Immunological diagnosis of tuberculosis: problems and strategies for success.
Tuberculosis remains a serious social and public health problem, affecting millions of people annually. The bacille Calmette-Guerin (BCG) vaccine, used prophylactically, does not impede the progression of the disease, which usually manifests as decreased cellular immunity. Early diagnosis, together with polychemotherapy, can control the dissemination of the tuberculosis infection. The current diagnostic methods present certain problems. Such problems include the low sensitivity of sputum smear microscopy, the fact that performing microbiological cultures is quite time-consuming, and the low specificity of the skin test with the purified protein derivative of M. tuberculosis. New diagnostic methods, which use specific antigens such as the early secreted antigenic target 6-kDa and culture filtrate protein 10kDa, are being evaluated. The genes that encode these antigens are located in the DNA region of difference 1 of M. tuberculosis, M. africanum and M. bovis. However, they are absent from the M. bovis (BCG) and from most environmental mycobacteria. Diagnostic methods such as QuantiFERON-TB(R) and T SPOT.TB(R), which are based on the production of interferon-gamma by T lymphocytes, in response to those antigens, are being tested and have been found to outstrip the purified protein derivative skin test in the following characteristics: greater sensitivity; lower cross-reactivity due to BCG vaccination or infection with environmental mycobacteria; and execution time. The introduction of diagnostic methods that are more specific and sensitive, together with gaining a better understanding of the molecular and cellular mechanisms that regulate the parasite-host interaction, can increase the efficiency of strategies devised to combat tuberculosis.


Potential and limitations of inferring ecosystem photosynthetic capacity from leaf functional traits.
The aim of this study was to systematically analyze the potential and limitations of using plant functional trait observations from global databases versus in situ data to improve our understanding of vegetation impacts on ecosystem functional properties (EFPs). Using ecosystem photosynthetic capacity as an example, we first provide an objective approach to derive robust EFP estimates from gross primary productivity (GPP) obtained from eddy covariance flux measurements. Second, we investigate the impact of synchronizing EFPs and plant functional traits in time and space to evaluate their relationships, and the extent to which we can benefit from global plant trait databases to explain the variability of ecosystem photosynthetic capacity. Finally, we identify a set of plant functional traits controlling ecosystem photosynthetic capacity at selected sites. Suitable estimates of the ecosystem photosynthetic capacity can be derived from light response curve of GPP responding to radiation (photosynthetically active radiation or absorbed photosynthetically active radiation). Although the effect of climate is minimized in these calculations, the estimates indicate substantial interannual variation of the photosynthetic capacity, even after removing site-years with confounding factors like disturbance such as fire events. The relationships between foliar nitrogen concentration and ecosystem photosynthetic capacity are tighter when both of the measurements are synchronized in space and time. When using multiple plant traits simultaneously as predictors for ecosystem photosynthetic capacity variation, the combination of leaf carbon to nitrogen ratio with leaf phosphorus content explains the variance of ecosystem photosynthetic capacity best (adjusted R2 = 0.55). Overall, this study provides an objective approach to identify links between leaf level traits and canopy level processes and highlights the relevance of the dynamic nature of ecosystems. Synchronizing measurements of eddy covariance fluxes and plant traits in time and space is shown to be highly relevant to better understand the importance of intra- and interspecific trait variation on ecosystem functioning.


Side-chain ionization states in a potassium channel.
KcsA is a bacterial K+ channel that is gated by pH. Continuum dielectric calculations on the crystal structure of the channel protein embedded in a low dielectric slab suggest that side chains E71 and D80 of each subunit, which lie adjacent to the selectivity filter region of the channel, form a proton-sharing pair in which E71 is neutral (protonated) and D80 is negatively charged at pH 7. When K+ ions are introduced into the system at their crystallographic positions the pattern of proton sharing is altered. The largest perturbation is for a K+ ion at site S3, i.e., interacting with the carbonyls of T75 and V76. The presence of multiple K+ ions in the filter increases the probability of E71 being ionized and of D80 remaining neutral (i.e., protonated). The ionization states of the protein side chains influence the potential energy profile experienced by a K+ ion as it is translated along the pore axis. In particular, the ionization state of the E71-D80 proton-sharing pair modulates the shape of the potential profile in the vicinity of the selectivity filter. Such reciprocal effects of ion occupancy on side-chain ionization states, and of side-chain ionization states on ion potential energy profiles will complicate molecular dynamics simulations and related studies designed to calculate ion permeation energetics.


Long Noncoding RNA SPRY4-IT1 Modulates Ketamine-Induced Neurotoxicity in Human Embryonic Stem Cell-Derived Neurons through EZH2.
This study aimed to investigate whether long noncoding RNA sprouty receptor tyrosine kinase signaling antagonist 4-intronic transcript 1 (SPRY4-IT1) is involved in the regulation of ketamine-induced neurotoxicity. Human embryonic stem cells (hESCs) were induced into neurons in vitro and treated with ketamine. Apoptosis and neurite degeneration assays were used to determine ketamine-induced neurotoxicity and qRT-PCR to determine SPRY4-IT1 expression. SPRY4-IT1 was downregulated in hESC-induced neurons to examine its regulation on ketamine-induced neurotoxicity. The correlation between enhancer of zeste homolog 2 (EZH2) and SPRY4-IT1 was also examined. EZH2 was upregulated in SPRY4-IT1-downregualted hESC-induced neurons to further examine its participation in SPRY4-IT1-mediated ketamine neurotoxicity. Ketamine-induced dose-dependent apoptosis, neurite degeneration, and SPRY4-IT1 upregulation in hESC-induced neurons. Lentivirus-mediated SPRY4-IT1 downregulation protected ketamine neurotoxicity. EZH2 expression was positively correlated with SPRY4-IT1 in hESC-induced neurons. EZH2 overexpression markedly reversed the protective effects of SPRY4-IT1 knockdown on ketamine neurotoxicity. SPRY4-IT1 is involved in anesthesia-induced neurotoxicity, possibly through the regulation on EZH2 gene.


Workplace phobia, workplace problems, and work ability among primary care patients with chronic mental disorders.
Work-related anxieties are frequent and have a negative effect on the occupational performance of patients and absence due to sickness. Most important is workplace phobia, that is, panic when approaching or even thinking of the workplace. This study is the first to estimate the prevalence of workplace phobia among primary care patients suffering from chronic mental disorders and to describe which illness-related or workplace-specific context factors are associated with workplace phobia. A convenience sample of 288 primary care patients with chronic mental disorders (70% women) seen by 40 primary care clinicians in Germany were assessed using a standardized diagnostic interview about mental disorders and workplace problems. Workplace phobia was assessed by the Workplace Phobia Scale and a structured Diagnostic and Statical Manual of Mental Disorders-based diagnostic interview. In addition, capacity and participation restrictions, illness severity, and sick leave were assessed. Workplace phobia was found in 10% of patients with chronic mental disorders, that is, approximately about 3% of all general practice patients. Patients with workplace phobia had longer durations of sick leave than patients without workplace phobia and were impaired to a higher degree in work-relevant capacities. They also had a higher degree of restrictions in participation in other areas of life. Workplace phobia seems to be a frequent problem in primary care. It may behoove primary care clinicians to consider workplace-related anxiety, including phobia, particularly when patients ask for a work excuse for nonspecific somatic complaints.


Social learning from humans or conspecifics: differences and similarities between wolves and dogs.
Most domestication hypotheses propose that dogs have been selected for enhanced communication and interactions with humans, including learning socially from human demonstrators. However, to what extent these skills are newly derived and to what extent they originate from wolf-wolf interactions is unclear. In order to test for the possible origins of dog social cognition, we need to compare the interactions of wolves and dogs with humans and with conspecifics. Here, we tested identically raised and kept juvenile wolves and dogs in a social learning task with human and conspecific demonstrators. Using a local enhancement task, we found that both wolves and dogs benefitted from a demonstration independent of the demonstrator species in comparison to a control, no demonstration condition. Interestingly, if the demonstrator only pretended to hide food at the target location, wolves and dogs reacted differently: while dogs differentiated between this without-food and with-food demonstration independent of the demonstrator species, wolves only did so in case of human demonstrators. We attribute this finding to wolves being more attentive toward behavioral details of the conspecific models than the dogs: although the demonstrator dogs were trained to execute the demonstration, they disliked the food reward, which might have decreased the interest of the wolves in finding the food reward. Overall, these results suggest that dogs but also wolves can use information provided by both human and conspecific demonstrators in a local enhancement task. Therefore we suggest that a more fine-scale analysis of dog and wolf social learning is needed to determine the effects of domestication.


Institutional experience of healthy pediatric patients presenting with atrial fibrillation who had an electrophysiology study.
Atrial fibrillation (AF) is a very common tachyarrhythmia with increasing prevalence with age, but uncommon in the pediatric population. Understanding that AF increases comorbidities make the need for investigation and potential elimination of alternate etiologies in pediatric AF patients critical. The objective of this study was to review our institutional data and compare our findings with previously documented adult AF risk factors to pediatric patients while also identifying which patients had alternate electrophysiology diagnoses amenable to transcatheter ablation. A retrospective chart review was performed identifying AF patients who were less than 21 years old, had no significant congenital cardiovascular anomalies, a documented episode of AF on electrocardiogram and underwent invasive electrophysiology study (EPS). Nineteen patients were identified over a 9-year period of time finding a male predominance (74%), the average age of 14.95 ± 4.17 years, the average weight of 78.5 ± 31.4 kg, and average body mass index of 26.8 ± 6.87 kg/m2 . Preprocedural left atrial volumes made on echocardiograms demonstrated a mean of 33.96 ± 16.35 mL/m2 (Z-scores -0.81 ± 1.50), indicating no dilation. Five of nineteen patients (26%) had additional electrophysiologic diagnoses during EPS, including atrioventricular reentrant tachycardia (n = 2, 10%) and atrioventricular nodal reentrant tachycardia (n = 3, 16%). Four patients underwent successful ablation with no documented or clinical AF recurrence. Adult risk factors of male predominance and obesity were seen in pediatric AF patients, while left atrial enlargement was not. Twenty-one percent of the pediatric AF patients who had additional electrophysiologic substrates and successful ablations resulted in no further clinical episodes of AF. This suggests that pediatric patients presenting with AF might benefit from an EPS as part of a complete evaluation.


Observations on the fine structure of the turtle atrium.
The general fine structure of the atrial musculature of the turtle heart is described, including; the nature of the sarcolemma; the cross-banded structure of the myofibrils; the character of the sarcoplasm, and the form and disposition of its organelles. An abundant granular component of the sarcoplasm in this species is tentatively identified as a particulate form of glycogen. The myocardium is composed of individual cells joined end to end at primitive intercalated discs, and side to side at sites of cohesion that resemble the desmosomes of epithelia. Transitional forms are found between desmosomes and intercalated discs. Both consist of a thickened area of the cell membrane with an accumulation of dense material in the subjacent cytoplasm. This dense amorphous component is often continuous with the Z substance of the myofibrils and may be of the same composition. The observations reported reemphasize the basic similarity between desmosomes and terminal bars of epithelia and intercalated discs of cardiac muscle. Numerous unmyelinated nerves are found beneath the endocardium. Some of these occupy recesses in the surface of Schwann cells; others are naked axons. No specialized nerve endings are found. Axons passing near the sarcolemma contain synaptic vesicles, and it is believed that this degree of proximity is sufficient to constitute a functioning myoneural junction.


Dopaminergic regulation of striatal acetylcholine release: importance of D1 and N-methyl-D-aspartate receptors.
The dopaminergic regulation of striatal cholinergic activity was studied using in vivo microdialysis to measure interstitial concentrations of acetylcholine (ACh) and choline in the striata of freely moving rats. The quaternary acetylcholinesterase inhibitor neostigmine (100 nM) was included in the perfusion solution to increase the recovery of ACh. d-Amphetamine (2 mg/kg, s.c.) and nomifensine (5 mg/kg, s.c.) increased the concentration of ACh in the striatal dialysate by 40 to 60%. Interstitial choline concentrations were reduced by both drugs. Administration of the selective D1 receptor antagonist SCH 23390 (0.3 mg/kg, s.c.) decreased the concentration of ACh in the striatal dialysate by 15 to 20%; in contrast, the selective D2 antagonist raclopride (1 mg/kg, s.c.) increased striatal ACh release by 50 to 60%. Raclopride also briefly increased the extracellular concentration of choline. Raclopride blocked the increase in locomotor activity produced by d-amphetamine, but did not further enhance ACh release. In contrast, SCH 23390 completely antagonized the increases in locomotion and striatal ACh release produced by d-amphetamine. These results indicate that d-amphetamine increases ACh release in the striatum via a D1 receptor mechanism. Consistent with this hypothesis, the selective D1 receptor agonist CY 208-243 (1 mg/kg, s.c.) increased striatal ACh release by approximately 60%. In contrast, local application of CY 208-243 (10 microM) and SCH 23390 (10 microM) failed to alter ACh concentrations in the striatal dialysate. Inclusion of the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (10 microM) in the striatal perfusion solution significantly attenuated the increase in striatal ACh release produced by systemic CY 208-243.(ABSTRACT TRUNCATED AT 250 WORDS)


Working and health conditions of Italian air traffic controllers.
The paper summarises some studies carried out in recent years on Italian air traffic controllers. Work stress appeared related mainly to mental charge, aggravated by time pressure and high responsibility. Shiftwork was not considered highly stressful, thanks to the good shift schedules adopted. The disorders most frequently complained of had a prevalent psychosomatic component, concerning the digestive, neuropsychic, and cardiovascular systems. However, their prevalence was lower than, or similar to, that recorded in general population. The controllers engaged in regional centres showed slightly worse health conditions than those engaged in airport towers; the same was found in the radar controllers compared to those working in non-radar positions.


New DNA Methylation Markers for Pancreatic Cancer: Discovery, Tissue Validation, and Pilot Testing in Pancreatic Juice.
Discriminant markers for pancreatic cancer detection are needed. We sought to identify and validate methylated DNA markers for pancreatic cancer using next-generation sequencing unbiased by known targets. At a referral center, we conducted four sequential case-control studies: discovery, technical validation, biologic validation, and clinical piloting. Candidate markers were identified using variance-inflated logistic regression on reduced-representation bisulfite DNA sequencing results from matched pancreatic cancers, benign pancreas, and normal colon tissues. Markers were validated technically on replicate discovery study DNA and biologically on independent, matched, blinded tissues by methylation-specific PCR. Clinical testing of six methylation candidates and mutant KRAS was performed on secretin-stimulated pancreatic juice samples from 61 patients with pancreatic cancer, 22 with chronic pancreatitis, and 19 with normal pancreas on endoscopic ultrasound. Areas under receiver-operating characteristics curves (AUC) for markers were calculated. Sequencing identified >500 differentially hyper-methylated regions. On independent tissues, AUC on 19 selected markers ranged between 0.73 and 0.97. Pancreatic juice AUC values for CD1D, KCNK12, CLEC11A, NDRG4, IKZF1, PKRCB, and KRAS were 0.92*, 0.88, 0.85, 0.85, 0.84, 0.83, and 0.75, respectively, for pancreatic cancer compared with normal pancreas and 0.92*, 0.73, 0.76, 0.85*, 0.73, 0.77, and 0.62 for pancreatic cancer compared with chronic pancreatitis (*, P = 0.001 vs. KRAS). We identified and validated novel DNA methylation markers strongly associated with pancreatic cancer. On pilot testing in pancreatic juice, best markers (especially CD1D) highly discriminated pancreatic cases from controls.


Extended stability of restriction enzymes at ambient temperatures.
The stability of restriction enzymes as supplied by manufacturers without any modification has been examined. No reduction in activity was observed for three enzymes (HindIII, EcoRI and Tsp509I) held at ambient temperature or 4 degrees C for the period of study (12 months), while activity was observed for up to 12 weeks after storage at 37 degrees C, which was considerably better than following desiccation with trehalose, a recognized preservation technique. A larger trial of 23 different restriction enzymes held at room temperature for one week showed that all enzymes retained significant activity. As a practical demonstration of the usefulness of this finding, enzymes were posted to Africa by conventional mail (cost $1 US) and shown to retain activity upon arrival after three weeks in transit (compared to a cost of $1000 US by cold-chain transportation). Supplying enzymes to third-world markets should now be possible by removing the necessity for cold-chain transport. After arrival, enzymes can simply be stored in a standard domestic refrigerator.


Cytotoxic and DNA-damaging properties of glyphosate and Roundup in human-derived buccal epithelial cells.
Glyphosate (G) is the largest selling herbicide worldwide; the most common formulations (Roundup, R) contain polyoxyethyleneamine as main surfactant. Recent findings indicate that G exposure may cause DNA damage and cancer in humans. Aim of this investigation was to study the cytotoxic and genotoxic properties of G and R (UltraMax) in a buccal epithelial cell line (TR146), as workers are exposed via inhalation to the herbicide. R induced acute cytotoxic effects at concentrations > 40 mg/l after 20 min, which were due to membrane damage and impairment of mitochondrial functions. With G, increased release of extracellular lactate dehydrogenase indicative for membrane damage was observed at doses > 80 mg/l. Both G and R induced DNA migration in single-cell gel electrophoresis assays at doses > 20 mg/l. Furthermore, an increase of nuclear aberrations that reflect DNA damage was observed. The frequencies of micronuclei and nuclear buds were elevated after 20-min exposure to 10-20 mg/l, while nucleoplasmatic bridges were only enhanced by R at the highest dose (20 mg/l). R was under all conditions more active than its active principle (G). Comparisons with results of earlier studies with lymphocytes and cells from internal organs indicate that epithelial cells are more susceptible to the cytotoxic and DNA-damaging properties of the herbicide and its formulation. Since we found genotoxic effects after short exposure to concentrations that correspond to a 450-fold dilution of spraying used in agriculture, our findings indicate that inhalation may cause DNA damage in exposed individuals.


[Validation of the Personal Wellbeing Index (PWI) in vulnerable users of health care services in Santiago, Chile].
Personal well-being calculates quality of life in terms of the necessary conditions required to live well. To validate the Personal Wellbeing Index (PWI) in a representative sample of vulnerable users of the public health system in Santiago, Chile. A probabilistic and multistage sample consisting of 400 individuals aged 44 ± 18 years (61% females) belonging to the lower income group of the National Health Fund (FONASA), residents of Gran Santiago was surveyed. Internal consistency and correlation between items and scale were examined. Structure was analyzed through confirmatory factor analysis. The seven-item PWI is a good indicator of subjective well-being in the population under study, considering internal consistency, factor loadings, relation with overall life satisfaction and goodness of fit. The indicators mostly associated with personal well-being are the socioeconomic level followed by relationships with the community, health conditions and achievements. The 7-item version of the PWI is suitable for application in vulnerable health service users.


Murine serum glycoprotein gp70 behaves as an acute phase reactant.
A single intraperitoneal injection of bacterial lipopolysaccharide (LPS) or its lipid A component induced high levels of glycoprotein, gp70, in sera of several strains of mice within 24 h. This serum gp70 response induced by LPS was independent of the activation of B cells and the presence of T cells. However, serological and immunohistochemical studies demonstrated the production of gp70 by hepatic parenchymal cells and its subsequent release into the circulating blood. The expression of gp70 in the serum was enhanced not only by LPS but also other inducers of acute phase reactants (APR) such as turpentine oil or polyriboinosinic-polyribocytidylic acid. Further, the serum gp70 response was kinetically identical to those of APR. These results strongly suggest that (a) the liver may be the major source for serum gp70, (b) serum gp70 behaves like an APR, (c) its expression may be controlled by a mechanism similar to that for other APR, and (d) this glycoprotein apparently behaves as a normal host constituent and not a product of a viral genome.


Scent variation and hybridization cause the displacement of a sexually deceptive orchid species.
In the sexually deceptive orchid genus Ophrys, reproductive isolation is based on the specific attraction of males of a single pollinator species, mostly bees, by mimicking the female sex pheromone of this species. Changes in the floral odor can lead to hybridization, introgression, and possibly speciation. We investigated hybrid swarms of O. lupercalis and O. iricolor on Sardinia using behavioral, electrophysiological (GC-EAD), chemical, morphological, and genetic methods (AFLPs). In behavioral experiments, approximately 20% of the flowers from both species and hybrids were attractive to the "wrong" or both pollinator species. Analysis of the EAD-active hydrocarbons in the floral odor showed an overlap in the two species, whereby hybrid individuals could not be separated from O. iricolor. The genetic analysis confirmed the hybridization of the species. Plants of O. iricolor and hybrids are genetically indistinguishable and form an O. iricolor × lupercalis hybrid population. Remaining plants of O. lupercalis will possibly be displaced by the O. iricolor × lupercalis hybrid population in the future. Our study showed that in deceptive orchids, variation in the pollinator attracting cues, in this case, scent, can be the first step for speciation and at the same time cause the displacement of a species.


Listeriolysin S: A bacteriocin from epidemic Listeria monocytogenes strains that targets the gut microbiota.
Listeria monocytogenes is a Gram-positive food-borne pathogen that in humans may traverse the intestinal, placental and blood/brain barriers, causing gastroenteritis, abortions and meningitis. Crossing of these barriers is dependent on the bacterial ability to enter host cells, and several L. monocytogenes surface and secreted virulence factors are known to facilitate entry and the intracellular lifecycle. The study of L. monocytogenes strains associated to human listeriosis epidemics has revealed the presence of novel virulence factors. One such factor is Listeriolysin S, a thiazole/oxazole modified microcin that displays bactericidal activity and modifies the host microbiota during infection. Our recent results therefore highlight the interaction of L. monocytogenes with gut microbes as a crucial step in epidemic listeriosis. In this article, we will discuss novel implications for this family of toxins in the pathogenesis of diverse medically relevant microorganisms.


CTLA-4 antibody-drug conjugate reveals autologous destruction of B-lymphocytes associated with regulatory T cell impairment.
Germline CTLA-4 deficiency causes severe autoimmune diseases characterized by dysregulation of Foxp3+ Tregs, hyper-activation of effector memory T cells, and variable forms autoimmune cytopenia including gradual loss of B cells. Cancer patients with severe immune-related adverse events (irAE) after receiving anti-CTLA-4/PD-1 combination immunotherapy also have markedly reduced peripheral B cells. The immunological basis for B cell loss remains unexplained. Here we probe the decline of B cells in human CTLA-4 knock-in mice by using antihuman CTLA-4 antibody Ipilimumab conjugated to a drug payload emtansine (Anti-CTLA-4 ADC). The anti-CTLA-4 ADC-treated mice have T cell hyper-proliferation and their differentiation into effector cells which results in B cell depletion. B cell depletion is mediated by both CD4 and CD8 T cells and at least partially rescued by anti-TNF-alpha antibody. These data revealed an unexpected antagonism between T and B cells and the importance of regulatory T cells in preserving B cells.


Standardizing Terminology and Assessment for Orofacial Conditions in Juvenile Idiopathic Arthritis: International, Multidisciplinary Consensus-based Recommendations.
To propose multidisciplinary, consensus-based, standardization of operational terminology and method of assessment for temporomandibular joint (TMJ) involvement in juvenile idiopathic arthritis (JIA). Using a sequential expert group-defined terminology and methods-of-assessment approach by (1) establishment of task force, (2) item generation, (3) working group consensus, (4) external expert content validity testing, and (5) multidisciplinary group of experts final Delphi survey consensus. Seven standardized operational terms were defined: TMJ arthritis, TMJ involvement, TMJ arthritis management, dentofacial deformity, TMJ deformity, TMJ symptoms, and TMJ dysfunction. CONCLUSION: Definition of 7 operational standardized terms provides an optimal platform for communication across healthcare providers involved in JIA-TMJ arthritis management.


Apoptin enhances radiation-induced cell death in poorly responding head and neck squamous cell carcinoma cells.
Treatment of head and neck cancers is still rather poor and worldwide new treatment options are sought. Sensitizing radioresistant tumours by combining irradiation with other therapeutics to induce apoptosis are widely investigated. We examined whether chicken anaemia virus-derived apoptin protein would have a beneficial effect on irradiation of radiosensitive SCC61 and radioresistant SQD9 human head and neck squamous carcinoma cell lines. In both cell lines, concurrent exposure to irradiation and apoptin resulted in analysed mitochondrial cytochrome c release and in cleavage of caspase-3, whereas irradiation alone of SQD9 cells under identical conditions did not. Moreover, in comparison with the irradiation, only the synchronized treatment of apoptin and irradiation resulted in increased cell death in especially the radioresistant SQD9 cells, as measured by means of a colony survival assay. Our data reveal that apoptin treatment represents an effective way for enhancing radiotherapy of tumours responding poorly to radiotherapy.


Regulation of reproduction and longevity by nutrient-sensing pathways.
Nutrients are necessary for life, as they are a crucial requirement for biological processes including reproduction, somatic growth, and tissue maintenance. Therefore, signaling systems involved in detecting and interpreting nutrient or energy levels-most notably, the insulin/insulin-like growth factor 1 (IGF-1) signaling pathway, mechanistic target of rapamycin (mTOR), and adenosine monophosphate-activated protein kinase (AMPK)-play important roles in regulating physiological decisions to reproduce, grow, and age. In this review, we discuss the connections between reproductive senescence and somatic aging and give an overview of the involvement of nutrient-sensing pathways in controlling both reproductive function and lifespan. Although the molecular mechanisms that affect these processes can be influenced by distinct tissue-, temporal-, and pathway-specific signaling events, the progression of reproductive aging and somatic aging is systemically coordinated by integrated nutrient-sensing signaling pathways regulating somatic tissue maintenance in conjunction with reproductive capacity.


KRAS and BRAF mutation analysis in metastatic colorectal cancer: a cost-effectiveness analysis from a Swiss perspective.
Monoclonal antibodies against the epidermal growth factor receptor (EGFR), such as cetuximab, have led to significant clinical benefits for metastatic colorectal cancer (mCRC) patients but have also increased treatment costs considerably. Recent evidence associates KRAS and BRAF mutations with resistance to EGFR antibodies. We assessed the cost-effectiveness of predictive testing for KRAS and BRAF mutations, prior to cetuximab treatment of chemorefractory mCRC patients. A life-long Markov simulation model was used to estimate direct medical costs (€) and clinical effectiveness [quality-adjusted life-years (QALY)] of the following strategies: KRAS testing, KRAS testing with subsequent BRAF testing of KRAS wild-types (KRAS/BRAF), cetuximab treatment without testing. Comparison was against no cetuximab treatment (reference strategy). In the testing strategies, cetuximab treatment was initiated if no mutations were detected. Best supportive care was given to all patients. Survival times/utilities were derived from published randomized clinical trials. Costs were assessed from the perspective of the Swiss health system. Average remaining lifetime costs ranged from €3,983 (no cetuximab) to €38,662 (no testing). Cetuximab treatment guided by KRAS/BRAF achieved gains of 0.491 QALYs compared with the reference strategy. The KRAS testing strategy achieved an additional gain of 0.002 QALYs compared with KRAS/BRAF. KRAS/BRAF testing was the most cost-effective approach when compared with the reference strategy (incremental cost-effectiveness ratio: €62,653/QALY). New predictive tests for KRAS and BRAF status are currently being introduced in pathology. Despite substantial costs of predictive testing, it is economically favorable to identify patients with KRAS and BRAF wild-type status.


The MSL3 chromodomain directs a key targeting step for dosage compensation of the Drosophila melanogaster X chromosome.
The male-specific lethal (MSL) complex upregulates the single male X chromosome to achieve dosage compensation in Drosophila melanogaster. We have proposed that MSL recognition of specific entry sites on the X is followed by local targeting of active genes marked by histone H3 trimethylation (H3K36me3). Here we analyze the role of the MSL3 chromodomain in the second targeting step. Using ChIP-chip analysis, we find that MSL3 chromodomain mutants retain binding to chromatin entry sites but show a clear disruption in the full pattern of MSL targeting in vivo, consistent with a loss of spreading. Furthermore, when compared to wild type, chromodomain mutants lack preferential affinity for nucleosomes containing H3K36me3 in vitro. Our results support a model in which activating complexes, similarly to their silencing counterparts, use the nucleosomal binding specificity of their respective chromodomains to spread from initiation sites to flanking chromatin.


PEEK versus titanium locking plates for proximal humerus fracture fixation: a comparative biomechanical study in two- and three-part fractures.
The high rigidity of metal implants may be a cause of failure after fixation of proximal humerus fractures. Carbon fiber-reinforced polyetheretherketone (PEEK) plates with a modulus similar to human cortical bone may help to overcome this problem. The present study assesses the biomechanical behavior of a PEEK plate compared with a titanium locking plate. Unstable two- and three-part fractures were simulated in 12 pairs of cadaveric humeri and were fixed with either a PEEK or a titanium locking plate using a pairwise comparison. With an optical motion capture system, the stiffness, failure load, plate bending, and the relative motion at the bone-implant interface and at the fracture site were evaluated. The mean load to failure for two- and three-part fracture fixations was, respectively, 191 N (range 102-356 N) and 142 N (range 102-169 N) in the PEEK plate group compared with 286 N (range 191-395 N) and 258 N (range 155-366 N) in the titanium locking plate group. The PEEK plate showed significantly more bending in both the two- and three-part fractures (p < 0.05), an increased relative motion at the bone-implant interface and lower stiffness values (p < 0.05). In this biomechanical study on unstable proximal humerus fractures, fixation with a PEEK plate showed lower fixation strength and increased motion at the bone-implant interface compared with a titanium locking plate.


An Orally Active Phenylaminotetralin-Chemotype Serotonin 5-HT7 and 5-HT1A Receptor Partial Agonist that Corrects Motor Stereotypy in Mouse Models.
Stereotypy (e.g., repetitive hand waving) is a key phenotype of autism spectrum disorder, Fragile X and Rett syndromes, and other neuropsychiatric disorders, and its severity correlates with cognitive and attention deficits. There are no effective treatments, however, for stereotypy. Perturbation of serotonin (5-HT) neurotransmission contributes to stereotypy, suggesting that distinct 5-HT receptors may be pharmacotherapeutic targets to treat stereotypy and related neuropsychiatric symptoms. For example, preclinical studies indicate that 5-HT7 receptor activation corrects deficits in mouse models of Fragile X and Rett syndromes, and clinical trials for autism are underway with buspirone, a 5-HT1A partial agonist with relevant affinity at 5-HT7 receptors. Herein, we report the synthesis, in vitro molecular pharmacology, behavioral pharmacology, and pharmacokinetic parameters in mice after subcutaneous and oral administration of (+)-5-(2'-fluorophenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine ((+)-5-FPT), a new, dual partial agonist targeting both 5-HT7 (Ki = 5.8 nM, EC50 = 34 nM) and 5-HT1A (Ki = 22 nM, EC50 = 40 nM) receptors. Three unique, heterogeneous mouse models were used to assess the efficacy of (+)-5-FPT to reduce stereotypy: idiopathic jumping in C58/J mice, repetitive body rotations in C57BL/6J mice treated with the NMDA antagonist, MK-801, and repetitive head twitching in C57BL/6J mice treated with the 5-HT2 agonist, DOI. Systemic (+)-5-FPT potently and efficaciously reduced or eliminated stereotypy in each of the mouse models without altering locomotor behavior on its own, and additional tests showed that (+)-5-FPT, at the highest behaviorally active dose tested, enhanced social interaction and did not cause behaviors indicative of serotonin syndrome. These data suggest that (+)-5-FPT is a promising medication for treating stereotypy in psychiatric disorders.


Epithelial expression of the hormone leptin by bovine skin.
Leptin (Lep) stimulates keratinocytes to proliferate, intervenes in the wound healing and participates to hair follicle morphogenesis and cycle. While it is secreted by skin structures including epidermis and hair follicles, intradermal adipose tissue also seems to have a role in Lep secretion and accordingly in the control of hair follicle growth in mice and humans. Lep was investigated in the skin of humans and laboratory animals but there are not data regarding bovine species. The aim of this work was to study the expression of Lep and its receptor (LepR) in the skin of bovine and, at the same time, to investigate the presence and extension of intradermal adipose tissue. A morphological evaluation of the skin was performed while the presence and localization of Lep and LepR were analyzed by RT-PCR and immunohistochemistry. A high and thick dermis without adipocytes was observed. Hair follicles and sebaceous and sweat glands were located in the proximal part of the skin while a thick layer of connective tissue, lacking adipose cells, separated these structures by subcutis. RT-PCR evidenced the transcripts for both molecules. By immunohistochemistry, Lep and LepR were observed in the epidermis and hair follicles. Based on the absence of intradermal adipose tissue and the presence of both Lep and LepR in the epidermis and in the hair follicle epithelium, it can be posited that in bovine skin Lep participates to the control of epidermis growth and hair follicle cycle through a paracrine and autocrine mechanisms.


Refinement: promoting the three Rs in practice.
Refinement of scientific procedures carried out on protected animals is an iterative process, which begins with a critical evaluation of practice. The process continues with objective assessment of the impact of the procedures, identification of areas for improvement, selection and implementation of an improvement strategy and evaluation of the results to determine whether there has been the desired effect, completing the refinement loop and resulting in the perpetuation of good practice. Refinements may be science-driven (those which facilitate getting high-quality results) or welfare-driven or may encompass both groups, but whatever the driver, refinements almost always result in benefits to both welfare and science. Refinements can be implemented in all aspects of animal use: improved methodology in invasive techniques, housing and husbandry, and even statistical analyses can all benefit animal welfare and scientific quality. If refinement is not actively sought, outdated and unnecessarily invasive techniques may not be replaced by better methods as they become available, and thus outdated information is passed down to the next generation, causing perpetuation of old-fashioned methods. This leads to a spiral of ignorance, leading ultimately to poor practice, poor animal welfare and poor-quality scientific data. Refinement is a legal and ethical requirement, yet refinements may not always be implemented. There are numerous obstacles to the implementation of refinement, which may be real or perceived. Either way, in order to take refinement forward, it is important to coordinate the approach to refinement, validate the science behind refinement, ensure there is adequate education and training in new techniques, improve liaison between users and make sure there is feedback on suitability of refinements for use. Overall, refinement requires a coordinated ongoing process of critical appraisal of practice and active scrutiny of resources for likely improvements. In the busy world of biomedical research, this process needs help. In order to develop these themes further, a workshop was held at the LASA Winter Meeting 2006, UK, to assist in identifying potential obstacles to refinement, and then to explore and develop strategies for overcoming these obstacles in key areas. A range of strategies appropriate to different circumstances was identified, which should facilitate the implementation of refinements.


Metabolic decisions in development and disease-a Keystone Symposia report.
There is an increasing appreciation for the role of metabolism in cell signaling and cell decision making. Precise metabolic control is essential in development, as evident by the disorders caused by mutations in metabolic enzymes. The metabolic profile of cells is often cell-type specific, changing as cells differentiate or during tumorigenesis. Recent evidence has shown that changes in metabolism are not merely a consequence of changes in cell state but that metabolites can serve to promote and/or inhibit these changes. Metabolites can link metabolic pathways with cell signaling pathways via several mechanisms, for example, by serving as substrates for protein post-translational modifications, by affecting enzyme activity via allosteric mechanisms, or by altering epigenetic markers. Unraveling the complex interactions governing metabolism, gene expression, and protein activity that ultimately govern a cell's fate will require new tools and interactions across disciplines. On March 24 and 25, 2021, experts in cell metabolism, developmental biology, and human disease met virtually for the Keystone eSymposium, "Metabolic Decisions in Development and Disease." The discussions explored how metabolites impact cellular and developmental decisions in a diverse range of model systems used to investigate normal development, developmental disorders, dietary effects, and cancer-mediated changes in metabolism.


Validation of the rapid assessment procedure for loiasis (RAPLOA) in the Democratic Republic of Congo.
A simple method called RAPLOA, to rapidly assess what proportion of people in a community are infected with L. loa and hence which communities are at high risk of severe adverse reactions following ivermectin treatment, was developed in Cameroon and Nigeria. The method needed further validation in other geographical and cultural contexts before its application in all endemic countries. The present study was designed to validate RAPLOA in two regions in the North East and South West of the Democratic Republic of Congo. In each study region, villages were selected from different bio-ecological zones in order to cover a wide range of loiasis endemicity. In each selected community, 80 people above the age of 15 years were interviewed for a history of eye worm (migration of adult L. loa under the conjunctiva of the eye) and parasitologically examined for the presence and intensity of L. loa infection. In total, 8100 individuals from 99 villages were enrolled into the study. The results confirmed the findings of the original RAPLOA study: i) the eye worm phenomenon was well-known in all endemic areas, ii) there was a clear relationship between the prevalence of eye worm history and the prevalence and intensity of L. loa microfilaraemia, and iii) using a threshold of 40%, the prevalence of eye worm history was a sensitive and specific indicator of high-risk communities. Following this successful validation, RAPLOA was recommended for the assessment of loiasis endemicity in areas targeted for ivermectin treatment by lymphatic filariasis and onchocerciasis control programmes.


The POSNA-COUR International Scholar Program. Results of the First 7 Years.
The Pediatric Orthopedic Society of North America (POSNA)-Children's Orthopedics in Underserved Regions (COUR) International Scholar Program was initiated in 2007 to provide educational opportunities for emerging leaders who treat children with orthopaedic conditions in resource-challenged environments worldwide. Financial support is available each year for 4 to 6 orthopaedic surgeons to attend either the POSNA Annual Meeting or the International Pediatric Orthopedic Symposium. The scholars are also encouraged to visit selected centers for observerships during their trip. Since 2007 there have been 41 international scholars who have participated in the program. We wished to assess the impact of the program and to obtain feedback to improve the experience for future participants. A 23-question web-based survey was created and sent to 38 past scholars from 22 countries who have participated in the program by July 2013. The responses were gathered online and the data were analyzed for the 24 (62%) respondents from 18 countries who completed the survey. Of the respondents, 16/24 (66%) reported that their current practice is comprised of at least 75% pediatrics. Twelve of 24 (52%) were fellowship trained in pediatric orthopaedics, typically outside of North America. All scholars found the meeting they attended to be very useful and have subsequently made changes to their clinical practice. Nineteen of 24 (82%) did a premeeting or postmeeting observership. Twenty-two of 24 (92%) participants have remained in contact with POSNA members they met at the meeting, with 86% of respondents stating that they have subsequently consulted POSNA members on management of patients. Sixty-two percent of the scholars had a POSNA member visit them following the scholarship and 29% have since returned to visit POSNA members for further clinical observerships. Twenty-one of 24 (91%) have had the opportunity to share the knowledge they gained with others in their region through lectures, surgical demonstrations, and/or clinical training. A common response from the scholars was that the scholarship program was a truly transformative life experience that provided them with an opportunity to receive the highest quality of professional education. The main challenges that these scholars report are lack of available fellowship/subspecialty training in their region, patients' inability to pay, and excessive physician workload. All of the respondents expressed interest in arranging a POSNA cosponsored regional meeting. Since 2007, the POSNA-COUR international scholar program has been a fruitful resource for orthopaedists practicing in resource-challenged environments worldwide. It has provided unique training for the scholars and has further enabled them to teach others in their region. The program has thus far succeeded in fostering lasting relationships that have led to continued educational exchanges. Level IV-case series.


The effects of whole-body vibration on muscle strength and power: a meta-analysis.
Exercise with whole-body vibration (WBV) is becoming popular as an alternative to conventional training or as supplementary training. However, despite increasing research efforts in this field, additive effects of WBV on muscle performance remain unclarified. In this review, we investigated the additive effects of long-term WBV on muscle strength and power. This meta-analysis was restricted to randomized controlled trials lasting for at least 5 weeks comparing exercise with and without WBV, or comparing only WBV exposure and control. Data from a total of 314 participants in 10 studies on knee extension muscle strength, and 249 participants in 7 studies on countermovement jump height were pooled using random-effect models. Meta-analysis showed significant additional effects of WBV on muscle strength (standardized mean difference [SMD]=0.76, 95% confidence interval [CI]=0.21-1.32; p=0.007) and countermovement jump (SMD=0.87, 95% CI=0.29-1.46; p=0.003). Based on these findings, we concluded that the use of WBV would lead to greater improvements in both knee extension muscle strength and countermovement jump than under identical conditions without WBV.


The effectiveness of exercise interventions for the management of frailty: a systematic review.
This systematic review examines the effectiveness of current exercise interventions for the management of frailty. Eight electronic databases were searched for randomized controlled trials that identified their participants as "frail" either in the title, abstract, and/or text and included exercise as an independent component of the intervention. Three of the 47 included studies utilized a validated definition of frailty to categorize participants. Emerging evidence suggests that exercise has a positive impact on some physical determinants and on all functional ability outcomes reported in this systematic review. Exercise programs that optimize the health of frail older adults seem to be different from those recommended for healthy older adults. There was a paucity of evidence to characterize the most beneficial exercise program for this population. However, multicomponent training interventions, of long duration (≥5 months), performed three times per week, for 30-45 minutes per session, generally had superior outcomes than other exercise programs. In conclusion, structured exercise training seems to have a positive impact on frail older adults and may be used for the management of frailty.


Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein (ACP) reductase: kinetic and chemical mechanisms.
Beta-ketoacyl-acyl carrier protein (ACP) reductase from Mycobacterium tuberculosis (MabA) is responsible for the second step of the type-II fatty acid elongation system of bacteria, plants, and apicomplexan organisms, catalyzing the NADPH-dependent reduction of beta-ketoacyl-ACP to generate beta-hydroxyacyl-ACP and NADP(+). In the present work, the mabA-encoded MabA has been cloned, expressed, and purified to homogeneity. Initial velocity studies, product inhibition, and primary deuterium kinetic isotope effects suggested a steady-state random bi-bi kinetic mechanism for the MabA-catalyzed reaction. The magnitudes of the primary deuterium kinetic isotope effect indicated that the C(4)-proS hydrogen is transferred from the pyridine nucleotide and that this transfer contributes modestly to the rate-limiting step of the reaction. The pH-rate profiles demonstrated groups with pK values of 6.9 and 8.0, important for binding of NADPH, and with pK values of 8.8 and 9.6, important for binding of AcAcCoA and for catalysis, respectively. Temperature studies were employed to determine the activation energy of the reaction. Solvent kinetic isotope effects and proton inventory analysis established that a single proton is transferred in a partially rate-limiting step and that the mechanism of carbonyl reduction is probably concerted. The observation of an inverse (D)2(O)V/K and an increase in (D)2(O)V when [4S-(2)H]NADPH was the varied substrate obscured the distinction between stepwise and concerted mechanisms; however, the latter was further supported by the pH dependence of the primary deuterium kinetic isotope effect. Kinetic and chemical mechanisms for the MabA-catalyzed reaction are proposed on the basis of the experimental data.


Gene Expression Profiling and Functional Characterization of Macrophages in Response to Circulatory Microparticles Produced during Trypanosoma cruzi Infection and Chagas Disease.
Chronic inflammation and oxidative stress are hallmarks of chagasic cardiomyopathy (CCM). In this study, we determined if microparticles (MPs) generated during Trypanosoma cruzi (Tc) infection carry the host's signature of the inflammatory/oxidative state and provide information regarding the progression of clinical disease. MPs were harvested from supernatants of human peripheral blood mononuclear cells in vitro incubated with Tc (control: LPS treated), plasma of seropositive humans with a clinically asymptomatic (CA) or symptomatic (CS) disease state (vs. normal/healthy [NH] controls), and plasma of mice immunized with a protective vaccine before challenge infection (control: unvaccinated/infected). Macrophages (mφs) were incubated with MPs, and we probed the gene expression profile using the inflammatory signaling cascade and cytokine/chemokine arrays, phenotypic markers of mφ activation by flow cytometry, cytokine profile by means of an ELISA and Bioplex assay, and oxidative/nitrosative stress and mitotoxicity by means of colorimetric and fluorometric assays. Tc- and LPS-induced MPs stimulated proliferation, inflammatory gene expression profile, and nitric oxide (∙NO) release in human THP-1 mφs. LPS-MPs were more immunostimulatory than Tc-MPs. Endothelial cells, T lymphocytes, and mφs were the major source of MPs shed in the plasma of chagasic humans and experimentally infected mice. The CS and CA (vs. NH) MPs elicited >2-fold increase in NO and mitochondrial oxidative stress in THP-1 mφs; however, CS (vs. CA) MPs elicited a more pronounced and disease-state-specific inflammatory gene expression profile (IKBKB, NR3C1, and TIRAP vs. CCR4, EGR2, and CCL3), cytokine release (IL-2 + IFN-γ > GCSF), and surface markers of mφ activation (CD14 and CD16). The circulatory MPs of nonvaccinated/infected mice induced 7.5-fold and 40% increases in ∙NO and IFN-γ production, respectively, while these responses were abolished when RAW264.7 mφs were incubated with circulatory MPs of vaccinated/infected mice. Circulating MPs reflect in vivo levels of an oxidative, nitrosative, and inflammatory state, and have potential utility in evaluating disease severity and the efficacy of vaccines and drug therapies against CCM.


The long form of the leptin receptor (OB-Rb) is widely expressed in the human brain.
Leptin exerts important effects on the regulation of food intake and energy expenditure by acting in the brain. Leptin action is mediated by the interaction with a receptor that is alternatively spliced, resulting in at least five different isoforms. The long form (OB-Rb) has a long intracellular domain that is essential for intracellular signal transduction. The specific aim of this study was to further investigate the role that the brain may play in the pathogenesis of obesity in humans. We studied the expression of OB-R mRNA (both short or common and long isoforms) in the brains of obese, lean and diabetic subjects, by in situ hybridization, semiquantitative RT-PCR and Northern blots analysis. We used two alternative probes: one that recognizes all known splice variants (OB-Ra) and a second that recognizes only the long form, OB-Rb. Several brain regions, including hypothalamus, cerebellum, neocortex, entorrhinal cortex, amygdala, and rostral medulla, were evaluated. In situ hybridization studies revealed that both OB-Ra and OB-Rb mRNAs are widely distributed in the human brain. The specific hybridization signal with both probes was detected exclusively in the cytoplasm of the cell body, dendrites and proximal axons of neurons. Hypothalamic nuclei, Purkinje cells and dentate nuclei of the cerebellum, inferior olivary and cranial nerves nuclei in the medulla, amygdala and neurons from both neocortex and entorrhinal cortex demonstrated positive signals. The hybridization signal obtained in ependyma was lower than that in neurons and no specific hybridization was detected in glial cells. No significant differences were identified among the regions or among the three groups studied. These results match those previously obtained by us [Couce et al.: Neuroendocrinology 1997;66:145] in which the distribution of the OB-R protein in the human brain was first described. RT-PCR indicated that the OB-Rb was highly expressed in the hypothalamus and cerebellum. No significant differences of OB-Ra or OB-Rb mRNA expression were identified in lean or obese individuals in these two cerebral regions. The levels of OB-Rb were significantly higher in cerebellum compared to hypothalamus in lean and obese individuals. The original hypothesis that OB-Rb is present only in the hypothalamus needs to be reconsidered. This OB-Rb isoform seems to be widely expressed in the human brain with highest levels in the cerebellum. Obesity and hyperleptinemia appears not to be associated with down-regulation of the OB-Rb in the human brain.


Informational needs of patients with melanoma and their views on the utility of investigative tests.
The aim of the study was to identify the informational needs of patients with melanoma on disease status and prognosis, and to ascertain their views on the utility of positron emission tomography (PET) and sentinel node biopsy (SNB). Patients attending the weekly melanoma outpatient clinic at St Thomas' Hospital London UK between February and August 2007 participated in this cross-sectional survey. Views of 106 melanoma patients were elicited using a face-to face semi-structured questionnaire. The majority of participants wanted to know everything about their disease (88%). Prognostic information (> 85%) and information on palliative care input (97%) were highly valued. More than 50% expected the doctor to impart this information without negotiation. Nearly 70% of the responders who had previously had a PET scan felt they should decide if and when the scans should be performed. Fifty three percentage had undergone the SNB because the doctor had suggested it. Patients with melanoma want detailed and prompt information about their disease including prognosis. Regular PET scans provide reassurance. The role of SNB is not clear to all patients.


MicroRNA-126 regulates HOXA9 by binding to the homeobox.
The PicTar program predicted that microRNA-126 (miR-126), miR-145, and let-7s target highly conserved sites within the Hoxa9 homeobox. There are increased nucleotide constraints in the three microRNA seed sites among Hoxa9 genes beyond that required to maintain protein identity, suggesting additional functional conservation. In preliminary experiments, forced expression of these microRNAs in Hoxa9-immortalized bone marrow cells downregulated the HOXA9 protein and caused loss of biological activity. The microRNAs were shown to target their predicted sites within the homeobox. miR-126 and Hoxa9 mRNA are coexpressed in hematopoietic stem cells and downregulated in parallel during progenitor cell differentiation; however, miR-145 is barely detectable in hematopoietic cells, and let-7s are highly expressed in bone marrow progenitors, suggesting that miR-126 may function in normal hematopoietic cells to modulate HOXA9 protein. In support of this hypothesis, expression of miR-126 alone in MLL-ENL-immortalized bone marrow cells decreased endogenous HOXA9 protein, while inhibition of endogenous miR-126 increased expression of HOXA9 in F9 cells.


Quantification of Trans-resveratrol in Red Wines Using QuEChERS Extraction Combined with Liquid Chromatography-Tandem Mass Spectrometry.
Resveratrol is one of representative ingredient in red wine, but its quantification is a challenge because of a complex and abundant matrix. In this study, two sample pretreatments, direct dilution and QuEChERS extraction, coupling LCMS analysis were examined for resveratrol quantification. Similar recoveries of 106.4 to 93.7% were obtained for direct dilution and QuEChERS, respectively. With the aid of condition optimization, QuEChERS extraction could concentrate the resveratrol from red wines to improve the detection sensitivity with a LOD value of 2.5 ng/mL, which is four-times greater than the direct dilution approach. As a result, the QuEChERS method can provide a high linearity within the concentration range of 5 - 500 ng/mL, in which direct dilution produced the linear calibration curve within the concentrations of 25 - 500 ng/mL. A high consistency was obtained for both approaches in which intra-day precisions were within 0.5 to 7.2% (n = 3), and the inter-day precisions were within 7.8 to 16.0% (n = 9). Overall, the sample pretreatment of QuEChERS can effectively reduce the matrix effect, which leads LCMS to quantify the low resveratrol abundance of 8.0 ppb in each red wine sample, which is not achieved with the direct dilution approach.


Research methods of Talking About The Smokes: an International Tobacco Control Policy Evaluation Project study with Aboriginal and Torres Strait Islander Australians.
To describe the research methods and baseline sample of the Talking About The Smokes (TATS) project. The TATS project is a collaboration between research institutions and Aboriginal community-controlled health services (ACCHSs) and their state and national representative bodies. It is one of the studies within the International Tobacco Control Policy Evaluation Project, enabling national and international comparisons. It includes a prospective longitudinal study of Aboriginal and Torres Strait Islander smokers and recent ex-smokers; a survey of non-smokers; repeated cross-sectional surveys of ACCHS staff; and descriptions of the tobacco policies and practices at the ACCHSs. Community members completed face-to-face surveys; staff completed surveys on paper or online. We compared potential biases and the distribution of variables common to the main community baseline sample and unweighted and weighted results of the 2008 National Aboriginal and Torres Strait Islander Social Survey (NATSISS). The baseline survey (Wave 1) was conducted between April 2012 and October 2013. 2522 Aboriginal and Torres Strait Islander people in 35 locations (the communities served by 34 ACCHSs and one community in the Torres Strait), and 645 staff in the ACCHSs. Sociodemographic and general health indicators, smoking status, number of cigarettes smoked per day and quit attempts. The main community baseline sample closely matched the distribution of the Aboriginal and Torres Strait Islander population in the weighted NATSISS by age, sex, jurisdiction and remoteness. There were inconsistent differences in some sociodemographic factors between our sample and the NATSISS: our sample had higher proportions of unemployed people, but also higher proportions who had completed Year 12 and who lived in more advantaged areas. In both surveys, similar percentages of smokers reported having attempted to quit in the past year, and daily smokers reported similar numbers of cigarettes smoked per day. The TATS project provides a detailed and nationally representative description of Aboriginal and Torres Strait Islander smoking behaviour, attitudes, knowledge and exposure to tobacco control activities and policies, and their association with quitting.


Chromatin barcodes as biomarkers for melanoma.
The major barrier to effective cancer therapy is the presence of genetic and phenotypic heterogeneity within cancer cell populations that provides a reservoir of therapeutically resistant cells. As the degree of heterogeneity present within tumours will be proportional to tumour burden, the development of rapid, robust, accurate and sensitive biomarkers for cancer progression that could detect clinically occult disease before substantial heterogeneity develops would provide a major therapeutic benefit. Here, we explore the application of chromatin conformation capture technology to generate a diagnostic epigenetic barcode for melanoma. The results indicate that binary states from chromatin conformations at 15 loci within five genes can be used to provide rapid, non-invasive multivariate test for the presence of melanoma using as little as 200 μl of patient blood.


Drosophila left/right asymmetry establishment is controlled by the Hox gene abdominal-B.
In Drosophila, left/right (LR) asymmetry is apparent in the directional looping of the gut and male genitalia. The dextral orientation of the organs depends on the activity of a single gene, MyosinID (myoID), whose mutation leads to a fully inverted LR axis, thus revealing the activity of a recessive sinistral pathway. Here, we present the identification of the Hox gene Abdominal-B (Abd-B) as an upstream regulator of LR determination. This role appears distinct from its function in anteroposterior patterning. We show that the Abd-Bm isoform binds to regulatory sequences of myoID and controls MyoID expression in the organ LR organizer. Abd-Bm is also required for the sinistral pathway. Thus, when Abd-B activity is missing, no symmetry breaking occurs and flies develop symmetrically. These findings identify the Hox gene Abd-B as directing the earliest events of LR asymmetry establishment in Drosophila.


Serum estrogen and androgen levels following treatment for cervical cancer.
Endogenous sex hormones seem to influence the risk of several common and debilitating diseases. With a view toward better understanding the effects of surgical removal of the ovaries and high-dose pelvic radiotherapy on plasma sex hormone levels, we measured estrogen and androgen concentrations cross-sectionally among 147 women who had been treated for cervical cancer 0.3-18.5 years previously. Pelvic radiotherapy (mean dose to ovaries, 50 Gy) and bilateral ovariectomy were associated with similarly reduced hormone concentrations relative to levels among nonirradiated women with intact ovaries, most of whom had had early-stage disease and were treated by hysterectomy. There was little evidence that radiotherapy in addition to ovariectomy further lowered concentrations below levels associated with ovariectomy alone, such as might be expected if radiation was suppressing adrenal endocrine function. Among women age 50 years or older at the time of blood drawing, the removal or irradiation of the ovaries was associated with approximately 45% lower concentrations of estradiol (mean ratio [MR], 0.55; 95% confidence interval [CI], 0.32-0.95) and testosterone (MR, 0.57; 95% CI, 0.32-0.99), and 25-30% lower concentrations of estrone (MR, 0.69; 95% CI, 0.44-1.09) and androstenedione (MR, 0.76; 95% CI, 0.47-1.23), relative to the hysterectomy-only group. Among women younger than 50, ovariectomy and radiotherapy, alone or in combination, were associated with 83% lower estradiol concentrations (MR, 0.17; 95% CI, 0.09-0.31), 46% lower estrone concentrations (MR, 0.54; 95% CI, 0.37-0.81), 23% lower androstenedione concentrations (MR, 0.77; 95% CI, 0.57-1.04), and 14% lower testosterone levels (MR, 0.86; 95% CI, 0.64-1.15).(ABSTRACT TRUNCATED AT 250 WORDS)


Autophagy provides metabolic substrates to maintain energy charge and nucleotide pools in Ras-driven lung cancer cells.
Autophagy degrades and is thought to recycle proteins, other macromolecules, and organelles. In genetically engineered mouse models (GEMMs) for Kras-driven lung cancer, autophagy prevents the accumulation of defective mitochondria and promotes malignancy. Autophagy-deficient tumor-derived cell lines are respiration-impaired and starvation-sensitive. However, to what extent their sensitivity to starvation arises from defective mitochondria or an impaired supply of metabolic substrates remains unclear. Here, we sequenced the mitochondrial genomes of wild-type or autophagy-deficient (Atg7(-/-)) Kras-driven lung tumors. Although Atg7 deletion resulted in increased mitochondrial mutations, there were too few nonsynonymous mutations to cause generalized mitochondrial dysfunction. In contrast, pulse-chase studies with isotope-labeled nutrients revealed impaired mitochondrial substrate supply during starvation of the autophagy-deficient cells. This was associated with increased reactive oxygen species (ROS), lower energy charge, and a dramatic drop in total nucleotide pools. While starvation survival of the autophagy-deficient cells was not rescued by the general antioxidant N-acetyl-cysteine, it was fully rescued by glutamine or glutamate (both amino acids that feed the TCA cycle and nucleotide synthesis) or nucleosides. Thus, maintenance of nucleotide pools is a critical challenge for starving Kras-driven tumor cells. By providing bioenergetic and biosynthetic substrates, autophagy supports nucleotide pools and thereby starvation survival.


Investigation of Au SAMs Photoclick Derivatization by PM-IRRAS.
In this work, we present a clean one-step process for modifying headgroups of self-assembled monolayers (SAMs) on gold using photo-enabled click chemistry. A thiolated, cyclopropenone-caged strained alkyne precursor was first functionalized onto a flat gold substrate through self-assembly. Exposure of the cyclopropenone SAM to UVA light initiated the efficient photochemical decarbonylation of the cyclopropenone moiety, revealing the strained alkyne capable of undergoing the interfacial strain-promoted alkyne-azide cycloaddition (SPAAC). Irradiated SAMs were derivatized with a series of model azides with varied hydrophobicity to demonstrate the generality of this chemical system for the modification and fine-tuning of the surface chemistry on gold substrates. SAMs were characterized at each step with polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS) to confirm the successful functionalization and reactivity. Furthermore, to showcase the compatibility of this approach with biochemical applications, cyclopropenone SAMs were irradiated and modified with azide-bearing cell adhesion peptides to promote human fibroblast cell adhesion, and then imaged by live-cell fluorescence microscopy. Thus, the "photoclick" methodology reported here represents an improved, versatile, catalyst-free protocol that allows for a high degree of control over the modification of material surfaces, with applicability in materials science as well as biochemistry.


Diversity and Distribution of Sulfur Oxidation-Related Genes in Thioalkalivibrio, a Genus of Chemolithoautotrophic and Haloalkaliphilic Sulfur-Oxidizing Bacteria.
Soda lakes are saline alkaline lakes characterized by high concentrations of sodium carbonate/bicarbonate which lead to a stable elevated pH (>9), and moderate to extremely high salinity. Despite this combination of extreme conditions, biodiversity in soda lakes is high, and the presence of diverse microbial communities provides a driving force for highly active biogeochemical cycles. The sulfur cycle is one of the most important of these and bacterial sulfur oxidation is dominated by members of the obligately chemolithoautotrophic genus Thioalkalivibrio. Currently, 10 species have been described in this genus, but over one hundred isolates have been obtained from soda lake samples. The genomes of 75 strains were sequenced and annotated previously, and used in this study to provide a comprehensive picture of the diversity and distribution of genes related to dissimilatory sulfur metabolism in Thioalkalivibrio. Initially, all annotated genes in 75 Thioalkalivibrio genomes were placed in ortholog groups and filtered by bi-directional best BLAST analysis. Investigation of the ortholog groups containing genes related to sulfur oxidation showed that flavocytochrome c (fcc), the truncated sox system, and sulfite:quinone oxidoreductase (soe) are present in all strains, whereas dissimilatory sulfite reductase (dsr; which catalyzes the oxidation of elemental sulfur) was found in only six strains. The heterodisulfide reductase system (hdr), which is proposed to oxidize sulfur to sulfite in strains lacking both dsr and soxCD, was detected in 73 genomes. Hierarchical clustering of strains based on sulfur gene repertoire correlated closely with previous phylogenomic analysis. The phylogenetic analysis of several sulfur oxidation genes showed a complex evolutionary history. All in all, this study presents a comprehensive investigation of sulfur metabolism-related genes in cultivated Thioalkalivibrio strains and provides several avenues for future research.


The incidence of congenital hypothyroidism (CH) in Guangxi, China and the predictors of permanent and transient CH.
The incidence of congenital hypothyroidism (CH) differs significantly among different ethnicities and regions, and early differentiation of transient CH is important to avoid unnecessary prolonged treatment with L-T4. To investigate the incidence of CH based on the newborn screening program in Guangxi Zhuang Autonomous Region, China, and to analyze the predictors that might allow for an early differentiation between permanent (P) and transient (T) CH. Data from newborn screening program over a seven-year period (January 2009 to January 2016) at Guangxi Maternal and Child Health Hospital are analyzed. Blood samples were collected on filter paper between 3 and 7 days after birth, and TSH level was measured by time-resolved fluorescence assay. Individuals with increased TSH (TSH ≥ 8 IU/L) levels detected by newborn screening were recalled for further evaluation. Serum TSH, FT3 and FT4 were determined by electrochemiluminescence assay using venous blood samples. Diagnosis of CH is based on elevated TSH levels (>10 IU/L) and decreased FT4 levels (<12 pmol/L). Patients with elevated TSH levels and normal FT4 levels were diagnosed as hyperthyrotropinemia. Permanent or transient CH was determined by using the results of thyroid function tests after temporary withdrawal of L-T4 therapy at approximately 2-3 years of age. Among 1,238,340 infants in the newborn screening program, 14,443 individuals were recalled for reevaluation (re-call rate 1.18%), 911 and 731 individuals were subsequently determined to have hyperthyrotropinemia and CH respectively; thus, a prevalence of 1:1359 and 1:1694 for hyperthyrotropinemia and CH. Of the 731 patients with CH, 161 patients were diagnosed with permanent CH (PCH), and 159 patients were diagnosed with transient CH (TCH), the other 411 patients are too young to determine their subtypes. Patients with PCH required an increasing dose of L-T4 during the first few years, whereas patients with TCH required a decreased dose of L-T4. The TSH levels at diagnosis and the dose of L-T4 used were significantly higher in PCH cases than in transient cases. The FT4 levels at diagnosis were significantly lower in PCH cases than in TCH cases. The TSH levels at diagnosis, FT4 levels at diagnosis and L-T4 doses at 90 days were evaluated as predictors for differentiating PCH and TCH, and their accuracy at their respective optimal cutoffs were determined to be 60.6%, 66.7% and 93.9%, respectively. The CH incidence in Guangxi Zhuang Autonomous Region is slightly higher (1:1694) compared to the worldwide levels (1/2000-1/4000). The PCH and TCH ratio is close to 1; thus, the estimated PCH incidence is 1/3388, which is similar to reported worldwide average incidence (1/3000). The L-T4 dose required at 90 days (>30 μg/day) has the highest predictive value for PCH. Earlier differentiation of PCH and TCH helps to determine appropriate treatment course.


A new species of Petroleuciscus (Teleostei: Cyprinidae) from the Büyük Menderes River, southwestern Anatolia, Turkey.
Petroleuciscus ninae sp. nov. is described from the Büyük Menderes River drainage. The new species is distinguished by having a black lateral stripe from head to base of caudal fin, stripe distinct anteriorly and posteriorly, wider than eye diameter; numerous black pigments on anal-fin rays; body depth at dorsal-fin origin 27-30% standard length (LS ); head width at posterior margin of eye 16-19% LS ; and eye diameter smaller than snout length. Petroleuciscus ninae is also distinguished from other species in adjacent waters by having six fixed diagnostic nucleotide substitutions in the mitochondrial DNA coI barcode region.


Carrier detection and prenatal diagnosis of hemoglobinopathies in Ontario.
The province of Ontario has a total population of approximately 10 million people, with approximately 20% being of African, Southeast Asian, East Indian, Mediterranean, or Middle Eastern ancestry in whom the gene frequency for hemoglobinopathies is relatively high. In 1989, the Ontario Ministry of Health funded the establishment of the Provincial Hemoglobinopathy DNA Diagnostic Laboratory located at the McMaster University Medical Centre in Hamilton, Ontario. The Laboratory provides DNA analysis to identify the globin gene mutations in carriers and affected individuals, and performs prenatal diagnosis for severe hemoglobinopathies. Annually, more than 400 patient samples are referred to the Laboratory for investigation, of which 25-35 are fetal samples from pregnancies at risk for either homozygous alpha-thalassemia, beta-thalassemia major, or sickling disorders. We have detected more than 70 different globin gene mutations, including several mutations not previously reported in the literature. Here we present examples of the approaches used to detect globin gene mutations in a heterogeneous "at risk" population such as in Ontario, and discuss the impact of this service on patient care, genetic counselling, and the incidence of severe hemoglobinopathies in Ontario.


Usefulness of red cell distribution width in predicting all-cause long-term mortality after non-ST-elevation myocardial infarction.
Red blood cell distribution width (RDW) is a strong predictor of adverse outcomes in patients with heart failure, stable coronary artery disease, stroke and acute myocardial infarction. The aim of our study was to explore the predictive value of RDW on all-cause mortality in patients with non-ST-segment elevation myocardial infarction (NSTEMI). This observational study includes 619 NSTEMI patients, discharged from Staten Island University Hospital between September 2004 and December 2006. Patients were divided into equal RDW tertiles and survival was evaluated in each tertile. Patients in the highest RDW tertile (RDW >14) had higher in-patient (7 vs. 1%) and 4-year (30 vs. 7%) mortality rates compared to those in the lowest tertile (RDW <13) (Wilcoxon χ(2) = 34.64, p < 0.0001). After controlling for Global Registry of Acute Coronary Events risk profile scores and other confounding variables, the RDW adjusted hazard ratio for 4-year all-cause mortality increased by 1.10 for each one unit increase in RDW (confidence interval 1.004-1.213, p = 0.042). RDW is an independent predictor of all-cause long-term mortality in NSTEMI patients. Further studies are needed to clarify the mechanisms of this association between RDW and adverse outcomes in patients with coronary artery disease.


EDAR mutation in autosomal dominant hypohidrotic ectodermal dysplasia in two Swedish families.
Hypohidrotic ectodermal dysplasia (HED) is a genetic disorder characterized by defective development of teeth, hair, nails and eccrine sweat glands. Both autosomal dominant and autosomal recessive forms of HED have previously been linked to mutations in the ectodysplasin 1 anhidrotic receptor (EDAR) protein that plays an important role during embryogenesis. The coding DNA sequence of the EDAR gene was analyzed in two large Swedish three-generational families with autosomal dominant HED. A non-sense C to T mutation in exon 12 was identified in both families. This disease-specific mutation changes an arginine amino acid in position 358 of the EDAR protein into a stop codon (p.Arg358X), thereby truncating the protein. In addition to the causative mutation two polymorphisms, not associated with the HED disorder, were also found in the EDAR gene. The finding of the p.Arg358X mutation in the Swedish families is the first corroboration of a previously described observation in an American family. Thus, our study strengthens the role of this particular mutation in the aetiology of autosomal dominant HED and confirms the importance of EDAR for the development of HED.


Cross-linked envelope-related markers for squamous differentiation in human lung cancer cell lines.
Lung carcinoma cell lines were analyzed in culture and in nude mouse xenograft for both morphological appearance and expression of specific proteins that participate in cross-linked envelope formation during normal squamous cell terminal differentiation. Cross-linked envelope formation, induced by artificial influx of millimolar Ca2+ into the cultured cells, was an exclusive trait of squamous, adenosquamous, and mucoepidermoid carcinomas. Small cell lung carcinoma and non-squamous non-small cell lung carcinoma lines, such as adenocarcinoma and large cell carcinoma, were uniformly negative for cross-linked envelope formation. Involucrin, which is incorporated into the cross-linked envelope by the enzyme transglutaminase, was expressed at highest levels in squamous tumors, but several of the non-squamous non-small cell lung carcinoma lines also expressed comparable amounts. On the other hand, transglutaminase activity was consistently higher in squamous as opposed to non-squamous lines, so that in cell culture, a clear contrast between the groups could be observed. A Mr 195,000 protein that is incorporated into cultured human epidermal cell cross-linked envelopes was also observed in some but not all of the squamous lines. Two forms of transglutaminase are expressed in cultured keratinocytes. One of them, tissue transglutaminase, was expressed in the majority of squamous cell lines even though it is not a normal product of squamous differentiation in vivo. Keratinocyte transglutaminase, which is distinct from the tissue form and is normally expressed during terminal differentiation in squamous epithelia. was measurably present in only one of the six squamous cell lines tested. In nude mouse xenografts, keratinocyte transglutaminase, localized immunohistochemically with a biotinylated mouse monoclonal antibody, was again present only in a minority of the squamous lines whereas involucrin was expressed in all. In contrast to involucrin, keratinocyte transglutaminase is not an obligatory component of squamous differentiation in the pulmonary carcinoma cell lines tested. Its expression may be of value in further refining their classification.


Raman spectroscopic analysis of high molecular weight proteins in solution - considerations for sample analysis and data pre-processing.
This study explores the potential of Raman spectroscopy, coupled with multivariate regression techniques and a protein separation technique (ion exchange chromatography), to quantitatively monitor diagnostically relevant changes in high molecular weight proteins in liquid plasma. Measurement protocols to detect the imbalances in plasma proteins as an indicator of various diseases using Raman spectroscopy are optimised, such that strategic clinical applications for early stage disease diagnostics can be evaluated. In a simulated plasma protein mixture, concentrations of two proteins of identified diagnostic potential (albumin and fibrinogen) were systematically varied within physiologically relevant ranges. Scattering from the poorly soluble fibrinogen fraction is identified as a significant impediment to the accuracy of measurement of mixed proteins in solution, although careful consideration of pre-processing methods allows construction of an accurate multivariate regression prediction model for detecting subtle changes in the protein concentration. Furthermore, ion exchange chromatography is utilised to separate fibrinogen from the rest of the proteins and mild sonication is used to improve the dispersion and therefore quality of the prediction. The proposed approach can be expeditiously employed for early detection of pathological disorders associated with high or low plasma/serum proteins.


Rigidity of the extracellular part of HER2: Evidence from engineering subdomain interfaces and shared-helix DARPin-DARPin fusions.
The second member of the human ErbB family of receptor tyrosine kinases, HER2/hErbB2, is regarded as an exceptional case: The four extracellular subdomains could so far only be found in one fixed overall conformation, designated "open" and resembling the ligand-bound form of the other ErbB receptors. It thus appears to be different from the extracellular domains of the other family members that show inter-subdomain flexibility and exist in a "tethered" form in the absence of ligand. For HER2, there was so far no direct evidence for such a tethered conformation on the cell surface. Nonetheless, alternative conformations of HER2 in vivo could so far not be excluded. We now demonstrate the rigidity of HER2 on the surface of tumor cells by employing two orthogonal approaches of protein engineering: To directly test the potential of the extracellular domain of HER2 to adopt a pseudo-tethered conformation on the cell surface, we first designed HER2 variants with a destabilized interface between extracellular subdomains I and III that would favor deviation from the "open" conformation. Secondly, we used differently shaped versions of a Designed Ankyrin Repeat Protein (DARPin) fusion, recognizing subdomain I of HER2, devised to work as probes for a putative pseudo-tethered extracellular domain of HER2. Combining our approaches, we exclude, on live cells and in vitro, that significant proportions of HER2 deviate from the "open" conformation.


Two coronaviruses isolated from central nervous system tissue of two multiple sclerosis patients.
Two coronaviruses were isolated from brain material obtained at autopsy from two multiple sclerosis patients. The viruses were neutralized by serum and spinal fluid from these patients. Although most of the population have antibody to these virus isolates, multiple sclerosis patients have slightly higher concentrations of serum antibody than controls. The results suggest that coronaviruses should be considered as one additional virus with a potential implication in the etiology of multiple sclerosis.


Timing a one-handed catch. II. Adaptation to telestereoscopic viewing.
A pre-exposure, exposure, post-exposure design was used to assess the adaptation of the timing of a one-handed catch during telestereoscopic viewing. More specifically, it was examined whether the adaptation involved: (1) ignoring binocular sources of information and selecting other information, or (2) a recalibration of the coupling between the effected binocular information and the catching movement, and (3), if it is recalibration, whether it is restricted to the manipulated binocular information. To test these hypotheses, subjects (n=16) were assigned to one of two groups, each group performing three blocks of 15 trials in the dark with only the ball visible. In the exposure condition, both groups were required to catch balls under binocular telestereoscopic viewing. In the pre-exposure and post-exposure conditions, subjects performed under binocular and monocular viewing, respectively. Kinematics of the grasping movement were recorded. It was predicted that, in the case of a selection process, no after effects would occur in the post-exposure condition, whereas, in the case of recalibration, aftereffects would occur. Moreover, if the recalibration is restricted to the manipulated information, only the group that was provided with binocular vision during the pre- exposure and post-exposure conditions would show aftereffects. Significant condition (pre-exposure, exposure, post-exposure) by block (first three trials, last three trials) effects were found for the moments of grasp onset, peak opening velocity and hand closure, indicating that the hand was opened and closed earlier in the first three trials of telestereoscopic viewing. This coincided with an increase in catching failures. In addition, for the moments of hand closure and peak closing velocity, negative aftereffects were found in the post-exposure condition. The hand was closed later in the first three trials after removal of telestereoscope. With respect to the presence of the aftereffects, no differences were found between the groups. It was concluded that adaptation to telestereoscopic viewing in the timing of a one-handed catch is due to the recalibration of the coupling between information and movement, rather than a selection of another source of information. Moreover, it is likely that the recalibration was not restricted to the single, manipulated information. Rather, the recalibration involves multiple binocular and monocular optical and oculomotor sources of information.


Prevalence of precancerous skin lesions and non-melanoma skin cancer in Japanese-Brazilians in Bauru, São Paulo State, Brazil.
Precancerous lesions and skin cancer are infrequent in Asians, and have received little documentation in the literature. Brazil has the world's largest contingent of Japanese immigrants and their descendants, and 70% live in the State of São Paulo. The prevalence of such skin lesions in Japanese-Brazilians is unknown. This study aimed to assess the prevalence of actinic keratoses and non-melanoma skin cancer in first and second-generation Japanese-Brazilians over 30 years of age, without miscegenation, living in the city of Bauru, São Paulo State, in 2006. Of the 567 Japanese-Brazilians that underwent dermatological examination, actinic keratosis was diagnosed in 76, with a mean age of 68.9 years, and a single case of basal cell carcinoma was detected in a 39-year-old female patient. In Japan, prevalence of actinic keratosis varies from 0.76% to 5%, and the incidence of non-melanoma skin cancer is 1.2 to 5.4/100 thousand. Japanese-Brazilians from Bauru showed a 13.4% prevalence of actinic keratoses and earlier age at onset. Proximity to the Equator and a history of farming contribute to these higher rates. Presence of solar melanosis was associated with a 1.9-fold risk of developing actinic keratosis.


Severe evolution of chronic hepatitis C in renal transplantation: a case control study.
To evaluate the impact of kidney transplantation on histopathological progression of hepatitis C virus (HCV)-related liver disease. In a retrospective study, 28 HCV-positive renal transplant patients, who underwent two sequential liver biopsies with a mean of 7.1+/-4.0 years, were compared with 28 matched immunocompetent controls. According to the Metavir score, the initial and final activity scores (from 0 to 3) increased from 0.2+/-0.4 to 1.4+/-1.1 (P<0.001) and those of fibrosis (from 0 to 4) from 0.5+/-0.5 to 2.0+/-1.4 (P<0.001) in the transplanted group, respectively, whereas the respective differences were not significant in the control group. The yearly progression rate of activity and fibrosis was significantly higher in the renal transplant group as compared with the immunocompetent group: 0.26+/-0.41 vs 0.01+/-0.19 (P<0.01) and 0.26+/-0.35 vs 0.05+/-0.21 (P<0.03), respectively. Twenty (71.5%) and 14 (50.0%) of the renal allograft recipients had activity and fibrosis progression as compared with four (16%) (P<0.001) and four (16%) (P<0.01) in immunocompetent patients; six kidney recipients (21.4%) evolved to cirrhosis vs only one in the control group (3.6%) (P=0.07). Liver-related mortality was significantly higher during the follow-up period in renal transplant patients than in the control group (10 vs 0%) (P<0.05). Using conventional immunosuppressive regimen, renal transplantation is associated with a more severe evolution of chronic hepatitis C as compared with HCV-infected immunocompetent subjects. Thus, the histopathological evaluation should be performed and anti-viral therapy discussed before renal transplantation.


Air trapping on chest CT is associated with worse ventilation distribution in infants with cystic fibrosis diagnosed following newborn screening.
In school-aged children with cystic fibrosis (CF) structural lung damage assessed using chest CT is associated with abnormal ventilation distribution. The primary objective of this analysis was to determine the relationships between ventilation distribution outcomes and the presence and extent of structural damage as assessed by chest CT in infants and young children with CF. Data of infants and young children with CF diagnosed following newborn screening consecutively reviewed between August 2005 and December 2009 were analysed. Ventilation distribution (lung clearance index and the first and second moment ratios [LCI, M(1)/M(0) and M(2)/M(0), respectively]), chest CT and airway pathology from bronchoalveolar lavage were determined at diagnosis and then annually. The chest CT scans were evaluated for the presence or absence of bronchiectasis and air trapping. Matched lung function, chest CT and pathology outcomes were available in 49 infants (31 male) with bronchiectasis and air trapping present in 13 (27%) and 24 (49%) infants, respectively. The presence of bronchiectasis or air trapping was associated with increased M(2)/M(0) but not LCI or M(1)/M(0). There was a weak, but statistically significant association between the extent of air trapping and all ventilation distribution outcomes. These findings suggest that in early CF lung disease there are weak associations between ventilation distribution and lung damage from chest CT. These finding are in contrast to those reported in older children. These findings suggest that assessments of LCI could not be used to replace a chest CT scan for the assessment of structural lung disease in the first two years of life. Further research in which both MBW and chest CT outcomes are obtained is required to assess the role of ventilation distribution in tracking the progression of lung damage in infants with CF.


Case finding and therapy for chronic viral hepatitis in primary care (HepFREE): a cluster-randomised controlled trial.
The prevalence of viral hepatitis (hepatitis B virus and hepatitis C virus) in migrants is higher than among the general population in many high-income countries. We aimed to determine whether incentivising and supporting primary-care physicians in areas with a high density of migrants increases the numbers of adult migrants screened for viral hepatitis. HepFREE was a multicentre, open, cluster-randomised controlled trial in general practices in areas of the UK with a high density of migrants (Bradford, Yorkshire, and northeast and southeast London). Participants were adult patients (aged 18 years or older) in primary care, who had been identified as a first or second generation migrant from a high-risk country. General practices were randomly assigned (1:2:2:2:2) to an opportunistic screening (control) group or to one of four targeted screening (interventional) groups: standard (ie, hospital-based) care and a standard invitation letter; standard care and an enhanced invitation letter; community care and a standard invitation letter; or community care and an enhanced invitation letter. In control screening, general practitioners (GPs) were given a teaching session on viral hepatitis and were asked to test all registered migrants. In the intervention, GPs were paid a nominal sum for setting up searches of records, reimbursed for signed consent forms, and supported by a dedicated clinician. Patients who were eligible for testing and tested positive for viral hepatitis in the intervention groups were eligible to enrol in a second embedded trial of community versus hospital based care. The primary outcomes were the proportion of patients eligible for screening, the proportion of those eligible who were sent an invitation letter in the intervention groups, the uptake of viral hepatitis screening (in the intention-to-treat population), the proportion of patients who tested positive for viral hepatitis, the proportion who complied with treatment, and the cost-effectiveness of the intervention. This trial is registered with ISRCTN, number ISRCTN54828633. Recruitment and testing ran from Oct 31, 2013, to Feb 4, 2017, and each practice recruited for 18 consecutive calendar months. We approached 70 general practices in three areas with a high density of migrants, of which 63 general practices agreed to participate. Five practices withdrew and 58 practices were randomly assigned: eight to control and 50 to an intervention. In control practices, 26 046 (38·4%) of 67 820 patients who were initially registered were eligible for testing, as were 152 321 (43·3%) of 351 710 patients in the interventional groups in London and Bradford. Of 51 773 randomly selected eligible patients in the intervention groups in London and Bradford, letters were sent to 43 585 (84·2%) patients. In the eight control general practices, screening was taken up by 543 (1·7%) of 31 738 eligible participants, which included 5692 newly registered patients. However, in the 50 general practices that used the intervention, screening was taken up by 11 386 (19·5%) of 58 512 eligible participants (including 6739 newly registered patients; incidence rate ratio 3·70, 95% CI 1·30-10·51; p=0·014) and this intervention was cost-effective. 720 (4·5%) of 15 844 patients who received a standard letter versus 1032 (3·7%) of 28 095 patients who received the enhanced letter were tested (0·70, 0·38-1·31; p=0·26). In the control group, 17 patients tested positive for viral hepatitis, as did 220 patients (one with a co-infection) in the intervention groups. In the embedded study, 220 patients were randomly assigned to either hospital-based care or community care; 80 (87·9%) of 91 patients in the hospital setting complied with treatment versus 105 (81·4%) of 129 patients in the community setting. The intervention was cost-effective at willingness to pay thresholds in excess of £8540. One serious adverse event (thyroiditis) was noted. Screening migrants for viral hepatitis in primary care is effective if doctors are incentivised and supported. Community care is expensive and there is no evidence that this offers benefits in this setting or that bespoke invitation letters add value. We suggest that bespoke invitation letters should not be used, and we suggest that outreach, community-based services for migrants should not be developed. National Institute for Health Research.


De novo mutation in the NOTCH3 gene causing CADASIL.
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) is one of the most common hereditary forms of stroke, and migraine with aura, mood disorders and dementia. CADASIL is caused by mutations of the NOTCH3 gene. This mutation is inherited as an autosomal dominant trait. Most individuals with CADASIL have a parent with the disorder. In extremely rare cases, CADASIL may occur due to a spontaneous genetic mutation that occurs for unknown reasons (de novo mutation). We report a new case of patient with de novo mutation of the NOTCH3 gene and a condition strongly suggestive of CADASIL (migraine, stroke, and white matter abnormalities), except that this patient did not have any first-degree relatives with similar symptoms.