Polymorphisms in the glucocorticoid receptor gene and their associations with metabolic parameters and body composition.
Most actions of glucocorticoids (GCs) are mediated by the glucocorticoid receptor (GR). The interindividual response to GCs varies considerably, as demonstrated by a variable suppressive response to 0.25-mg dexamethasone (DEX). Several polymorphisms in the gene coding for the GR have been described. It is unclear to what extent the observed response variability is due to GR polymorphisms or to other factors. However, at least three polymorphisms seem to be associated with altered GC sensitivity and changes in body composition and metabolic parameters. The N363S polymorphism has been associated with increased sensitivity to GCs, increased insulin response to DEX, a tendency towards lower bone mineral density, and increased body mass index (BMI). However, other reports found no associations with BMI. Another polymorphism, previously described as a BclI restriction fragment length polymorphism, recently was identified as a C --> G nucleotide change. The G allele also was associated with increased sensitivity to GCs. In middle-aged subjects, the G allele of this BclI polymorphism was associated with increased abdominal obesity, while at older age, a lower BMI was found, accompanied by a tendency towards lower lean body mass. A third polymorphism consists of two linked, single-nucleotide mutations in codons 22 and 23, of which the second mutation results in an amino acid change from arginine (R) to lysine (K). In contrast to the other polymorphisms, this ER22/23EK polymorphism was associated with a relative resistance to GCs. In line with this, ER22/23EK carriers had lower total cholesterol and low-density lipoprotein cholesterol levels as well as lower fasting insulin concentrations and a better insulin sensitivity. C-reactive protein levels were lower in ER22/23EK carriers, as was found in a different population of elderly males. In accordance with this healthy metabolic profile, we found in this population a significantly better survival in ER22/23EK carriers after a 4-year follow-up. GCs also affect the brain. Although a certain level of cortisol is essential for proper brain functioning, excessive GC levels have been shown to negatively affect brain morphology and functions. At older age, we found that the risk of dementia and white matter lesions was lower in ER22/23EK carriers. GCs are also important in the regulation of body fat distribution. At young age, we observed sex-specific differences in body composition. Male ER22/23EK carriers were taller, had more muscle mass, and were stronger than noncarriers. In young females, ER22/23EK carriers had tendencies towards smaller waist and hip circumferences and lower body weight. Another polymorphism (TthIIII) was not associated with altered GC sensitivity. In conclusion, these polymorphisms in the GR gene may contribute considerably to the observed variability in GC sensitivity. As a result, they are associated with several differences in body composition and metabolic factors.


Association of the CpG methylation pattern of the proximal insulin gene promoter with type 1 diabetes.
The insulin (INS) region is the second most important locus associated with Type 1 Diabetes (T1D). The study of the DNA methylation pattern of the 7 CpGs proximal to the TSS in the INS gene promoter revealed that T1D patients have a lower level of methylation of CpG -19, -135 and -234 (p = 2.10(-16)) and a higher methylation of CpG -180 than controls, while methylation was comparable for CpG -69, -102, -206. The magnitude of the hypomethylation relative to a control population was 8-15% of the corresponding levels in controls and was correlated in CpGs -19 and -135 (r = 0.77) and CpG -135 and -234 (r = 0.65). 70/485 (14%) of T1D patients had a simultaneous decrease in methylation of CpG -19, -135, -234 versus none in 317 controls. CpG methylation did not correlate with glycated hemoglobin or with T1D duration. The methylation of CpG -69, -102, -180, -206, but not CpG -19, -135, -234 was strongly influenced by the cis-genotype at rs689, a SNP known to show a strong association with T1D. We hypothesize that part of this genetic association could in fact be mediated at the statistical and functional level by the underlying changes in neighboring CpG methylation. Our observation of a CpG-specific, locus-specific methylation pattern, although it can provide an epigenetic biomarker of a multifactorial disease, does not indicate whether the reported epigenetic pattern preexists or follows the establishment of T1D. To explore the effect of chronic hyperglycemia on CpG methylation, we studied non obese patients with type 2 diabetes (T2D) who were found to have decreased CpG-19 methylation versus age-matched controls, similar to T1D (p = 2.10(-6)) but increased CpG-234 methylation (p = 5.10(-8)), the opposite of T1D. The causality and natural history of the different epigenetic changes associated with T1D or T2D remain to be determined.


Diabetes, Associated Clinical Spectrum, Long-term Prognosis, and Genotype/Phenotype Correlations in 201 Adult Patients With Hepatocyte Nuclear Factor 1B (HNF1B) Molecular Defects.
Molecular defects of hepatocyte nuclear factor 1B (HNF1B) are associated with a multiorgan disease, including diabetes (maturity-onset diabetes of the young 5) and kidney abnormalities. The HNF1B syndrome is related to HNF1B mutations or to a 17q12 deletion spanning 15 genes, including HNF1B. Here, we described HNF1B-related diabetes and associated phenotypes and assessed genotype/phenotype correlations at diagnosis and in the long-term. This multicenter retrospective cohort study included 201 patients, aged 18 years or older at follow-up, with HNF1B mutations (n = 101) or deletion (n = 100). Diabetes was present in 159 patients. At diagnosis, clinical symptoms of diabetes were present in 67 of 144 patients and HNF1B renal disease in 64 of 102. Although responsiveness to sulfonylureas/repaglinide was observed in 29 of the 51 tested, 111 of 140 patients (79%) were treated with insulin at follow-up. Diabetic retinopathy and/or neuropathy were present in 46 of 114 patients. Renal cysts were present in 122 of 166 patients, chronic kidney disease stages 3-4 (CKD3-4) in 75 of 169 (44%), and end-stage renal disease (ESRD) in 36 of 169 (21%). Compared with the patients with mutations, those with HNF1B deletion less often had CKD3-4/ESRD at diagnosis (11 of 43 vs. 27 of 35, P < 10-4) and in the long term (40 of 78 vs. 71 of 91, P = 0.0003). They were leaner and more frequently treated with insulin. In patients with HNF1B syndrome, diabetes complications, cardiovascular risk factors, CKD3-4, and ESRD are highly prevalent. At diabetes diagnosis, the presence of morphological and/or functional kidney disease may help etiological diagnosis. Genotype/phenotype correlations may have implications for the care and the prognosis of these patients.


Dynamical analysis of gene networks requires both mRNA and protein expression information.
One of the important goals of biology is to understand the relationship between DNA sequence information and nonlinear cellular responses. This relationship is central to the ability to effectively engineer cellular phenotypes, pathways, and characteristics. Expression arrays for monitoring total gene expression based on mRNA can provide quantitative insight into which gene or genes are on or off; but this information is insufficient to fully predict dynamic biological phenomena. Using nonlinear stability analysis we show that a combination of gene expression information at the message level and at the protein level is required to describe even simple models of gene networks. To help illustrate the need for such information we consider a mechanistic model for circadian rhythmicity which shows agreement with experimental observations when protein and mRNA information are included and we propose a framework for acquiring and analyzing experimental and mathematically derived information about gene networks.


Structure of the DNA repair helicase hel308 reveals DNA binding and autoinhibitory domains.
Hel308 is a superfamily 2 helicase conserved in eukaryotes and archaea. It is thought to function in the early stages of recombination following replication fork arrest and has a specificity for removal of the lagging strand in model replication forks. A homologous helicase constitutes the N-terminal domain of human DNA polymerase Q. The Drosophila homologue mus301 is implicated in double strand break repair and meiotic recombination. We have solved the high resolution crystal structure of Hel308 from the crenarchaeon Sulfolobus solfataricus, revealing a five-domain structure with a central pore lined with essential DNA binding residues. The fifth domain is shown to act as an autoinhibitory domain or molecular brake, clamping the single-stranded DNA extruded through the central pore of the helicase structure to limit the helicase activity of the enzyme. This provides an elegant mechanism to tune the processivity of the enzyme to its functional role. Hel308 can displace streptavidin from a biotinylated DNA molecule, and this activity is only partially inhibited when the DNA is pre-bound with abundant DNA-binding proteins RPA or Alba1, whereas pre-binding with the recombinase RadA has no effect on activity. These data suggest that one function of the enzyme may be in the removal of bound proteins at stalled replication forks and recombination intermediates.


Autofluorescence of the cells in human subretinal fluid.
The origin of autofluorescence in the subretinal space and the autofluorescence properties of the cells were investigated in surgically collected subretinal fluid. Subretinal fluid was surgically collected from four eyes of patients with rhegmatogenous retinal detachment (three eyes) and Coats' disease (one eye). After cytocentrifuge preparation of the cells in the fluid and immunofluorescence staining, a cytologic examination was conducted by using confocal scanning laser microscopy. The autofluorescence of the cells was elucidated by measuring the fluorescence spectra with spectroscopy, to obtain different excitation laser light emission fingerprints. The cells from the subretinal fluid were classified into three types: CD68-negative cells containing numerous pigmented granules, CD68-positive cells containing few pigments, and CD68-negative cells with no pigmented granules. Autofluorescence was observed in the inclusions of the cells classified into the former two types. When the cells were excited by a 458- or 488-nm laser light, emission spectra in autofluorescence showed little difference between CD68-positive and -negative cells. Peak analysis confirmed that the two types of cells showed the same emission peaks within this range of excitation light. Autofluorescent inclusions appeared in the CD68-positive and -negative cells in the subretinal fluid. The macrophages in the subretinal fluid possess autofluorescence that is spectroscopically similar to lipofuscin. Autofluorescence of macrophages can be attributed to degenerated outer segments and debris from apoptotic photoreceptors. Clinicians should consider migration of macrophages, in addition to retinal pigment epithelium, as the possible source when abnormal fundus autofluorescence is observed using an ordinary set of fluorescence filters.


Label-free detection of Giardia lamblia cysts using a deep learning-enabled portable imaging flow cytometer.
We report a field-portable and cost-effective imaging flow cytometer that uses deep learning and holography to accurately detect Giardia lamblia cysts in water samples at a volumetric throughput of 100 mL h-1. This flow cytometer uses lens free color holographic imaging to capture and reconstruct phase and intensity images of microscopic objects in a continuously flowing sample, and automatically identifies Giardia lamblia cysts in real-time without the use of any labels or fluorophores. The imaging flow cytometer is housed in an environmentally-sealed enclosure with dimensions of 19 cm × 19 cm × 16 cm and weighs 1.6 kg. We demonstrate that this portable imaging flow cytometer coupled to a laptop computer can detect and quantify, in real-time, low levels of Giardia contamination (e.g., <10 cysts per 50 mL) in both freshwater and seawater samples. The field-portable and label-free nature of this method has the potential to allow rapid and automated screening of drinking water supplies in resource limited settings in order to detect waterborne parasites and monitor the integrity of the filters used for water treatment.


A Qualitative Analysis of Family Dynamics and Motivation in Sessions With 15 Women in Drug Treatment Court.
Women with substance use disorders (SUDs) often experience inadequate health care, mental and physical health problems, trauma, lack of social support, and undermining of support for psychological needs of autonomy, competence, and relatedness needed for motivation and well-being. For women with SUD trying to reclaim sobriety and a healthy life, family can present both barriers and support. The aim of this study is to gain a deeper understanding of the intersection of family relationships with motivation of women in Drug Treatment Court (DTC) to attain their health goals. Data consist of transcribed intervention sessions between trained peer interventionists and 15 DTC participants from The Women's Initiative Supporting Health DTC Intervention Study. This analysis uses a qualitative framework approach to analyze the data. The Self-determination Theory of human motivation and Family Systems Theory provide the conceptual framework to understand how participants' expressions of motivation-related basic needs of autonomy, competence, and relatedness and change-related behaviors interfaced with family support. Analysis revealed more mentions of family in motivation-supportive contexts than in motivation-thwarting contexts, but highlighted complex roles families can play in health of women in recovery from SUD. Providers may be able to incorporate this knowledge to address the needs of this challenging population.


Critical Findings on Magnetic Resonance Arthrograms in Posterior Shoulder Instability Compared With an Age-Matched Controlled Cohort.
Posterior shoulder instability is less common and potentially more difficult to diagnose clinically and radiographically compared with anterior shoulder instability. Radiographic findings including posterior labral tears, increased retroversion, presence of glenoid dysplasia, and increased capsular area are associated with symptomatic recurrent posterior shoulder instability. This study aimed to determine the prevalence and severity of associated radiographic parameters found on magnetic resonance arthrograms (MRAs) in patients with arthroscopically confirmed isolated posterior labral tears and symptomatic recurrent posterior shoulder instability, compared with an age-matched cohort of patients without posterior instability or labral injury confirmed with shoulder arthroscopy. Cross-sectional study, Level of evidence, 3. Patients who received a preoperative standard shoulder MRA at an academic institution over a 5-year period and had symptomatic posterior instability and received a repair of an arthroscopically confirmed posterior labral tear (n = 63) were identified. These patients were compared with an age-matched control group of patients without posterior instability (n = 49) who underwent an isolated arthroscopic distal clavicle resection that included an arthroscopic glenohumeral joint evaluation. Glenoid version, posterior humeral head subluxation, glenoid dysplasia, and linear and capsular area measurements were evaluated between the 2 groups. Interobserver reliability for continuous and categorical variables was assessed for all measurements. Multivariate logistic regression revealed that the presence of increased glenoid retroversion (P = .0018), glenoid dysplasia (P = .03), and increased axial posterior capsular cross-sectional area (P = .05) were significantly associated with posterior labral tears and symptomatic posterior shoulder instability compared with the age-matched control group. Posterior humeral head subluxation was found to be a statistically significant variable with univariate analysis (P = .001) for posterior shoulder instability but not with multivariate logistic regression (P = .53). Interobserver reliability was good to very good for all measurements (intraclass correlation coefficient [ICC] = 0.74-0.85; κ = 0.64) but was moderate for total capsular area and sagittal capsular area measurements (ICC = 0.43-0.56). The presence of increased glenoid retroversion, glenoid dysplasia, and increased posterior capsular area on MRA are significantly associated with posterior labral tears and symptomatic posterior shoulder instability. Identification of these critical radiographic variables on magnetic resonance arthrography assists in the accurate diagnosis and management of clinically significant posterior shoulder instability.


Propensity of Stroke in Standard versus Various Aortic Arch Variants: A 200 Patients Study.
Abnormal origin of arteries from the aortic arch could alter the hemodynamics. Therefore, aortic arch variations might predispose patients to atherosclerosis, which would increase the stroke risk by impending thrombus formation. To investigate the prevalence of various types of the anatomy of the aortic arch in ischemic stroke patients and determine if aortic morphology has any effect on early-onset strokes. Observational study including 200 imaging-confirmed (Non Contrast Computed Tomography(NCCT)/magnetic resonance imaging [MRI] of the brain) acute ischemic stroke patients. This was followed by computed tomography [CT]/MRI angiography of the arch of the aorta, neck vessels, and intracranial arteries. The occurrence of various types of standard and other aortic arch variants was studied. The prevalence of stroke and its characteristics were analyzed for demographics, types, location, and the predominant side of involvement among standard arch variants and standard versus various aortic arch variants. A P value < 0.05 was considered significant. Standard arch Type I was the most common (P < 0.0001). Age at stroke onset in Type 1 was 61.83 years ± 2.78 years, in Type 2 was 59.8 years ± 3.55 years, and in Type 3 was 60.96 years ± 3.56 years (P = 0.0012). Among the bovine aortic arch, age at stroke presentation in Type A was 53.33 years ± 8.35 years, in Type B was 53.36 years ± 7.4 years, and in Type C was 63.25 years ± 9.25 years (P < 0.0001). Standard aortic arch Type 2, bovine aortic arch Type A, and Type B are associated with an early age at stroke presentation. During routine carotid evaluation by CT or MR angiography in stroke patients, it would be better to evaluate the aortic arch as well, especially in young patients.


Gut microbiota confers host resistance to obesity by metabolizing dietary polyunsaturated fatty acids.
Gut microbiota mediates the effects of diet, thereby modifying host metabolism and the incidence of metabolic disorders. Increased consumption of omega-6 polyunsaturated fatty acid (PUFA) that is abundant in Western diet contributes to obesity and related diseases. Although gut-microbiota-related metabolic pathways of dietary PUFAs were recently elucidated, the effects on host physiological function remain unclear. Here, we demonstrate that gut microbiota confers host resistance to high-fat diet (HFD)-induced obesity by modulating dietary PUFAs metabolism. Supplementation of 10-hydroxy-cis-12-octadecenoic acid (HYA), an initial linoleic acid-related gut-microbial metabolite, attenuates HFD-induced obesity in mice without eliciting arachidonic acid-mediated adipose inflammation and by improving metabolic condition via free fatty acid receptors. Moreover, Lactobacillus-colonized mice show similar effects with elevated HYA levels. Our findings illustrate the interplay between gut microbiota and host energy metabolism via the metabolites of dietary omega-6-FAs thereby shedding light on the prevention and treatment of metabolic disorders by targeting gut microbial metabolites.


A new twist on an old problem: a case of pediatric meningitis caused by multidrug-resistant Streptococcus pneumoniae serotype 19A.
Recent studies show that multidrug-resistant Streptococcus pneumoniae serotype 19A continues to emerge as a cause of invasive pneumococcal disease after the introduction of Prevnar. We report a case of multidrug-resistant S. pneumoniae serotype 19A meningitis successfully treated with vancomycin and levofloxacin. This case reinforces the need for the empiric use of vancomycin in meningitis and the need for alternative treatments.


WGCNA: an R package for weighted correlation network analysis.
Correlation networks are increasingly being used in bioinformatics applications. For example, weighted gene co-expression network analysis is a systems biology method for describing the correlation patterns among genes across microarray samples. Weighted correlation network analysis (WGCNA) can be used for finding clusters (modules) of highly correlated genes, for summarizing such clusters using the module eigengene or an intramodular hub gene, for relating modules to one another and to external sample traits (using eigengene network methodology), and for calculating module membership measures. Correlation networks facilitate network based gene screening methods that can be used to identify candidate biomarkers or therapeutic targets. These methods have been successfully applied in various biological contexts, e.g. cancer, mouse genetics, yeast genetics, and analysis of brain imaging data. While parts of the correlation network methodology have been described in separate publications, there is a need to provide a user-friendly, comprehensive, and consistent software implementation and an accompanying tutorial. The WGCNA R software package is a comprehensive collection of R functions for performing various aspects of weighted correlation network analysis. The package includes functions for network construction, module detection, gene selection, calculations of topological properties, data simulation, visualization, and interfacing with external software. Along with the R package we also present R software tutorials. While the methods development was motivated by gene expression data, the underlying data mining approach can be applied to a variety of different settings. The WGCNA package provides R functions for weighted correlation network analysis, e.g. co-expression network analysis of gene expression data. The R package along with its source code and additional material are freely available at http://www.genetics.ucla.edu/labs/horvath/CoexpressionNetwork/Rpackages/WGCNA.


A structural investigation of complex I and I+III2 supercomplex from Zea mays at 11-13 A resolution: assignment of the carbonic anhydrase domain and evidence for structural heterogeneity within complex I.
The projection structures of complex I and the I+III2 supercomplex from the C4 plant Zea mays were determined by electron microscopy and single particle image analysis to a resolution of up to 11 A. Maize complex I has a typical L-shape. Additionally, it has a large hydrophilic extra-domain attached to the centre of the membrane arm on its matrix-exposed side, which previously was described for Arabidopsis and which was reported to include carbonic anhydrase subunits. A comparison with the X-ray structure of homotrimeric gamma-carbonic anhydrase from the archaebacterium Methanosarcina thermophila indicates that this domain is also composed of a trimer. Mass spectrometry analyses allowed to identify two different carbonic anhydrase isoforms, suggesting that the gamma-carbonic anhydrase domain of maize complex I most likely is a heterotrimer. Statistical analysis indicates that the maize complex I structure is heterogeneous: a less-abundant "type II" particle has a 15 A shorter membrane arm and an additional small protrusion on the intermembrane-side of the membrane arm if compared to the more abundant "type I" particle. The I+III2 supercomplex was found to be a rigid structure which did not break down into subcomplexes at the interface between the hydrophilic and the hydrophobic arms of complex I. The complex I moiety of the supercomplex appears to be only of "type I". This would mean that the "type II" particles are not involved in the supercomplex formation and, hence, could have a different physiological role.


The Not-so-Sterile Womb: Evidence That the Human Fetus Is Exposed to Bacteria Prior to Birth.
The human microbiome includes trillions of bacteria, many of which play a vital role in host physiology. Numerous studies have now detected bacterial DNA in first-pass meconium and amniotic fluid samples, suggesting that the human microbiome may commence in utero. However, these data have remained contentious due to underlying contamination issues. Here, we have used a previously described method for reducing contamination in microbiome workflows to determine if there is a fetal bacterial microbiome beyond the level of background contamination. We recruited 50 women undergoing non-emergency cesarean section deliveries with no evidence of intra-uterine infection and collected first-pass meconium and amniotic fluid samples. Full-length 16S rRNA gene sequencing was performed using PacBio SMRT cell technology, to allow high resolution profiling of the fetal gut and amniotic fluid bacterial microbiomes. Levels of inflammatory cytokines were measured in amniotic fluid, and levels of immunomodulatory short chain fatty acids (SCFAs) were quantified in meconium. All meconium samples and most amniotic fluid samples (36/43) contained bacterial DNA. The meconium microbiome was dominated by reads that mapped to Pelomonas puraquae. Aside from this species, the meconium microbiome was remarkably heterogeneous between patients. The amniotic fluid microbiome was more diverse and contained mainly reads that mapped to typical skin commensals, including Propionibacterium acnes and Staphylococcus spp. All meconium samples contained acetate and propionate, at ratios similar to those previously reported in infants. P. puraquae reads were inversely correlated with meconium propionate levels. Amniotic fluid cytokine levels were associated with the amniotic fluid microbiome. Our results demonstrate that bacterial DNA and SCFAs are present in utero, and have the potential to influence the developing fetal immune system.


Vital Signs: Preventing Antibiotic-Resistant Infections in Hospitals - United States, 2014.
Health care-associated antibiotic-resistant (AR) infections increase patient morbidity and mortality and might be impossible to successfully treat with any antibiotic. CDC assessed health care-associated infections (HAI), including Clostridium difficile infections (CDI), and the role of six AR bacteria of highest concern nationwide in several types of health care facilities. During 2014, approximately 4,000 short-term acute care hospitals, 501 long-term acute care hospitals, and 1,135 inpatient rehabilitation facilities in all 50 states reported data on specific infections to the National Healthcare Safety Network. National standardized infection ratios and their percentage reduction from a baseline year for each HAI type, by facility type, were calculated. The proportions of AR pathogens and HAIs caused by any of six resistant bacteria highlighted by CDC in 2013 as urgent or serious threats were determined. In 2014, the reductions in incidence in short-term acute care hospitals and long-term acute care hospitals were 50% and 9%, respectively, for central line-associated bloodstream infection; 0% (short-term acute care hospitals), 11% (long-term acute care hospitals), and 14% (inpatient rehabilitation facilities) for catheter-associated urinary tract infection; 17% (short-term acute care hospitals) for surgical site infection, and 8% (short-term acute care hospitals) for CDI. Combining HAIs other than CDI across all settings, 47.9% of Staphylococcus aureus isolates were methicillin resistant, 29.5% of enterococci were vancomycin-resistant, 17.8% of Enterobacteriaceae were extended-spectrum beta-lactamase phenotype, 3.6% of Enterobacteriaceae were carbapenem resistant, 15.9% of Pseudomonas aeruginosa isolates were multidrug resistant, and 52.6% of Acinetobacter species were multidrug resistant. The likelihood of HAIs caused by any of the six resistant bacteria ranged from 12% in inpatient rehabilitation facilities to 29% in long-term acute care hospitals. Although there has been considerable progress in preventing some HAIs, many remaining infections could be prevented with implementation of existing recommended practices. Depending upon the setting, more than one in four of HAIs excluding CDI are caused by AR bacteria. Physicians, nurses, and health care leaders need to consistently and comprehensively follow all recommendations to prevent catheter- and procedure-related infections and reduce the impact of AR bacteria through antimicrobial stewardship and measures to prevent spread.


Cur2004-8, a synthetic curcumin derivative, extends lifespan and modulates age-related physiological changes in Caenorhabditis elegans.
Curcumin, a compound found in Indian yellow curry, is known to possess various biological activities, including anti-oxidant, anti-inflammatory, and anti-cancer activities. Cur2004-8 is a synthetic curcumin derivative having symmetrical bis-alkynyl pyridines that shows a strong anti-angiogenic activity. In the present study, we examined the effect of dietary supplementation with Cur2004-8 on response to environmental stresses and aging using Caenorhabditis elegans as a model system. Dietary intervention with Cur2004-8 significantly increased resistance of C. elegans to oxidative stress. Its anti-oxidative-stress effect was greater than curcumin. However, response of C. elegans to heat stress or ultraviolet irradiation was not significantly affected by Cur2004-8. Next, we examined the effect of Cur2004-8 on aging. Cur2004-8 significantly extended both mean and maximum lifespan, accompanying a shift in time-course distribution of progeny production. Age-related decline in motility was also delayed by supplementation with Cur2004-8. In addition, Cur2004-8 prevented amyloid-beta-induced toxicity in Alzheimer's disease model animals which required a forkhead box (FOXO) transcription factor DAF-16. Dietary supplementation with Cur2004-8 also reversed the increase of mortality observed in worms treated with high-glucose-diet. These results suggest that Cur2004-8 has higher anti-oxidant and anti-aging activities than curcumin. It can be used for the development of novel anti-aging product.


Sickle hemoglobin (HbS) allele and sickle cell disease: a HuGE review.
Sickle cell disease is caused by a variant of the beta-globin gene called sickle hemoglobin (Hb S). Inherited autosomal recessively, either two copies of Hb S or one copy of Hb S plus another beta-globin variant (such as Hb C) are required for disease expression. Hb S carriers are protected from malaria infection, and this protection probably led to the high frequency of Hb S in individuals of African and Mediterranean ancestry. Despite this advantage, individuals with sickle cell disease exhibit significant morbidity and mortality. Symptoms include chronic anemia, acute chest syndrome, stroke, splenic and renal dysfunction, pain crises, and susceptibility to bacterial infections. Pediatric mortality is primarily due to bacterial infection and stroke. In adults, specific causes of mortality are more varied, but individuals with more symptomatic disease may exhibit early mortality. Disease expression is variable and is modified by several factors, the most influential being genotype. Other factors include beta-globin cluster haplotypes, alpha-globin gene number, and fetal hemoglobin expression. In recent years, newborn screening, better medical care, parent education, and penicillin prophylaxis have successfully reduced morbidity and mortality due to Hb S.


Hepatitis C in urban and rural public safety workers.
A sample of 719 Oregon public safety personnel (police officers, firefighters, and corrections officers) was tested for hepatitis C virus (HCV) antibody after completing a risk questionnaire. Seven of nine positive enzyme immunoassay tests (78%) were confirmed with recombinant immunoblot assay, yielding confirmed prevalence estimates of 1.2% (95% confidence interval, 0.4 to 2.8%) among the 406 firefighters and emergency medical technicians, and 0.7% (95% confidence interval, 0.1 to 2.6%) in 274 corrections personnel. No cases were observed in the 29 participating police officers. Self-reports of the number of workplace exposures to blood were not associated with HCV positivity, and the number of years of public safety employment seemed to be slightly less for HCV-positive subjects. Two of the seven (28.6%) HCV-positive individuals reported having at least one nonoccupational risk factor (odds ratio, 4.3; 95% confidence interval, 0.4 to 27.1), suggesting the greater relative importance of nonoccupational exposures.


The association of meniscal damage with joint effusion in persons without radiographic osteoarthritis: the Framingham and MOST osteoarthritis studies.
To assess the cross-sectional association between meniscal status and joint effusion on magnetic resonance imaging (MRI) in knees without radiographic osteoarthritis (OA). Knees without OA (Kellgren/Lawrence grade 0) from the Framingham and MOST studies were examined by MRI. Meniscal status was assessed with a score of 0-4 in the anterior horn/body/posterior horn of the medial/lateral meniscus and effusion was assessed using a score of 0-3. The odds ratios (ORs) of joint effusion in those with meniscal damage were estimated using a logistic regression model. A subanalysis was performed for knees without MRI-detected cartilage damage. Of 1368 knees, 296 (21.6%) showed meniscal pathology in at least one subregion. Effusion was present in 133 (44.9%) of knees with meniscal damage vs 328 (30.6%) in those without meniscal damage. The adjusted OR of effusion in a knee with meniscal damage was 1.8, 95% confidence intervals (CI) [1.4, 2.4]. The OR of effusion for the group with meniscal pathology in two compartments was 5.4, 95% CI [2.1, 14.3]. For knees without any cartilage lesions but with meniscal damage in any compartment the OR was 2.3, 95% CI [1.1, 4.5]. Knees without OA but with meniscal pathology exhibit joint effusion to a significantly higher degree than knees without meniscal damage. The association persists for knees without cartilage damage. The prevalence of effusion is further increased when present in two compartments. Concomitant occurrence of synovial activation and meniscal damage contributes to understanding the pathophysiology of early degenerative joint disease.


Enhanced UHPLC-MS/MS screening of selective androgen receptor modulators following urine hydrolysis.
Selective androgen receptor modulators (SARMs) represent non-steroidal agents commonly abused in human and animal (i.e. equine, canine) sports, with potential for further misuse as growth promoting agents in livestock-based farming. As a direct response to the real and possible implications of illicit application in both sport as well as food production systems, this study incorporated enzymatic hydrolysis (β-glucuronidase/arylsulfatase) into a previously established protocol while maintaining the minimal volume (200 µL) of urine sample required to detect SARMs encompassing various pharmacophores in urine from a range of species (i.e. equine, bovine, human, canine and rodent). The newly presented semi-quantitative UHPLC-MS/MS-based assay is shown to be fit-for-purpose, being rapid and offering high-throughput, with validation findings fulfilling criteria stipulated within relevant doping and food control legislation.•CCβ values determined at 1 ng mL-1 for majority of analytes.•Deconjugation step included in the method led to significantly increased relative abundance of ostarine in analysed incurred urine samples demonstrating the requirement for hydrolysis to detect a total form of emerging SARMs.•Assay amenable for use within routine testing to ensure fair play in animal and human sports and that animal-derived food is free from contamination with SARM residues.


Structural origin of proton mobility in a protic ionic liquid/imidazole mixture: insights from computational and experimental results.
The structure, dynamics, and phase behavior of a binary mixture based on the protic ionic liquid 1-ethylimidazolium bis(trifluoromethanesulfonyl)imide (C2HImTFSI) and imidazole are investigated by (1)H NMR spectroscopy, vibrational spectroscopy, diffusion NMR, calorimetric measurements, and molecular dynamics simulations. Particular attention is given to the nature of the H-bonds established and the consequent occurrence of the Grotthuss mechanism of proton transfer. We find that due to their structural similarity, the imidazolium cation and the imidazole molecule behave as interchangeable and competing sites of interaction for the TFSI anion. All investigated properties, that is the phase behavior, strength of ion-ion and ion-imidazole interactions, number of specific H-bonds, density, and self-diffusivity, are composition dependent and show trend changes at mole fractions of imidazole (χ) approximately equal to 0.2 and 0.5. Beyond χ = 0.8 imidazole is not miscible in C2HImTFSI at room temperature. We find that at the equimolar composition (χ ≈ 0.5) a structural transition occurs from an ionic network mainly stabilized by coulombic forces to a mixed phase held together by site specific H-bonds. The same composition also marks a steeper decrease in density and increase in diffusivity, resulting from the preference of imidazole molecules to H-bond to each other in a chain-like manner. As a result of these structural features the Grotthuss mechanism of proton transfer is less favored at the equimolar composition where H-bonds are too stable. By contrast, the Grotthuss mechanism is more pronounced in the low concentration range where imidazole acts as a base pulling the proton of the imidazolium cation. At high imidazole concentrations the contribution from the vehicular mechanism dominates.


Protein release through nonlethal oncotic pores as an alternative nonclassical secretory pathway.
Nonclassical (unconventional) protein secretion is thought to represent the primary secretion mechanism for several cytosolic proteins, such as HIV-Tat, galectin 1, interleukin-1β, and several proteins that shuttle between the nucleus and cytosol, such as fibroblast growth factor 1 (FGF1), FGF2, and nucleolin. Four nonclassical secretory pathways have been described including direct transport (presumably through transporters in the plasma membrane), secretion via exosomes, lysosomal secretion, and blebbing. The purpose of this study was to gain mechanistic insight into nonclassical protein secretion using phosphoglycerate kinase 1 (PGK1), a previously identified nonclassical secretory protein, as a reporter protein. Upon shifting HeLa cells into serum-free media PGK1 was released as a free soluble protein without cell loss. Release occurred in two phases: a rapid early phase and a slow late phase. Using a repertory of inhibitors, PGK1 release was shown not to rely on the classical secretory pathway. However, components of the cytoskeleton partially contributed to its release. Significantly, the presence of serum or bovine serum albumin in the media inhibited PGK1 release. These results are consistent with a novel model of protein release termed oncotic release, in which a change in the colloidal osmotic pressure (oncotic pressure) upon serum withdrawal creates nonlethal oncotic pores in the plasma membrane through which PGK1 - and likely other nearby proteins - are released before the pores are rapidly resealed. These findings identify an alternative mechanism of release for FGF1, HIV-Tat, and galectin 1 whose reported nonclassical secretion is induced by serum withdrawal. Oncotic release may occur in routine cell biological experiments during which cells are washed with serum-free buffers or media and in pathophysiological conditions, such as edema, during which extracellular protein concentrations change.


Secondary osteon and Haversian canal dimensions as behavioral indicators.
Variation in the size of structures within mature cortical bone is relevant to our understanding of apparent differences between human samples, and it is relevant to the development of histologically based age-estimation methods. It was proposed that variation may reflect effects of physical activity, through biomechanical and/or metabolic mechanisms. If these factors are local, femoral osteon area (On.Ar) should be more histologically variable than On.Ar in ribs. Ribs should show a higher variation in Haversian canal area (H.Ar) if they are sites of more remodeling activity and hence of arrested refilling of secondary osteons at time of death. This study compares On.Ar and H.Ar of secondary osteons from femora (15) and ribs (29) from 44 Holocene (Later Stone Age) foragers from South Africa (M = 19, F = 25) to values from paired femora and ribs from historic samples (Spitalfields and St. Thomas, 20 pairs from each). Fixed-effects analysis of variance demonstrates rib On.Ar to be significantly smaller than femur, but with no sex or age effects. The femur-to-rib On.Ar ratio is lower for the Holocene foragers than for the two modern samples because of relatively large rib On.Ar. Femora and ribs from the same skeleton normally show femoral On.Ar larger than rib On.Ar (37/44 pairs). Mean femoral values of On.Ar are more diverse than rib On.Ar values, but within-sample coefficients of variation are similar. Values for H.Ar are highly variable and do not reflect anatomical site, age, sex, or population effects. The patterning of osteon size does not appear to be linked to physical activity or to different rates of metabolic activity within the skeleton, at least not in a straightforward way.


Interventions to prevent or reduce the level of frailty in community-dwelling older adults: a protocol for a scoping review of the literature and international policies.
With ageing comes increased vulnerability such that older adults' ability to recover from acute illnesses, fall-related injuries and other stresses related to the physical ageing processes declines. This increased vulnerability, also known as frailty, is common in older adults and associated with increased healthcare service use and adverse health outcomes. Currently, there is no overview of available interventions to prevent or reduce the level of frailty (as defined by study's authors) which will help healthcare providers in community settings caring for older adults. We will address this gap by reviewing interventions and international policies that are designed to prevent or reduce the level of frailty in community-dwelling older adults. We will conduct a scoping review using the updated guidelines of Arksey and O'Malley to systematically search the peer-reviewed journal articles to identify interventions that aimed to prevent or reduce the level of frailty. We will search grey literature for international policies. The 6-stage scoping review model involves: (1) identifying the research question; (2) identifying relevant studies; (3) selecting studies; (4) charting the data; (5) collating, summarising and reporting the results and (6) consulting with key stakeholders. Our scoping review will use robust methodology to search for available interventions focused on preventing or reducing the level of frailty in community-dwelling older adults. We will consult with stakeholders to find out whether they find the frailty interventions/policies useful and to identify the barriers and facilitators to their implementation in Canada. We will disseminate our findings to relevant stakeholders at local, national and international levels by presenting at relevant meetings and publishing the findings. Our review will identify gaps in research and provide healthcare providers and policymakers with an overview of interventions that can be implemented to prevent or postpone frailty.


The effect of substituted laminin A chain-derived peptides on the conformation and activation kinetics of plasminogen.
Conversion of the zymogen plasminogen (Pg) to the active enzyme plasmin is catalyzed by proteinases such as tissue-type plasminogen activator (t-PA). Interaction of Pg with small ligands such as lysine or macromolecular ligands such as fibrin induces a dramatic conformational change in the zymogen which enhances its efficacy as a t-PA substrate, thereby increasing catalytic efficiency of the activation reaction. We have previously demonstrated that a synthetic peptide derived from amino acids 2091-2108 of the laminin A chain (designated LamA2091-2108) can significantly enhance t-PA-catalyzed Pg activation. To probe the mechanism of this stimulatory reaction, we have determined the effect of substituted LamA2091-2108 derivatives on Pg activation by t-PA. Substitution of charged residues in LamA2091-2108 with neutral amino acids decreases the kcat/Km observed in the presence of native LamA2091-2108. Furthermore, fluorescence-quenching experiments demonstrate that whereas LamA2091-2108 alters the solvent accessibility of Pg Trp residues, charge-substituted peptides have little effect on Pg conformation. These data suggest that LamA2091-2108 stimulates Pg activation by inducing a conformational change in the zymogen similar to that observed upon binding of other ligands such as lysine and fibrin.


Patients' priorities in selecting chronic lymphocytic leukemia treatments.
Currently, in the United States, 130 000 people live with chronic lymphocytic leukemia (CLL), and almost 20 000 new cases of CLL are diagnosed each year. Little is known about the value patients place upon the attributes of available CLL treatments, which vary in efficacy, side effects, and mode of administration. We used a discrete-choice experiment (DCE) to investigate patients' preferences for treatment attributes and the impact of out-of-pocket cost on patients' choices. DCE surveys pose a series of choices between hypothetical treatment options, each defined by a set of attributes, and the responses provide quantitative estimates of the average relative preferences for treatment attribute. Each hypothetical treatment in this survey was defined by 5 attributes with predefined levels for efficacy, adverse events, and mode administration. A patient advocacy organization recruited 384 patients with a self-reported physician diagnosis of CLL to complete the online survey. Respondents placed the highest relative importance on longer progression-free survival (PFS). However, the risk of adverse events also was important, as significant additional PFS was needed to offset patients' acceptance of worsening adverse events. A supplemental question with 2 treatments and varying costs was included to assess the impact of cost on choice. When costs were included, a large proportion of patients changed their choices between the 2 treatments. Given the available treatments and the high cost of some treatments, physicians may want to explore their patients' preferences for different treatment features, including benefit-risk tradeoffs and out-of-pocket cost, when selecting the best treatment strategies for patients.


Presynaptic inhibition of spinal sensory feedback ensures smooth movement.
The precision of skilled movement depends on sensory feedback and its refinement by local inhibitory microcircuits. One specialized set of spinal GABAergic interneurons forms axo-axonic contacts with the central terminals of sensory afferents, exerting presynaptic inhibitory control over sensory-motor transmission. The inability to achieve selective access to the GABAergic neurons responsible for this unorthodox inhibitory mechanism has left unresolved the contribution of presynaptic inhibition to motor behaviour. We used Gad2 as a genetic entry point to manipulate the interneurons that contact sensory terminals, and show that activation of these interneurons in mice elicits the defining physiological characteristics of presynaptic inhibition. Selective genetic ablation of Gad2-expressing interneurons severely perturbs goal-directed reaching movements, uncovering a pronounced and stereotypic forelimb motor oscillation, the core features of which are captured by modelling the consequences of sensory feedback at high gain. Our findings define the neural substrate of a genetically hardwired gain control system crucial for the smooth execution of movement.


Association Between Early-Life Antibiotic Use and the Risk of Islet or Celiac Disease Autoimmunity.
Evidence is lacking regarding the consequences of antibiotic use in early life and the risk of certain autoimmune diseases. To test the association between early-life antibiotic use and islet or celiac disease (CD) autoimmunity in genetically at-risk children prospectively followed up for type 1 diabetes (T1D) or CD. HLA-genotyped newborns from Finland, Germany, Sweden, and the United States were enrolled in the prospective birth cohort of The Environmental Determinants of Diabetes in the Young (TEDDY) study between November 20, 2004, and July 8, 2010. The dates of analysis were November 20, 2004, to August 31, 2014. Individuals from the general population and those having a first-degree relative with T1D were enrolled if they had 1 of 9 HLA genotypes associated with a risk for T1D. Parental reports of the most common antibiotics (cephalosporins, penicillins, and macrolides) used between age 3 months and age 4 years were recorded prospectively. Islet autoimmunity and CD autoimmunity were defined as being positive for islet or tissue transglutaminase autoantibodies at 2 consecutive clinic visits at least 3 months apart. Hazard ratios and 95% CIs calculated from Cox proportional hazards regression models were used to assess the relationship between antibiotic use in early life before seroconversion and the development of autoimmunity. Participants were 8495 children (49.0% female) and 6558 children (48.7% female) enrolled in the TEDDY study who were tested for islet and tissue transglutaminase autoantibodies, respectively. Exposure to and frequency of use of any antibiotic assessed in this study in early life or before seroconversion did not influence the risk of developing islet autoimmunity or CD autoimmunity. Cumulative use of any antibiotic during the first 4 years of life was not associated with the appearance of any autoantibody (hazard ratio [HR], 0.98; 95% CI, 0.95-1.01), multiple islet autoantibodies (HR, 0.99; 95% CI, 0.95-1.03), or the transglutaminase autoantibody (HR, 1.00; 95% CI, 0.98-1.02). The use of the most prescribed antibiotics during the first 4 years of life, regardless of geographic region, was not associated with the development of autoimmunity for T1D or CD. These results suggest that a risk of islet or tissue transglutaminase autoimmunity need not influence the recommendations for clinical use of antibiotics in young children at risk for T1D or CD.


Equipping tomorrow's doctors for the patients of today.
As the proportion of older patients with frailty presenting to health services increases, so does the need for doctors to be adequately trained to meet their needs. The presentations seen in such patients, the evidence-based models of care and skillsets required to deliver them are different than for younger patient groups-so specific training is required. Several research programmes have used detailed and explicit methods to establish evidence-based expert-validated curricula outlining learning outcomes for undergraduates in geriatric medicine-there is now broad-consensus on what newly qualified doctors need to know. There are, despite this, shortcomings in the teaching of undergraduates about geriatric medicine. National and international surveys from the UK, EU, USA, Canada, Austria and the Netherlands have all shown shortcomings in the content and amount of undergraduate teaching. Mechanisms to improve this situation, aside from specifying curricula, include developing academic departments and professorships in geriatric medicine, providing grants to develop teaching in geriatric medicine and developing novel teaching interventions to make the best of existing resources. Under the last of these headings, innovations have been shown to improve outcomes by: using technology to ensure the most effective allocation of teaching time and resources; using inter-professional education as a means of improving attitudes towards care of older patients; focusing teaching specifically on attitudes towards older patients and those who work with them; and trying to engage patients in teaching. Research areas going forward include how to incentivise medical schools to deliver specified curricula, how to choose from an ever-expanding array of teaching technologies, how to implement interprofessional education in a sustainable way and how to design teaching interventions using a qualitative understanding of attitudes towards older patients and the teams that care for them.


Polyglutamine Fibrils: New Insights into Antiparallel β-Sheet Conformational Preference and Side Chain Structure.
Understanding the structure of polyglutamine (polyQ) amyloid-like fibril aggregates is crucial to gaining insights into the etiology of at least ten neurodegenerative disorders, including Huntington's disease. Here, we determine the structure of D2Q10K2 (Q10) fibrils using ultraviolet resonance Raman (UVRR) spectroscopy and molecular dynamics (MD). Using UVRR, we determine the fibril peptide backbone Ψ and glutamine (Gln) side chain χ3 dihedral angles. We find that most of the fibril peptide bonds adopt antiparallel β-sheet conformations; however, a small population of peptide bonds exist in parallel β-sheet structures. Using MD, we simulate three different potential fibril structural models that consist of either β-strands or β-hairpins. Comparing the experimentally measured Ψ and χ3 angle distributions to those obtained from the MD simulated models, we conclude that the basic structural motif of Q10 fibrils is an extended β-strand structure. Importantly, we determine from our MD simulations that Q10 fibril antiparallel β-sheets are thermodynamically more stable than parallel β-sheets. This accounts for why polyQ fibrils preferentially adopt antiparallel β-sheet conformations instead of in-register parallel β-sheets like most amyloidogenic peptides. In addition, we directly determine, for the first time, the structures of Gln side chains. Our structural data give new insights into the role that the Gln side chains play in the stabilization of polyQ fibrils. Finally, our work demonstrates the synergistic power and utility of combining UVRR measurements and MD modeling to determine the structure of amyloid-like fibrils.


Plants Developed by New Genetic Modification Techniques-Comparison of Existing Regulatory Frameworks in the EU and Non-EU Countries.
The development of new genetic modification techniques (nGMs), also referred to as "new (breeding) techniques" in other sources, has raised worldwide discussions regarding their regulation. Different existing regulatory frameworks for genetically modified organisms (GMO) cover nGMs to varying degrees. Coverage of nGMs depends mostly on the regulatory trigger. In general two different trigger systems can be distinguished, taking into account either the process applied during development or the characteristics of the resulting product. A key question is whether regulatory frameworks either based on process- or product-oriented triggers are more advantageous for the regulation of nGM applications. We analyzed regulatory frameworks for GMO from different countries covering both trigger systems with a focus on their applicability to plants developed by various nGMs. The study is based on a literature analysis and qualitative interviews with regulatory experts and risk assessors of GMO in the respective countries. The applied principles of risk assessment are very similar in all investigated countries independent of the applied trigger for regulation. Even though the regulatory trigger is either process- or product-oriented, both triggers systems show features of the respective other in practice. In addition our analysis shows that both trigger systems have a number of generic advantages and disadvantages, but neither system can be regarded as superior at a general level. More decisive for the regulation of organisms or products, especially nGM applications, are the variable criteria and exceptions used to implement the triggers in the different regulatory frameworks. There are discussions and consultations in some countries about whether changes in legislation are necessary to establish a desired level of regulation of nGMs. We identified five strategies for countries that desire to regulate nGM applications for biosafety-ranging from applying existing biosafety frameworks without further amendments to establishing new stand-alone legislation. Due to varying degrees of nGM regulation, international harmonization will supposedly not be achieved in the near future. In the context of international trade, transparency of the regulatory status of individual nGM products is a crucial issue. We therefore propose to introduce an international public registry listing all biotechnology products commercially used in agriculture.


[The prevalence of Chagas' disease in puerperal women and congenital transmission in an endemic area of Peru].
To determine the prevalence of antibodies against Trypanosoma cruzi in puerperal women and to assess possible congenital transmission of Chagas' disease in the department of Arequipa, Peru, where the disease is endemic. Women who had given birth between December 2001 and July 2002 in three hospitals (two urban and one rural) and four health centers (three rural and one urban) of the department of Arequipa, Peru, were studied. The serological study included screening all the puerperal women in order to detect antibodies against T. cruzi through indirect immunofluorescence (IIF), with confirmatory testing done with enzyme-linked immunosorbent assay (ELISA) testing and the titration of immunoglobulin G (IgG) antibodies by IIF. IIF tests to screen for immunoglobulin M (IgM) antibodies were done with the seropositive women and their newborns, and infection was evaluated through xenodiagnosis (evaluated at 30 and 60 days) and the direct micromethod of Freilij et al. The results were analyzed in terms of the presence of the vector and of cases of Chagas' disease in the places where the puerperal women had been born and where they were living. Two neonatologists clinically evaluated the newborns in order to detect abnormalities and signs of congenital Chagas' disease. The overall prevalence of Chagas' disease in the 3 000 puerperal women studied was 0.73%. Prevalence was highest in two health centers located in rural areas (2.2% in El Pedregal and 4.1% in La Joya) (P=0.018). The disease was associated with previous direct contact with the vector (P<0.05) and with having been born in an area considered endemic (P<0.01). Four (20%) of the 20 seropositive puerperal women were also positive by xenodiagnosis. However, none of the women was aware of her infectious carrier state, and none showed the characteristic symptoms or signs of acute or chronic Chagas' disease. IgM antibodies were not detected in any of the puerperal women. One neonate (whose mother did not have evidence of parasitemia) presented an IgM titer of 1/8, but in later controls neither IgM nor IgG antibodies were detected. Parasites were not detected in the blood of the neonates by either of the two testing methods used. Of the 20 neonates evaluated, one presented microcephaly and hepatosplenomegaly; although the child had specific IgG antibodies against T. cruzi at birth, the antibodies were not present at the age of two months. The growth and development of the other 19 newborns were normal. The prevalence of Chagas' disease in puerperal women of the department of Arequipa, Peru, is low. No cases of intrauterine congenital transmission were found. We recommend carrying out studies on prenatal detection that evaluate more mothers and in which women who give birth at home also participate.


Proinsulin binds with high affinity the insulin receptor isoform A and predominantly activates the mitogenic pathway.
Proinsulin is generally regarded as an inactive prohormone because of its low metabolic activity. However, proinsulin appears to regulate embryo development in animal models. In this study, we evaluated whether proinsulin may differentially bind to and activate the two insulin receptor (IR) isoforms (IR-A and IR-B), because IR-A is a relatively low-specificity receptor that is prevalent in fetal and cancer cells and is able to mediate the growth effects of IGF-II. Mouse R(-) fibroblasts devoid of IGF-I receptor (IGF-IR) and stably transfected with cDNA encoding either human IR-A or IR-B (R(-) /IR-A and R(-) /IR-B cells) were used. Three human cancer cell lines were also studied. We found that proinsulin stimulated phosphorylation of IR-A with an EC(50) of 4.5 ± 0.6 nm and displaced [(125)I]insulin from IR-A with a similar EC(50). In contrast, proinsulin EC(50) values for stimulation of IR-B phosphorylation and for [(125)I]insulin displacement from IR-B were approximately 7-fold higher. Proinsulin did not bind or activate IGF-IR or IR/IGF-IR hybrids. Via IR-A, proinsulin activated the ERK/p70S6K pathway to a similar degree as insulin but elicited a weaker Akt response. Despite its low metabolic activity, proinsulin was almost equipotent as insulin in inducing cell proliferation and migration in cells expressing various IR-A levels. In conclusion, proinsulin is a selective IR-A ligand and may induce biological effects through this IR isoform.


Polymorphism of brain derived neurotrophic factor influences β amyloid load in cognitively intact apolipoprotein E ε4 carriers.
Aside from apolipoprotein E (APOE), genetic risk factors for β amyloid deposition in cognitively intact individuals remain to be identified. Brain derived neurotrophic factor (BDNF) modulates neural plasticity, which has been implicated in Alzheimer's disease. We examined in cognitively normal older adults whether the BDNF codon 66 polymorphism affects β amyloid burden and the relationship between β amyloid burden and cognitive scores, and how this relates to the effect of APOE. Amyloid load was measured by means of (18)F-flutemetamol PET in 64 community-recruited cognitively intact individuals (mean age 66, S.D. 5.1). Recruitment was stratified according to a factorial design with APOE (ε4 allele present vs absent) and BDNF (met allele at codon 66 present vs absent) as factors. Individuals in the four resulting cells were matched by the number of cases, age, and gender. Among the APOE ε4 carriers, BDNF met positive subjects had a significantly higher amyloid load than BDNF met negative subjects, while BDNF met carrier status did not have an effect in APOE ε4 noncarriers. This interaction effect was localized to precuneus, orbitofrontal cortex, gyrus rectus, and lateral prefrontal cortex. In the APOE ε4/BDNF met carriers, a significant inverse relationship existed between episodic memory scores and amyloid burden but not in any of the other groups. This hypothesis-generating experiment highlights a potential role of BDNF polymorphisms in the preclinical phase of β amyloid deposition and also suggests that BDNF codon 66 polymorphisms may influence resilience against β amyloid-related effects on cognition.


Pantoea species sepsis associated with sickle cell crisis in a pregnant woman with a history of pica.
Bacteria in the Pantoea genus are plant and soil associated Gram-negative rods described as nosocomial pathogens and as rare causes of community-acquired infections. The latter have been classically associated with gardening and plant thorn injuries and immunocompromised states are additional risk factors. We report a patient with pica and geophagia, Pantoea sepsis, and sickle cell crisis, associations not previously described. A 23-year-old pregnant female presented to the emergency department with sickle cell pain crisis. On the third day of hospitalization the patient developed fever subsequently determined to be caused by Pantoea bacteremia and sepsis. She was successfully treated with a two-week course of ceftriaxone. The patient admitted to a habit of frequently eating large amounts of soil and this geophagia had increased since she became pregnant. She had marked clinical improvement with treatment and she was counseled to stop eating soil. This is the first reported case of Pantoea infection possibly associated with geophagia and the first reported case of Pantoea bacteremia and sepsis related to an episode of sickle cell crisis.


A cautionary tale on ancient migration detection: mitochondrial DNA variation in Santa Cruz Islands, Solomon Islands.
Over the past decade, the origin of the first Malayo-Polynesian settlers of the island Pacific has become a contentious issue in molecular anthropology as well as in archaeology and historical linguistics. Whether the descendants of the ancestral Malayo-Polynesian speakers moved rapidly through Indonesia and Island Melanesia in a few hundred years, or whether they were the product of considerable intermingling within the more westerly part of the latter region, it is widely accepted that they were the first humans to colonize the distant Pacific islands beyond the central Solomon Islands approximately 3,000 years ago. The Santa Cruz Islands in the Eastern Solomons would have most likely been the first in Remote Oceania to be colonized by them. Archaeologically, the first Oceanic Austronesian settlement of this region appears to have been overlain by various later influences from groups farther west in a complex manner. Molecular anthropologists have tended to equate the spread of various Austronesian-speaking groups with a particular mitochondrial variant (a 9-base-pair [bp] deletion with specific D-loop variants). We have shown before that this is an oversimplified picture, and assumed that the Santa Cruz situation, with its series of intrusions, would be informative as to the power of mitochondrial DNA haplotype interpretations. In the Santa Cruz Islands, the 9-bp deletion is associated with a small number of very closely related hypervariable D-loop haplotypes resulting in a star-shaped Bandelt median network, suggesting a recent population expansion. This network is similar to Polynesian median networks. In a pairwise mismatch comparison, the Santa Cruz haplotypes have a bimodal distribution, with the first cluster being composed almost entirely of the 9-bp-deleted haplotypes-again attesting to their recent origins. Conversely, the nondeleted haplogroups bear signatures of more ancient origins within the general region. Therefore, while the profiles of the two sets of haplotypes indicate very distinctive origins in different populations with divergent expansion histories, the sequence of their introduction into the Santa Cruz Islands clearly does not follow simply.


Tumor induction by human adenovirus type 12 in hamsters: loss of the viral genome from adenovirus type 12-induced tumor cells is compatible with tumor formation.
The patterns of integration of adenovirus type 12 (Ad12) DNA in 39 virus-induced hamster tumors were determined. Both the amount of Ad12 DNA persisting and the apparent sites of insertion differed from tumor to tumor. In 30 tumors, the intact Ad12 genome persisted in colinear arrangement and in multiple copies. In nine tumors, only the left- or the left- and right-hand parts of the Ad12 genome persisted in the tumor cells. In three other cell lines the Ad12 genomes were lost completely during continuous passage in culture. A shift from epithelioid to fibroblastic morphology correlated with loss of Adl2 genomes. The cell line H1111(1) derived from an Ad12-induced tumor had lost all viral DNA by the thirteenth subpassage, but was still oncogenic when reinjected into animals. This finding raises the question, to what extent persistence of the Ad12 genome is essential for the oncogenic phenotype. Tumor cells could be detected histologically inside local lymphatic vessels. In those experiments in which Ad12 preparations were used which contained sizeable proportions of the symmetric recombinant between Ad12 and KB cellular DNA (Deuring et al., 1981), tumors were observed in the nuchal region of the animals.


Danish Observational Study of Eldercare work and musculoskeletal disorderS (DOSES): a prospective study at 20 nursing homes in Denmark.
Musculoskeletal disorders (MSDs), sickness absence and premature retirement are highly prevalent among eldercare workers. We conducted a prospective observational workplace study with the main purpose to investigate longitudinal associations between physical and psychosocial working conditions and occurrence of MSD and its consequences (pain-related interference with daily work activities and sickness absence) among Danish eldercare workers. At 20 Danish nursing homes, a total of 941 eldercare workers employed in day and evening shifts were invited to the study. Of those, 553 participated in the baseline measurements, and 441 completed the total period of 12 months follow-up. Data were collected from September 2013 to January 2016. Physical and psychosocial working conditions were assessed with multiple methods (observations, accelerometer measurements and work schedules), and multiple levels of information (nursing home, ward, resident and eldercare worker) were incorporated in the data collection. MSD and the consequences hereof were assessed monthly during a 1-year follow-up. Study participants and non-participants were comparable on most of the 27 sociodemographic, health and working condition characteristics at baseline. The exceptions were higher neck-shoulder pain intensity, less sickness absence, more exposure to negative behaviour from residents and a higher percentage of working day shifts and fewer evening shifts among participants compared with non-participants. The first publications will report on the associations of physical and psychosocial working conditions with occurrence of MSD and its consequences. In addition, the cohort gives the opportunity to investigate the importance of organisational, management and team factors for distribution of physical work demands and development of MSD among the workers. This will provide important knowledge for future workplace interventions to reduce MSD and sickness absence.


Exploring quantitative group-wise differentiation of Alzheimer's disease and behavioural variant frontotemporal dementia using tract-specific microstructural white matter and functional connectivity measures at multiple time points.
This study explored group-wise quantitative measures of tract-specific white matter (WM) microstructure and functional default mode network (DMN) connectivity to establish an initial indication of their clinical applicability for early-stage and follow-up differential diagnosis of Alzheimer's disease (AD) and behavioural variant frontotemporal dementia (bvFTD). Eleven AD and 12 bvFTD early-stage patients and 18 controls underwent diffusion tensor imaging and resting state functional magnetic resonance imaging at 3 T. All AD and 6 bvFTD patients underwent the same protocol at 1-year follow-up. Functional connectivity measures of DMN and WM tract-specific diffusivity measures were determined for all groups. Exploratory analyses were performed to compare all measures between the three groups at baseline and between patients at follow-up. Additionally, the difference between baseline and follow-up diffusivity measures in AD and bvFTD patients was compared. Functional connectivity of the DMN was not different between groups at baseline and at follow-up. Diffusion abnormalities were observed widely in bvFTD and regionally in the hippocampal cingulum in AD. The extent of the differences between bvFTD and AD was diminished at follow-up, yet abnormalities were still more pronounced in bvFTD. The rate of change was similar in bvFTD and AD. This study provides a tentative indication that quantitative tract-specific microstructural WM abnormalities, but not quantitative functional connectivity of the DMN, may aid early-stage and follow-up differential diagnosis of bvFTD and AD. Specifically, pronounced microstructural changes in anterior WM tracts may characterise bvFTD, whereas microstructural abnormalities of the hippocampal cingulum may characterise AD. • The clinical applicability of quantitative brain imaging measures for early-stage and follow-up differential diagnosis of dementia subtypes was explored using a group-wise approach. • Quantitative tract-specific microstructural white matter abnormalities, but not quantitative functional connectivity of the default mode network, may aid early-stage and follow-up differential diagnosis of behavioural variant frontotemporal dementia and Alzheimer's disease. • Pronounced microstructural white matter (WM) changes in anterior WM tracts characterise behavioural variant frontotemporal dementia, whereas microstructural WM abnormalities of the hippocampal cingulum in the absence of other WM changes characterise Alzheimer's disease.


The myeloid-specific sialic acid-binding receptor, CD33, associates with the protein-tyrosine phosphatases, SHP-1 and SHP-2.
The myeloid restricted membrane glycoprotein, CD33, is a member of the recently characterized "sialic acid-binding immunoglobulin-related lectin" family. Although CD33 can mediate sialic acid-dependent cell interactions as a recombinant protein, its function in myeloid cells has yet to be determined. Since CD33 contains two potential immunoreceptor tyrosine-based inhibition motifs in its cytoplasmic tail, we investigated whether it might act as a signaling receptor in myeloid cells. Tyrosine phosphorylation of CD33 in myeloid cell lines was stimulated by cell surface cross-linking or by pervanadate, and inhibited by PP2, a specific inhibitor of Src family tyrosine kinases. Phosphorylated CD33 recruited both the protein-tyrosine phosphatases, SHP-1 and SHP-2. CD33 was dephosphorylated in vitro by the co-immunoprecipitated tyrosine phosphatases, suggesting that it might also be an in vivo substrate. The first CD33 phosphotyrosine motif is dominant in CD33-SHP-1/SHP-2 interactions, since mutating tyrosine 340 in a CD33-cytoplasmic tail fusion protein significantly reduced binding to SHP-1 and SHP-2 in THP-1 lysates, while mutation of tyrosine 358 had no effect. Furthermore, the NH2-terminal Src homology 2 domain of SHP-1 and SHP-2, believed to be essential for phosphatase activation, selectively bound a CD33 phosphopeptide containing tyrosine 340 but not one containing tyrosine 358. Finally, mutation of tyrosine 340 increased red blood cell binding by CD33 expressed in COS cells. Hence, CD33 signaling through selective recruitment of SHP-1/SHP-2 may modulate its ligand(s) binding activity.


Thymic-independent T cell regeneration occurs via antigen-driven expansion of peripheral T cells resulting in a repertoire that is limited in diversity and prone to skewing.
Thymic regenerative capacity in humans decreases with age, suggesting that thymic-independent pathways of T cell regeneration may predominate during adulthood. Using a murine bone marrow transplantation model, we present evidence that thymic-independent T cell regeneration occurs primarily via expansion of peripheral T cells and is Ag driven since significant expansion of CD4+ or CD8+ transgenic (Tg+)/TCR-bearing cells occurs only in the presence of Ag specific for the TCR. Such expansion resulted in skewing of the regenerated repertoire with 40 to 65% of the regenerated CD4+ or CD8+ T cells expressing the Tg+/TCR in thymectomized hosts after bone marrow transplantation. In experiments in which nontransgenic population are used as T cell inocula, we noted decreased CD4 expansion when Class II MHC was blocked by mAb treatment in vivo, an CD8 expansion failed to occur in Class I MHC-deficient hosts providing evidence that T cell regeneration in thymic-deficient hosts largely occurs via TCR-MHC-mediated selection of peripheral T cell populations. This process results in a T cell repertoire comprised exclusively of T cells recently activated by the antigenic milieu of the host, with negligible numbers of residual "naive" cells bearing TCRs for Ags absent at the time of expansion. These findings have important implications for approached to enhance T cell regeneration in humans and provide evidence that vaccine strategies could skew the T cell repertoire toward a specific antigenic target if administered to thymic-deficient hosts during immune reconstitution.


Mesoderm is required for coordinated cell movements within zebrafish neural plate in vivo.
Morphogenesis of the zebrafish neural tube requires the coordinated movement of many cells in both time and space. A good example of this is the movement of the cells in the zebrafish neural plate as they converge towards the dorsal midline before internalizing to form a neural keel. How these cells are regulated to ensure that they move together as a coherent tissue is unknown. Previous work in other systems has suggested that the underlying mesoderm may play a role in this process but this has not been shown directly in vivo. Here we analyze the roles of subjacent mesoderm in the coordination of neural cell movements during convergence of the zebrafish neural plate and neural keel formation. Live imaging demonstrates that the normal highly coordinated movements of neural plate cells are lost in the absence of underlying mesoderm and the movements of internalization and neural tube formation are severely disrupted. Despite this, neuroepithelial polarity develops in the abnormal neural primordium but the resulting tissue architecture is very disorganized. We show that the movements of cells in the zebrafish neural plate are highly coordinated during the convergence and internalization movements of neurulation. Our results demonstrate that the underlying mesoderm is required for these coordinated cell movements in the zebrafish neural plate in vivo.


Lay and peer counsellors to reduce leprosy-related stigma--lessons learnt in Cirebon, Indonesia.
Counselling has been identified as a promising strategy to reduce stigma. Lay and peer counsellors have provided counselling in various fields, but this has not yet been studied in the field of leprosy. The Stigma Assessment and Reduction of Impact (SARI) project in Cirebon District, Indonesia took up this endeavour. This paper describes the initial experiences based on the perspectives of the lay and peer counsellors and aims to provide lessons learnt for future initiatives. The selection of lay and peer counsellors was based upon pre-defined criteria such as completed junior high school and level of confidence. This study draws on the notes of seven monitoring and evaluation meetings and 21 group discussions the main researcher facilitated with the lay and peer counsellors and the notes written by the lay and peer counsellors on the sessions with their clients. In total, 198 people affected by leprosy were offered counselling by the 11 lay and 12 peer counsellors; 145 accepted this offer. The other 53 either did not need counselling or did not want to participate for example due to worries about disclosure. Effective communication skills such as listening and asking effective questions were important, but also difficult to acquire for the lay and peer counsellors. Sharing personal experiences was highly appreciated by clients and stimulated a deepened reflection. Challenges related to concealment and effective skills exist, but some people affected by leprosy and others can become effective counsellors making it at the outset a challenging but nevertheless promising intervention.


The population structure of Plasmodium falciparum and Plasmodium vivax during an epidemic of malaria in the Eastern Highlands of Papua New Guinea.
Although most of the Papua New Guinea highlands are too high for stable malaria transmission, local epidemics are a regular feature of the region. Few detailed descriptions of such epidemics are available, however. We describe the investigation of a malaria epidemic in the Obura Valley, Eastern Highlands Province, Papua New Guinea. Of the 244 samples examined by microscopy, 6.6% were positive for Plasmodium falciparum only, 9.4% were positive for Plasmodium vivax only, and 1.2% were mixed infections. MSP2 and MSP3alpha genotyping and AMA1 sequencing were used to determine the genetic variation present in a sample of P. falciparum and P. vivax infections. The P. vivax infections were found to be genetically highly diverse. In contrast, all P. falciparum samples were of a single genotype. This striking difference in genetic diversity suggests endemic, low-level local transmission for P. vivax but an outside introduction of P. falciparum as the most likely source of the epidemic.


Monitoring quality of care in residential care for the elderly: the information challenge.
This study assesses the administrative data compiled on residential care facilities for the elderly (RCFEs) by the state of California and considers the feasibility of their adaptation into a comprehensive information system. Required state RCFE reporting forms were reviewed for potential data elements. Recording and reporting variation was evaluated using a stratified probability sample of 340 facilities licensed in Northern and Central California. Stratification was by facility size and state district office. Data collection included a 5-year retrospective review of forms and documents in each facility's public file. Little of the information required from RCFEs is computerized. Most of it is maintained at the individual facility and not included in public files. Basic information, while included in the public file, is commonly either not available or not current. Resident characteristics and outcomes are not compiled, except indirectly in citations. The information required from RCFEs, if appropriately compiled and maintained, would produce a comprehensive quality assurance system and more effectively support consumer information and policy needs.


The design and synthesis of inhibitors of pantothenate synthetase.
Pantothenate synthetase catalyses the ATP-dependent condensation of D-pantoate and beta-alanine to form pantothenate. Ten analogues of the reaction intermediate pantoyl adenylate, in which the phosphodiester is replaced by either an ester or sulfamoyl group, were designed as potential inhibitors of the enzyme. The esters were all modest competitive inhibitors, the sulfamoyls were more potent, consistent with their closer structural similarity to the pantoyl adenylate intermediate.


Devalued, deskilled and diversified: explaining the proliferation of the strip industry in the UK.
This paper looks beyond the debates that focus on the objectification of the female body to examine the question as to why strip clubs have proliferated and found a permanent place in the night-time economy in the UK. Using empirical qualitative and quantitative data from the largest study into the strip industry in the UK to date, we challenge the common assumption that 'demand' is responsible for the rise in erotic dance. Instead, we argue that the proliferation of strip clubs is largely due to the internal economic structures of the industry which have developed partly in response to the financial crisis beginning in 2008. First, we argue that clubs profit from individual dancers through an exploitative system of fees and fines, rendering a strip club business a low cost investment with high returns and little risk to club owners. Second, we note that the last decade has seen diversification of the industry accompanied by deskilling and devaluing of dancing and dancers' labour. Third, we demonstrate that despite the negative effects of these changes on workers, there has been an expansion of the industry as the ability to make profit, even during a financial crisis was ensured through the transferral of risk to workers. Overall, we suggest that far from proliferating as a response to demand, the industry has maintained its market presence due to its ability to establish highly financially exploitation employment relationships with dancers at a time of economic fragility.


Visual Prognosis in USH2A-Associated Retinitis Pigmentosa Is Worse for Patients with Usher Syndrome Type IIa Than for Those with Nonsyndromic Retinitis Pigmentosa.
USH2A mutations are an important cause of retinitis pigmentosa (RP) with or without congenital sensorineural hearing impairment. We studied genotype-phenotype correlations and compared visual prognosis in Usher syndrome type IIa and nonsyndromic RP. Clinic-based, longitudinal, multicenter study. Consecutive patients with Usher syndrome type IIa (n = 152) and nonsyndromic RP (n = 73) resulting from USH2A mutations from ophthalmogenetic clinics in the Netherlands and Belgium. Data on clinical characteristics, visual acuity, visual field measurements, retinal imaging, and electrophysiologic features were extracted from medical charts over a mean follow-up of 9 years. Cumulative lifetime risks of low vision and blindness were estimated using Kaplan-Meier survival analysis. Low vision and blindness. Participant groups had similar distributions of gender (48% vs. 45% males in Usher syndrome type IIa vs. nonsydromic RP; P = 0.8), ethnicity (97% vs. 99% European; P = 0.3), and median follow-up time (6.5 years vs. 3 years; P = 0.3). Usher syndrome type IIa patients demonstrated symptoms at a younger age (median age, 15 years vs. 25 years; P < 0.001), were diagnosed earlier (median age, 26 years vs. 36.5 years; P < 0.001), and became visually impaired 13 years earlier (median age, 41 years vs. 54 years; P < 0.001) based on VF and 18 years earlier based on VA (median age, 54 years vs. 72 years; P < 0.001) than nonsyndromic RP patients. The presence of 2 truncating mutations in USH2A was associated mostly with the syndromic phenotype, whereas other combinations were present in both groups. We found novel variants in Usher syndrome type IIa (25%) and nonsyndromic RP (19%): 29 missense mutations, 10 indels, 14 nonsense mutations, 9 frameshift mutations, and 5 splice-site mutations. Most patients with USH2A-associated RP have severe visual impairment by age 50. However, those with Usher syndrome type IIa have an earlier decline of visual function and a higher cumulative risk of visual impairment than those without nonsyndromic RP. Complete loss of function of the USH2A protein predisposes to Usher syndrome type IIa, but remnant protein function can lead to RP with or without hearing loss.


Diabetes insipidus.
Diabetes insipidus is a heterogeneous condition characterized by polyuria and polydipsia caused by a lack of secretion of vasopressin, its physiological suppression following excessive water intake, or kidney resistance to its action. In many patients, it is caused by the destruction or degeneration of the neurons that originate in the supraoptic and paraventricular nuclei of the hypothalamus. Known causes of these lesions include: germinoma or craniopharyngioma; Langerhans cell histiocytosis and sarcoidosis of the central nervous system; local inflammatory, autoimmune or vascular diseases; trauma following surgery or accident; and, rarely, genetic defects in vasopressin biosynthesis inherited as autosomal dominant or X-linked recessive traits. Thirty to fifty percent of cases are considered idiopathic. Magnetic resonance imaging (MRI) allows identification of the posterior pituitary hyperintensity and of hypothalamic-pituitary abnormalities. Thickening of the pituitary stalk is the second most common finding on MRI scans in several local inflammatory pathologies and autoimmune diseases or germinoma, but it is not specific to any single subtype. A progressive increase in the size of the anterior pituitary gland should alert physicians to the possibility that a germinoma is present, whereas a decrease can suggest the presence of an inflammatory or autoimmune process. Most children with acquired central diabetes insipidus and a thickened pituitary stalk have anterior pituitary hormone deficiencies during follow-up. Biopsy of enlarged pituitary stalk should be reserved for patients with a hypothalamic-pituitary mass and progressive thickening of the pituitary stalk, since spontaneous recovery may occur.


Wheat domestication accelerated evolution and triggered positive selection in the beta-xylosidase enzyme of Mycosphaerella graminicola.
Plant cell wall degrading enzymes (PCWDEs) of plant pathogens are receiving increasing interest for their potential to trigger plant defense reactions. In an antagonistic co-evolutionary arms race between host and pathogen, PCWDEs could be under strong selection. Here, we tested the hypothesis that PCWDEs in the fungal wheat pathogen Mycosphaerella graminicola have been positively selected by analyzing ratios of non-synonymous and synonymous nucleotide changes in the genes encoding these enzymes. Analyses of five PCWDEs demonstrated that one (beta-xylosidase) has been under strong positive selection and experienced an accelerated rate of evolution. In contrast, PCWDEs in the closest relatives of M. graminicola collected from wild grasses did not show evidence for selection or deviation from a molecular clock. Since the genealogical divergence of M. graminicola from these latter species coincided with the onset of agriculture, we hypothesize that the recent domestication of the host plant and/or agricultural practices triggered positive selection in beta-xylosidase and that this enzyme played a key role in the emergence of a host-specialized pathogen.


Construction and characterization of attenuated delta aroA delta virG Shigella flexneri 2a strain CVD 1203, a prototype live oral vaccine.
We engineered an oral Shigella vaccine prototype that can invade intestinal epithelial cells but cannot undergo extensive intracellular replication or extend to adjacent epithelial cells. Strain CVD 1203, derived from wild-type Shigella flexneri 2a by introducing deletions in chromosomal aroA and invasion plasmid virG, was highly attenuated in the Sereny test. Two 10(9)-CFU orogastric doses (2 weeks apart) stimulated production of secretory immunoglobulin A antibodies to S. flexneri 2a and protected against conjunctival sac challenge with virulent S. flexneri 2a.


Nontuberculous mycobacteria in hospital water systems: application of HPLC for identification of environmental mycobacteria.
Nontuberculous mycobacteria (NTM), ubiquitous in water environments, are increasingly recognized as nosocomial pathogens. Our study reports a one-year survey of the water system of two hospitals, A and B, in a small town near Florence, Italy. NTM were found throughout the study period in both settings, but B showed a significantly higher mycobacterial load. Mycobacterium gordonae and Mycobacterium fortuitum were the most frequent species isolated. Identification was carried out by conventional techniques and by high performance liquid chromatography (HPLC) analysis of cell wall mycolic acids. HPLC profiling could be used as a first-choice method for identification of environmental mycobacteria.


Combined Effect of Levels in Personal Self-Regulation and Regulatory Teaching on Meta-Cognitive, on Meta-Motivational, and on Academic Achievement Variables in Undergraduate Students.
The Theory of Self- vs. Externally-Regulated Learning™ (SRL vs. ERL) proposed different types of relationships among levels of variables in Personal Self-Regulation (PSR) and Regulatory Teaching (RT) to predict the meta-cognitive, meta-motivational and -emotional variables of learning, and of Academic Achievement in Higher Education. The aim of this investigation was empirical in order to validate the model of the combined effect of low-medium-high levels in PSR and RT on the dependent variables. For the analysis of combinations, a selected sample of 544 undergraduate students from two Spanish universities was used. Data collection was obtained from validated instruments, in Spanish versions. Using an ex-post-facto design, different Univariate and Multivariate Analyses (3 × 1, 3 × 3, and 4 × 1) were conducted. Results provide evidence for a consistent effect of low-medium-high levels of PSR and of RT, thus giving significant partial confirmation of the proposed rational model. As predicted, (1) the levels of PSR and positively and significantly effected the levels of learning approaches, resilience, engagement, academic confidence, test anxiety, and procedural and attitudinal academic achievement; (2) the most favorable type of interaction was a high level of PSR with a high level RT process. The limitations and implications of these results in the design of effective teaching are analyzed, to improve university teaching-learning processes.


Integrating Deoxyribozymes into Colorimetric Sensing Platforms.
Biosensors are analytical devices that have found a variety of applications in medical diagnostics, food quality control, environmental monitoring and biodefense. In recent years, functional nucleic acids, such as aptamers and nucleic acid enzymes, have shown great potential in biosensor development due to their excellent ability in target recognition and catalysis. Deoxyribozymes (or DNAzymes) are single-stranded DNA molecules with catalytic activity and can be isolated to recognize a wide range of analytes through the process of in vitro selection. By using various signal transduction mechanisms, DNAzymes can be engineered into fluorescent, colorimetric, electrochemical and chemiluminescent biosensors. Among them, colorimetric sensors represent an attractive option as the signal can be easily detected by the naked eye. This reduces reliance on complex and expensive equipment. In this review, we will discuss the recent progress in the development of colorimetric biosensors that make use of DNAzymes and the prospect of employing these sensors in a range of chemical and biological applications.


Reversible thiol oxidation in the H2O2-dependent activation of the transcription factor Pap1.
Reversible thiol oxidation is both a mark of hydrogen peroxide (H2O2) toxicity and an initiator of signalling events. H2O2 sensors contain exposed and reactive cysteine residues, which become transiently oxidized as an activation mechanism. In fission yeast, the Pap1 (pombe AP-1) transcription factor is normally cytosolic, and upon H2O2 stress it undergoes post-translational modifications impairing its nuclear export; genetic evidences suggested the formation of a disulphide bond in Pap1 as a triggering activation event. Nuclear Pap1 is then recruited to about 50-80 promoters and induces an adaptation response. We have now dissected the role of all seven cysteine residues in Pap1 using genetic and proteomic techniques, and we show that four of them are required for Pap1 to be activated by H2O2 stress. Thus, mutants lacking each one of these cysteine residues display sensitivity to peroxides. Furthermore, these mutant proteins do not become oxidized by H2O2 and cannot bind to promoters or trigger the Pap1-dependent gene expression program. We also demonstrate, by proteomic analysis of reduced and oxidized Pap1, that these four cysteine residues are reversibly oxidized upon H2O2 stress. Our study suggests that not just one but probably two disulphide bonds are required to promote the important conformational changes that trigger Pap1 activation and nuclear accumulation.


The prevalence and cost of unapproved uses of top-selling orphan drugs.
The Orphan Drug Act encourages drug development for rare conditions. However, some orphan drugs become top sellers for unclear reasons. We sought to evaluate the extent and cost of approved and unapproved uses of orphan drugs with the highest unit sales. We assessed prescription patterns for four top-selling orphan drugs: lidocaine patch (Lidoderm) approved for post-herpetic neuralgia, modafinil (Provigil) approved for narcolepsy, cinacalcet (Sensipar) approved for hypercalcemia of parathyroid carcinoma, and imatinib (Gleevec) approved for chronic myelogenous leukemia and gastrointestinal stromal tumor. We pooled patient-specific diagnosis and prescription data from two large US state pharmaceutical benefit programs for the elderly. We analyzed the number of new and total patients using each drug and patterns of reimbursement for approved and unapproved uses. For lidocaine patch, we subcategorized approved prescriptions into two subtypes of unapproved uses: neuropathic pain, for which some evidence of efficacy exists, and non-neuropathic pain. We found that prescriptions for lidocaine patch, modafinil, and cinacalcet associated with non-orphan diagnoses rose at substantially higher rates (average monthly increases in number of patients of 14.6, 1.45, and 1.58) than prescriptions associated with their orphan diagnoses (3.12, 0.24, and 0.03, respectively (p<0.001 for all)). By contrast, for imatinib, approved uses increased significantly over off-label (0.97 vs. 0.47 patients, p<0.001). Spending on off-label uses was highest for lidocaine patch and modafinil (>75%). Increases in lidocaine patch use for non-neuropathic pain far exceeded neuropathic pain (10.2 vs. 3.6 patients, p<0.001). In our sample, three of four top-selling orphan drugs were used more commonly for non-orphan indications. These orphan drugs treated common clinical symptoms (pain and fatigue) or laboratory abnormalities. We should continue to monitor orphan drug use after approval to identify products that come to be widely used for non-FDA approved indications, particularly those without adequate evidence of efficacy.


Prescription drug abuse - A timely update.
Prescription drug abuse is a rising problem in Australia and pharmaceutical drugs have been the most frequent contributors to overdose deaths in Victoria in recent years. The objectives of this article are to examine the main prescription drugs contributing to overdose deaths and to consider how doctors may help in reducing this problem. Data from the Coroners Court of Victoria list the main drugs that contributed to drug-related deaths in 2009-15. Analysis of the data reveals that pharmaceutical drugs contributed to 80% of overdose deaths; benzodiazepines and opioids were the main drug groups involved. Strategies for reducing and managing prescription drug abuse in primary care settings are outlined in this article, including references to published evidence-based clinical guidelines from The Royal Australian College of General Practitioners (RACGP). The safety profile of buprenorphine/ naloxone over methadone is noted and raised as a consideration for clinicians when assessing a patient for opioid replacement therapy.