Jointly Inferring the Dynamics of Population Size and Sampling Intensity from Molecular Sequences.
Estimating past population dynamics from molecular sequences that have been sampled longitudinally through time is an important problem in infectious disease epidemiology, molecular ecology, and macroevolution. Popular solutions, such as the skyline and skygrid methods, infer past effective population sizes from the coalescent event times of phylogenies reconstructed from sampled sequences but assume that sequence sampling times are uninformative about population size changes. Recent work has started to question this assumption by exploring how sampling time information can aid coalescent inference. Here, we develop, investigate, and implement a new skyline method, termed the epoch sampling skyline plot (ESP), to jointly estimate the dynamics of population size and sampling rate through time. The ESP is inspired by real-world data collection practices and comprises a flexible model in which the sequence sampling rate is proportional to the population size within an epoch but can change discontinuously between epochs. We show that the ESP is accurate under several realistic sampling protocols and we prove analytically that it can at least double the best precision achievable by standard approaches. We generalize the ESP to incorporate phylogenetic uncertainty in a new Bayesian package (BESP) in BEAST2. We re-examine two well-studied empirical data sets from virus epidemiology and molecular evolution and find that the BESP improves upon previous coalescent estimators and generates new, biologically useful insights into the sampling protocols underpinning these data sets. Sequence sampling times provide a rich source of information for coalescent inference that will become increasingly important as sequence collection intensifies and becomes more formalized.


Prison health care: is contracting out healthy?
US prison health care has recently been in the news and in the courts. A particular issue is whether prisons should contract out for health care. Contracting out has been growing over the past few decades. The stated motivation for this change ranges from a desire to improve the prison healthcare system, sometimes in response to a court mandate, to a desire to reduce costs. This study is a first attempt to quantify the impact of this change on inmate health. As morbidity measures are not readily obtainable, we focus on mortality. More specifically, we use a panel of state prisons from 1979 to 1990 and a fixed effects Poisson model to estimate the change in mortality associated with increase in the percentage of medical personnel employed under contract. In contrast to the first stated aim of contracting, we find that a 13% increase in percentage of medical personnel employed under contract increases mortality by 1.3%.


Risk factors profile of young and older patients with myocardial infarction.
Myocardial infarction (MI) among young adults (<45 years) represents a considerable proportion of the total heart attack incidents. The underlying pathophysiologic characteristics, atherosclerotic plaque features, and risk factors profile differ between young and older patients with MI. This review article discusses the main differences between the younger and elderly MI patients as well as the different pathogenic mechanisms underlying the development of MI in the younger. Young patients with MI often have eccentric atherosclerotic plaques with inflammatory features but fewer lesions, and are more likely to be smokers, obese, and have poor lifestyle, such as inactivity and alcohol intake. Compared to older MI patients, younger are more likely to be men, have familial-combined hyperlipidaemia and increased levels of lipoprotein-a. In addition, MI in younger patients may be related to use of cannabis, cocaine use, and androgenic anabolic steroids. Genomic differences especially in the pathways of coagulation and lipid metabolism have also been identified between young and older patients with MI. Better understanding of the risk factors and the anatomic and pathophysiologic processes in young adults can improve MI prevention and treatment strategies in this patient group. Awareness could help identify young subjects at increased risk and guide primary prevention strategies. Additional studies focusing on gene pathways related to lipid metabolism, inflammation, and coagulation are needed.


Illumination invariant hyperspectral image unmixing based on a digital surface model.
Although many spectral unmixing models have been developed to address spectral variability caused by variable incident illuminations, the mechanism of the spectral variability is still unclear. This paper proposes an unmixing model, named illumination invariant spectral unmixing (IISU). IISU makes the first attempt to use the radiance hyperspectral data and a LiDAR-derived digital surface model (DSM) in order to physically explain variable illuminations and shadows in the unmixing framework. Incident angles, sky factors, visibility from the sun derived from the LiDAR-derived DSM support the explicit explanation of endmember variability in the unmixing process from radiance perspective. The proposed model was efficiently solved by a straightforward optimization procedure. The unmixing results showed that the other state-of-the-art unmixing models did not work well especially in the shaded pixels. On the other hand, the proposed model estimated more accurate abundances and shadow compensated reflectance than the existing models.


Profiling Ssb-Nascent Chain Interactions Reveals Principles of Hsp70-Assisted Folding.
The yeast Hsp70 chaperone Ssb interacts with ribosomes and nascent polypeptides to assist protein folding. To reveal its working principle, we determined the nascent chain-binding pattern of Ssb at near-residue resolution by in vivo selective ribosome profiling. Ssb associates broadly with cytosolic, nuclear, and hitherto unknown substrate classes of mitochondrial and endoplasmic reticulum (ER) nascent proteins, supporting its general chaperone function. Ssb engages most substrates by multiple binding-release cycles to a degenerate sequence enriched in positively charged and aromatic amino acids. Timely association with this motif upon emergence at the ribosomal tunnel exit requires ribosome-associated complex (RAC) but not nascent polypeptide-associated complex (NAC). Ribosome footprint densities along orfs reveal faster translation at times of Ssb binding, mainly imposed by biases in mRNA secondary structure, codon usage, and Ssb action. Ssb thus employs substrate-tailored dynamic nascent chain associations to coordinate co-translational protein folding, facilitate accelerated translation, and support membrane targeting of organellar proteins.


Efficient genotype elimination via adaptive allele consolidation.
We propose the technique of Adaptive Allele Consolidation, that greatly improves the performance of the Lange-Goradia algorithm for genotype elimination in pedigrees, while still producing equivalent output. Genotype elimination consists in removing from a pedigree those genotypes that are impossible according to the Mendelian law of inheritance. This is used to find errors in genetic data and is useful as a preprocessing step in other analyses (such as linkage analysis or haplotype imputation). The problem of genotype elimination is intrinsically combinatorial, and Allele Consolidation is an existing technique where several alleles are replaced by a single "lumped" allele in order to reduce the number of combinations of genotypes that have to be considered, possibly at the expense of precision. In existing Allele Consolidation techniques, alleles are lumped once and for all before performing genotype elimination. The idea of Adaptive Allele Consolidation is to dynamically change the set of alleles that are lumped together during the execution of the Lange-Goradia algorithm, so that both high performance and precision are achieved. We have implemented the technique in a tool called Celer and evaluated it on a large set of scenarios, with good results.


Racial or ethnic differences in allele frequencies of single-nucleotide polymorphisms in the methylenetetrahydrofolate reductase gene and their influence on response to methotrexate in rheumatoid arthritis.
The anti-folate drug methotrexate (MTX) is commonly used to treat rheumatoid arthritis. To determine the allele frequencies of five common coding single-nucleotide polymorphisms (SNPs) in the methylenetetrahydrofolate reductase (MTHFR) gene in African-Americans and Caucasians with rheumatoid arthritis and controls to assess whether there are differences in allele frequencies among these ethnic or racial groups and whether these SNPs differentially affect the efficacy or toxicity of MTX. Allele frequencies in the 677, 1298 and 3 additional SNPs in the MTHFR coding region in 223 (193 Caucasians and 30 African-Americans) patients with rheumatoid arthritis who previously participated in one of two prospective clinical trials were characterised, and genotypes were correlated with the efficacy and toxicity of MTX. Another 308 subjects with rheumatoid arthritis who participated in observational studies, one group predominantly Caucasian and the other African-American, as well as 103 normal controls (53 African-Americans and 50 Caucasians) were used to characterise allele frequencies of these SNPs and their associated haplotypes. Significantly different allele frequencies were seen in three of the five SNPs and haplotype frequencies between Caucasians and African-Americans. Allele frequencies were similar between patients with rheumatoid arthritis and controls of the same racial or ethnic group. Frequencies of the rs4846051C, 677T and 1298C alleles were 0.33, 0.11 and 0.13, respectively, among African-Americans with rheumatoid arthritis. Among Caucasians with rheumatoid arthritis, these allele frequencies were 0.08 (p<0.001 compared with African-Americans with rheumatoid arthritis), 0.30 (p = 0.002) and 0.34 (p<0.001), respectively. There was no association between SNP alleles or haplotypes and response to MTX as measured by the mean change in the 28-joint Disease Activity Score from baseline values. In Caucasians, the 1298 A (major) allele was associated with a significant increase in MTX-related adverse events characteristic of a recessive genetic effect (odds ratio 15.86, 95% confidence interval 1.51 to 167.01; p = 0.021), confirming previous reports. There was an association between scores of MTX toxicity and the rs4846051 C allele, and haplotypes containing this allele, in African-Americans, but not in Caucasians. : These results, although preliminary, highlight racial or ethnic differences in frequencies of common MTHFR SNPs. The MTHFR 1298 A and the rs4846051 C alleles were associated with MTX-related adverse events in Caucasians and African-Americans, respectively, but these findings should be replicated in larger studies. The rs4846051 SNP, which is far more common in African-Americans than in Caucasians, can also be proved to be a useful ancestry informative marker in future studies on genetic admixture.


Fat tissue, aging, and cellular senescence.
Fat tissue, frequently the largest organ in humans, is at the nexus of mechanisms involved in longevity and age-related metabolic dysfunction. Fat distribution and function change dramatically throughout life. Obesity is associated with accelerated onset of diseases common in old age, while fat ablation and certain mutations affecting fat increase life span. Fat cells turn over throughout the life span. Fat cell progenitors, preadipocytes, are abundant, closely related to macrophages, and dysdifferentiate in old age, switching into a pro-inflammatory, tissue-remodeling, senescent-like state. Other mesenchymal progenitors also can acquire a pro-inflammatory, adipocyte-like phenotype with aging. We propose a hypothetical model in which cellular stress and preadipocyte overutilization with aging induce cellular senescence, leading to impaired adipogenesis, failure to sequester lipotoxic fatty acids, inflammatory cytokine and chemokine generation, and innate and adaptive immune response activation. These pro-inflammatory processes may amplify each other and have systemic consequences. This model is consistent with recent concepts about cellular senescence as a stress-responsive, adaptive phenotype that develops through multiple stages, including major metabolic and secretory readjustments, which can spread from cell to cell and can occur at any point during life. Senescence could be an alternative cell fate that develops in response to injury or metabolic dysfunction and might occur in nondividing as well as dividing cells. Consistent with this, a senescent-like state can develop in preadipocytes and fat cells from young obese individuals. Senescent, pro-inflammatory cells in fat could have profound clinical consequences because of the large size of the fat organ and its central metabolic role.


Sleep deprivation predisposes gujarati Indian adolescents to obesity.
Recent studies on various populations indicate that lack of sleep is one of the potential risk factors predisposing the youth to obesity. Since there is a significant rise in obesity among Indian youth and because research indicating the role of sleep in development of obesity among Indian population is scant, the current study was undertaken to assess the effect of sleep duration on adiposity among Gujarati Indian adolescents. A randomized cross-sectional study was done on 489 voluntarily participating Indian adolescents in the age group of 16-19 years. Participants were grouped into two categories 1). Adequate Sleep Duration at Night (more than seven hours, ASDN) and 2) Inadequate Sleep Duration at Night (less than seven hours, IASDN) as reported by the participants. The participants were later assessed for adiposity in terms of BMI, BF %, FM, FMI and waist circumference, meal frequency per day and physical activity status. In both boys as well as girls, the BMI, BF%, FM and FMI were significantly lower in the ASDN group than the IASDN group. However, there was an insignificant difference in the meal frequency and physical activity status between the ASDN and IASDN group. Inadequate sleep duration increases adiposity among Gujarati Indian adolescents but further studies are required to find out the mechanisms through which sleep affects adiposity in this population.


Acute Effect of Ankle Kinesio™ Taping on Lower-Limb Biomechanics During Single-Legged Drop Landing.
Chronic ankle instability is documented to be followed by a recurrence of giving away episodes due to impairments in mechanical support. The application of ankle Kinesiotaping (KT) as a therapeutic intervention has been increasingly raised among athletes and physiotherapists. This study aimed to investigate the impacts of ankle KT on the lower-limb kinematics, kinetics, dynamic balance, and muscle activity of college athletes with chronic ankle instability. A crossover study design. Twenty-eight college athletes with chronic ankle sprain (11 females and 17 males, 23.46 [2.65] y, 175.36 [11.49] cm, 70.12 [14.11] kg) participated in this study. The participants executed 3 single-leg drop landings under nontaped and ankle Kinesio-taped conditions. Ankle, knee, and hip kinematics, kinetics, and dynamic balance status and the lateral gastrocnemius, medial gastrocnemius, tibialis anterior, and peroneus longus muscle activity were recorded and analyzed. The application of ankle KT decreased ankle joint range of motion (P = .039) and angular velocities (P = .044) in the sagittal plane, ground reaction force rate of loading (P = .019), and mediolateral time to stability (P = .035). The lateral gastrocnemius (0.002) and peroneus longus (0.046) activity amplitudes also experienced a significant decrease after initial ground contact when the participants' ankles were taped, while the application of ankle KT resulted in an increase in the peroneus longus (0.014) activity amplitudes before initial ground contact. Ankle lateral supports provided by KT potentially decreases mechanical stresses applied to the lower limbs, aids in dynamic balance, and lowers calf muscle energy consumption; therefore, it could be offered as a suitable supportive means for acute usage in athletes with chronic ankle instability.


Concave reflective SU-8 photoresist gratings for flat-field integrated spectrometers.
On-chip spectrometry will play a leading role in the development of micro-optofluidic systems for analytical chemistry. Integrated spectrometers fabricated using a polymer-on-silicon platform have been designed, fabricated, and characterized. Reflective grating designs have been implemented using a recursive algorithm to calculate the facet positions as described by McGreer [Appl. Opt. 35, 5904 (1996)]. It is shown that the free spectral range, the output focal plane geometry, and the linear dispersion can be selected with a high degree of control independently of the chosen grating order. The polymer-on-silicon platform facilitates integration with microfluidic circuits and cost-efficient batch fabrication.


Splenic marginal zone lymphoma: a prognostic model for clinical use.
The Integruppo Italiano Linfomi (IIL) carried out a study to assess the outcomes of splenic marginal zone lymphoma and to identify prognostic factors in 309 patients. The 5-year cause-specific survival (CSS) rate was 76%. In univariate analysis, the parameters predictive of shorter CSS were hemoglobin levels below 12 g/dL (P < .001), albumin levels below 3.5 g/dL (P = .001), International Prognostic Index (IPI) scores of 2 to 3 (P < .001), lactate dehydrogenase (LDH) levels above normal (P < .001), age older than 60 years (P = .01), platelet counts below 100,000/microL (P = .04), HbsAg-positivity (P = .01), and no splenectomy at diagnosis (P = .006). Values that maintained a negative influence on CSS in multivariate analysis were hemoglobin level less than 12 g/dL, LDH level greater than normal, and albumin level less than 3.5 g/dL. Using these 3 variables, we grouped patients into 3 prognostic categories: low-risk group (41%) with no adverse factors, intermediate-risk group (34%) with one adverse factor, and high-risk group (25%) with 2 or 3 adverse factors. The 5-year CSS rate was 88% for the low-risk group, 73% for the intermediate-risk group, and 50% for the high-risk group. The cause-specific mortality rate (x 1000 person-years) was 20 for the low-risk group, 47 for the intermediate-risk group, and 174 for the high-risk group. This latter group accounted for 54% of all lymphoma-related deaths. In conclusion, with the use of readily available factors, this prognostic index may be an effective tool for evaluating the need for treatment and the intensity of therapy in an individual patient.


Mindfulness training improves attentional task performance in incarcerated youth: a group randomized controlled intervention trial.
We investigated the impact of cognitive behavioral therapy and mindfulness training (CBT/MT) on attentional task performance in incarcerated adolescents. Attention is a cognitive system necessary for managing cognitive demands and regulating emotions. Yet persistent and intensive demands, such as those experienced during high-stress intervals like incarceration and the events leading to incarceration, may deplete attention resulting in cognitive failures, emotional disturbances, and impulsive behavior. We hypothesized that CBT/MT may mitigate these deleterious effects of high stress and protect against degradation in attention over the high-stress interval of incarceration. Using a quasi-experimental, group randomized controlled trial design, we randomly assigned dormitories of incarcerated youth, ages 16-18, to a CBT/MT intervention (youth n = 147) or an active control intervention (youth n = 117). Both arms received approximately 750 min of intervention in a small-group setting over a 3-5 week period. Youth in the CBT/MT arm also logged the amount of out-of-session time spent practicing MT exercises. The Attention Network Test was used to index attentional task performance at baseline and 4 months post-baseline. Overall, task performance degraded over time in all participants. The magnitude of performance degradation was significantly less in the CBT/MT vs. control arm. Further, within the CBT/MT arm, performance degraded over time in those with no outside-of-class practice time, but remained stable over time in those who practiced mindfulness exercises outside of the session meetings. Thus, these findings suggest that sufficient CBT/MT practice may protect against functional attentional impairments associated with high-stress intervals.


Ipsilesional versus contralesional postural deficits induced by unilateral brain trauma: a side reversal by opioid mechanism.
Unilateral traumatic brain injury and stroke result in asymmetric postural and motor deficits including contralateral hemiplegia and hemiparesis. In animals, a localized unilateral brain injury recapitulates the human upper motor neuron syndrome in the formation of hindlimb postural asymmetry with contralesional limb flexion and the asymmetry of hindlimb nociceptive withdrawal reflexes. The current view is that these effects are developed due to aberrant activity of motor pathways that descend from the brain into the spinal cord. These pathways and their target spinal circuits may be regulated by local neurohormonal systems that may also mediate effects of brain injury. Here, we evaluate if a unilateral traumatic brain injury induces hindlimb postural asymmetry, a model of postural deficits, and if this asymmetry is spinally encoded and mediated by the endogenous opioid system in rats. A unilateral right-sided controlled cortical impact, a model of clinical focal traumatic brain injury was centred over the sensorimotor cortex and was observed to induce hindlimb postural asymmetry with contralateral limb flexion. The asymmetry persisted after complete spinal cord transection, implicating local neurocircuitry in the development of the deficits. Administration of the general opioid antagonist naloxone and μ-antagonist β-funaltrexamine blocked the formation of postural asymmetry. Surprisingly, κ-antagonists nor-binaltorphimine and LY2444296 did not affect the asymmetry magnitude but reversed the flexion side; instead of contralesional (left) hindlimb flexion the ipsilesional (right) limb was flexed. The postural effects of the right-side cortical injury were mimicked in animals with intact brain via intrathecal administration of the opioid κ-agonist (2)-(trans)-3,4-Dichloro-N-methyl-N-[2-(1-pyrrolidiny)-cyclohexyl]benzeneacetamide that induced hindlimb postural asymmetry with left limb flexion. The δ-antagonist naltrindole produced no effect on the contralesional (left) flexion but inhibited the formation of the ipsilesional (right) limb flexion in brain-injured rats that were treated with κ-antagonist. The effects of the antagonists were evident before and after spinal cord transection. We concluded that the focal traumatic brain injury-induced postural asymmetry was encoded at the spinal level, and was blocked or its side was reversed by administration of opioid antagonists. The findings suggest that the balance in activity of the mirror symmetric spinal neural circuits regulating contraction of the left and right hindlimb muscles is controlled by different subtypes of opioid receptors; and that this equilibrium is impaired after unilateral brain trauma through side-specific opioid mechanism.


Long-pulse generation with a stable-relaxation-oscillation Nd:YLF laser.
A simple technique for long-pulse (0.2-2 microsec) generation with neodymium lasers has been demonstrated. Under the proper conditions, a normal-mode oscillator, operated in a single transverse mode, produces well-defined relaxation oscillations from which a single subpulse can be isolated for amplification. The characteristic subpulse temporal profile is ideal for saturated amplification without pulse shortening. Data are presented for a Nd:YLF system consisting of an oscillator followed by a 64-mm-long amplifier. Pulse energies in excess of 100 mJ were achieved with a small-signal gain of 630.


Incidence of childhood cancer in twins.
The incidence of childhood cancer in twins was evaluated by linking a roster of 30,925 twins born in Connecticut (United States) between 1930 and 1969 with the Connecticut Tumor Registry. Cancer, exclusive of nonmelanoma skin cancer, was identified in 19 females and 12 males under 15 years of age. The incidence rate among twins was 7.9 cancers per 100,000 person-years (PY) overall, and 9.7 and 6.1 per 100,000 PYs for females and males, respectively. Four of 13 leukemias occurred in two female twin pairs, representing concordance rates of 18 percent overall and 29 percent for like-sex pairs, which are somewhat higher than values reported previously. The number of cancers expected was computed on the assumption that twins experienced the same sex-, age-, and calendar time-specific cancer rates as recorded for all Connecticut-born children. Because active follow-up of individuals was not conducted, an adjustment to person-years of observation was made to account for childhood mortality, including the high perinatal mortality characteristic of twins. Childhood cancer was 30 percent less frequent than expected (standardized incidence ratio [SIR] = 0.7; 95 percent confidence interval [CI] = 0.5-0.9), a deficit that is marginally greater than those found in previous studies. Both leukemia (SIR = 0.8; CI = 0.4-1.4), and all other cancers combined (SIR = 0.6; CI = 0.3-0.9) occurred less often than expected. The deficit was greater among males (SIR = 0.5; CI = 0.2-0.8) than among females (SIR = 0.9; CI = 0.5-1.4) and was especially pronounced among males younger than five years (SIR = 0.2; CI = 0.0-0.7). The data support the view that twins, particularly male twins, have a lower risk of childhood cancer than single-born children. Any added risk for twins associated with their greater frequency of exposure to prenatal X-rays appears to have been insufficient to offset an 'effect' of twinning per se. Possible explanations for this finding include (i) the low birthweight distribution of twins, or (ii) selective early mortality of twin fetuses or neonates who would otherwise have developed a clinical cancer.


Mutations in a novel gene, NPHP3, cause adolescent nephronophthisis, tapeto-retinal degeneration and hepatic fibrosis.
Nephronophthisis (NPHP), a group of autosomal recessive cystic kidney disorders, is the most common genetic cause of progressive renal failure in children and young adults. NPHP may be associated with Leber congenital amaurosis, tapeto-retinal degeneration, cerebellar ataxia, cone-shaped epiphyses, congenital oculomotor apraxia and hepatic fibrosis. Loci associated with an infantile type of NPHP on 9q22-q31 (NPHP2), juvenile types of NPHP on chromosomes 2q12-q13 (NPHP1) and 1p36 (NPHP4) and an adolescent type of NPHP on 3q21-q22 (NPHP3) have been mapped. NPHP1 and NPHP4 have been identified, and interaction of the respective encoded proteins nephrocystin and nephrocystin-4 has been shown. Here we report the identification of NPHP3, encoding a novel 1,330-amino acid protein that interacts with nephrocystin. We describe mutations in NPHP3 in families with isolated NPHP and in families with NPHP with associated hepatic fibrosis or tapeto-retinal degeneration. We show that the mouse ortholog Nphp3 is expressed in the node, kidney tubules, retina, respiratory epithelium, liver, biliary tract and neural tissues. In addition, we show that a homozygous missense mutation in Nphp3 is probably responsible for the polycystic kidney disease (pcy) mouse phenotype. Interventional studies in the pcy mouse have shown beneficial effects by modification of protein intake and administration of methylprednisolone, suggesting therapeutic strategies for treating individuals with NPHP3.


Enzymatic Cascades for Tailored 13C6 and 15N Enriched Human Milk Oligosaccharides.
Several health benefits, associated with human milk oligosaccharides (HMOS), have been revealed in the last decades. Further progress, however, requires not only the establishment of a simple "routine" method for absolute quantification of complex HMOS mixtures but also the development of novel synthesis strategies to improve access to tailored HMOS. Here, we introduce a combination of salvage-like nucleotide sugar-producing enzyme cascades with Leloir-glycosyltransferases in a sequential pattern for the convenient tailoring of stable isotope-labeled HMOS. We demonstrate the assembly of [13C6]galactose into lacto-N- and lacto-N-neo-type HMOS structures up to octaoses. Further, we present the enzymatic production of UDP-[15N]GlcNAc and its application for the enzymatic synthesis of [13C6/15N]lacto-N-neo-tetraose for the first time. An exemplary application was selected-analysis of tetraose in complex biological mixtures-to show the potential of tailored stable isotope reference standards for the mass spectrometry-based quantification, using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) as a fast and straightforward method for absolute quantification of HMOS. Together with the newly available well-defined tailored isotopic HMOS, this can make a crucial contribution to prospective research aiming for a more profound understanding of HMOS structure-function relations.


Structural and thermodynamic insight into phenylalanine hydroxylase from the human pathogen Legionella pneumophila.
Phenylalanine hydroxylase from Legionella pneumophila (lpPAH) has a major functional role in the synthesis of the pigment pyomelanin, which is a potential virulence factor. We present here the crystal structure of lpPAH, which is a dimeric enzyme that shows high thermostability, with a midpoint denaturation temperature of 79 °C, and low substrate affinity. The structure revealed a dimerization motif that includes ionic interactions and a hydrophobic core, composed of both β-structure and a C-terminal region, with the specific residues (P255, P256, Y257 and F258) interacting with the same residues from the adjacent subunit within the dimer. This unique dimerization interface, together with a number of aromatic clusters, appears to contribute to the high thermal stability of lpPAH. The crystal structure also explains the increased aggregation of the enzyme in the presence of salt. Moreover, the low affinity for substrate l-Phe could be explained from three consecutive glycine residues (G181, 182, 183) located at the substrate-binding site. This is the first structure of a dimeric bacterial PAH and provides a framework for interpreting the molecular and kinetic properties of lpPAH and for further investigating the regulation of the enzyme.


Mitochondrial function in Leydig cell steroidogenesis.
The first and rate-limiting step in the biosynthesis of steroid hormones is the transfer of cholesterol into mitochondria, which is facilitated by the steroidogenic acute regulatory (StAR) protein. Recent studies of Leydig cell function have focused on the molecular events controlling steroidogenesis; however, few studies have examined the importance of the mitochondria. The purpose of this investigation was to determine which aspects of mitochondrial function are necessary for Leydig cell steroidogenesis. MA-10 tumor Leydig cells were treated with 8-bromo-cAMP (cAMP) and site-specific mitochondrial disrupters, pro-oxidants, and their effects on progesterone synthesis, StAR expression, mitochondrial membrane potential (delta psi(m)) and ATP synthesis were determined. Dissipating delta psi(m) with CCCP inhibited progesterone synthesis, even in the presence of newly synthesized StAR protein. The electron transport inhibitor antimycin A significantly reduced cellular ATP, inhibited steroidogenesis, and reduced StAR protein expression. The F0/F1 ATPase inhibitor oligomycin reduced cellular ATP and inhibited progesterone synthesis and StAR protein expression, but had no effect on delta psi(m). Disruption of pH with nigericin significantly reduced progesterone production and StAR protein, but had minimal effects on delta psi(m). Sodium arsenite at low concentrations inhibited StAR protein but not mRNA expression and inhibited progesterone without disrupting delta psi(m). The mitochondrial Ca2+ inhibitor Ru360 also inhibited StAR protein expression. These results demonstrate that delta psi(m), ATP synthesis, delta pH and [Ca2+]mt are all required for steroid biosynthesis, and that mitochondria are sensitive to oxidative stress. These results suggest that mitochondria must be energized, polarized, and actively respiring to support Leydig cell steroidogenesis and alterations in the state of mitochondria may be involved in regulating steroid biosynthesis.


Internet Based Obesity Prevention Program for Thai School Children- A Randomized Control Trial.
Internet based obesity prevention program is one approach in learning strategies to improve healthy behaviour. It has been advocated as one strategy to address the rising prevalence of childhood obesity; however, their efficacy is not seen consistently. The purpose of this study was to assess the efficacy of internet based obesity prevention program in Thai school children. Healthy children studying in public schools in one township of central Thailand were randomly assigned to either the intervention (internet based) program or the control group. Anthropometric characteristics were recorded at baseline and for the next four following months at monthly intervals. Changes in the percentage of overweight/obese children and changes in BMI at the end of study were considered as the primary and secondary outcome, respectively. A total of 217 children, mean age of 10.7 years, were included into the final analysis. Baseline anthropometric parameters and percentages of overweight/obesity were not significantly different between groups. At the end of the study, the control group had a higher percentage of overweight/obesity than the intervention group (56.6% vs. 39.6%, respectively; p-value=0.009). Children in the control group had a significantly higher increase in net BMI gains than those in the intervention group (1.24kg/m2 vs. 0.40kg/m2, p-value=0.027). The intervention group had no changes in BMI z-score (-0.001, 95%CI -0.19 to 0.18, p-value=0.988), contrary to those in the control group, which had significant gain of BMI z-score at the end of study (0.45, 95%CI 0.27 to 0.63, p-value<0.001). Internet the based obesity prevention program was effective in modifying anthropometric outcome and helped to address the rising prevalence of overweight and obese status in Thai children.


Construction of a replication-competent murine retrovirus vector expressing the human immunodeficiency virus type 1 tat transactivator protein.
A replication-competent Akv murine leukemia virus-based vector encoding the human immunodeficiency virus tat cDNA under control of the simian virus 40 early promoter sequences was constructed. The simian virus 40 tat sequences were placed within the U3 region of the 3' long terminal repeat. The resulting virus, derived by transfection, replicated efficiently in mouse NIH 3T3 cells and maintained the tat cDNA insert. It has been suggested that Tat function requires the presence of a human-specific cofactor, which is absent in murine cells. However, infection of murine cells with the Akv virus encoding tat resulted in significant transactivation of a human immunodeficiency virus long terminal repeat-driven reporter gene, indicating that human cofactors are not always required for Tat function. The vector system described may be useful for introduction of foreign genes in vivo and in whole animals when virus spread is required for efficient infection and levels of gene expression.


[Circulating interferon in patients with systemic lupus erythematosus. A prospective study].
The serum levels of interferon (IFN) were measured in 31 patients with systemic lupus erythematosus (SLE). Among the 31 patients, 8 (25%) showed IFN in the sera: in 4 patients IFN titers of 16 U/ml, and in the other 4 patients titers of greater than or equal to 32 U/ml became detectable. Whereas antibodies to native DNA (nDNA) were present in 6 of the 8 IFN positive patients, anti-nDNA antibodies were only positive in 6 of the 23 IFN negative individuals (p less than 0.002). Eight months after the first search for IFN in the patients' sera, 6 of the 8 IFN positive patients showed reduced IFN serum levels and one each had an increased or stable IFN titer. A good correlation between IFN titers and both the anti-nDNA antibody titers and the disease activity was observed. However, there were patients with active disease, high anti-nDNA titers but no IFN in the serum.


Dynamic double layer effects on ac-induced dipoles of dielectric nanocolloids.
Normal and tangential surface ionic currents around low-permittivity nanocolloids with surface charges are shown to produce three different conductive mechanisms for ac-induced dipoles, all involving dynamic space charge accumulation at the double layerbulk interface with a conductivity jump. However, the distinct capacitor dimensions and diffusive contributions produce three disparate crossover frequencies at which the induced dipole reverses direction relative to the bulk field. A highly conducting collapsed diffuse layer, with bulk ion mobility, renders the particle conductive and produces an ionic strength independent crossover frequency for weak electrolytes. A precipitous drop in crossover frequency occurs at high ionic strengths when charging occurs only at the poles through field focusing around the insulated colloid. A peculiar maximum in crossover frequency exists between these two asymptotes for colloids smaller than a critical size when normal charging of the diffuse layer occurs over the entire surface. The crossover frequency data for latex nanocolloids of various sizes in different electrolytes of wide ranging ionic strengths are collapsed by explicit theoretical predictions without empirical parameters.


Role of ficolin-A and lectin complement pathway in the innate defense against pathogenic Aspergillus species.
Aspergillus species are saprophytic molds causing life-threatening invasive fungal infections in the immunocompromised host. Innate immune recognition, in particular, the mechanisms of opsonization and complement activation, has been reported to be an integral part of the defense against fungi. We have shown that the complement component ficolin-A significantly binds to Aspergillus conidia and hyphae in a concentration-dependent manner and was inhibited by N-acetylglucosamine and N-acetylgalactosamine. Calcium-independent binding to Aspergillus fumigatus and A. terreus was observed, but binding to A. flavus and A. niger was calcium dependent. Ficolin-A binding to conidia was increased under low-pH conditions, and opsonization led to enhanced binding of conidia to A549 airway epithelial cells. In investigations of the lectin pathway of complement activation, ficolin-A-opsonized conidia did not lead to lectin pathway-specific C4 deposition. In contrast, the collectin mannose binding lectin C (MBL-C) but not MBL-A led to efficient lectin pathway activation on A. fumigatus in the absence of ficolin-A. In addition, ficolin-A opsonization led to a modulation of the proinflammatory cytokine interleukin-8. We conclude that ficolin-A may play an important role in the innate defense against Aspergillus by opsonizing conidia, immobilizing this fungus through enhanced adherence to epithelial cells and modulation of inflammation. However, it appears that other immune pattern recognition molecules, i.e., those of the collectin MBL-C, are involved in the Aspergillus-lectin complement pathway activation rather than ficolin-A.


Long noncoding RNA expression signatures of bladder cancer revealed by microarray.
Dysregulation of long noncoding RNAs (lncRNAs) has been regarded as a primary feature of several human cancers. However, the genome-wide expression and functional significance of lncRNAs in bladder cancer remains unclear. The aim of this study was to identify aberrantly expressed lncRNAs that may play an important role in contributing to bladder cancer pathogenesis. In this study, we described lncRNAs profiles in four pairs of human bladder cancer and matched normal bladder tissues by microarray. We finally determined 3,324 differentially expressed human lncRNAs and 2,120 differentially expressed mRNAs (≥2-fold change). A total of 110 lncRNAs were significantly differentially expressed between the tumor and the control groups (≥8-fold change). Four lncRNAs (TNXA, CTA-134P22.2, CTC-276P9.1 and KRT19P3) were selected for further confirmation of microarray results using quantitative PCR (qPCR), and a strong correlation was identified between the qPCR results and microarray data. We also observed that numerous lncRNA expression levels were significantly correlated with the expression of tens of protein coding genes by construction of the lncRNA-mRNA co-expression network. Kyoto Encyclopedia of Genes and Genomes annotation showed a significant association with p53, bladder cancer, cell cycle and propanoate metabolism pathway gene expression in the bladder cancer group compared with the normal tissue group, indicating that deregulated lncRNAs may act by regulating protein-coding genes in these pathways. We demonstrated the expression profiles of human lncRNAs in bladder cancer by microarray. We identified a collection of aberrantly expressed lncRNAs in bladder cancer compared with matched normal tissue. It is likely that these deregulated lncRNAs play a key or partial role in the development and/or progression of bladder cancer.


Sedation in palliative care - a critical analysis of 7 years experience.
BACKGROUND: The administration of sedatives in terminally ill patients becomes an increasingly feasible medical option in end-of-life care. However, sedation for intractable distress has raised considerable medical and ethical concerns. In our study we provide a critical analysis of seven years experience with the application of sedation in the final phase of life in our palliative care unit. METHODS: Medical records of 548 patients, who died in the Palliative Care Unit of GK Havelhoehe between 1995-2002, were retrospectively analysed with regard to sedation in the last 48 hrs of life. The parameters of investigation included indication, choice and kind of sedation, prevalence of intolerable symptoms, patients' requests for sedation, state of consciousness and communication abilities during sedation. Critical evaluation included a comparison of the period between 1995-1999 and 2000-2002. RESULTS: 14.6% (n = 80) of the patients in palliative care had sedation given by the intravenous route in the last 48 hrs of their life according to internal guidelines. The annual frequency to apply sedation increased continuously from 7% in 1995 to 19% in 2002. Main indications shifted from refractory control of physical symptoms (dyspnoea, gastrointestinal, pain, bleeding and agitated delirium) to more psychological distress (panic-stricken fear, severe depression, refractory insomnia and other forms of affective decompensation). Patients' and relatives' requests for sedation in the final phase were significantly more frequent during the period 2000-2002. CONCLUSION: Sedation in the terminal or final phase of life plays an increasing role in the management of intractable physical and psychological distress. Ethical concerns are raised by patients' requests and needs on the one hand, and the physicians' self-understanding on the other hand. Hence, ethically acceptable criteria and guidelines for the decision making are needed with special regard to the nature of refractory and intolerable symptoms, patients' informed consent and personal needs, the goals and aims of medical sedation in end-of-life care.


Improved estimation of subject-level functional connectivity using full and partial correlation with empirical Bayes shrinkage.
Reliability of subject-level resting-state functional connectivity (FC) is determined in part by the statistical techniques employed in its estimation. Methods that pool information across subjects to inform estimation of subject-level effects (e.g., Bayesian approaches) have been shown to enhance reliability of subject-level FC. However, fully Bayesian approaches are computationally demanding, while empirical Bayesian approaches typically rely on using repeated measures to estimate the variance components in the model. Here, we avoid the need for repeated measures by proposing a novel measurement error model for FC describing the different sources of variance and error, which we use to perform empirical Bayes shrinkage of subject-level FC towards the group average. In addition, since the traditional intra-class correlation coefficient (ICC) is inappropriate for biased estimates, we propose a new reliability measure denoted the mean squared error intra-class correlation coefficient (ICCMSE) to properly assess the reliability of the resulting (biased) estimates. We apply the proposed techniques to test-retest resting-state fMRI data on 461 subjects from the Human Connectome Project to estimate connectivity between 100 regions identified through independent components analysis (ICA). We consider both correlation and partial correlation as the measure of FC and assess the benefit of shrinkage for each measure, as well as the effects of scan duration. We find that shrinkage estimates of subject-level FC exhibit substantially greater reliability than traditional estimates across various scan durations, even for the most reliable connections and regardless of connectivity measure. Additionally, we find partial correlation reliability to be highly sensitive to the choice of penalty term, and to be generally worse than that of full correlations except for certain connections and a narrow range of penalty values. This suggests that the penalty needs to be chosen carefully when using partial correlations.


The properties of gold nanospheres studied with dark field optical trapping.
We demonstrate trapping and characterization of multiple gold nanospheres with a setup composed of dark field imaging and optical tweezers. The number of trapped nanospheres is quantified by the overall dark-field scattering intensity. The spectra of the scattering intensity show that there is no interparticle coupling among trapped nanospheres when the density of nanospheres in the trap is low enough (less than 10 particles), while the density of nanosphere increases the interparticle coupling of nanospheres becomes obvious. In addition, the trapping potential of a single gold nanosphere is obtained by trapping an ensemble of gold nanospheres.


Relation of prediagnostic serum estrogen and androgen levels to breast cancer risk.
To evaluate the relation of serum sex hormones to breast cancer risk, we conducted a prospective nested case-control study using the Breast Cancer Serum Bank (Columbia, MO). This bank included serum from 3375 postmenopausal women free of cancer and not taking replacement estrogens when they donated blood between 1977 and 1987. Of these, 71 were diagnosed subsequently with breast cancer. For each case, two women alive and free of cancer at the age of the case's diagnosis and matched to the case on age and on date and time of day of blood collection were selected as controls. The median age of subjects at blood collection was 62 years, and the time from blood collection to diagnosis ranged from less than 1 to 9.5 years, with a median of 2.9 years. Postmenopausal women with elevated serum levels of total and non-sex hormone-binding globulin-bound E2 were at an increased risk of developing breast cancer. For non-sex hormone-binding globulin-bound E2, risks were elevated 4-5 fold for women in the upper three quartiles relative to those in the lowest quartile. Although breast cancer was not related to estrone or estrone sulfate concentration, the ratio of estrone sulfate to estrone was significantly inversely associated with risk, suggesting that women who develop breast cancer may be less able to metabolize estrone to its less active form. Serum testosterone was significantly positively associated with postmenopausal breast cancer; the relative risk for women in the highest versus the lowest quartile was 6.2 (95% confidence interval, 2.0-19.0). Our results support the hypothesis that prediagnostic serum estrogens and androgens are related to the subsequent diagnosis of breast cancer in postmenopausal women.


Bacterial diversity in the haloalkaline Lake Elmenteita, Kenya.
Lake Elmenteita is one of the alkaline saline lakes within the Kenyan Rift valley. The lake is situated on the floor of the Kenyan Rift Valley at 1,776 m above sea level and has no direct outlet. The microbial diversity of the lake was investigated using a culture-independent approach. Five different sampling points were selected randomly within the lake. Wet sediments and water samples were collected from each sampling point. In addition, dry mud cake was collected from three points where the lake had dried. DNA was extracted from the samples and the 16S rRNA genes amplified using universal primers for Bacteria. Thirteen clone libraries were constructed using the PCR amplified 16S rRNA genes. A total of 1,663 clones were picked. Representative clones were selected using ARDRA technique for sequencing. 655 partial and non-chimeric clone sequences indicated the presence of 37 orders in the Domain Bacteria. Cyanobacteria were the most abundant clones in terms of numbers whereas members of the phylum Firmicutes group were the second in terms of numbers but the most diverse in terms of genera represented. All clones affiliated to the class Betaproteobacteria originated from DNA obtained from the water samples. Analysis using BLAST showed that 93.1% of the sequenced clones had similarity values below 98% to both cultured and as yet uncultured bacteria, resulting in 596 phylotypes. Therefore, it can be concluded that Lake Elmenteita harbours phylogenetically diverse groups of bacteria involved in complex metabolic interactions within the Lake's ecosystem.


Exploring possible DNA structures in real-time polymerase kinetics using Pacific Biosciences sequencer data.
Pausing of DNA polymerase can indicate the presence of a DNA structure that differs from the canonical double-helix. Here we detail a method to investigate how polymerase pausing in the Pacific Biosciences sequencer reads can be related to DNA sequences. The Pacific Biosciences sequencer uses optics to view a polymerase and its interaction with a single DNA molecule in real-time, offering a unique way to detect potential alternative DNA structures. We have developed a new way to examine polymerase kinetics data and relate it to the DNA sequence by using a wavelet transform of read information from the sequencer. We use this method to examine how polymerase kinetics are related to nucleotide base composition. We then examine tandem repeat sequences known for their ability to form different DNA structures: (CGG)n and (CG)n repeats which can, respectively, form G-quadruplex DNA and Z-DNA. We find pausing around the (CGG)n repeat that may indicate the presence of G-quadruplexes in some of the sequencer reads. The (CG)n repeat does not appear to cause polymerase pausing, but its kinetics signature nevertheless suggests the possibility that alternative nucleotide conformations may sometimes be present. We discuss the implications of using our method to discover DNA sequences capable of forming alternative structures. The analyses presented here can be reproduced on any Pacific Biosciences kinetics data for any DNA pattern of interest using an R package that we have made publicly available.


Development of measures of the quality of emergency department care for children using a structured panel process.
Performance measures are essential components of public reporting and quality improvement. To date, few such measures exist to provide a comprehensive assessment of the quality of emergency department services for children. Our goal was to use a systematic process to develop measures of emergency department care for children (0-19 years) that are (1) based on research evidence and expert opinion, (2) representative of a range of conditions treated in most emergency departments, (3) related to links between processes and outcomes, and (4) feasible to measure. We presented a panel of providers and managers data from emergency department use to identify common conditions across levels of patient acuity, which could be targets for quality improvement. We used a structured panel process informed by a literature review to (1) identify condition-specific links between processes of care and defined outcomes and (2) select indicators to assess these process-outcome links. We determined the feasibility of calculating these indicators using an administrative data set of emergency department visits for Ontario, Canada. The panel identified 18 clinical conditions for indicator development and 61 condition-specific links between processes of care and outcomes. After 2 rounds of ratings, the panel defined 68 specific clinical indicators for the following conditions: adolescent mental health problems, ankle injury, asthma, bronchiolitis, croup, diabetes, fever, gastroenteritis, minor head injury, neonatal jaundice, seizures, and urinary tract infections. Visits for these conditions account for 23% of all pediatric emergency department use. Using an administrative data set, we were able to calculate 19 indicators, covering 9 conditions, representing 20% of all emergency department visits by children. Using a structured panel process, data on emergency department use, and literature review, it was possible to define indicators of emergency department care for children. The feasibility of these indicators will depend on the availability of high-quality data.


Discovering and understanding oncogenic gene fusions through data intensive computational approaches.
Although gene fusions have been recognized as important drivers of cancer for decades, our understanding of the prevalence and function of gene fusions has been revolutionized by the rise of next-generation sequencing, advances in bioinformatics theory and an increasing capacity for large-scale computational biology. The computational work on gene fusions has been vastly diverse, and the present state of the literature is fragmented. It will be fruitful to merge three camps of gene fusion bioinformatics that appear to rarely cross over: (i) data-intensive computational work characterizing the molecular biology of gene fusions; (ii) development research on fusion detection tools, candidate fusion prioritization algorithms and dedicated fusion databases and (iii) clinical research that seeks to either therapeutically target fusion transcripts and proteins or leverages advances in detection tools to perform large-scale surveys of gene fusion landscapes in specific cancer types. In this review, we unify these different-yet highly complementary and symbiotic-approaches with the view that increased synergy will catalyze advancements in gene fusion identification, characterization and significance evaluation.


Insensitivity to anaesthetic agents conferred by a class of GABA(A) receptor subunit.
A common feature of general anaesthetic agents is their ability to potentiate neuronal inhibition through GABA(A) (gamma-aminobutyric acid) receptors. At concentrations relevant to clinical anaesthesia, these agents cause a dramatic stimulation of the chloride currents that are evoked by the binding of the natural ligand, GABA. Although there is widespread evidence that the sensitivity of GABA(A) receptors to anaesthetic agents is heterogeneous, the structural basis of these differences is largely unknown. Variations in subunit composition can have profound effects on the sensitivity of GABA(A) receptors to modulatory agents such as benzodiazepines. However, strict subunit specificity has not been demonstrated for the potentiating effects of anaesthetic agents. Here we describe a new class of human GABA(A) receptor subunit (epsilon) that can assemble with alpha- and beta-subunits and confer an insensitivity to the potentiating effects of intravenous anaesthetic agents. The epsilon-subunit also abolishes the normal outward rectification of recombinant receptors in which it assembles. The expression pattern of this subunit in the brain suggests a new target for manipulation of neuronal pathways within the basal ganglia.


Ab initio and experimental study on thermally degradable polycarbonates: the effect of substituents on the reaction rates.
Thermal elimination reactions on polycarbonates are investigated from both theoretical and experimental points of view, to obtain insight into the microscopic aspects that influence the reaction mechanism and rates. In particular, attention is focused on the influence of the type of substituents in the polymer chain on the reaction rates. Ab initio density functional theory calculations are performed on a series of model compound systems for the polycarbonates under study, in particular carbonates differing by the groups attached at the alpha and beta carbon atoms. Reactants, products, and transition states are optimized at the B3LYP/6-311g level of theory. The structures of the activated complex give insight into the mechanistic details of this type of E(i) elimination reactions. The C(alpha)-O bond dissociates before the C(beta)-H bond, developing some carbocation character in the transition state on the C(alpha) atom. The kinematics of the thermal decomposition reactions have been studied by means of transition state theory by construction of the microscopic partition functions. It turns out that the rates of the E(i) elimination reactions are increased by the presence of those substituents on the C(alpha) and C(beta) carbon atoms which are stabilizing the carbocation character in the transition state. In a second part, degradation temperatures have been experimentally measured for some polycarbonates through thermogravimetric analysis. It is investigated whether the relative rates of the model compound carbonate systems are representative of the behavior of the thermal degradation temperatures in polycarbonates. The study as presented here proves that ab initio calculations on small model systems, which are representative for the active area of the degradation process in polycarbonates, can provide insight into the principal ingredients that govern the reaction rates.


Topographic and age-dependent expression of heme oxygenase-1 and catalase in the human retinal pigment epithelium.
To investigate the hypothesis that there are topographic and age-related changes in the expression of heme oxygenase (HO)-1 and catalase in the RPE. Cryosections of the macula and periphery of human eyes (n = 18; aged 27-87 years) were subjected to a high-sensitivity digoxigenin (DIG)-labeled cRNA in situ hybridization protocol to determine the expression of HO-1 and catalase. The immunoreactivity of HO-1 and catalase were also investigated in the same sample set. Specimens were examined by light microscopy, and images were captured with a digital camera. The total number of RPE cells and HO-1- and catalase-labeled RPE cells was counted in each section, and the ratio of labeled RPE cells to total RPE cells was calculated in both the macular and the peripheral regions of each donor eye. There was a mosaic pattern of mRNA and protein expression of HO-1 and catalase in macular and peripheral RPE. Topographical differences in the expression of HO-1 at the mRNA level and catalase at both the mRNA and protein levels was also observed. The topographical differences between the expression of HO-1 in the macula and periphery protein were not statistically significant but showed similar trends. For HO-1, the only significant age-related decline in expression was observed in the macula and periphery. Expression of HO-1 at the protein level and that of catalase at both the mRNA and protein levels showed no significant decline with age. There is a possible age-related decline in HO-1 expression, whereas catalase expression remains unchanged with aging. Both exhibit mosaic patterns in the RPE monolayer.


Wound Healing Finally Enters the Age of Molecular Diagnostic Medicine.
Many wounds are difficult to heal because of the large, complex community of microbes present within the wound. Classical laboratory culture methods do not provide an accurate picture of the microbial interactions or representation of microorganisms within a wound. There is an inherent bias in diagnosis based upon classical culture stemming from the ability of certain organisms to thrive in culture while others are underrepresented or fail to be identified in culture altogether. Chronic wounds also contain polymicrobial infections existing as a cooperative community that is resistant to antibiotic therapy. New methods in molecular diagnostic medicine allow the identification of nearly all organisms present in a wound irrespective of the ability of these organisms to be grown in culture. Advances in DNA analyses allow absolute identification of microorganisms from very small clinical specimens. These new methods also provide a quantitative representation of all microorganisms contributing to these polymicrobial infections. Technological advances in laboratory diagnostics can significantly shorten the time required to heal chronic wounds. Identification of the genetic signatures of organisms present within a wound allows clinicians to identify and treat the primary organisms responsible for nonhealing wounds. Advanced genetic technologies targeting the specific needs of wound care patients are now accessible to all wound care clinicians.


Transmission of equine influenza virus to dogs.
Molecular and antigenic analyses of three influenza viruses isolated from outbreaks of severe respiratory disease in racing greyhounds revealed that they are closely related to H3N8 equine influenza virus. Phylogenetic analysis indicated that the canine influenza virus genomes form a monophyletic group, consistent with a single interspecies virus transfer. Molecular changes in the hemagglutinin suggested adaptive evolution in the new host. The etiologic role of this virus in respiratory disease was supported by the temporal association of rising antibody titers with disease and by experimental inoculation studies. The geographic expansion of the infection and its persistence for several years indicate efficient transmission of canine influenza virus among greyhounds. Evidence of infection in pet dogs suggests that this infection may also become enzootic in this population.


Treatment of chronic hepatitis C virus infection in Rwanda with ledipasvir-sofosbuvir (SHARED): a single-arm trial.
Limited treatment data are available for hepatitis C virus (HCV) in sub-Saharan Africa, especially for genotype 4. Our objective was to establish the safety and efficacy of ledipasvir-sofosbuvir for chronic HCV genotype 1 or 4 infection in adults in Rwanda. We did a single-arm trial to evaluate the safety and efficacy of ledipasvir-sofosbuvir in Rwandan adults with chronic HCV infection at a single study site (Rwanda Military Hospital, Kigali, Rwanda). We enrolled individuals aged 18 years or older with HCV genotype 1 or 4 infection and a plasma HCV RNA concentration of more than 1000 IU/mL at screening. All participants were given ledipasvir (90 mg) and sofosbuvir (400 mg) in a single combination tablet once daily for 12 weeks. We established HCV genotype using an Abbott platform, and HCV subtype with PCR amplification. The primary endpoint was the proportion of participants with a sustained virological response 12 weeks after therapy (SVR12). All patients enrolled in the study were included in the primary endpoint analyses. This study is registered with ClinicalTrials.gov, number NCT02964091. 300 participants were enrolled between Feb 6, 2017, and Sept 18, 2017, and the follow-up period was completed on March 1, 2018. On genotyping, 248 (83%) participants were reported as having genotype 4, four (1%) genotype 1, and 48 (16%) both genotype 1 and genotype 4. Subsequent viral sequencing showed all participants actually had genotype 4 infection with subtype 4k (134 [45%]), subtype 4r (48 [16%]), subtype 4q (42 [14%]), and subtype 4v (24 [8%]) predominating. Overall, 261 (87%, 95% CI 83-91) participants achieved SVR12. In participants with genotype 4r, SVR12 was observed in 27 (56%, 95% CI 41-71) participants versus 234 (93%, 90-96) individuals with other subtypes. There were no drug-related treatment discontinuations due to ledipasvir-sofosbuvir. The most common adverse events were hypertension (97 [32%]), headache (78 [26%]), dizziness (61 [20%]), and fatigue (56 [19%]). There were six serious adverse events; none were assessed to be due to the study drug. 296 participants had data for pill counts at week 4 and 8; 271 (92%) had 100% adherence and only one (<1%) had an adherence of less than 90%. This is the first large-scale prospective study reporting direct-acting antiviral outcomes in sub-Saharan Africa. The high adherence and treatment success without intensive support measures or highly specialised clinical providers, and lack of treatment discontinuations due to adverse events support the feasibility of HCV treatment decentralisation and scale-up in sub-Saharan Africa. Genotype 4r is uniquely expressed in this region and associated with high rates of treatment failure, suggesting a need for rigorous test-of-cure in clinical practice and consideration of the use of newer pangenotypic direct-acting antiviral regimens in this region. Gilead Sciences.


Impacts of soil and water pollution on food safety and health risks in China.
Environmental pollution and food safety are two of the most important issues of our time. Soil and water pollution, in particular, have historically impacted on food safety which represents an important threat to human health. Nowhere has that situation been more complex and challenging than in China, where a combination of pollution and an increasing food safety risk have affected a large part of the population. Water scarcity, pesticide over-application, and chemical pollutants are considered to be the most important factors impacting on food safety in China. Inadequate quantity and quality of surface water resources in China have led to the long-term use of waste-water irrigation to fulfill the water requirements for agricultural production. In some regions this has caused serious agricultural land and food pollution, especially for heavy metals. It is important, therefore, that issues threatening food safety such as combined pesticide residues and heavy metal pollution are addressed to reduce risks to human health. The increasing negative effects on food safety from water and soil pollution have put more people at risk of carcinogenic diseases, potentially contributing to 'cancer villages' which appear to correlate strongly with the main food producing areas. Currently in China, food safety policies are not integrated with soil and water pollution management policies. Here, a comprehensive map of both soil and water pollution threats to food safety in China is presented and integrated policies addressing soil and water pollution for achieving food safety are suggested to provide a holistic approach.


Functional expression of potassium channels in cardiomyocytes derived from embryonic stem cells.
Royan B(1) stem cell can be differentiated to specialized cell types including cardiomyocytes. This developmental change is accompanied with expression of various K(+) channel types. The aim of this study was to detect functional expression of K(+) currents from stem cell stage and one week and two weeks after differentiation into cardiomyocyte. Mouse stem cell derived cardiomyocytes (ES-cardiomyocytes) were isolated to single cell suspension for K(+) current recording using whole cell patch-clamp technique. The predominant depolarizing current in ES-cardiomyocytes was a tetraethylammonium (TEA) (10 mM) sensitive current which was partially blocked by nifedipine (1 μM) and attenuated by increasing concentration of EGTA (10 mM) in the pipette solution. Pharmacology and electrophysiological properties of this oscillatory sustained current very well matched with characteristics of Ca(2+) activated K(+) current. In addition there was another kind of sustained outward K(+) current which was resistance to TEA but was inhibited by 3,4-diaminopyridine. The characteristic features of this current indicate that this current was due to activation of delayed rectifier K(+) channels. RT-PCR study also confirmed expression of these two types of K(+) channels in ES-cardiomyocytes. Therefore, present study shows functional expression of two types of K(+) ionic current in ES-cardiomyocytes.


Brainstem motor nuclei respond differentially to degenerative disease in the mutant mouse wobbler.
Degenerative motoneurone diseases, whether in humans, animals, or transgenic mouse models, do not affect all types of motoneurone to the same degree. Understanding the relative differences in vulnerability of certain motor pools may be the key to developing therapies. Expression of calbindin (CB) and parvalbumin (PV) immunoreactivity, which are potentially neuroprotective calcium-binding proteins, and NADPH-diaphorase (NADPH-d) histochemical reactivity, a marker for neurodegeneration, was studied in brainstem sections from mutant wobbler mice and their normal littermates during the motoneurone degeneration phase (3-8 weeks of age). The motor trigeminal and facial nuclei reacted in a manner previously observed in spinal somatic motoneurones in the wobbler. Many motoneurones expressed moderate NADPH-d reactivity, correlated with the appearance of vacuolated motoneurones in Nissl-stained sections. This was not observed in littermate controls. Motoneurone counts from Nissl-stained sections from 14-month-old wobblers and littermates revealed significantly fewer (approximately 27%) motoneurones in the trigeminal nucleus of wobblers. In contrast, the wobbler hypoglossal nucleus contained neither vacuolated nor NADPH-d reactive motoneurones. However, expression of CB immunoreactivity by the majority of wobbler hypoglossal motoneurones was observed but not in littermate controls or in any other motor nucleus. Counts in older animals showed a smaller but still significant difference in motoneurone number between wobblers and controls (approximately 9% reduction). Finally, the wobbler abducens nucleus displayed neither vacuolated neurones, nor NADPH-d reactivity nor CB immunoreactivity. Motor nuclei innervating extraocular muscles appear to be protected in many forms of motoneurone disease in man and other species. However, there were still markedly fewer abducens motoneurones in the old wobblers compared to controls (approximately 29% reduction). Sparing of oculomotor neurones in other diseases has been attributed to their relatively high PV expression, which we also observed in the abducens nucleus of both wobblers and littermates, and to a lesser extent in the other motor nuclei too. In conclusion, our results suggest that, in the wobbler mouse, motoneurone degeneration may occur without overt signs such as cell body vacuolation and NADPH-d expression. Induced CB expression may be neuroprotective but that constitutive expression of PV may not.


Baseline Factors Associated With Improvements in Decompensated Cirrhosis After Direct-Acting Antiviral Therapy for Hepatitis C Virus Infection.
Treatment with direct-acting antiviral (DAA) agents can reduce Model for End-Stage Liver Disease and Child-Pugh-Turcotte (CPT) scores in patients with decompensated cirrhosis caused by hepatitis C virus. However, many of these patients still die or require liver transplantation. We collected data on baseline features of patients and aimed to develop a scoring system to predict response to DAA therapy. We performed a retrospective analysis of data from 4 trials on the effects of sofosbuvir-based therapy in patients with hepatitis C virus-associated decompensated cirrhosis (502 of CPT class B and 120 of CPT class C). In these trials, patients were given 12 or 24 weeks of treatment with ledipasvir, sofosbuvir, and ribavirin or velpatasvir, sofosbuvir, and/or ribavirin, or 48 weeks of treatment with sofosbuvir and ribavirin. We collected demographic, clinical, treatment response, and laboratory data from patients and tested their associations with patient outcomes at 36 weeks. The primary outcome was factors associated with reduction of CPT score to class A. The presence of ascites or encephalopathy, serum level of albumin <3.5 g/dL or alanine aminotransferase <60 U/L, and body mass index >25 kg/m2 were associated with an increased risk of not achieving a reduction in CPT to class A, independent of sustained viral response to therapy. Serum level of albumin <2.8 g/dL and abnormal level of bilirubin were associated with an increased risk of liver transplantation or death. We developed a scoring system based on 5 baseline factors (body mass index, encephalopathy, ascites, and serum levels of alanine aminotransferase and albumin) associated significantly with patient outcomes, which we called the "BE3A score." For patients with scores of 4-5, the hazard ratio for reduction of CPT score to class A was 52.3 (95% confidence interval, 15.2-179.7). We identified 5 baseline factors (body mass index, encephalopathy, ascites, and serum levels of alanine aminotransferase and albumin) associated with a reduction of CPT score to class A in patients with hepatitis C virus-associated decompensated cirrhosis receiving DAA therapy. We developed a predictive score using these factors, called the BE3A score, which can be used as a shared decision-making tool, quantifying the potential benefits of DAA therapy for patients with decompensated cirrhosis.


Multicenter evaluation of the Cepheid Xpert methicillin-resistant Staphylococcus aureus (MRSA) test as a rapid screening method for detection of MRSA in nares.
The first U.S. multicenter clinical trial to assess the performance of the Cepheid Xpert MRSA assay (Xpert MRSA) was conducted. The assay is a qualitative test designed for the rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) directly from nares swabs. This novel test combines integrated nucleic acid extraction and automated real-time PCR for the detection of a MRSA-specific signature sequence. A total of 1,077 nares specimens were collected from seven geographically distinct health care sites across the United States with prevalence rates ranging from 5.2% to 44%. Nares specimens were tested by (i) the Xpert MRSA assay, (ii) direct culture on CHROMagar MRSA medium (direct CM culture), and (iii) broth-enriched culture (Trypticase soy broth with 6.5% sodium chloride) followed by plating onto CHROMagar MRSA medium (broth-enriched CM culture). When direct CM culture was designated the reference method, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the Xpert MRSA assay were 94.3%, 93.2%, 73.0%, and 98.8%, respectively. When broth-enriched CM culture was used as the reference method, the clinical sensitivity, specificity, PPV, and NPV of the Xpert MRSA assay were 86.3%, 94.9%, 80.5%, and 96.6%, respectively. The BD GeneOhm MRSA (BDGO) assay was performed as a comparative molecular method. No statistical performance differences were observed between the Xpert MRSA and BDGO assays when they were compared to culture methods. From this large-scale, multicenter clinical comparison, we conclude that the Xpert MRSA assay is a simple, rapid, and accurate method for performing active surveillance for MRSA in a variety of health care populations.


Impacts of using rainwater tanks on stormwater harvesting and runoff quality.
The popularity of rainwater use in Australia depends completely on the individual householder's preference. The quality of reticulated water supplies in major cities of Australia is far superior to water stored in rainwater tanks. However, due to persistent drought and the implementation of stringent water restrictions, cities such as Melbourne have encouraged the use of rainwater harvesting within the property. The benefits of trapping stormwater within a property and using it effectively also reduce polluted runoff excess reaching receiving water. The study reported herein focuses on the effectiveness of rainwater tanks as a potential water sensitive urban design element used to manage stormwater using the MUSIC model. The study shows that the installation of a 3 kL tank reduces hydraulic loading by 75%, Total Suspended Solids by 97%, Total Phosphorous by 90% and Total Nitrogen by 81% if the rainwater stored in the tank is used to meet the indoor demand (toilet flushing and laundry use) as well as the outdoor demand (garden watering).


Tyler asbestos workers: A mortality update in a cohort exposed to amosite.
The Tyler asbestos plant produced pipe insulation from 1954 to 1972 and exclusively used amosite asbestos. There were 1130 former workers of this plant during the period of operation. A death certificate mortality analysis was published regarding this plant in 1998 for the period through 1993. This study represents an update of the mortality analysis with additional certificates collected for deaths occurring through 2011.Searches of the National Death Index database were conducted in 2004 and again in 2013. At the time of the latter search, only deaths occurring through 2011 were available. In total, 265 distinct additional death certificates were secured and added to 304 available from the original study. After the new certificates were coded (ICD-9), data were analyzed using the Centers for Disease Control and Prevention Life Table Analysis System (LTAS) and standard mortality ratios (SMR) generated with 95% confidence limits (CL). LTAS constructs cause-specific mortality rates by age, gender, race, and person-time at risk, and compares observed rates with a referent population in order to derive SMR. A significant excess number of deaths due to nonmalignant respiratory disease (asbestosis) and from select malignant neoplasms were identified. There were in total 23 mesothelioma deaths (4% of deaths), with 16 pleural and 7 peritoneal. The SMR for malignant neoplasms of the trachea, bronchus, and lung was 244 (with 95% CL 196, 300), suggesting that exposed workers from this cohort were nearly 2.5-fold (244 %) more likely to die from this cause as the general referent population. The analysis also showed that exposures of short duration (<6 mo) produced significantly elevated SMR for all respiratory cancers, lung cancer, and pleural mesothelioma. There was a significant difference in median duration of exposure for mesothelioma types, confirming association of peritoneal mesothelioma with longer duration of exposure. Deaths due to intestinal cancer (predominantly colon; not including rectum) were also found in excess. The mortality experience of the Tyler cohort continues to be followed with great interest, given the exclusivity of exposure to amosite. Data confirm the inherent pathogenicity of this fiber type for nonmalignant disease as well as select cancers, particularly relevant given the importance of this amphibole's use in the United States.


Recyclable scavengers for photo-cyclopropanation via an in situ crystallization process.
The palladium(ii) cyclophane systems, constructed by previously reported proof-of-concept self-assembly, represent a crucial landmark in the field of effective and recyclable scavenging of triiodide (I3-) in the photo-cyclopropanation of alkenes with CH2I2. The scavenger's driving force behind photo-cyclopropanation is the efficient in situ crystallization of triiodide-exchanged species. The exact quantitative photoreaction yields according to the mole ratios of the cyclophane system are impressive. The recycling behavior can be ascribed to the rigidity and stability of the four-layered tripalladium(ii)cyclophane.


S100-A9 protein in exosomes from chronic lymphocytic leukemia cells promotes NF-κB activity during disease progression.
Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by accumulation of clonal B lymphocytes, resulting from a complex balance between cell proliferation and apoptotic death. Continuous crosstalk between cancer cells and local/distant host environment is required for effective tumor growth. Among the main actors of this dynamic interplay between tumoral cells and their microenvironment are the nano-sized vesicles called exosomes. Emerging evidence indicates that secretion, composition, and functional capacity of exosomes are altered as tumors progress to an aggressive phenotype. In CLL, no data exist exploring the specific changes in the proteomic profile of plasma-derived exosomes from patients during disease evolution. We hereby report for the first time different proteomic profiles of plasma exosomes, both between indolent and progressive CLLs as well as within the individual patients at the onset of disease and during its progression. Next, we focus on the changes of the exosome protein cargoes, which are found exclusively in patients with progressive CLL after disease progression. The alterations in the proteomic cargoes underline different networks specific for leukemia progression related to inflammation, oxidative stress, and NF-κB and phosphatidylinositol 3-kinase/AKT pathway activation. Finally, our results suggest a preponderant role for the protein S100-A9 as an activator of the NFκB pathway during CLL progression and suggest that the leukemic clone can generate an autoactivation loop through S100-A9 expression, NF-κB activation, and exosome secretion. Collectively, our data propose a new pathway for NF-κB activation in CLL and highlight the importance of exosomes as extracellular mediators promoting tumor progression in CLL.


Sequence effects of single base loops in intramolecular quadruplex DNA.
We have examined the properties of intramolecular G-quadruplexes in which the G3 tracts are separated by single base loops. The most stable complex contained 1',2'-dideoxyribose in all three loops, while loops containing T and C were slightly less stable (by about 2 degrees C). Quadruplexes containing loops with single A residues were less stable by 8 degrees C for each T to A substitution. These folded sequences display similar CD spectra, which are consistent with the formation of parallel stranded complexes with double-chain reversal loops. These results demonstrate that loop sequence, and not just length, affects quadruplex stability.


Effect of two different chemomechanical caries removal agents on dentin microhardness: An in vitro study.
The aim of this in vitro study was to evaluate the effect of two different chemomechanical caries removal (CMCR) agents on dentin microhardness. In this study, the crown portion of ten carious-free and ten caries-affected teeth were selected. In carious-free samples, the teeth were decoronated at the level of cemento - enamel junction. Only the crown portion of the teeth was selected. Occlusal one-third of the crowns were cross-sectioned and discarded to expose the dentin, and it was divided into two groups, five teeth in each group. Then, they were further sectioned longitudinally through the centre. In one group, no agent was applied on one half and Carisolv was applied on other half. In another group, no agent was applied on one-half and Carie-Care was applied on the other half for 1 min. In carious samples, the crowns were sectioned through the centre of carious lesion. Carisolv was applied on one-half and Carie-Care was applied on the other half. After using CMCR agents, surface hardness of dentin was examined using Vickers hardness number (VHN). The data were analyzed using t-test and one-way analysis of variance (ANOVA). There were no significant difference among normal dentin (62.91 ± 2.76), Carisolv-treated normal dentin (61.72 ± 2.89), and Carie-Care-treated normal dentin (61.90 ± 3.19). In carious samples, the results of Carisolv-treated dentin (58.57 ± 2.62) was not statistically significantly different from those of the Carie-Care-treated dentin (56.77 ± 4.41). In conclusion, neither of the CMCR methods caused a significant change in the microhardness of normal dentin and the treated carious dentin.


Cutaneous leishmaniasis in southern Sri Lanka.
Cutaneous leishmaniasis is an established disease in Sri Lanka. The majority of cases have been reported from the North-Central Province, with the southern parts of the country considered less affected. However, during 2004, when the services of a dermatologist were available, a considerable number of patients were referred from the Southern Province, which formed the basis for this study. The clinical pattern, detailed geographical distribution within the Southern Province and periodicity of the cases were studied over a period of 12 months. Of the 113 patients diagnosed, the highest number was within the 10-19 years age group. Most patients were from densely populated rural areas around Matara, a large town within this province. There was a notable increase in the number of cases presenting during February-March and August-September, which are periods following monsoonal rains. Exposed areas of the skin were commonly affected, with the majority of patients having single lesions. Females and males were equally affected. This is in contrast to our previous findings in the North-Central Province where the majority of patients were male soldiers with multiple lesions.


Carbohydrate Mouth Rinse Improves 1.5 h Run Performance: Is There A Dose-Effect?
There is a substantial body of recent evidence showing ergogenic effects of carbohydrate (CHO) mouth rinsing on endurance performance. However, there is a lack of research on the dose-effect and the aim of this study was to investigate the effect of two different concentrations (6% and 12% weight/volume, w/v) on 90 minute treadmill running performance. Seven active males took part in one familiarization trial and three experimental trials (90-minute self-paced performance trials). Solutions (placebo, 6% or 12% CHO-electrolyte solution, CHO-E) were rinsed in the mouth at the beginning, and at 15, 30 and 45 minutes during the run. The total distance covered was greater during the CHO-E trials (6%, 14.6 ± 1.7 km; 12%, 14.9 ± 1.6 km) compared to the placebo trial (13.9 ± 1.7 km, P < 0.05). There was no significant difference between the 6% and 12% trials (P > 0.05). There were no between trial differences (P > 0.05) in ratings of perceived exertion (RPE) and feeling or arousal ratings suggesting that the same subjective ratings were associated with higher speeds in the CHO-E trials. Enhanced performance in the CHO-E trials was due to higher speeds in the last 30 minutes even though rinses were not provided during the final 45 minutes, suggesting the effects persist for at least 20-45 minutes after rinsing. In conclusion, mouth rinsing with a CHO-E solution enhanced endurance running performance but there does not appear to be a dose-response effect with the higher concentration (12%) compared to a standard 6% solution.


Patients' experiences of pain in the 48 hours following total knee arthroplasty.
Previous research indicates that both pain severity and interference caused by pain should be addressed in the recovery period following total knee arthroplasty. This research describes the range of pain severity of patients in the first 48 hours postoperation, ways in which pain interferes with activities, and the observed differences in pain experience by patients' age, level of education, race, and gender, using the Brief Pain Inventory. Descriptive statistics and multivariate analysis were used to assess the data. Of the participants, 12% reported severe pain and 35% reported severe interference with general activities. Pain interfered most with walking, activities, and physical therapy. There were no differences by race, gender, or education. Older participants reported less pain and less interference. Participants reporting nonsurgical pain reported more interference with mood, walking, relationships with other people, and sleep. Pain evaluation should include an assessment of pain unrelated to the surgical procedure. Additional research exploring the impact of nonpostoperative surgical pain during the postoperative period is needed.


Cardiopulmonary exercise testing early after stroke using feedback-controlled robotics-assisted treadmill exercise: test-retest reliability and repeatability.
Exercise capacity is seriously reduced after stroke. While cardiopulmonary assessment and intervention strategies have been validated for the mildly and moderately impaired populations post-stroke, there is a lack of effective concepts for stroke survivors suffering from severe motor limitations. This study investigated the test-retest reliability and repeatability of cardiopulmonary exercise testing (CPET) using feedback-controlled robotics-assisted treadmill exercise (FC-RATE) in severely motor impaired individuals early after stroke. 20 subjects (age 44-84 years, <6 month post-stroke) with severe motor limitations (Functional Ambulatory Classification 0-2) were selected for consecutive constant load testing (CLT) and incremental exercise testing (IET) within a powered exoskeleton, synchronised with a treadmill and a body weight support system. A manual human-in-the-loop feedback system was used to guide individual work rate levels. Outcome variables focussed on standard cardiopulmonary performance parameters. Relative and absolute test-retest reliability were assessed by intraclass correlation coefficients (ICC), standard error of the measurement (SEM), and minimal detectable change (MDC). Mean difference, limits of agreement, and coefficient of variation (CoV) were estimated to assess repeatability. Peak performance parameters during IET yielded good to excellent relative reliability: absolute peak oxygen uptake (ICC =0.82), relative peak oxygen uptake (ICC =0.72), peak work rate (ICC =0.91), peak heart rate (ICC =0.80), absolute gas exchange threshold (ICC =0.91), relative gas exchange threshold (ICC =0.88), oxygen cost of work (ICC =0.87), oxygen pulse at peak oxygen uptake (ICC =0.92), ventilation rate versus carbon dioxide output slope (ICC =0.78). For these variables, SEM was 4-13%, MDC 12-36%, and CoV 0.10-0.36. CLT revealed high mean differences and insufficient test-retest reliability for all variables studied. This study presents first evidence on reliability and repeatability for CPET in severely motor impaired individuals early after stroke using a feedback-controlled robotics-assisted treadmill. The results demonstrate good to excellent test-retest reliability and appropriate repeatability for the most important peak cardiopulmonary performance parameters. These findings have important implications for the design and implementation of cardiovascular exercise interventions in severely impaired populations. Future research needs to develop advanced control strategies to enable the true limit of functional exercise capacity to be reached and to further assess test-retest reliability and repeatability in larger samples.


Subretinal hemorrhages in proliferative diabetic retinopathy.
To describe the clinical features of patients with advanced proliferative diabetic retinopathy who underwent vitrectomy and were found to have subretinal hemorrhages. The authors conducted a retrospective study of 49 patients with complications of proliferative diabetic retinopathy requiring pars plana vitrectomy and demonstrating the presence of subretinal hemorrhage. Preoperative, intraoperative, and postoperative clinical characteristics were evaluated. Patients were observed for a minimum of 6 months. The location, size, and clearance of subretinal hemorrhages revealed wide variation. Forty-two patients had focal subretinal hemorrhages, and 14 patients within this group had submacular hemorrhages. A retinal break was observed in 15 patients (31%). Only one patient required drainage of the subretinal hemorrhage to achieve retinal reattachment. Vitreous surgery resulted in 59% of patients achieving a visual acuity > or = 5/200. Seventy-nine percent had stable or improved vision, whereas 20% had worse vision after surgery. Subretinal hemorrhages appear to be an uncommon feature associated with long-term, advanced proliferative diabetic retinopathy and portend a guarded visual prognosis. These hemorrhages may occur spontaneously in previously untreated eyes and are often unsuspected until observed at the time of vitreous surgery. In general, removal of subretinal hemorrhages was not necessary to achieve macular anatomic attachment, and most patients experienced improved visual function after surgery. Diabetic subretinal hemorrhages may indicate a retinal break, and, therefore, careful ophthalmic inspection should be performed in these patients.


Mass spectrometric analysis of unknown haemoglobin fractions identified by high-performance liquid chromatography in an antenatal screening programme.
The majority of haematology laboratories use automated high-performance liquid chromatography (HPLC) as a primary method in the screening of samples for haemoglobin variants and thalassaemia. HPLC detects adducted fractions, such as HbA1C and other peaks of unknown significance. The National Health Service (NHS) Sickle and Thalassaemia Screening Programme does not mandate further assessment of these unknown peaks, but their presence may cause concern or precipitate detailed investigations. Mass spectrometry (MS) is being increasingly used in NHS Laboratories and is an effective method of characterising variant haemoglobins. Antenatal blood samples accepted for NHS Sickle and Thalassaemia Screening Programme from 2005 to 2013 were assessed. Those samples with unidentified peaks on HPLC were further analysed by Liquid chromatography tandem MS (LCMSMS). 58,493 samples were processed. 966 (1.6%) significant haemoglobinopathies were identified. 68 patients (0.11%) were found to have unidentified peaks on HPLC. α chain variants: two were hyperunstable α variants, Hb Adana and Hb H/Constant Spring. The patient with HbH/Constant Spring required transfusion during pregnancy. Other abnormalities include 24 unstable α chain variants of minimal clinical significance and 17 α chain variants of no clinical significance. The frequency of β chain abnormalities was lower, with 10 patients having an insignificant β chain variant, 4 mildly unstable β chain variants (2 of whom had increased oxygen affinity) and 4 with variants associated with increased oxygen affinity. The prevalence of unidentified variants on automated HPLC within our population is 0.1%. LCMSMS is an effective technique to assist in the characterisation of unknown haemoglobin variants. α chain variants of limited clinical significance were the most commonly detected abnormality.


Functional maturation of the human antibody response to rotavirus.
Infant Abs induced by viruses exhibit poor functional activity compared with those of adults. The human B cell response to rotavirus is dominated by use of the V(H)1-46 gene segment in both adults and infants, but only adult sequences are highly mutated. We investigated in detail the kinetic, structural, and functional advantage conferred by individual naturally occurring somatic mutations in rotavirus-specific human Abs encoded by the immunodominant V(H)1-46 gene segment. Adult Abs achieved enhanced binding through naturally occurring somatic mutations in the H chain CDR2 region that conferred a markedly prolonged off-rate and a desirable increase in antiviral potency. Three-dimensional cryoelectron microscopy studies of Ag-Ab complexes revealed the mechanism of viral inhibition to be the binding of high-affinity Abs at the viral RNA release pore in the double-layer particle. These structure-function studies suggest a molecular basis for the poor quality of Abs made in infancy following virus infection or immunization.


Changes in perfectionism following cognitive-behavioral treatment for social phobia.
Previous studies have found that social phobia (social anxiety disorder) is associated with elevated levels of perfectionism, particularly concerns over making mistakes (CM) and doubts about actions (DA). This study investigated the extent to which various dimensions of perfectionism change as a result of participating in a 12-session cognitive-behavioral group treatment for social phobia. One hundred seven individuals completed the Frost Multidimensional Perfectionism Scale before and after treatment. Participants improved on several measures of social anxiety, generalized anxiety, and depression. With respect to perfectionism, significant reductions were seen on total perfectionism scores and scores on particular dimensions (CM, DA, organization), but not on other dimensions (personal standards, parental expectations, parental criticism). Furthermore, changes in DA and to some extent CM predicted posttreatment levels of social anxiety after controlling for pretreatment levels of social anxiety and changes in anxiety and depression. Implications of these findings are discussed.