Crystal Structure of an Integron Gene Cassette-Associated Protein from Vibrio cholerae Identifies a Cationic Drug-Binding Module

Background The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes. Methodology/Principal Findings We report the 1.8 A crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators. Conclusions/Significance Genetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds.National Health and Medical Research Council (Australia) (NHMRC grant 488502)National Institutes of Health (U.S.) (Grant GM62414-0 )Ontario. Ministry of Revenue (Challenge Fund

Quantum Measurement Theory in Gravitational-Wave Detectors

The fast progress in improving the sensitivity of the gravitational-wave (GW) detectors, we all have witnessed in the recent years, has propelled the scientific community to the point, when quantum behaviour of such immense measurement devices as kilometer-long interferometers starts to matter. The time, when their sensitivity will be mainly limited by the quantum noise of light is round the corner, and finding the ways to reduce it will become a necessity. Therefore, the primary goal we pursued in this review was to familiarize a broad spectrum of readers with the theory of quantum measurements in the very form it finds application in the area of gravitational-wave detection. We focus on how quantum noise arises in gravitational-wave interferometers and what limitations it imposes on the achievable sensitivity. We start from the very basic concepts and gradually advance to the general linear quantum measurement theory and its application to the calculation of quantum noise in the contemporary and planned interferometric detectors of gravitational radiation of the first and second generation. Special attention is paid to the concept of Standard Quantum Limit and the methods of its surmounting.Comment: 147 pages, 46 figures, 1 table. Published in Living Reviews in Relativit

Biomechanical spinal growth modulation and progressive adolescent scoliosis – a test of the 'vicious cycle' pathogenetic hypothesis: Summary of an electronic focus group debate of the IBSE

There is no generally accepted scientific theory for the causes of adolescent idiopathic scoliosis (AIS). As part of its mission to widen understanding of scoliosis etiology, the International Federated Body on Scoliosis Etiology (IBSE) introduced the electronic focus group (EFG) as a means of increasing debate on knowledge of important topics. This has been designated as an on-line Delphi discussion. The text for this debate was written by Dr Ian A Stokes. It evaluates the hypothesis that in progressive scoliosis vertebral body wedging during adolescent growth results from asymmetric muscular loading in a "vicious cycle" (vicious cycle hypothesis of pathogenesis) by affecting vertebral body growth plates (endplate physes). A frontal plane mathematical simulation tested whether the calculated loading asymmetry created by muscles in a scoliotic spine could explain the observed rate of scoliosis increase by measuring the vertebral growth modulation by altered compression. The model deals only with vertebral (not disc) wedging. It assumes that a pre-existing scoliosis curve initiates the mechanically-modulated alteration of vertebral body growth that in turn causes worsening of the scoliosis, while everything else is anatomically and physiologically 'normal' The results provide quantitative data consistent with the vicious cycle hypothesis. Dr Stokes' biomechanical research engenders controversy. A new speculative concept is proposed of vertebral symphyseal dysplasia with implications for Dr Stokes' research and the etiology of AIS. What is not controversial is the need to test this hypothesis using additional factors in his current model and in three-dimensional quantitative models that incorporate intervertebral discs and simulate thoracic as well as lumbar scoliosis. The growth modulation process in the vertebral body can be viewed as one type of the biologic phenomenon of mechanotransduction. In certain connective tissues this involves the effects of mechanical strain on chondrocytic metabolism a possible target for novel therapeutic intervention

EGFR interacts with the fusion protein of respiratory syncytial virus strain 2-20 and mediates infection and mucin expression.

Respiratory syncytial virus (RSV) is the major cause of viral lower respiratory tract illness in children. In contrast to the RSV prototypic strain A2, clinical isolate RSV 2-20 induces airway mucin expression in mice, a clinically relevant phenotype dependent on the fusion (F) protein of the RSV strain. Epidermal growth factor receptor (EGFR) plays a role in airway mucin expression in other systems; therefore, we hypothesized that the RSV 2-20 F protein stimulates EGFR signaling. Infection of cells with chimeric strains RSV A2-2-20F and A2-2-20GF or over-expression of 2-20 F protein resulted in greater phosphorylation of EGFR than infection with RSV A2 or over-expression of A2 F, respectively. Chemical inhibition of EGFR signaling or knockdown of EGFR resulted in diminished infectivity of RSV A2-2-20F but not RSV A2. Over-expression of EGFR enhanced the fusion activity of 2-20 F protein in trans. EGFR co-immunoprecipitated most efficiently with RSV F proteins derived from "mucogenic" strains. RSV 2-20 F and EGFR co-localized in H292 cells, and A2-2-20GF-induced MUC5AC expression was ablated by EGFR inhibitors in these cells. Treatment of BALB/c mice with the EGFR inhibitor erlotinib significantly reduced the amount of RSV A2-2-20F-induced airway mucin expression. Our results demonstrate that RSV F interacts with EGFR in a strain-specific manner, EGFR is a co-factor for infection, and EGFR plays a role in RSV-induced mucin expression, suggesting EGFR is a potential target for RSV disease

Abstract Reasoning and Friendship in High Functioning Preadolescents with Autism Spectrum Disorders

To investigate the relationship between cognitive and social functioning, 20 Israeli individuals with HFASD aged 8–12 and 22 age, maternal education, and receptive vocabulary–matched preadolescents with typical development (TYP) came to the lab with a close friend. Measures of abstract reasoning, friendship quality, and dyadic interaction during a play session were obtained. As hypothesized, individuals with HFASD were significantly impaired in abstract reasoning, and there were significant group differences in friend and observer reports of friendship quality. There also was consistency in reports between friends. Two factors—“relationship appearance” and “relationship quality” described positive aspects of the relationships. Disability status and age related to relationship appearance. Proband abstract reasoning was related to relationship quality

Physiological and molecular responses to an acute bout of reduced-exertion high-intensity interval training (REHIT)

PurposeWe have previously shown that 6 weeks of reduced-exertion high-intensity interval training (REHIT) improves V˙O2V˙O2 max in sedentary men and women and insulin sensitivity in men. Here, we present two studies examining the acute physiological and molecular responses to REHIT.MethodsIn Study 1, five men and six women (age: 26 ± 7 year, BMI: 23 ± 3 kg m−2, V˙O2V˙O2 max: 51 ± 11 ml kg−1 min−1) performed a single 10-min REHIT cycling session (60 W and two 20-s ‘all-out’ sprints), with vastus lateralis biopsies taken before and 0, 30, and 180 min post-exercise for analysis of glycogen content, phosphorylation of AMPK, p38 MAPK and ACC, and gene expression of PGC1α and GLUT4. In Study 2, eight men (21 ± 2 year; 25 ± 4 kg·m−2; 39 ± 10 ml kg−1 min−1) performed three trials (REHIT, 30-min cycling at 50 % of V˙O2V˙O2 max, and a resting control condition) in a randomised cross-over design. Expired air, venous blood samples, and subjective measures of appetite and fatigue were collected before and 0, 15, 30, and 90 min post-exercise.ResultsAcutely, REHIT was associated with a decrease in muscle glycogen, increased ACC phosphorylation, and activation of PGC1α. When compared to aerobic exercise, changes in V˙O2V˙O2 , RER, plasma volume, and plasma lactate and ghrelin were significantly more pronounced with REHIT, whereas plasma glucose, NEFAs, PYY, and measures of appetite were unaffected.ConclusionsCollectively, these data demonstrate that REHIT is associated with a pronounced disturbance of physiological homeostasis and associated activation of signalling pathways, which together may help explain previously observed adaptations once considered exclusive to aerobic exercise

Early Markers of Glycaemic Control in Children with Type 1 Diabetes Mellitus

Background: Type 1 diabetes mellitus (T1DM) may lead to severe long-term health consequences. In a longitudinal study, we aimed to identify factors present at diagnosis and 6 months later that were associated with glycosylated haemoglobin (HbA 1c) levels at 24 months after T1DM diagnosis, so that diabetic children at risk of poor glycaemic control may be identified. Methods: 229 children,15 years of age diagnosed with T1DM in the Auckland region were studied. Data collected at diagnosis were: age, sex, weight, height, ethnicity, family living arrangement, socio-economic status (SES), T1DM antibody titre, venous pH and bicarbonate. At 6 and 24 months after diagnosis we collected data on weight, height, HbA 1c level, and insulin dose. Results: Factors at diagnosis that were associated with higher HbA1c levels at 6 months: female sex (p,0.05), lower SES (p,0.01), non-European ethnicity (p,0.01) and younger age (p,0.05). At 24 months, higher HbA1c was associated with lower SES (p,0.001), Pacific Island ethnicity (p,0.001), not living with both biological parents (p,0.05), and greater BMI SDS (p,0.05). A regression equation to predict HbA1c at 24 months was consequently developed. Conclusions: Deterioration in glycaemic control shortly after diagnosis in diabetic children is particularly marked in Pacific Island children and in those not living with both biological parents. Clinicians need to be aware of factors associated wit

Identification of the calcitonin receptor in osteoarthritic chondrocytes

<p>Abstract</p> <p>Background</p> <p>Preclinical and clinical studies have shown that salmon calcitonin has cartilage protective effects in joint degenerative diseases, such as osteoarthritis (OA). However, the presence of the calcitonin receptor (CTR) in articular cartilage chondrocytes is yet to be identified. In this study, we sought to further investigate the expression of the CTR in naïve human OA articular chondrocytes to gain further confirmation of the existents of the CTR in articular cartilage.</p> <p>Methods</p> <p>Total RNA was purified from primary chondrocytes from articular cartilage biopsies from four OA patients undergoing total knee replacement. High quality cDNA was produced using a dedicated reverse transcription polymerase chain reaction (RT-PCR) protocol. From this a nested PCR assay amplifying the full coding region of the CTR mRNA was completed. Western blotting and immunohistochemistry were used to characterize CTR protein on protein level in chondrocytes.</p> <p>Results</p> <p>The full coding transcript of the CTR isoform 2 was identified in all four individuals. DNA sequencing revealed a number of allelic variants of the gene including two potentially novel polymorphisms: a frame shift mutation, +473del, producing a shorter form of the receptor protein, and a single nucleotide polymorphism in the 3' non coding region of the transcript, +1443 C>T. A 53 kDa protein band, consistent with non-glycosylated CTR isoform 2, was detected in chondrocytes with a similar size to that expressed in osteoclasts. Moreover the CTR was identified in the plasma membrane and the chondrocyte lacuna of both primary chondrocytes and OA cartilage section.</p> <p>Conclusions</p> <p>Human OA articular cartilage chondrocytes do indeed express the CTR, which makes the articular a pharmacological target of salmon calcitonin. In addition, the results support previous findings suggesting that calcitonin has a direct anabolic effect on articular cartilage.</p

What's New Is Old: Resolving the Identity of Leptothrix ochracea Using Single Cell Genomics, Pyrosequencing and FISH

Leptothrix ochracea is a common inhabitant of freshwater iron seeps and iron-rich wetlands. Its defining characteristic is copious production of extracellular sheaths encrusted with iron oxyhydroxides. Surprisingly, over 90% of these sheaths are empty, hence, what appears to be an abundant population of iron-oxidizing bacteria, consists of relatively few cells. Because L. ochracea has proven difficult to cultivate, its identification is based solely on habitat preference and morphology. We utilized cultivation-independent techniques to resolve this long-standing enigma. By selecting the actively growing edge of a Leptothrix-containing iron mat, a conventional SSU rRNA gene clone library was obtained that had 29 clones (42% of the total library) related to the Leptothrix/Sphaerotilus group (≤96% identical to cultured representatives). A pyrotagged library of the V4 hypervariable region constructed from the bulk mat showed that 7.2% of the total sequences also belonged to the Leptothrix/Sphaerotilus group. Sorting of individual L. ochracea sheaths, followed by whole genome amplification (WGA) and PCR identified a SSU rRNA sequence that clustered closely with the putative Leptothrix clones and pyrotags. Using these data, a fluorescence in-situ hybridization (FISH) probe, Lepto175, was designed that bound to ensheathed cells. Quantitative use of this probe demonstrated that up to 35% of microbial cells in an actively accreting iron mat were L. ochracea. The SSU rRNA gene of L. ochracea shares 96% homology with its closet cultivated relative, L. cholodnii, This establishes that L. ochracea is indeed related to this group of morphologically similar, filamentous, sheathed microorganisms