Organization of butyrate synthetic genes in human colonic bacteria: phylogenetic conservation and horizontal gene transfer

Butyrate producers constitute an important bacterial group in the human large intestine. Butyryl-CoA is formed from two molecules of acetyl-CoA in a process resembling beta-oxidation in reverse. Three different arrangements of the six genes coding for this pathway have been found in low mol% G+C-content gram-positive human colonic bacteria using DNA sequencing and degenerate PCR. Gene arrangements were strongly conserved within phylogenetic groups defined by 16S rRNA gene sequence relationships. In the case of one of the genes, encoding beta-hydroxybutyryl-CoA dehydrogenase, however, sequence relationships were strongly suggestive of horizontal gene transfer between lineages. The newly identified gene for butyryl-CoA CoA-transferase, which performs the final step in butyrate formation in most known human colonic bacteria, was not closely linked to these central pathway genes.     

Behavioral phenotypes of Disc1 missense mutations in mice

To support the role of DISC1 in human psychiatric disorders, we identified and analyzed two independently derived ENU-induced mutations in Exon 2 of mouse Disc1. Mice with mutation Q31L showed depressive-like behavior with deficits in the forced swim test and other measures that were reversed by the antidepressant bupropion, but not by rolipram, a phosphodiesterase-4 (PDE4) inhibitor. In contrast, L100P mutant mice exhibited schizophrenic-like behavior, with profound deficits in prepulse inhibition and latent inhibition that were reversed by antipsychotic treatment. Both mutant DISC1 proteins exhibited reduced binding to the known DISC1 binding partner PDE4B. Q31L mutants had lower PDE4B activity, consistent with their resistance to rolipram, suggesting decreased PDE4 activity as a contributory factor in depression. This study demonstrates that Disc1 missense mutations in mice give rise to phenotypes related to depression and schizophrenia, thus supporting the role of DISC1 in major mental illness.     

Pathogenesis of infantile haemangioma: new molecular and cellular insights

Infantile haemangioma is the most common tumour of infancy, yet the origin of these lesions remains controversial and the predictable life cycle is poorly understood. Much new information on infantile haemangiomas has emerged over the past decade, but experts continue to debate fundamental features, including cell of origin, nonrandom distribution, and mechanisms regulating the sometimes explosive growth and slow involution. The development of useful laboratory models has been difficult, in turn restricting the development of treatment options available to the clinician. Despite this, new research and creative thinking has spawned several hypotheses on the origin of these tumours and their interesting clinical behaviour, including suggestions of an intrinsic defect in local endothelial cells, a contribution of circulating endothelial progenitors or haemangioblasts, embolisation of shed placental cells and developmental field defects. While no single hypothesis seems to describe all features of infantile haemangioma, continued research seeks to integrate these ideas, create a better understanding of these important tumours and bring new treatments to the clinic.     

DarkHorse: a method for genome-wide prediction of horizontal gene transfer

A new approach to rapid, genome-wide identification and ranking of horizontal transfer candidate proteins is presented. The method is quantitative, reproducible, and computationally undemanding. It can be combined with genomic signature and/or phylogenetic tree-building procedures to improve accuracy and efficiency. The method is also useful for retrospective assessments of horizontal transfer prediction reliability, recognizing orthologous sequences that may have been previously overlooked or unavailable. These features are demonstrated in bacterial, archaeal, and eukaryotic examples.     

Home-based exercise rehabilitation in addition to specialist heart failure nurse care: design, rationale and recruitment to the Birmingham Rehabilitation Uptake Maximisation study for patients with congestive heart failure (BRUM-CHF): a randomised controlled trial

Background

We aimed to assess whether we could identify a graded association between increasing number of components of the metabolic syndrome and cardiac structural and functional abnormalities independently of predicted risk of coronary heart disease by the Framingham risk score.

Methods

We conducted a cross-sectional study on a random sample of the urban population of Porto aged 45 years or over. Six hundred and eighty-four participants were included. Data were collected by a structured clinical interview with a physician, ECG and a transthoracic M-mode and 2D echocardiogram. The metabolic syndrome was defined according to ATPIII-NCEP. The association between the number of features of the metabolic syndrome and the cardiac structural and functional abnormalities was assessed by 3 multivariate regression models: adjusting for age and gender, adjusting for the 10-year predicted risk of coronary heart disease by Framingham risk score and adjusting for age, gender and systolic blood pressure.

Results

There was a positive association between the number of features of metabolic syndrome and parameters of cardiac structure and function, with a consistent and statistically significant trend for all cardiac variables considered when adjusting for age and gender. Parameters of left ventricular geometry patterns, left atrial diameter and diastolic dysfunction maintained this trend when taking into account the 10-year predicted risk of coronary heart disease by the Framingham score as an independent variable, while left ventricular systolic dysfunction did not. The prevalence of left ventricular diastolic dysfunction, and the mean left ventricular mass, left ventricular diameter and left atrial diameter increased significantly with the number of features of the metabolic syndrome when additionally adjusting for systolic blood pressure as a continuous variable.

Conclusion

Increasing severity of metabolic syndrome was associated with increasingly compromised structure and function of the heart. This association was independent of Framingham risk score for indirect indices of diastolic dysfunction but not systolic dysfunction, and was not explained by blood pressure level.     

Fragment-based identification of determinants of conformational and spectroscopic change at the ricin active site

Background  

Ricin is a potent toxin and known bioterrorism threat with no available antidote. The ricin A-chain (RTA) acts enzymatically to cleave a specific adenine base from ribosomal RNA, thereby blocking translation. To understand better the relationship between ligand binding and RTA active site conformational change, we used a fragment-based approach to find a minimal set of bonding interactions able to induce rearrangements in critical side-chain positions.     

CYP116B5: a new class VII catalytically self‐sufficient cytochrome P450 from Acinetobacter radioresistens that enables growth on alkanes

A gene coding for a class VII cytochrome P450 monooxygenase (CYP116B5) was identified from Acinetobacter radioresistens S13 growing on media with medium (C14, C16) and long (C24, C36) chain alkanes as the sole energy source. Phylogenetic analysis of its N‐ and C‐terminal domains suggests an evolutionary model involving a plasmid‐mediated horizontal gene transfer from the donor Rhodococcus jostii RHA1 to the receiving A. radioresistens S13. This event was followed by fusion and integration of the new gene in A. radioresistens chromosome. Heterologous expression of CYP116B5 in Escherichia coli BL21, together with the A. radioresistens Baeyer–Villiger monooxygenase, allowed the recombinant bacteria to grow on long‐ and medium‐chain alkanes, showing that CYP116B5 is involved in the first step of terminal oxidation of medium‐chain alkanes overlapping AlkB and in the first step of sub‐terminal oxidation of long‐chain alkanes. It was also demonstrated that CYP116B5 is a self‐sufficient cytochrome P450 consisting of a heme domain (aa 1–392) involved in the oxidation step of n‐alkanes degradation, and its reductase domain (aa 444–758) comprising the NADPH‐, FMN‐ and [2Fe2S]‐binding sites. To our knowledge, CYP116B5 is the first member of this class to have its natural substrate and function identified.     

AFN-1252 is a potent inhibitor of enoyl-ACP reductase from Burkholderia pseudomallei—Crystal structure,mode of action,and biological activity

Melioidosis is a tropical bacterial infection caused by Burkholderia pseudomallei (B. pseudomallei; Bpm), a Gram-negative bacterium. Current therapeutic options are largely limited to trimethoprim-sulfamethoxazole and β-lactam drugs, and the treatment duration is about 4 months. Moreover, resistance has been reported to these drugs. Hence, there is a pressing need to develop new antibiotics for Melioidosis. Inhibition of enoyl-ACP reducatase (FabI), a key enzyme in the fatty acid biosynthesis pathway has shown significant promise for antibacterial drug development. FabI has been identified as the major enoyl-ACP reductase present in B. pseudomallei. In this study, we evaluated AFN-1252, a Staphylococcus aureus FabI inhibitor currently in clinical development, for its potential to bind to BpmFabI enzyme and inhibit B. pseudomallei bacterial growth. AFN-1252 stabilized BpmFabI and inhibited the enzyme activity with an IC₅₀ of 9.6 nM. It showed good antibacterial activity against B. pseudomallei R15 strain, isolated from a melioidosis patient (MIC of 2.35 mg/L). X-ray structure of BpmFabI with AFN-1252 was determined at a resolution of 2.3 Å. Complex of BpmFabI with AFN-1252 formed a symmetrical tetrameric structure with one molecule of AFN-1252 bound to each monomeric subunit. The kinetic and thermal melting studies supported the finding that AFN-1252 can bind to BpmFabI independent of cofactor. The structural and mechanistic insights from these studies might help the rational design and development of new FabI inhibitors.     

The Impact of Collagen Fibril Polarity on Second Harmonic Generation Microscopy

In this work, we report the implementation of interferometric second harmonic generation (SHG) microscopy with femtosecond pulses. As a proof of concept, we imaged the phase distribution of SHG signal from the complex collagen architecture of juvenile equine growth cartilage. The results are analyzed in respect to numerical simulations to extract the relative orientation of collagen fibrils within the tissue. Our results reveal large domains of constant phase together with regions of quasi-random phase, which are correlated to respectively high- and low-intensity regions in the standard SHG images. A comparison with polarization-resolved SHG highlights the crucial role of relative fibril polarity in determining the SHG signal intensity. Indeed, it appears that even a well-organized noncentrosymmetric structure emits low SHG signal intensity if it has no predominant local polarity. This work illustrates how the complex architecture of noncentrosymmetric scatterers at the nanoscale governs the coherent building of SHG signal within the focal volume and is a key advance toward a complete understanding of the structural origin of SHG signals from tissues.