory effects of CGNs.

Recent literature implicates gut epithelia mucosa and intestinal microbiota as important players in post-stroke morbidity and mortality. As most studies have focused on the acute effects of stroke on gut dysbiosis, our study objective was to measure chronic, longitudinal changes in the gut microbiota and intestinal pathology following ischemic stroke. We hypothesized that mice with experimental ischemic stroke would exhibit chronic gut dysbiosis and intestinal pathology up to 36 days post-stroke compared to sham controls. Male C57BL/6J mice were subjected to 60 minutes of transient middle cerebral artery occlusion (tMCAO) or sham surgery. To determine the long-term effects of tMCAO on gut dysbiosis, fecal boli were collected pre- and post-tMCAO on days 0, 3, 14, and 28. Bioinformatics analysis demonstrate significant differences in abundance among Firmicutes and Bacteroidetes taxa at the phylum, family, and species levels in tMCAO compared to sham mice that persisted up to one month post-stroke. The most persistent changes in post-stroke microbial abundance were a decrease in bacteria family S24-7 and significant increases inRuminococcaceae. Overall, these changes resulted in a persistently increased Firmicutes:Bacteroidetes ratio in stroke animals. Intestinal histopathology showed evidence of chronic intestinal inflammation that included marked increases in immune cell infiltration with mild-moderate epithelial hyperplasia and villous blunting. Increased astrocyte and microglial activity were also detected one-month post-stroke. These results demonstrate that acute, post-stroke disruption of the gut-brain-microbiota axis progresses to chronic gut dysbiosis, intestinal inflammation, and chronic neuroinflammation.Clinical PerspectivesThe microbiota-gut-brain axis, recently implicated in several neurological disorders, remains largely unexplored at chronic time points post-tMCAO.Our results demonstrate chronic gut dysbiosis, prolonged behavioral deficits, and persistent cerebral and intestinal inflammation post-tMCAO in male C57BL/6J mice.These results suggest that manipulation of microbiota may help reduce poor outcomes after stroke and lead to improved post-stroke functional recovery.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor. Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype. TNBC expresses AHR and AHR ligands have anti-cancer activity in TNBC. The aggressiveness of TNBC is due in part to JAG1-NOTCH1 signaling. ITE is a putative endogenous AHR ligand. We show that ITE reduces the expression of JAG1 the amount of Notch 1 intracellular domain (NICD1) and the phosphorylation of STAT3 (at tyrosine 705) in TNBC MDA-MB-231 cells. The STAT3 inhibitor STATTIC also reduced JAG1. STAT3, thus, mediates regulation of JAG1 in MDA-MB-231 cells. Reducing the expression of JAG1 with short interfering RNA decreases the growth, migration and invasiveness of MDA-MB-231 cells. JAG1, therefore, has cellular effects in MDA-MB-231 cells under basal conditions. We consequently evaluated if exposing cells to greater amounts of JAG1 would counteract ITE cellular effects in MDA-MB-231 cells. The results show that JAG1 does not counteract the cellular effects of ITE. JAG1, thus, has no effect on growth or invasiveness in MDA-MB-231 cells treated with ITE. JAG1, therefore, has context dependent roles in MDA-MB-231 cells (basal versus ITE treatment). The results also show that other pathways, not inhibition of the JAG1-NOTCH1 pathway, are important for mediating the growth and invasive inhibitory effect of ITE on MDA-MB-231 cells.

Th17 cells contribute to host defense on mucosal surfaces but also provoke autoimmune diseases when directed against self-antigens. Identifying therapeutic targets that regulate Th17 cell differentiation and/or cytokine production has considerable value. Here, we study the aryl hydrocarbon receptor (AhR)-dependent transcriptome in human CD4 T cells treated with Th17-inducing cytokines. We show that the AhR reciprocally regulates IL-17 and IL-22 production in human CD4 T cells. Global gene expression analysis revealed that AhR ligation decreased IL21 expression, correlating with delayed upregulation of RORC during culture with Th17-inducing cytokines. Several of the AhR-dependent genes have known roles in cellular assembly, organization, development, growth and proliferation. We further show that expression of GPR15, GPR55 and GPR68 positively correlates with IL-22 production in the presence of the AhR agonist FICZ. Activation of GPR68 with the lorazepam derivative ogerin resulted in suppression of IL-22 and IL-10 secretion by T cells, with no effect on IL-17. Under neutral Th0 conditions, ogerin and the Gq/11 receptor inhibitor YM254890 blunted IL-22 induction by FICZ. These data reveal the AhR-dependent transcriptome in human CD4 T cells and suggest the mechanism through which the AhR regulates T cell function may be partially dependent on Gq-coupled receptors including GPR68.

Cytochrome P450 (CYP) enzyme 2B6 plays a significant role in the stereo-selective metabolism of (S)-methadone to 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolidine, an inactive methadone metabolite. Elevated (S)-methadone can cause cardiotoxicity by prolonging the QT interval of the heart's electrical cycle. Large inter-individual variability of methadone pharmacokinetics causes discordance in the relationship between dose, plasma concentrations and side effects. The purpose of this study was to determine if one or more single nucleotide polymorphisms (SNPs) located within the CYP2B6 gene contributes to a poor metabolizer phenotype for methadone in these fatal cases. The genetic analysis was conducted on 125 Caucasian methadone-only fatalities obtained from the West Virginia and Kentucky Offices of the Chief Medical Examiner. The frequency of eight exonic and intronic SNPs (rs2279344, rs3211371, rs3745274, rs4803419, rs8192709, rs8192719, rs12721655 and rs35979566) was determined. The frequencies of SNPs rs3745274 (*9, c516G > T, Q172H), and rs8192719 (21563 C > T) were enhanced in the methadone-only group. Higher blood methadone concentrations were observed in individuals who were genotyped homozygous for SNP rs3211371 (*5, c1459C > T, R487C). These results indicate that these three CYP2B6 SNPs are associated with methadone fatalities.

Mouse models of antibiotic-induced ablation of the intestinal microbiome have been used to study the microbiome in health and disease. The fecal microbiomes of mice treated with broad-spectrum antibiotics while being fed different laboratory chows were analyzed by Gram stain, quantitative flow cytometry, bacterial cell culture, next generation sequencing of the V3 regions of the 16S ribosomal RNA (rRNA) gene, microscopy, and sequence analysis of the tuf gene. Noncultivatable gram-positive cocci and cultivatable yeast were the microorganisms most readily detected in feces of antibiotic-treated mice fed a defined diet that utilizes casein as a protein source, maltodextrin 10 and sucrose as sources of carbohydrates, and lard as the major source of fat. High-throughput sequencing of the variable regions of the 16S rRNA gene and tuf gene sequencing identified the major bacterial phylotype as Lactococcus. The mouse chow was heavily laden with noncultivatable Lactococcus, which dominated the intestinal flora after consumption. The microbiome of antibiotic-treated mice fed a grain-based diet (mainly wheat, corn and alfalfa) consisted predominantly of a member of the Enterobacteriaceae identified as Escherichia coli, and yeast was not detected by culture or Gram stain. Appearance of intestinal yeast by culture and Gram stain was dependent on the specific chow, although yeast was not detected by culture or Gram stain in the chow. We conclude that bacteria found in food sources can influence qualitative and quantitative assessments of the fecal microbiome, at least in the context of antibiotic therapy, and potentially confound molecular studies that assess the effects of diet on the intestinal ecology. Not surprisingly, different food sources can influence the microbiome, particularly in the context of antibiotic-mediated ablation of the intestinal microbiome. Whether and how the food-derived dead bacteria alter intestinal physiology needs to be determined.

This article contains supporting data for the research paper entitled: ‘Dietary walnut altered gene expressions related to tumor growth, survival, and metastasis in breast cancer patients: a pilot clinical trial’ [1] Hardman et al., 2019. Included are tables for all mapped genes and all unmapped loci identifications that were significantly changed in breast cancers by consumption of walnut for about 2 weeks. All gene networks that were identified by Ingenuity Pathway Analyses as modified are shown in table 3. Files containing the raw reads, along with a shell script describing the complete data analysis pipeline, were deposited to the Gene Expression Omnibus (GEO) at the National Center for Biotechnology Information (NCBI) and can be obtained via accession number GSE111073. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE111073. Keywords: Breast cancer, Walnut consumption, mRNA expression

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that is regulated by environmental toxicants that function as AHR agonists such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). L-Type Amino Acid Transporter 1 (LAT1) is a leucine transporter that is overexpressed in cancer. The regulation of LAT1 by AHR in MCF-7 and MDA-MB-231 breast cancer cells (BCCs) was investigated in this report. Ingenuity pathway analysis (IPA) revealed a significant association between TCDD-regulated genes (TRGs) and molecular transport. Overlapping the TCDD-RNA-Seq dataset obtained in this study with a published TCDD-ChIP-seq dataset identified LAT1 as a primary target of AHR-dependent TCDD induction. Short interfering RNA (siRNA)-directed knockdown of AHR confirmed that TCDD-stimulated increases in LAT1 mRNA and protein required AHR expression. TCDD-stimulated increases in LAT1 mRNA were also inhibited by the AHR antagonist CH-223191. Upregulation of LAT1 by TCDD coincided with increases in leucine uptake by MCF-7 cells in response to TCDD. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assays revealed increases in AHR, AHR nuclear translocator (ARNT) and p300 binding and histone H3 acetylation at an AHR binding site in the LAT1 gene in response to TCDD. In MCF-7 and MDA-MB-231 cells, endogenous levels of LAT1 mRNA and protein were reduced in response to knockdown of AHR expression. Knockdown experiments demonstrated that proliferation of MCF-7 and MDA-MB-231 cells is dependent on both LAT1 and AHR. Collectively, these findings confirm the dependence of cancer cells on leucine uptake and establish a mechanism for extrinsic and intrinsic regulation of LAT1 by AHR.

Background: Early onset dementias have variable clinical presentations and are often difficult to diagnose. We established a family pedigree that demonstrated consistent recurrence of very early onset dementia in successive generations. Objective and Method: In order to refine the diagnosis in this family, we sequenced the exomes of two affected family members and relied on discrete filtering to identify disease genes and the corresponding causal variants. Results: Among the 720 nonsynonymous single nucleotide polymorphisms (SNPs) shared by two affected members, we found a C to T transition that gives rise to a Thr147Ile missense substitution in the presenilin 1 (PS1) protein. The presence of this same mutation in a French early-onset Alzheimer’s disease family, other affected members of the family, and the predicted high pathogenicity of the substitution strongly suggest that it is the causal variant. In addition to exceptionally young age of onset, we also observed significant limb spasticity and early loss of speech, concurrent with progression of dementia in affected family members. These findings extend the clinical presentation associated with the Thr147Ile variant. Lastly, one member with the Thr147Ile variant was treated with the PKC epsilon activator, bryostatin, in a compassionate use trial after successful FDA review. Initial improvements with this treatment were unexpectedly clear, including return of some speech, increased attentional focus, ability to swallow, and some apparent decrease in limb spasticity. Conclusions: Our findings confirm the role of the PS1 Thr147Ile substitution in Alzheimer’s disease and expand the clinical phenotype to include expressive aphasia and very early onset of dementia.

Summary The vertebrate extinction rate over the past century is approximately 22–100 times greater than background extinction rates [1], and large mammals are particularly at risk [2, 3]. Quaternary megafaunal extinctions have been attributed to climate change [4], overexploitation [5], or a combination of the two [6]. Rhinoceroses (Family: Rhinocerotidae) have a rich fossil history replete with iconic examples of climate-induced extinctions [7], but current pressures threaten to eliminate this group entirely. The Sumatran rhinoceros ( Dicerorhinus sumatrensis ) is among the most imperiled mammals on earth. The 2011 population was estimated at ≤216 wild individuals [8], and currently the species is extirpated, or nearly so, throughout the majority of its former range [8–12]. Understanding demographic history is important in placing current population status into a broader ecological and evolutionary context. Analysis of the Sumatran rhinoceros genome reveals extreme changes in effective population size throughout the Pleistocene. Population expansion during the early to middle Pleistocene was followed by decline. Ecological niche modeling indicated that changing climate most likely played a role in the decline of the Sumatran rhinoceros, as less suitable habitat on an emergent Sundaland corridor isolated Sumatran rhinoceros populations. By the end of the Pleistocene, the Sundaland corridor was submerged, and populations were fragmented and consequently reduced to low Holocene levels from which they would never recover. Past events denuded the Sumatran rhinoceros of genetic diversity through population decline, fragmentation, or some combination of the two and most likely made the species even more susceptible to later exploitation and habitat loss. Video Abstract

Francisella tularensis is an extremely virulent bacterium that can be transmitted naturally by blood sucking arthropods. During mammalian infection, F. tularensis infects numerous types of host cells, including erythrocytes. As erythrocytes do not undergo phagocytosis or endocytosis, it remains unknown how F. tularensis invades these cells. Furthermore, the consequence of inhabiting the intracellular space of red blood cells (RBCs) has not been determined. Here, we provide evidence indicating that residing within an erythrocyte enhances the ability of F. tularensis to colonize ticks following a blood meal. Erythrocyte residence protected F. tularensis from a low pH environment similar to that of gut cells of a feeding tick. Mechanistic studies revealed that the F. tularensis type VI secretion system (T6SS) was required for erythrocyte invasion as mutation of mglA (a transcriptional regulator of T6SS genes), dotU, or iglC (two genes encoding T6SS machinery) severely diminished bacterial entry into RBCs. Invasion was also inhibited upon treatment of erythrocytes with venom from the Blue-bellied black snake (Pseudechis guttatus), which aggregates spectrin in the cytoskeleton, but not inhibitors of actin polymerization and depolymerization. These data suggest that erythrocyte invasion by F. tularensis is dependent on spectrin utilization which is likely mediated by effectors delivered through the T6SS. Our results begin to elucidate the mechanism of a unique biological process facilitated by F. tularensis to invade erythrocytes, allowing for enhanced colonization of ticks.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that upon activation by the toxicant 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) stimulates gene expression and toxicity. AHR is also important for normal mouse physiology and may play a role in cancer progression in the absence of environmental toxicants. The objective of this report was to identify AHR-dependent genes (ADGs) whose expression is regulated by AHR in the absence of toxicants. RNA-Seq analysis revealed that AHR regulated the expression of over 600 genes at an FDR

Background The TALLYHO/Jng (TH) mouse is a polygenic model for obesity and type 2 diabetes first described in the literature in 2001. The origin of the TH strain is an outbred colony of the Theiler Original strain and mice derived from this source were selectively bred for male hyperglycemia establishing an inbred strain at The Jackson Laboratory. TH mice manifest many of the disease phenotypes observed in human obesity and type 2 diabetes. Results We sequenced the whole genome of TH mice maintained at Marshall University to a depth of approximately 64.8X coverage using data from three next generation sequencing runs. Genome-wide, we found approximately 4.31 million homozygous single nucleotide polymorphisms (SNPs) and 1.10 million homozygous small insertions and deletions (indels) of which 98,899 SNPs and 163,720 indels were unique to the TH strain compared to 28 previously sequenced inbred mouse strains. In order to identify potentially clinically-relevant genes, we intersected our list of SNP and indel variants with human orthologous genes in which variants were associated in GWAS studies with obesity, diabetes, and metabolic syndrome, and with genes previously shown to confer a monogenic obesity phenotype in humans, and found several candidate variants that could be functionally tested using TH mice. Further, we filtered our list of variants to those occurring in an obesity quantitative trait locus, tabw2, identified in TH mice and found a missense polymorphism in the Cidec gene and characterized this variant’s effect on protein function. Conclusions We generated a complete catalog of variants in TH mice using the data from whole genome sequencing. Our findings will facilitate the identification of causal variants that underlie metabolic diseases in TH mice and will enable identification of candidate susceptibility genes for complex human obesity and type 2 diabetes. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3245-6) contains supplementary material, which is available to authorized users.

Targeting the nuclear factor kappa B (NFκB) pathway is proposed as therapy for chronic lymphocytic leukemia (CLL). We hypothesized that an omega-3 fatty acids (n-3) supplement would suppress NFκB activation in lymphocytes of Rai Stage 0-1 CLL patients. The initial dose of 2.4 g n-3/day was gradually increased to 7.2 g n-3/day. After n-3 consumption: 1) plasma n-3 increased; 2) NFκB activation was suppressed in lymphocytes; 3) in vitro sensitivity of lymphocytes to doxorubicin was increased; and 4) expression of 32 genes in lymphocytes was significantly decreased.

Th17 cells contribute to host defense on mucosal surfaces but also provoke autoimmune diseases when directed against self-antigens. Identifying therapeutic targets that regulate Th17 cell differentiation and/or cytokine production has considerable value. Here, we study the aryl hydrocarbon receptor (AhR)-dependent transcriptome in human CD4 T cells treated with Th17-inducing cytokines. We show that the AhR reciprocally regulates IL-17 and IL-22 production in human CD4 T cells. Global gene expression analysis revealed that AhR ligation decreased IL21 expression, correlating with delayed upregulation of RORC during culture with Th17-inducing cytokines. We further show that expression of GPR15, GPR55 and GPR68 positively correlates with IL-22 production in the presence of the AhR agonist FICZ. Activation of GPR68 with the lorazepam-derivative ogerin resulted in suppression of IL-22 and IL-10 secretion by T cells, with no effect on IL-17. These data reveal a novel feedback inhibitory pathway on IL-22 and IL-10 production in the presence of AhR agonists.

More than 40 years after the discovery of vasoactive intestinal peptide (VIP), its transcriptome in the immune system has still not been completely elucidated. In an attempt to understand the biological role of this neuropeptide in immunity, we chose CD4 T cells as a cellular system. Agilent Mouse Whole Genome microarrays were hybridized with fluorescently labeled total RNA isolated from resting CD4 T cells cultured −/+ 10−7 M VIP for five hours or PMA/ionomycin activated CD4 T cells cultured −/+ 10−7 M VIP for five hours . These VIP-regulated transcriptomes were analyzed by Significance Analysis of Microarrays (SAM) and Ingenuity Pathway Analysis (IPA) software to identify relevant signaling pathways modulated by VIP in the absence and presence of T cell activation. In resting CD4 T cells, VIP modulated 368 genes, ranging from 3.49 to − 4.78 fold. In the PMA/ionomycin activated CD4 T cells, 326 gene expression levels were changed by VIP, ranging from 2.94 to −1.66 fold. IPA analysis revealed that VIP exposure alters cellular function through EGFR signaling in resting CD4 T cells, and modulates immediate early genes, Fos and CREM/ICER, in activated CD4 T cells. These gene expression changes are suggested to explain at a molecular level how VIP can regulate T cell homing to the gut and induce regulatory T cell generation.

Pituitary homeobox 2 (PITX2) is a homeodomain transcription factor that has a substantial role in cell proliferation and differentiation in various tissues. In this report, we have conducted a systematic study, using proteomic and genomic approaches, to characterize PITX2-interacting proteins and PITX2-regulating genes. We identified four novel PITX2-associated protein partners Y box binding factor-1, heterogeneous ribonucleoprotein K, nucleolin and heterogeneous nuclear ribonucleoprotein U in mass spectrometry analysis. We also found that overexpression of PITX2 upregulated 868 genes (2–25-fold) and downregulated 191 genes (2–15-fold) in DNA microarray analysis. These data provide an insightful perspective for further studying PITX2 function and mechanism of action.Structured summaryMINT-6823857: PITX2 (uniprotkb: Q99697) physically interacts (MI:0218) with hnRNP K (uniprotkb:P61978), YB-1 (uniprotkb:P67809), Nucleolin (uniprotkb:P19338) and hnRNP U (uniprotkb:Q00839) by anti tag coimmunoprecipitation (MI:0007)MINT-6823877: PITX2 (uniprotkb:Q99697) physically interacts (MI:0218) with beta catenin (uniprotkb:P35222), hnRNP U (uniprotkb:Q00839), Nucleolin (uniprotkb:P19338), YB-1 (uniprotkb:P67809) and hnRNP K (uniprotkb:P61978) by anti tag coimmunoprecipitation (MI:0007)MINT-6823902: YB-1 (uniprotkb:P67809) physically interacts (MI:0218) with PITX2 (uniprotkb: Q99697) by anti bait coimmunoprecipitation (MI:0006)

DNA damage blocks DNA polymerase progression and increases miscoding. In this study, we assessed the effects of specific lesions on Taq DNA polymerase fidelity and amplification efficiency. In the presence of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), Taq DNA polymerase inserted dCMP and to a lesser extent dAMP. 8-Oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodA) instructed the incorporation of dTMP and caused a pronounced n-1 deletion not observed in other systems. The presence of an abasic lesion led to dAMP incorporation and n-1 deletions. In addition, we introduce the mean modified efficiency (MME) as a more precise method for determining PCR amplification efficiency of damaged templates. Using this method, we were able to quantify reductions in amplification efficiency of templates containing 8-oxodG (single or multiple), 8-oxodA, or abasic sites. Because the MME method can detect small reductions in amplification efficiency, it may be useful in comparing the extent of damage in environmentally degraded or archival DNA specimens.

Methadone is difficult to administer as a therapeutic agent because of a wide range of interindividual pharmacokinetics, likely due to genetic variability of the CYP450 enzymes responsible for metabolism to its principal metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). CYP3A4 is one of the primary CYP450 isoforms responsible for the metabolism of methadone to EDDP in humans. The purpose of this study was to evaluate the role of CYP3A4 genetic polymorphisms in accidental methadone fatalities. A study cohort consisting of 136 methadone-only and 92 combined methadone/benzodiazepine fatalities was selected from cases investigated at the West Virginia and Kentucky Offices of the Chief Medical Examiner. Seven single nucleotide polymorphisms (SNPs) were genotyped within the CYP3A4 gene. Observed allelic and genotypic frequencies were compared with expected frequencies obtained from The National Center for Biotechnology Information dbSNP database. SNPs rs2242480 and rs2740574 demonstrated an apparent enrichment within the methadone-only overdose fatalities compared with the control group and the general population. This enrichment was not apparent in the methadone/benzodiazepine cases for these two SNPs. Our findings indicate that there may be two or more SNPs on the CYP3A4 gene that cause or contribute to the methadone poor metabolizer phenotype.

Excess weight is a known risk factor for coronary artery disease (CAD) and a large percentage of overweight and obese individuals ultimately develop CAD. The objective of this study was to identify human genes associated with CAD in a subgroup of overweight and obese individuals using population-based association methods. Logistic regression analyses were used to test the association between single nucleotide polymorphisms (SNPs) in 34 candidate genes and the CAD phenotype with age, gender, and BMI as covariates. Two SNPs in the Apolipoprotein B (Apo B) gene [rs1042031 and rs1800479], one in the Cholesterol Ester Transfer Protein (CETP) gene [rs5880], and one in the Low Density Lipoprotein Receptor (LDLR) gene [rs2569538] met the 0.01 significance level for association with CAD. Based on these findings, we conclude that variants within the CETP and Apo B genes conferred susceptibility to CAD in overweight individuals and that a variant with the LDLR gene conferred susceptibility in an obese group.

To define the functions of retinoids and their receptors in insulin secretion, we tested the effects of all-trans-retinoic acid (ATRA) and retinoic acid receptor (RAR) expression on cell growth, differentiation, and secretion using insulin-secreting RINm5F cells. Wild-type cells with a low abundance of mRNA for RAR beta were transfected with RAR beta or chloramphenicol acetyltransferase (CAT control). Cells were cultured for 2-7 days in media without (A-def) or with ATRA, 1, 10, 100, and 1,000 nM. At day 2 of culture, ATRA stimulated insulin release in wild-type and transfected cells, and this effect was dose dependent. At 7 days, ATRA stimulated insulin secretion from wild-type cells twofold at glucose concentrations of 0.5 mM (A-def, 5.1 +/- 0.27; ATRA, 1,000 nM, 10.5 +/- 1.43 ng/10(6) cells) and at 11.0 mM (A-def, 6.9 +/- 0.24; ATRA, 1,000 nM, 13.6 +/- 1.86 ng/10(6) cells). The cellular insulin content was increased about threefold (A-def, 39.2 +/- 2.95; ATRA, 1,000 nM, 118 +/- 8.54 ng/10(6) cells). ATRA inhibited growth of wild-type cells as early as 3 days, and this effect was dose dependent. Whereas in the absence of ATRA, the cell number increased over fivefold between day 3 and day 5, ATRA, 1,000 nM, inhibited cell growth completely. ATRA, 1,000 nM, increased apoptotic RINm5F cells (day 3 A-def, 0.53 +/- 0.27% of total cells, and ATRA, 2.30 +/- 1.44; day 5 A-def, 0.38 +/- 0.23, and ATRA, 2.14 +/- 0.59; day 7 A-def, 0.90 +/- 0.29, and ATRA, 6.02 +/- 1.64). RAR beta-transfected cells showed overexpression of mRNA to RAR beta and dose-dependent inhibition of growth, with almost-complete inhibition at ATRA concentrations as low as 100 nM. Overexpression of RAR beta increased insulin secretion at ATRA, 100-1,000 nM. In summary, ATRA increased the insulin secretion and content of RINm5F cells, while inhibiting growth and increasing apoptosis. Increased expression of RAR beta facilitated these effects on growth and secretion. These findings may reflect the known effect of ATRA on differentiation of cells and mediation through RAR beta.

Retinoid-X receptors (RXRs) are 9-cis-retinoic acid (9CRA)-dependent gene transcription factors, which modulate the action of all-trans-retinoic acid (ATRA), fatty acids, thyroid hormone (TH), and vitamin D (VD) by forming dimers with themselves or ATRA, TH, peroxisome proliferator activator receptors (PPARs), or VD receptors (VDRs). To determine if 9CRA and RXRs have a role in secretion, RINm5F cells were assayed for RXR transcripts and effects of 9CRA and ATRA on secretion. A single RXR alpha transcript and two RXR beta transcripts, but not RXR gamma, were evident by Northern blot. Cells were cultured for 48 hours without and with 9CRA 1 to 1,000 nmol/L and then stimulated with glucose 0, 0.5, 2.8, 7, and 11 mmol/L 9CRA increased secretion at each glucose concentration, 9CRA increased secretion by 50% to 100% (ANOVA, P.001) with consistent concentration-dependent responses (eg. at glucose 2.8 mmol/L 9CRA: 0 nmol/L, 5.02 +/- .20 ng/(10(6) cells.h); 1 nmol/L, 6.97 +/- .30; 10 nmol/L, 8.36 +/- .18; 100 nmol/L, 9.15 +/- .28; 1,000 nmol/L, 10.24 +/- .24; n = 6). Although RINm5F cells respond slightly if at all to glucose, 9CRA facilitated glucose-induced insulin release (eg, at 9CRA 100 nmol/L, glucose: 0.5 mmol/L, 7.47 +/- .22 ng/(10(6) cells.h); 2.8 mmol/L, 9.15 +/- .27; 7 mmol/L, 9.81 +/- .19; 11 mmol/L, 11.16 +/- .23; n = 6). ATRA increased secretion by 28% to 57% (ANOVA, P.001: at glucose 2.8 mmol/L, ATRA: 0 nmol/L, 6.17 +/- .32 ng/(10(6) cells.h); 1 nmol/L, 7.91 +/- .29; 10 nmol/L, 9.75 +/- .14; 100 nmol/L, 9.66 +/- .33; n = 6). 9CRA was more potent than ATRA (eg, at 2.8 mmol/L; baseline, 8.17 +/- .32 ng/(10(8) cells.h); ATRA 100 nmol/L, 9.66 +/- .33; 9CRA 100 nmol/L, 10.81 +/- .15; P.05, n = 6). When 9CRA was combined with ATRA, the combination was not additive or synergistic (eg, at 2.8 mmol/L: ATRA 100 nmol/L, 9.66 +/- .33 ng/(10(6) cells.h); 9CRA 100 nmol/L, 10.81 +/- .15; ATRA 100 nmol/L + 9CRA 100 nmol/L, 10.79 +/- .28; P.05, n = 6). These studies show that (1) 9CRA stimulates insulin secretion from RINm5F cells. This effect appears to be at least equal to if not greater than that observed with ATRA, but additive or synergistic effects with ATRA were not evident; (2) 9CRA may facilitate glucose-induced release; and (3) multiple RXR transcripts are present in insulin-secreting cells, implying specific functions. Our findings support the idea that the effects of 9CRA on insulin secretion are mediated through RXR homodimers or heterodimers with retinoic acid receptors (RARs) or possibly other nuclear receptors. Retinoid deficiency or alterations in retinoid receptor function could lead to abnormalities of cell growth or secretion.

Hematopoietic reconstitution, following bone marrow or stem cell transplantation, requires a microenvironment niche capable of supporting both immature progenitors and stem cells with the capacity to differentiate and expand. Osteoblasts comprise one important component of this niche. We determined that treatment of human primary osteoblasts (HOB) with melphalan or VP-16 resulted in increased phospho-Smad2, consistent with increased TGF-β1 activity. This increase was coincident with reduced HOB capacity to support immature B lineage cell chemotaxis and adherence. The supportive deficit was not limited to committed progenitor cells, as human embryonic stem cells (hESC) or human CD34+ bone marrow cells co-cultured with HOB pre-exposed to melphalan, VP-16 or rTGF-β1 had profiles distinct from the same populations co-cultured with untreated HOB. Functional support deficits were downstream of changes in HOB gene expression profiles following chemotherapy exposure. Melphalan and VP-16 induced damage of HOB suggests vulnerability of this critical niche to therapeutic agents frequently utilized in pre-transplant regimens and suggests that dose escalated chemotherapy may contribute to post-transplantation hematopoietic deficits by damaging structural components of this supportive niche.

To define the mechanism of vitamin A action at the β-cell level, we tested for the presence of messenger RNA for retinoic acid receptors α, β, and γ; cytosolic retinol-binding protein; and cytosolic retinoic acid-binding protein in RINm5F cells, an insulin-secreting cell line, and determined whether cytosolic retinol-binding protein and cytosolic retinoic acid-binding protein are present in isolated purified normal rat β-cells. Northern blot analyses showed two transcripts of retinoic acid receptor α messenger RNA (3.8 and 2.4 kb), one transcript of retinoic acid receptor messenger RNA (3.8 kb), and one transcript of cytosolic retinol-binding protein (0.9 kb) in RINm5F cells. Ribonuclease protection assays also showed the presence of cytosolic retinol-binding protein and cytosolic retinoic acid-binding protein in RINm5F cells. Quantitatively, cytosolic retinol-binding protein levels were 0.10 ± 0.02 pg/micrograms total RNA. Using specific radioimmunoassays, normal isolated purified rat β-cells contained CRBP (19.2 ± 2.38) and cytosolic retinoic acid-binding protein (16 ± 0.53 ng/10(6) cells). The presence of message for retinoic acid receptors α and γ, cytosolic retinol-binding protein, cytosolic retinoic acid-binding protein, and the gene products of cytosolic retinol-binding protein and cytosolic retinoic acid-binding protein in insulin-secreting cells support a mechanism of vitamin A action and role for cytosolic and nuclear receptors at the β-cell level similar to that suggested in nonendocrine cells. The presence of nuclear retinoic acid receptor a. and 7 suggests that vitamin A may affect insulin secretion through gene expression in the β-cell.

Cancer stem cells (CSCs) are considered a subset of the bulk tumor responsible for initiating and maintaining the disease. Several surface cellular markers have been recently used to identify CSCs. Among those is CD133, which is expressed by hematopoietic progenitor cells as well as embryonic stem cells and various cancers. We have recently isolated and cultured CD133 positive [CD133(+)] cells from various cancer cell lines using a NASA developed Hydrodynamic Focusing Bioreactor (HFB) (Celdyne, Houston, TX). For comparison, another bioreactor, the rotary cell culture system (RCCS) manufactured by Synthecon (Houston, TX) was used. Both the HFB and the RCCS bioreactors simulate aspects of hypogravity. In our study, the HFB increased CD133(+) cell growth from various cell lines compared to the RCCS vessel and to normal gravity control. We observed a (+)15-fold proliferation of the CD133(+) cellular fraction with cancer cells that were cultured for 7-days at optimized conditions. The RCCS vessel instead yielded a (−)4.8-fold decrease in the CD133(+)cellular fraction respect to the HFB after 7-days of culture. Interestingly, we also found that the hypogravity environment of the HFB greatly sensitized the CD133(+) cancer cells, which are normally resistant to chemo treatment, to become susceptible to various chemotherapeutic agents, paving the way to less toxic and more effective chemotherapeutic treatment in patients. To be able to test the efficacy of cytotoxic agents in vitro prior to their use in clinical setting on cancer cells as well as on cancer stem cells may pave the way to more effective chemotherapeutic strategies in patients. This could be an important advancement in the therapeutic options of oncologic patients, allowing for more targeted and personalized chemotherapy regimens as well as for higher response rates.

Human aortic endothelial cells (HAEC) exposed to 50 microg/ml oxidized L-A-phosphatidylcholine B-arachidonoyl-gamma-palmitoyl (ox-PAPC) for 6h increased in interleukin-8 mRNA and protein levels. Preincubation of HAEC with the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) inhibitor, (20 microM), significantly inhibited ox-PAPC-stimulated interleukin-8 mRNA and protein levels. Mevalonate (200 microM) reversed the inhibition of ox-PAPC-stimulated mRNA and protein levels by lovastatin, indicating the inhibitory effect of lovastatin was due to inhibition of mevalonate synthesis. Addition of the geranylgeraniol (GGOL, 10 microM) but not farnesol (FOL, 10 microM), reversed the inhibitory effect of lovastatin on interleukin-8 mRNA and protein levels stimulated by ox-PAPC, indicating that lovastatin exerted its effect by inhibiting stores of geranylgeranyl pyrophosphate (GGPP) which are necessary for geranylgeranylation of proteins. These results suggest a new mechanism for lovastatin in preventing atherosclerosis by inhibiting the inflammatory response that takes place in the vascular wall.

Polymerase stop assays used to quantify DNA damage assume that single lesions are sufficient to block polymerase progression. To test the effect of specific lesions on PCR amplification efficiency, we amplified synthetic 90 base oligonucleotides containing normal or modified DNA bases using real-time PCR and determined the relative threshold cycle amplification efficiency of each template. We found that while the amplification efficiencies of templates containing a single 8-oxo-7,8-dihydro-2 0 -deoxyguanosine (8-oxodG) were not significantly perturbed, the presence of a single 8-oxo-7,8-dihydro-2 0 -deoxyadenosine, abasic site, or a cis–syn thymidine dimer dramatically reduced amplification efficiency. In addition, while templates containing two 8-oxodGs separated by 13 bases amplified as well as the unmodified template, the presence of two tandem 8-oxodGs substantially hindered amplification. From these findings, we conclude that the reduction in polymerase progression is dependent on the type of damage and the relative position of lesions within the template. � 2004 Elsevier Inc. All rights reserved.

Hereditary pancreatitis (HP) is the second most common cause of chronic childhood pancreatitis in the United States. Mutations in the cationic trypsinogen gene on chromosome 7 are known to cause HP. We identified four families in West Virginia with symptoms consistent with HP. To determine whether members of these families had defects in the trypsinogen gene, we tested for linkage between the HP gene and simple tandem repeat markers on chromosome 7q and screened for a specific mutation in the cationic trypsinogen gene. Two-point linkage analysis indicated that the disease gene is closely linked to three 7q markers (D7S661, D7S2511, and D7S1805). Restriction fragment length polymorphism analysis showed that all clinically affected members and nonpenetrant carriers from the four families carried a G to A mutation in the third exon of the trypsinogen gene. These findings indicate that this mutation is the cause of HP in the families in our study. The observation that most individuals who carry the mutation have symptoms of HP is consistent with the high but incomplete penetrance of the trait. The presence of a single mutation and a common linked haplotype indicates that the defective allele arose in an ancestor common to all four families.

Using intact rat islets, hamster In-R1-G9 cells, and mouse alphaTC-1 clone 9 transgenic tumoral glucagon-secreting cells, we determined the effects of retinol (ROH) and retinoic acid (RA) on glucagon secretion. Since vitamin A effects may be mediated through nuclear RA receptors (RARs) and cytoplasmic ROH- and RA-binding proteins (CRBP and CRABP), cells were also assayed for RARs, CRBP, and CRABP mRNA by Northern blot analyses. Islets and cells were cultured in 2.8 mmol/L glucose and vitamin A-deficient (A-def) medium or in different concentrations of ROH and RA. Using intact islets, RA 10 and 100 nmol/L inhibited glucagon secretion to approximately 60% of control levels. Using In-R1-G9 cells, ROH 0.175 to 5.0 micromol/L inhibited glucagon secretion to 60% to 83% of control levels, and RA 100 and 1,000 nmol/L inhibited glucagon secretion from 72% to 43% of control levels, respectively. Using alphaTC-1 cells, ROH 1.75 micromol/L inhibited glucagon secretion to 80% of control levels, and RA 1 to 100 nmol/L inhibited secretion from 83% to 68% of control levels. Inhibition of secretion was dose-dependent. RARalpha RNA transcripts were detected in alpha TC-1 and In-R1-G9 total RNA extracts; RAR gamma transcripts were detected in alphaTC-1 cells. We conclude the following: (1) ROH and RA inhibit glucagon secretion in cultured rat islets and glucagon-secreting cell lines, and in cell lines the effect of RA is dose-dependent; (2) on a molar basis, RA is on the order of 10- to 100-fold more potent than ROH, a finding consistent with RA being the active metabolite of ROH at the alpha-cell level; and (3) this inhibition may be mediated through classic pathways of retinoid action involving nuclear RARs and gene expression of specific proteins.

Background To investigate the potential association between oral health and cognitive function, a pilot study was conducted to evaluate high throughput DNA sequencing of the V3 region of the 16S ribosomal RNA gene for determining the relative abundance of bacterial taxa in subgingival plaque from older adults with or without dementia. Methods Subgingival plaque samples were obtained from ten individuals at least 70 years old who participated in a study to assess oral health and cognitive function. DNA was isolated from the samples and a gene segment from the V3 portion of the 16S bacterial ribosomal RNA gene was amplified and sequenced using an Illumina HiSeq1000 DNA sequencer. Bacterial populations found in the subgingival plaque were identified and assessed with respect to the cognitive status and oral health of the participants who provided the samples. Results More than two million high quality DNA sequences were obtained from each sample. Individuals differed greatly in the mix of phylotypes, but different sites from different subgingival depths in the same subject were usually similar. No consistent differences were observed in this small sample between subjects separated by levels of oral health, sex, or age; however a consistently higher level of Fusobacteriaceae and a generally lower level of Prevotellaceae was seen in subjects without dementia, although the difference did not reach statistical significance, possibly because of the small sample size. Conclusions The results from this pilot study provide suggestive evidence that alterations in the subgingival microbiome are associated with changes in cognitive function, and provide support for an expanded analysis of the role of the oral microbiome in dementia.

Salmonella typhimurium strain DU501, which was found to be deficient in acetohydroxy acid synthase II (AHAS II) and to possess elevated levels of transaminase B and biosynthetic threonine deaminase, required isoleucine, methionine, or pantothenate for growth. This strain accumulated α-ketobutyrate and, to a lesser extent, α-aminobutyrate. We found that α-ketobutyrate was a competitive substrate for ketopantoate hydroxymethyltransferase, the first enzyme in pantothenate biosynthesis. This competition with the normal substrate, α-ketoisovalerate, limited the supply of pantothenate, which resulted in a requirement for methionine. Evidence is presented to support the conclusion that the ambivalent requirement for either pantothenate or methionine is related to a decrease in succinyl coenzyme A, which is produced from pantothenate and which is an obligatory precursor of methionine biosynthesis. The autointoxification by endogenously produced α-ketobutyrate could be mimicked in wild-type S. typhimurium by exogenously supplied α-ketobutyrate or salicylate, a known inhibitor of pantothenate biosynthesis. The accumulation of α-ketobutyrate was initiated by the inability of the residual AHAS activity provided by AHAS I to efficiently remove the α-ketobutyrate produced by biosynthetic threonine deaminase. The accumulation of α-ketobutyrate was amplified by the action of transaminase B, which decreased the isoleucine pool by catalyzing the formation of α-keto-β-methylvalerate and aminobutyrate from isoleucine and α-ketobutyrate; this resulted in release of threonine deaminase from end product inhibition and unbridled production of α-ketobutyrate. Isoleucine satisfied the auxotrophic requirement of the AHAS II-deficient strain by curtailing the activity of threonine deaminase. Additional lines of evidence based on genetic and physiological experiments are presented to support the basis for the autointoxification of strain DU501 as well as other nonpolarigenic ilvG mutant strains.