Distinct chromatin structures at the monoamine oxidase-A promoter correlate with allele-specific expression in SH-SY5Y cells.

Monoamine oxidase-A (MAOA) metabolises monoamines and is implicated in the pathophysiology of psychiatric disorders. A polymorphic repetitive DNA domain, termed the uVNTR (upstream variable number tandem repeat), located at the promoter of the MAOA gene is a risk factor for many of these disorders. MAOA is on the X chromosome suggesting gender could play a role in regulation. We analysed MAOA regulation in the human female cell line, SH-SY5Y, which is polymorphic for the uVNTR. This heterozygosity allowed us to correlate allele-specific gene expression with allele-specific transcription factor binding and epigenetic marks for MAOA. Gene regulation was analysed under basal conditions and in response to the mood stabiliser sodium valproate. Both alleles were transcriptionally active under basal growth conditions; however, the alleles showed distinct transcription factor binding and epigenetic marks at their respective promoters. Exposure of the cells to sodium valproate resulted in differential allelic expression which correlated with allele-specific changes in distinct transcription factor binding and epigenetic marks at the region encompassing the uVNTR. Biochemically our model for MAOA promoter function has implications for gender differences in gene × environment responses in which the uVNTR has been implicated as a genetic risk.

A TOMM40 poly-T variant modulates gene expression and is associated with vocabulary ability and decline in nonpathologic aging.

The Translocase of Outer Mitochondrial Membrane 40 Homolog and Apolipoprotein E (TOMM40-APOE) locus has been associated with a number of age-related phenotypes in humans including nonpathologic cognitive aging, late-onset Alzheimer's disease, and longevity. Here, we investigate the influence of the TOMM40 intron 6 poly-T variant (rs10524523) on TOMM40 gene expression and cognitive abilities and decline in a cohort of 1613 community-dwelling elderly volunteers who had been followed for changes in cognitive functioning over a period of 14 years (range = 12-18 years). We showed that the shorter length poly-T variants were found to act as a repressor of luciferase gene expression in reporter gene constructs. Expression was reduced to approximately half of that observed for the very long variant. We further observed that the shorter poly-T variant was significantly associated with reduced vocabulary ability and a slower rate of vocabulary decline with age compared to the very long poly-T variants. No significant associations were observed for memory, fluid intelligence or processing speed, although the direction of effect, where the short variant was correlated with reduced ability and slower rate of decline was observed for all tests. Our results indicate that the poly-T variant has the ability to interact with transcription machinery and differentially modulate reporter gene expression and influence vocabulary ability and decline with age.

Analysis of the effects of depression associated polymorphisms on the activity of the BICC1 promoter in amygdala neurones.

The Bicaudal C Homolog 1 (BICC1) gene, which encodes an RNA binding protein, has been identified by genome wide association studies (GWAS) as a candidate gene associated with major depressive disorder (MDD). We explored the hypothesis that MDD associated single-nucleotide polymorphisms (SNPs) affected the ability of cis-regulatory elements within intron 3 of the BICC1 gene to modulate the activity of the BICC1 promoter region. We initially established that the BICC1 promoter drove BICC1 mRNA expression in amygdala, hippocampus and hypothalamus. Intriguingly, we provide evidence that MDD associated polymorphisms alter the ability of the BICC1 promoter to respond to PKA signalling within amygdala neurones. Considering the known role of amygdala PKA pathways in fear learning and mood these observations suggest a possible mechanism through which allelic changes in the regulation of the BICC1 gene in amygdala neurones may contribute to mood disorders. Our findings also suggest a novel direction for the identification of novel drug targets and the design of future personalised therapeutics.The Pharmacogenomics Journal advance online publication, 6 October 2015; doi:10.1038/tpj.2015.62.

Effects of prenatal and postnatal depression, and maternal stroking, at the glucocorticoid receptor gene.

In animal models, prenatal and postnatal stress is associated with elevated hypothalamic-pituitary axis (HPA) reactivity mediated via altered glucocorticoid receptor (GR) gene expression. Postnatal tactile stimulation is associated with reduced HPA reactivity mediated via increased GR gene expression. In this first study in humans to examine the joint effects of prenatal and postnatal environmental exposures, we report that GR gene (NR3C1) 1-F promoter methylation in infants is elevated in the presence of increased maternal postnatal depression following low prenatal depression, and that this effect is reversed by self-reported stroking of the infants by their mothers over the first weeks of life.

Allele-specific transcriptional activity of the variable number of tandem repeats in 5' region of the DRD4 gene is stimulus specific in human neuronal cells.

The dopamine receptor D4 (DRD4) gene includes several variable number of tandem repeat loci that have been suggested to modulate DRD4 gene expression patterns. Previous studies showed differential basal activity of the two most common variants of a tandem repeat (120 bp per repeat unit) located in the 5' region adjacent to the DRD4 promoter in human cell lines. In this communication, we further characterized the ability of this polymorphic repeat to elicit tissue-, allele- and stimuli-specific transcriptional activity in vitro. The short and long variants of the DRD4 5' tandem repeat were cloned into a luciferase reporter gene construct containing the SV40 promoter. The luciferase constructs were cotransfected with expression vectors of two ubiquitously expressed human transcription factors (TFs), CCCTC-binding factor (CTCF) and upstream stimulatory factor 2 (USF2), into human cell lines and primary cultures of neonate rat cortex and luciferase activity measured. Overexpression with these TFs resulted in differential cell- and allele-specific transcriptional activities of the luciferase constructs. The results of our experiments show that variants of this tandem repeat in the 5' promoter of the DRD4 gene will direct differential reporter gene transcriptional activity in a cell-type-specific manner dependent on the signal pathways activated.

Behavioural genetics of the serotonin transporter.

The serotonin transporter is a key regulator of the bioavailability of serotonin and therefore any modulation in the expression or action of the transporter would be expected to have consequences on behaviour. The transporter has therefore become a target for pharmaceutical intervention in behavioural and mood disorders. The search for polymorphic variants in the transporter that would associate with neurological disorders has been extensive but has become focused on two domains which are both termed variable number tandem repeat (VNTR)polymorphisms. Both of these VNTRs are in non-coding DNA and therefore proposed to be mechanistically involved in a disorder through their ability to modulate transcriptional or post-transcriptional regulation of the transporter. The most extensively studied is in the promoter and is a bi-allelic insertion/deletion found in the 50 promoter region of the gene 1.2 kb upstream of the transcriptional start site. This VNTR, termed, 5-HTTLPR was initially identified as two variants containing either, 14 (short/deletion) or 16 (long/insertion) copies of a 22 bp repeat. A second widely studied VNTR found in the non-coding region of the transporter is located within intron 2 and comprises 9, 10 or 12 copies of a16­17 bp repeat termed, STin2.9, STin2.10 and STin2.12, respectively. These VNTR polymorphisms have been associated with a range of behavioural and psychiatric disorders including depression, OCD, anxiety and schizophrenia, however often the lack of reproducibility in different cohorts has led to debate on the actual association of the polymorphisms with this extensive range of neurological conditions. Here we review these two polymorphic VNTRs in depth and relate that to pharmaceutical response, their ability to regulate differential transporter expression, their core involvement in gene-environment interaction and their genetic association with specific disorders.

Phosphonoacetate biosynthesis: in vitro detection of a novel NADP(+)-dependent phosphonoacetaldehyde-oxidizing activity in cell-extracts of the marine Roseovarius nubinhibens ISM.

A novel phosphonoacetaldehyde-oxidizing activity was detected in cell-extracts of the marine bacterium Roseovarius nubinhibens ISM grown on 2-aminoethylphosphonic acid (2-AEP; ciliatine). Extracts also contained 2-AEP transaminase and phosphonoacetate hydrolase activities. These findings indicate the existence of a biological route from 2-AEP via phosphonoacetaldehyde for the production of phosphonoacetate, which has not previously been shown to be a natural product. The three enzymes appear to constitute a previously-unreported pathway for the mineralization of 2-AEP which is a potentially important source of phosphorus in the nutrient-stressed marine environment.

Stimulation of phosphate uptake and polyphosphate accumulation by activated sludge microorganisms in response to sulfite addition.

Enhanced phosphate removal from wastewaters is dependent on the synthesis and intracellular accumulation of polyphosphate by sludge microorganisms. However the role played by polyphosphate in microbial metabolism and the factors that trigger its formation remain poorly-understood. Many examples of the accumulation of the biopolymer by environmental microorganisms are documented; these include a recent report of the presence of large polyphosphate inclusions in sulfur-oxidizing marine bacteria. To investigate whether any link might exist outside the marine environment between the presence of reduced sulfur compounds and enhanced levels of microbial phosphate uptake and polyphosphate accumulation, activated sludge cultures were grown under laboratory conditions in media that contained sulfite, thiosulfate, hydrosulfite or tetrathionate. Only in the presence of sulfite was there any evidence of a stimulatory effect; in medium that contained 0.5 mM sodium sulfite some 17% more phosphate was removed by the sludge, whilst there was an almost two-fold increase in intracellular polyphosphate levels. No indications of sulfite toxicity were observed.

Intercontinental spread from Israel to Colombia of a KPC-3-producing Klebsiella pneumoniae strain.

In 2008, an increase in the prevalence of carbapenem-resistant Klebsiella pneumoniae was noted in a 286-bed tertiary case hospital in Colombia, where 84 patients (32 infected and 52 colonized) had positive cultures. The identified index patient came from Israel for a liver transplantation. High level carbapenem resistance was observed. Polymyxin B and tigecycline were the only two antibiotics that remained active. PCR-restriction fragment length polymorphism analysis and sequencing revealed blaKPC-3 in the major clone, which was indistinguishable from the K. pneumoniae carbapenemase-3-producing clone described previously in Israel. This exemplifies the threat posed by the global spread of K. pneumoniae carbapenemase-producing pathogens.

A long AAAG repeat allele in the 5' UTR of the ERR-γ gene is correlated with breast cancer predisposition and drives promoter activity in MCF-7 breast cancer cells.

We sequenced the 5' UTR of the estrogen-related receptor gamma gene (ERR-γ) in ~500 patient and volunteer samples and found that longer alleles of the (AAAG)(n) microsatellite were statistically and significantly more likely to exist in the germlines of breast cancer patients when compared to healthy volunteers. This microsatellite region contains multiple binding sites for a number of transcription factors, and we hypothesized that the polymorphic AAAG-containing sequence in the 5' UTR region of ERR-γ might modulate expression of ERR-γ. We found that the 369 bp PCR product containing the AAAG repeat drove expression of a reporter gene in estrogen receptor positive breast cancer cells. Our results support a role for the 5' UTR region in ERR-γ expression, which is potentially mediated via binding to the variable tandem AAAG repeat, the length of which correlates with breast cancer pre-disposition. Our study indicates that the AAAG tetranucleotide repeat polymorphism in ERR-γ gene 5' UTR region may be a new biomarker for genetic susceptibility to breast cancer.

Modeling antibiotic resistance to project future rates: quinolone resistance in Escherichia coli.

While the development of resistance to a new antibiotic is expected, the time course and degree of resistance that will develop are uncertain. Some best projections of the future extent of resistance can be highly impactful for activities, such as antimicrobial development, that require significant lead time. We focus on the surge among hospital isolates in fluoroquinolone-resistant Escherichia coli and use data on resistance and consumption to explore and quantify trends in increasing resistance and their relationship to antibiotic use from 2001 to 2007. A mixed-effects logistic regression model produced a good fit to the observed resistance rates during this period in the United States and Europe. The model contained significant effects of time, consumption, and country on developing fluoroquinolone resistance in E. coli. There was a larger projected increase in resistance for high fluoroquinolone-consuming countries projected to 2013: 45% (95% confidence interval [CI]: 38%, 53%) for high consumers vs. 33% (95% CI: 25%, 41%) for low consumers. The model was also used to obtain regional projections of resistance that can be used by local prescribers. In order to better understand and predict trends in antimicrobial resistance, it is vital to implement and expand current surveillance systems.

Evidence for phosphonate usage in the coral holobiont.

Phosphonates are characterized by a stable carbon-phosphorus bond and commonly occur as lipid conjugates in invertebrate cell membranes. Phosphonoacetate hydrolase encoded by the phnA gene, catalyses the cleavage of phosphonoacetate to acetate and phosphate. In this study, we demonstrate the unusually high phnA diversity in coral-associated bacteria. The holobiont of eight coral species tested positive when screened for phnA using degenerate primers. In two soft coral species, Sinularia and Discosoma, sequencing of the phnA gene showed 13 distinct groups on the basis of 90% sequence identity across 100% of the sequence. A total of 16 bacterial taxa capable of using phosphonoacetate as the sole carbon and phosphorus source were isolated; 8 of which had a phnA+ genotype. This study enhances our understanding of the wide taxonomic and environmental distribution of phnA, and highlights the importance of phosphonates in marine ecosystems.

The human neurokinin B gene, TAC3, and its promoter are regulated by Neuron Restrictive Silencing Factor (NRSF) transcription factor family.

We have previously shown that one of the major determinants directing the expression of the preprotachykinin-A (TAC1) gene, which encodes the neuropeptide substance P, is the transcription factor Neuronal Restrictive Silencer Factor (NSRF), which is also termed Repressor Element-1 Silencing Factor (REST). In rodent models of epilepsy, NRSF and its truncated isoform short NRSF (sNRSF), also termed REST4, are increased as an immediate response to seizure. In similar models the neurokinin B (NKB) gene (TAC3) is also induced and NKB has also been shown to be proconvulsant. In this communication we have demonstrated that both the TAC3 endogenous gene and its promoter are regulated, directly or indirectly, by the NRSF transcription factors resulting in both the increased expression of the endogenous gene and increased reporter gene activity. We demonstrate by chromatin immunoprecipitation analysis that NRSF and sNRSF will bind to the NKB promoter in vivo. Consistent with a model in which NRSF modulation of TAC3 gene expression is a mechanism that operates during epilepsy, the observed increases in both the level of the endogenous gene and the activity of the NKB promoter by these NRSF variants, were diminished by the action of the anticonvulsant drug, carbamazepine.

New developments in carbapenems.

Antibiotic resistance among Gram-negative pathogens in hospitals is a growing threat to patients and is driving the increased use of carbapenems. Carbapenems are potent members of the beta-lactam family of antibiotics, with a history of safety and efficacy for serious infections that exceeds 20 years. Original and review articles were identified from a Medline search (1979-2008). Reference citations from identified publications, abstracts from the Interscience Conferences on Antimicrobial Agents and Chemotherapy and package inserts were also used. Carbapenems are effective in treating severe infections at diverse sites, with relatively low resistance rates and a favourable safety profile. Carbapenems are the beta-lactams of choice for the treatment of infections caused by multidrug-resistant organisms. Optimized dosing of carbapenems should limit the emergence of resistance and prolong the utility of these agents. The newly approved doripenem should prove to be a valuable addition to the currently available carbapenems: imipenem, meropenem and ertapenem.

Mechanical stimulation induces preprotachykinin gene expression in osteoarthritic chondrocytes which is correlated with modulation of the transcription factor neuron restrictive silence factor.

We have previously demonstrated that the transcription factor termed neuron restrictive silencer factor (NRSF) and the truncated splice variant, NRSF short form (sNRSF) are major modulators of preprotachykinin A (TAC1) gene expression. In this communication we addressed whether TAC1 gene expression would be effected in response to mechanical stimulation of both normal and osteoarthritic (OA) chondrocytes. Chondrocytes were mechanically stimulated for 20 min, and then incubated under normal tissue culture conditions for 1 or 3h. RT-PCR and quantitative PCR (qPCR) were used to investigate expression of TAC1, NRSF and sNRSF mRNA at these time points. Western blotting was used to validate and confirm expression of sNRSF protein in chondrocytes in response to mechanical stimulation. We observed that TAC1 was expressed in normal chondrocytes, with no evidence of NRSF or sNRSF expression. TAC1 mRNA expression did not significantly change following mechanical stimulation in normal cells. OA chondrocytes expressed TAC1 and sNRSF mRNA, though not NRSF, and following mechanical stimulation there was a significant upregulation of both TAC1 and sNRSF mRNA, which returned to baseline levels 3h post-stimulation. sNRSF protein was upregulated at 1 and 2h following stimulation of OA chondrocytes. In summary, differential expression of TAC1 and sNRSF in OA chondrocytes associates their expression with the disease. The change in expression of sNRSF and TAC1 mRNA following mechanical stimulation in OA but not normal chondrocytes suggests that sNRSF may be involved in the regulation of SP production in OA cartilage. These differences between normal and OA mechanotransduction responses may be important in the production of phenotypic changes present in diseased cartilage.