Balancing redox cofactor generation and ATP synthesis: Key microaerobic responses in thermophilic fermentations

Geobacillus thermoglucosidasius is a Gram‐positive, thermophilic bacterium capable of ethanologenic fermentation of both C5 and C6 sugars and may have possible use for commercial bioethanol production [Tang et al., 2009; Taylor et al. (2009) Trends Biotechnol 27(7): 398–405]. Little is known about the physiological changes that accompany a switch from aerobic (high redox) to microaerobic/fermentative (low redox) conditions in thermophilic organisms. The changes in the central metabolic pathways in response to a switch in redox potential were analyzed using quantitative real‐time PCR and proteomics. During low redox (fermentative) states, results indicated that glycolysis was uniformly up‐regulated, the Krebs (tricarboxylic acid or TCA) cycle non‐uniformly down‐regulated and that there was little to no change in the pentose phosphate pathway. Acetate accumulation was accounted for by strong down‐regulation of the acetate CoA ligase gene (acs) in addition to up‐regulation of the pta and ackA genes (involved in acetate production), thus conserving ATP while reducing flux through the TCA cycle. Substitution of an NADH dehydrogenase (down‐regulated) by an up‐regulated NADH:FAD oxidoreductase and up‐regulation of an ATP synthase subunit, alongside the observed shifts in the TCA cycle, suggested that an oxygen‐scavenging electron transport chain likely remained active during low redox conditions. Together with the observed up‐regulation of a glyoxalase and down‐regulation of superoxide dismutase, thought to provide protection against the accumulation of toxic phosphorylated glycolytic intermediates and reactive oxygen species, respectively, the changes observed in G. thermoglucosidasius NCIMB 11955 under conditions of aerobic‐to‐microaerobic switching were consistent with responses to low pO₂ stress. Biotechnol. Bioeng. 2013; 110: 1057–1065. © 2012 Wiley Periodicals, Inc.     

Somatoform disorders among patients attending walk-in clinics in Trinidad: prevalence and association with depression and anxiety

Objectives Somatoform disorders are common in international primary care settings, but have been little studied in the developing world. The objective of this study was to determine the prevalence of severe undifferentiated somatoform disorder, and its relationship to depression and anxiety, among patients attending walk-in clinics in Trinidad.Methods The study participants, who were all aged 18 years or older and attending walk-in clinics at 16 randomly selected health centres, were surveyed between May and August 2007 using the PRIME-MD questionnaire.Results There were 594 participants (the response rate was 92%), of whom 72.7% were female. Their ages ranged from 18 to 93 years, and 54.5% were over 50 years of age. In total, 37.2% were married and 25.9% were single. Indo-Trinidadians represented 43.1% and Afro-Trinidadians represented 36% of the study sample; 56.5% of the participants reported that their income was less than US$ 400 per month, and 65.7% were unemployed. At walk-in clinics in Trinidad, the estimated prevalence of severe undifferentiated somatoform disorder was 10.3% (95% CI: 7.86–12.74), that of hypochondriasis was 28.5% (95% CI: 24.9–32.1), and that of body dysmorphic disorder was 15.8% (95% CI: 11.9–18.7). Severe undifferentiated somatoform disorder was statistically significantly associated with gender and ethnicity but not with age, level of education, employment status or income. Chi-square testing found significant associations between the presence of severe undifferentiated somatoform disorder and both depression and anxiety (P < 0.05), between hypochondriasis and both anxiety and depression (P < 0.05), and between body dysmorphic disorder and depression (P < 0.05) but not anxiety. Regression analysis suggested that the demographic features that predicted severe undifferentiated somatoform disorder were being female or Indo-Trinidadian.Conclusions Walk-in clinics in Trinidad that serve older patients on a lower income have a high proportion of patients with somatoform disorders as measured by the PRIME-MD scale. These patients exhibit many features of anxiety and depression. These findings have implications for medical training and service delivery.     

Kronecker积在分析多因子非平衡实验中的应用

以Kroneckert积和效应矩阵为工具,阐明了一种分析多因子缺值实验的严格、有效、方便的新方法。     

Are parents’ working patterns associated with their child’s sleep? An analysis of dualparent families in Australia

Insufficient sleep in children predicts emotional and behavioral problems, poorer school performance, and health problems. Child sleep durations have declined in recent decades, suggesting a need to identify and understand predictors of short sleep. The present study investigated whether aspects of parental employment (i.e. parental work hours, and non-standard work hours) were associated with sleep in children. Data collected from 2477 children aged 6-7 years as part of the Longitudinal Study of Australian Children were used in this paper. Child sleep duration, bedtimes, and wake times were determined from parent self-report using time-use diaries. Parents completed a survey assessing their work patterns as well as a range of other demographic and social factors. The results indicated that long mother work hours were associated with later bedtimes and increased odds of <9.5 h sleep in children. Long father work hours were associated with earlier waketimes, earlier bedtimes, and reduced odds of long sleep. Non-standard work hours were associated with longer sleep and earlier bedtimes. The present results indicate the need to develop strategies to limit any adverse effects of parental work on child sleep, perhaps by promoting earlier and regular bedtimes. These findings warrant further investigation given the importance of sleep in healthy child development.

     

A novel, extremely alkaliphilic and cold-active esterase from Antarctic desert soil

A novel, cold-active and highly alkaliphilic esterase was isolated from an Antarctic desert soil metagenomic library by functional screening. The 1,044 bp gene sequence contained several conserved regions common to lipases/esterases, but lacked clear classification based on sequence analysis alone. Moderate (<40%) amino acid sequence similarity to known esterases was apparent (the closest neighbour being a hypothetical protein from Chitinophaga pinensis), despite phylogenetic distance to many of the lipolytic “families”. The enzyme functionally demonstrated activity towards shorter chain p-nitrophenyl esters with the optimal activity recorded towards p-nitrophenyl propionate (C3). The enzyme possessed an apparent T_opt at 20°C and a pH optimum at pH 11. Esterases possessing such extreme alkaliphily are rare and so this enzyme represents an intriguing novel locus in protein sequence space. A metagenomic approach has been shown, in this case, to yield an enzyme with quite different sequential/structural properties to known lipases. It serves as an excellent candidate for analysis of the molecular mechanisms responsible for both cold and alkaline activity and novel structure–function relationships of esterase activity.     

A SURVEY FOR 1,3,6,7-TETRAHYDROXY-C-GLYCOSYLXANTHONES EMPHASIZING THE “PRIMITIVE” LEPTOSPORANGIATE FERNS AND THEIR ALLIES

A survey for 1,3,6,7-tetrahydroxy-C-glycosylxanthones of representative species within the primitive vascular plants, emphasizing the leptosporangiate ferns, has indicated a limited distribution of these compounds within three leptosporangiate families: Hymenophyllaceae, Aspleniaceae and Marsileaceae. In the Hymenophyllaceae the distribution of these compounds appears to be a useful criterion for segregating species of Mecodium from other species of Hymenophyllum (sensu lato) and suggests that the tubulate vs. the valvate indusial condition may not be an ideal character for separating all species of Hymenophyllum (s.l.) from those of Trichomanes (s.l.). These compounds appear useful for delimiting several species of Elaphoglossum section Pachyglossa and support a relationship among the Aspleniaceae, Athyriaceae, and Elaphoglossaceae. Their presence in Marsilea also raises questions as to the origin of this group of plants.     

Threonine phosphorylation prevents promoter DNA binding of the Group B Streptococcus response regulator CovR

All living organisms communicate with the external environment for their survival and existence. In prokaryotes, communication is achieved by two-component systems (TCS) comprising histidine kinases and response regulators. In eukaryotes, signalling is accomplished by serine/threonine and tyrosine kinases. Although TCS and serine/threonine kinases coexist in prokaryotes, direct cross-talk between these families was first described in Group B Streptococcus (GBS). A serine/threonine kinase (Stk1) and a TCS (CovR/CovS) co-regulate toxin expression in GBS. Typically, promoter binding of regulators like CovR is controlled by phosphorylation of the conserved active site aspartate (D53). In this study, we show that Stk1 phosphorylates CovR at threonine 65. The functional consequence of threonine phosphorylation of CovR in GBS was evaluated using phosphomimetic and silencing substitutions. GBS encoding the phosphomimetic T65E allele are deficient for CovR regulation unlike strains encoding the non-phosphorylated T65A allele. Further, compared with wild-type or T65A CovR, the T65E CovR is unable to bind promoter DNA and is decreased for phosphorylation at D53, similar to Stk1-phosphorylated CovR. Collectively, we provide evidence for a novel mechanism of response regulator control that enables GBS (and possibly other prokaryotes) to fine-tune gene expression for environmental adaptation.     

Assessing the significance of conserved genomic aberrations using high resolution genomic microarrays

Genomic aberrations recurrent in a particular cancer type can be important prognostic markers for tumor progression. Typically in early tumorigenesis, cells incur a breakdown of the DNA replication machinery that results in an accumulation of genomic aberrations in the form of duplications, deletions, translocations, and other genomic alterations. Microarray methods allow for finer mapping of these aberrations than has previously been possible; however, data processing and analysis methods have not taken full advantage of this higher resolution. Attention has primarily been given to analysis on the single sample level, where multiple adjacent probes are necessarily used as replicates for the local region containing their target sequences. However, regions of concordant aberration can be short enough to be detected by only one, or very few, array elements. We describe a method called Multiple Sample Analysis for assessing the significance of concordant genomic aberrations across multiple experiments that does not require a-priori definition of aberration calls for each sample. If there are multiple samples, representing a class, then by exploiting the replication across samples our method can detect concordant aberrations at much higher resolution than can be derived from current single sample approaches. Additionally, this method provides a meaningful approach to addressing population-based questions such as determining important regions for a cancer subtype of interest or determining regions of copy number variation in a population. Multiple Sample Analysis also provides single sample aberration calls in the locations of significant concordance, producing high resolution calls per sample, in concordant regions. The approach is demonstrated on a dataset representing a challenging but important resource: breast tumors that have been formalin-fixed, paraffin-embedded, archived, and subsequently UV-laser capture microdissected and hybridized to two-channel BAC arrays using an amplification protocol. We demonstrate the accurate detection on simulated data, and on real datasets involving known regions of aberration within subtypes of breast cancer at a resolution consistent with that of the array. Similarly, we apply our method to previously published datasets, including a 250K SNP array, and verify known results as well as detect novel regions of concordant aberration. The algorithm has been fully implemented and tested and is freely available as a Java application at http://www.cbil.upenn.edu/MSA.     

A chimeric protein of simian immunodeficiency virus envelope glycoprotein gp140 and Escherichia coli aspartate transcarbamoylase

The envelope glycoproteins of the human immunodeficiency virus and the related simian immunodeficiency virus (SIV) mediate viral entry into host cells by fusing viral and target cell membranes. We have reported expression, purification, and characterization of gp140 (also called gp160e), the soluble, trimeric ectodomain of the SIV envelope glycoprotein, gp160 (B. Chen et al., J. Biol. Chem. 275:34946-34953, 2000). We have now expressed and purified chimeric proteins of SIV gp140 and its variants with the catalytic subunit (C) of Escherichia coli aspartate transcarbamoylase (ATCase). The fusion proteins (SIV gp140-ATC) bind viral receptor CD4 and a number of monoclonal antibodies specific for SIV gp140. The chimeric molecule also has ATCase activity, which requires trimerization of the ATCase C chains. Thus, the fusion protein is trimeric. When ATCase regulatory subunit dimers (R(2)) are added, the fusion protein assembles into dimers of trimers as expected from the structure of C(6)R(6) ATCase. Negative-stain electron microscopy reveals spikey features of both SIV gp140 and SIV gp140-ATC. The production of the fusion proteins may enhance the possibilities for structure determination of the envelope glycoprotein either by electron cryomicroscopy or X-ray crystallography.     

Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae

Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes ∼8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature.