RT-qPCR reveals opsin gene upregulation associated with age and sex in guppies (Poecilia reticulata) - a species with color-based sexual selection and 11 visual-opsin genes

Background  

PCR-based surveys have shown that guppies (Poecilia reticulata) have an unusually large visual-opsin gene repertoire. This has led to speculation that opsin duplication and divergence has enhanced the evolution of elaborate male coloration because it improves spectral sensitivity and/or discrimination in females. However, this conjecture on evolutionary connections between opsin repertoire, vision, mate choice, and male coloration was generated with little data on gene expression. Here, we used RT-qPCR to survey visual-opsin gene expression in the eyes of males, females, and juveniles in order to further understand color-based sexual selection from the perspective of the visual system.     

Attitudinal and demographic predictors of measles-mumps-rubella vaccine (MMR) uptake during the UK catch-up campaign 2008-09: cross-sectional survey

Background and Objective

Continued suboptimal measles-mumps-rubella (MMR) vaccine uptake has re-established measles epidemic risk, prompting a UK catch-up campaign in 2008–09 for children who missed MMR doses at scheduled age. Predictors of vaccine uptake during catch-ups are poorly understood, however evidence from routine schedule uptake suggests demographics and attitudes may be central. This work explored this hypothesis using a robust evidence-based measure.

Design

Cross-sectional self-administered questionnaire with objective behavioural outcome.

Setting and Participants

365 UK parents, whose children were aged 5–18 years and had received <2 MMR doses before the 2008–09 UK catch-up started.

Main Outcome Measures

Parents'' attitudes and demographics, parent-reported receipt of invitation to receive catch-up MMR dose(s), and catch-up MMR uptake according to child''s medical record (receipt of MMR doses during year 1 of the catch-up).

Results

Perceived social desirability/benefit of MMR uptake (OR = 1.76, 95% CI = 1.09–2.87) and younger child age (OR = 0.78, 95% CI = 0.68–0.89) were the only independent predictors of catch-up MMR uptake in the sample overall. Uptake predictors differed by whether the child had received 0 MMR doses or 1 MMR dose before the catch-up. Receipt of catch-up invitation predicted uptake only in the 0 dose group (OR = 3.45, 95% CI = 1.18–10.05), whilst perceived social desirability/benefit of MMR uptake predicted uptake only in the 1 dose group (OR = 9.61, 95% CI = 2.57–35.97). Attitudes and demographics explained only 28% of MMR uptake in the 0 dose group compared with 61% in the 1 dose group.

Conclusions

Catch-up MMR invitations may effectively move children from 0 to 1 MMR doses (unimmunised to partially immunised), whilst attitudinal interventions highlighting social benefits of MMR may effectively move children from 1 to 2 MMR doses (partially to fully immunised). Older children may be best targeted through school-based programmes. A formal evaluation element should be incorporated into future catch-up campaigns to inform their continuing improvement.     

Frizzled-7 receptor ectodomain expression in a colon cancer cell line induces morphological change and attenuates tumor growth

Frizzled (FZD) receptors have a conserved N-terminal extracellular cysteine-rich domain that interacts with Wnts and co-expression of the receptor ectodomain can antagonize FZD-mediated signalling. Using the ectodomain as an antagonist we have modulated endogenous FZD7 signalling in the moderately differentiated colon adenocarcinoma cell line, SK-CO-1. Unlike the parental cell line, which grows as tightly associated adherent cell clusters, the FZD7 ectodomain expressing cells display a spread out morphology and grow as a monolayer in tissue culture. This transition in morphology was associated with decreased levels of plasma membrane-associated E-cadherin and beta-catenin, localized increased levels of vimentin and redistribution of alpha6 integrin to cellular processes in the FZD7 ectodomain expressing cells. The morphological and phenotype changes induced by FZD7 ectodomain expression in SK-CO-1 cells is thus consistent with the cells undergoing an epithelial-to-mesenchymal-like transition. Furthermore, initiation of tumor formation in a xenograft tumor growth assay was attenuated in the FZD7 ectodomain expressing cells. Our results indicate a pivotal role for endogenous FZD7 in morphology transitions that are associated with colon tumor initiation and progression.     

Contribution of manganese peroxidase and laccase to dye decoloration by Trametes versicolor

During dye decoloration by Trametes versicolor ATCC 20869 in modified Kirk’s medium, manganese peroxidase (MnP) and laccase were produced, but not lignin peroxidase, cellobiose dehydrogenase or manganese-independent peroxidase. Purified MnP decolorized azo dyes [amaranth, reactive black 5 (RB5) and Cibacron brilliant yellow] in Mn²⁺-dependent reactions but did not decolorize an anthraquinone dye [Remazol brilliant blue R (RBBR)]. However, the purified laccase decolorized RBBR five to ten times faster than the azo dyes and the addition of a redox mediator, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), did not alter decoloration rates. Amaranth and RB5 were decolorized the most rapidly by MnP since they have a hydroxyl group in an ortho position and a sulfonate group in the meta position relative to the azo bond. During a typical batch decoloration with the fungal culture, the ratio of laccase:MnP was 10:1 to 20:1 (based on enzyme activity) and increased to greater than 30:1 after decoloration was complete. Since MnP decolorized amaranth about 30 times more rapidly than laccase per unit of enzyme activity, MnP should have contributed more to decoloration than laccase in batch cultures.     

Description of a technique for anaesthetizing pregnant ewes for fetal surgery

General anaesthesia in 12 pregnant ewes undergoing surgery for fetal physiological research was supplemented with an intravenous infusion of remifentanil. This allowed us to employ a lighter plane of surgical anaesthesia and to use intermittent positive pressure ventilation. Our aim was to improve fetomaternal outcome. We monitored maternal pulse, blood pressure, transcutaneous oxygen saturation and end-tidal carbon dioxide levels. Remifentanil doses of 0.75-2.0 microg/kg/min were needed and typically this allowed halothane concentrations of 1-1.5% to be used for maintenance of anaesthesia. Surgery lasted up to 2.5 h. All 12 ewes and their singleton fetuses survived the peri- and postoperative period in good condition.     

Conformational changes in monoamine oxidase A in response to ligand binding or reduction

The structure of monoamine oxidase B revealed three aromatic amino acid residues within contact distance of the flavin cofactor and a large number of aromatic residues in the substrate binding site. Circular dichroism (CD) spectroscopy can detect alterations in the environment of aromatic residues as a result of ligand binding or redox changes. CD spectra of MAO A indicate that a small inhibitor such d-amphetamine perturbs the aromatic residues very little, but binding of the larger pirlindole (2,3,3a,4,5,6-hexahydro-8-methyl-1H-pyrazino[3,2,1-j,k]carbazole hydrochloride) causes spectral changes consistent with the alteration of the environment of tyrosine and tryptophan residues in particular. Reduction of the flavin cofactor induces large enhancement of the CD signals in the aromatic region (260-310 nm). When covalent modification of the flavin by clorgyline accompanies reduction, the perturbation is even greater. In contrast to the static picture offered by crystallography, this study reveals changes in the aromatic cage on ligand binding and suggests that reduction of the cofactor substantially alters the environment of aromatic residues presumably near the flavin. In addition, the covalently modified reduced MAO A shows significant differences from the substrate-reduced enzyme.     

High potency silencing by single-stranded boranophosphate siRNA

In RNA interference (RNAi), double-stranded short interfering RNA (ds-siRNA) inhibits expression from complementary mRNAs. Recently, it was demonstrated that short, single-stranded antisense RNA (ss-siRNA) can also induce RNAi. While ss-siRNA may offer several advantages in both clinical and research applications, its overall poor activity compared with ds-siRNA has prevented its widespread use. In contrast to the poor gene silencing activity of native ss-siRNA, we found that the silencing activity of boranophosphate-modified ss-siRNA is comparable with that of unmodified ds-siRNA. Boranophosphate ss-siRNA has excellent maximum silencing activity and is highly effective at low concentrations. The silencing activity of boranophosphate ss-siRNA is also durable, with significant silencing up to 1 week after transfection. Thus, we have demonstrated that boranophosphate-modified ss-siRNA can silence gene expression as well as native ds-siRNA, suggesting that boranophosphate-modified ss-siRNAs should be investigated as a potential new class of therapeutic agents.     

The structure of MbtI from Mycobacterium tuberculosis, the first enzyme in the biosynthesis of the siderophore mycobactin, reveals it to be a salicylate synthase

The ability to acquire iron from the extracellular environment is a key determinant of pathogenicity in mycobacteria. Mycobacterium tuberculosis acquires iron exclusively via the siderophore mycobactin T, the biosynthesis of which depends on the production of salicylate from chorismate. Salicylate production in other bacteria is either a two-step process involving an isochorismate synthase (chorismate isomerase) and a pyruvate lyase, as observed for Pseudomonas aeruginosa, or a single-step conversion catalyzed by a salicylate synthase, as with Yersinia enterocolitica. Here we present the structure of the enzyme MbtI (Rv2386c) from M. tuberculosis, solved by multiwavelength anomalous diffraction at a resolution of 1.8 A, and biochemical evidence that it is the salicylate synthase necessary for mycobactin biosynthesis. The enzyme is critically dependent on Mg2+ for activity and produces salicylate via an isochorismate intermediate. MbtI is structurally similar to salicylate synthase (Irp9) from Y. enterocolitica and the large subunit of anthranilate synthase (TrpE) and shares the overall architecture of other chorismate-utilizing enzymes, such as the related aminodeoxychorismate synthase PabB. Like Irp9, but unlike TrpE or PabB, MbtI is neither regulated by nor structurally stabilized by bound tryptophan. The structure of MbtI is the starting point for the design of inhibitors of siderophore biosynthesis, which may make useful lead compounds for the production of new antituberculosis drugs, given the strong dependence of pathogenesis on iron acquisition in M. tuberculosis.     

On-line estimation of dissolved methane concentration during methanotrophic fermentations

Knowledge of the aqueous phase methane concentration is critical to understanding and controlling process kinetics in methanotrophic bioreactors. Unfortunately since no dissolved methane probe is commercially available, this data must be obtained off-line by the time-consuming gas-liquid partition method. In this study we demonstrate how knowledge of the reactor's k(L)a for oxygen combined with gas phase methane analysis can be used to continuously estimate the aqueous phase concentration of dissolved methane. The on-line estimation was verified in two reactor systems with greatly different values of k (L)a. In both systems the measured and calculated dissolved methane concentrations were in good agreement although dissolved methane was underestimated in both cases. The utility of this methodology was demonstrated by revealing a possible metabolic bottleneck in the model system.