Virulence systems of Pseudomonas syringae pv. tomato promote bacterial speck disease in tomato by targeting the jasmonate signaling pathway.
Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000) causes bacterial speck disease on tomato. The pathogenicity of Pst DC3000 depends on both the type III secretion system that delivers virulence effector proteins into host cells and the phytotoxin coronatine (COR), which is thought to mimic the action of the plant hormone jasmonic acid (JA). We found that a JA-insensitive mutant (jai1) of tomato was unresponsive to COR and highly resistant to Pst DC3000, whereas host genotypes that are defective in JA biosynthesis were as susceptible to Pst DC3000 as wild-type (WT) plants. Treatment of WT plants with exogenous methyl-JA (MeJA) complemented the virulence defect of a bacterial mutant deficient in COR production, but not a mutant defective in the type III secretion system. Analysis of host gene expression using cDNA microarrays revealed that COR works through Jai1 to induce the massive expression of JA and wound response genes that have been implicated in defense against herbivores. Concomitant with the induction of JA and wound response genes, the type III secretion system and COR repressed the expression of pathogenesis-related (PR) genes in Pst DC3000-infected WT plants. Resistance of jai1 plants to Pst DC3000 was correlated with a high level of PR gene expression and reduced expression of JA/wound response genes. These results indicate that COR promotes bacterial virulence by activating the host's JA signaling pathway, and further suggest that the type III secretion system might also modify host defense by targeting the JA signaling pathway in susceptible tomato plants.


Use of drugs subject to controlled prescriptions: a retrospective analysis.
Use of drugs that may lead to abuse or dependence are subject to controlled prescriptions (CPs) in many countries, and these are closely monitored by health authorities. According to national regulations in Turkey, CPs may be red coloured (RCPs) or green coloured (GCPs). The aim of this study was to evaluate the use of such drugs in Istanbul. Retrospective case-control study. During the study period (01/01-31/12 2009), 502874 CPs were reported. Among these, 4000 CPs each month were randomly selected and evaluated. The majority of GCPs were issued to women (55.6%), while the majority of RCPs were issued to men (68.4%). GCPs were most frequently prescribed by physicians working in private hospitals (33.6%) while RCPs by physicians working in university hospitals (39.7%). GCPs were mostly prescribed by psychiatrists (37.6%) while for RCPs were child and adolescent psychiatrists (35.9%). Psycholeptics (ATC code N05) were the most prescribed controlled drugs (CDs) (43.8%). Methylphenidate (53.9%) was the mostly prescribed on RCPs and alprazolam (39.6%) was on GCPs. We demonstrate that utilization of CDs shows demographical and institutional differences. These data could be of help to improve surveillance of CDs as well as to train prescribers and patients.


Meta Networks.
Neural networks have been successfully applied in applications with a large amount of labeled data. However, the task of rapid generalization on new concepts with small training data while preserving performances on previously learned ones still presents a significant challenge to neural network models. In this work, we introduce a novel meta learning method, Meta Networks (MetaNet), that learns a meta-level knowledge across tasks and shifts its inductive biases via fast parameterization for rapid generalization. When evaluated on Omniglot and Mini-ImageNet benchmarks, our MetaNet models achieve a near human-level performance and outperform the baseline approaches by up to 6% accuracy. We demonstrate several appealing properties of MetaNet relating to generalization and continual learning.


Ferroelectric liquid-crystal tunable filter.
A new discrete-channel tunable Lyot filter that uses ferroelectric liquid-crystal (FLC) smectic C* wave plates is described and demonstrated. The device is tuned by changing the retardation of each birefringent element. This is achieved by electrically rotating the FLC wave plates in each stage by 45 degrees , which effectively changes the design wavelength of the Lyot filter. The transmission characteristics of such a device are demonstrated in a three-stage filter and show good agreement with theory. Advantages of the FLC tunable filter over existing filter structures include low switching voltages (+/-10 V), rapid tunability (~100 kHz), potentially high transmission and wide field of view, and large entrance aperture.


Regulatory T cells reinforce intestinal homeostasis.
Regulatory T cells help maintain intestinal homeostasis by preventing inappropriate innate and adaptive immune responses. CD4(+) T cells that express Foxp3 and Tr1-like cells that produce IL-10 comprise the major regulatory populations in the intestine. CD4(+)Foxp3(+) T cells play an important functional role in promoting tolerance of the flora and dietary proteins. Tr1-like cells can be generated in conditions that also promote effector T cell responses and may serve a similar function. In this review, we discuss the signals specific to the gastrointestinal tract that support both regulatory cell types and their distinct modes of action in the mesenteric lymph nodes and intestinal tissues. Dysregulation of intestinal immune homeostasis occurs in inflammatory bowel disease and can also be observed in graft-versus-host disease, tumor immunotherapy regimens, and acute HIV infection.


Shunt age-related complications in adult patients with suspected shunt dysfunction. A recommended diagnostic workup.
Patients admitted for suspicion of shunt dysfunction (SD) often show unspecific symptoms and require time-consuming, expensive and even invasive diagnostics involving significant radiation exposure. The purpose of this retrospective study was to analyse the current diagnostic procedures and to propose a process optimisation. As all patients admitted for suspicion of SD receive imaging studies, we searched for adult patients receiving neuroimaging in the period from January 2010 to July 2013, analysing referring diagnosis, clinical signs, products, diagnostic process and final diagnosis. Recursive partitioning was used to define time intervals for differentiating types of SD. A total of 148 patients, aged 18-89 (mean, 54) years, were studied. Forty-two percent were referred by a hospital or rehabilitation centre, 30% by general practitioners and 24% were self-referrals. The admission diagnosis was in the majority "shunt dysfunction" only. Further differentiations were rarely made. An SD was confirmed in 46% of the patients. In 17%, the symptoms were based on another cause and in 37% they could not be clearly attributed to any specific disorder. Abdominal dislocations (2%) and shunt infections (5%) were found within the first 6 months. Over- (3%) and under-drainage (14%) were the most frequent complications during the first 4 years. Disconnections (13%) occurred generally 4 years or more after implantation. Only shunt obstruction (9%) showed no temporal pattern. Symptoms of SD remain mostly unspecific. This study showed that the type of SD depends on the time interval from implantation. We propose a workup strategy for patients with SD based on the temporal profile.


Localized hypoxia within the subgranular zone determines the early survival of newborn hippocampal granule cells.
The majority of adult hippocampal newborn cells die during early differentiation from intermediate progenitors (IPCs) to immature neurons. Neural stem cells in vivo are located in a relative hypoxic environment, and hypoxia enhances their survival, proliferation and stemness in vitro. Thus, we hypothesized that migration of IPCs away from hypoxic zones within the SGZ might result in oxidative damage, thus triggering cell death. Hypoxic niches were observed along the SGZ, composed of adult NSCs and early IPCs, and oxidative byproducts were present in adjacent late IPCs and neuroblasts. Stabilizing hypoxia inducible factor-1α with dimethyloxallyl glycine increased early survival, but not proliferation or differentiation, in neurospheres in vitro and in newly born SGZ cells in vivo. Rescue experiments in Bax(fl/fl) mutants supported these results. We propose that localized hypoxia within the SGZ contributes to the neurogenic microenvironment and determines the early, activity-independent survival of adult hippocampal newborn cells.


DJ-1/PARK7 is an important mediator of hypoxia-induced cellular responses.
In cancer, DJ-1/PARK7 acts as an oncogene that drives Akt-mediated cell survival. Although amplification of DJ-1 has been described in several types of tumors, the mechanistic basis of DJ-1's oncogenic effect remains incompletely understood. A tumor's ability to adapt to hypoxia is absolutely critical for its survival and progression, and this adaptation is largely mediated by the transcription factor HIF1. The stabilization of HIF1 subunits during hypoxia is at least partly dependent on the PI3K/Akt/mTOR pathway. We hypothesized that DJ-1, a positive regulator of Akt when over-expressed, might be involved in regulating HIF1 transcriptional activity under hypoxic conditions. Our results show that loss of DJ-1 in human cell lines and transformed mouse fibroblasts decreases the transcription of a variety of HIF1-responsive genes during hypoxia. Moreover, DJ-1 expression is critical for the Akt and mTOR activities that sustain HIF1 stability. Surprisingly, DJ-1 also regulates the activity of the metabolic sensor AMPK, especially during hypoxia. Finally, DJ-1 appears to protect cells against hypoxia-induced cell death and is required for their adaptation to severe hypoxic stress. Our work positions DJ-1 as an upstream activator of HIF1 function in cancer cells and establishes that DJ-1's oncogenic activity stems from its ability to increase a cell's resistance to hypoxic stress through DJ-1's regulatory effects on mTOR and AMPK. The discovery of these functions of DJ-1 strengthens the case for the development of therapeutics that target DJ-1 activity in cancer cells.


Molecular mechanism of SLC5A8 inactivation in breast cancer.
SLC5A8 is a putative tumor suppressor that is inactivated in more than 10 different types of cancer, but neither the oncogenic signaling responsible for SLC5A8 inactivation nor the functional relevance of SLC5A8 loss to tumor growth has been elucidated. Here, we identify oncogenic HRAS (HRAS(G12V)) as a potent mediator of SLC5A8 silencing in human nontransformed normal mammary epithelial cell lines and in mouse mammary tumors through DNMT1. Further, we demonstrate that loss of Slc5a8 increases cancer-initiating stem cell formation and promotes mammary tumorigenesis and lung metastasis in an HRAS-driven murine model of mammary tumors. Mammary-gland-specific overexpression of Slc5a8 (mouse mammary tumor virus-Slc5a8 transgenic mice), as well as induction of endogenous Slc5a8 in mice with inhibitors of DNA methylation, protects against HRAS-driven mammary tumors. Collectively, our results provide the tumor-suppressive role of SLC5A8 and identify the oncogenic HRAS as a mediator of tumor-associated silencing of this tumor suppressor in mammary glands. These findings suggest that pharmacological approaches to reactivate SLC5A8 expression in tumor cells have potential as a novel therapeutic strategy for breast cancer treatment.


HMGA1-dependent and independent 7SK RNA gene regulatory activity.
The small nuclear 7SK RNA negatively controls transcription by inactivating positive transcription elongation factor b (P-TEFb) and is an integral component of Tat-dependent and independent HIV-1 transcription initiation complexes. 7SK RNA has recently been shown to also directly control HMGA1 transcription activity. HMGA1 is a master regulator of gene expression and its deregulation is associated with virtually any type of human cancer. The degree of HMGA1 over-expression thereby correlates with tumor malignancy and metastatic potential. 7SK snRNA directly interacts through its loop2 (7SK L2) with the first A/T-hook DNA binding motif of HMGA1. We have developed several 7SK L2 RNA chimera with the Epstein Barr Virus expressed RNA 2 (EBER2) to target HMGA1 function in transcription regulation. The efficiency of interfering with HMGA1 transcription activity by the chimeric 7SK L2-EBER2 fusions by large exceeds the efficiency of 7SK wild-type RNA due to the stronger EBER2 promoter activity. Furthermore, the 7SK L2-EBER2 chimera do not interfere with P-TEFb controlled transcription elongation or the formation of 7SK sn/hnRNPs. The comparison of the effects of wild-type 7SK RNA on cellular transcriptome dynamics with those induced by the two 7SK L2 mutants as well as the changes in gene expression following inhibition of HMGA1 allow the identification and characterization of HMGA1-dependent and independent effects of 7SK snRNA. We furthermore also present evidence for P-TEFb and HMGA1-independent 7SK RNA L2 regulatory activity.


The diversity of Campylobacter spp. throughout the poultry processing plant.
Campylobacteriosis is the leading food-borne disease in developed countries, and poultry are a major source for human infection. The diversity of Campylobacter on chicken carcasses during processing may lead to isolates that are able to survive abattoir processing. This has important implications for public health and adds a further layer to the complexity of the epidemiology of campylobacteriosis. The diversity of the Campylobacter spp. populations on broiler carcasses was studied at three different stages of processing (post-bleed, post-scald and post-chill) in three UK processing plants, using the pulsed-field gel electrophoresis (PFGE) KpnI enzyme. One hundred and sixty Campylobacter strains from 3 processing plants were identified as C. jejuni (92.3%) with 27 PFGE subtype profiles recovered from carcasses at the post-bleed point. Change in populations was identified when carcasses move towards the end of poultry processing. Seven C. jejuni genotypes were able to survive the scalding tank stage process, and 5 genotypes surviving the entire poultry process. Confirmation by PFGE gives information on the genotypic profiles of C. jejuni on chicken carcasses and how they change according to the temperatures exposed to during processing. Diversity within C. jejuni populations produces genotypes that adapt to tolerate the processing environment, and these may be capable of causing human disease. Understanding more about the genotypes that survive the processing will have important implications for public health.


Appreciation of research information in patients with bipolar disorder.
Ethicists have debated whether patients with serious mental illness can appreciate the risks of research participation and make autonomous decisions. We compared the abilities of euthymic and manic bipolar patients to appreciate and make voluntary decisions regarding research participation. Twenty-six subjects with mania and 25 euthymic subjects reviewed hypothetical consent forms for three research studies of varying risk. We assessed subjects' appreciation of: their diagnosis and need for treatment; the researcher's role; the risks of participation; and the degree of influence of family, the treating clinician, and payment on decisions to participate. Most subjects (92%) agreed they had bipolar disorder requiring medication treatment. Subjects were less likely to participate in riskier studies. About half of subjects erroneously believed that researchers would make decisions based solely on what would be the best care for them (therapeutic misconception); and in randomized medication trials, they mistakenly believed they had improved chances of receiving one treatment over another. There were no differences between mood groups on these measures. Over half of subjects (59%) indicated that their mental health provider might influence them to participate in a study even when they did not want to, but most rejected a role for family in decision making. Payment was rated as having little impact on decisions to participate in research. Mania does not substantially influence appreciation of research participation. Subjects with bipolar disorder, regardless of mood state, are at risk for therapeutic misconception and optimistic bias. Special protections may be needed when mental health professionals approach their own patients to participate in research.


Tracking the Evolution of Transiently Transfected Individual Cells in a Microfluidic Platform.
Transient gene expression (TGE) technology enables the rapid production of large amount of recombinant proteins, without the need of fastidious screening of the producing cells required for stable transfection (ST). However, several barriers must be overcome before reaching the production yields using ST. For optimizing the production yields from suspended cells using TGE, a better understanding of the transfection conditions at the single cell level are required. In this study, a universal droplet microfluidic platform was used to assess the heterogeneities of CHO-S population transiently transfected with cationic liposomes (CL) (lipoplexes) complexed with GFP-coding plasmid DNA (pDNA). A single cell analysis of GFP production kinetics revealed the presence of a subpopulation producing higher levels of GFP compared with the main population. The size of high producing (HP) cells, their relative abundance, and their specific productivity were dependent on the charge and the pDNA content of the different lipoplexes: HPs showed increased cell size in comparison to the average population, lipoplexes with positive charge produced more HPs, and lipoplexes carrying a larger amount of pDNA yielded a higher specific productivity of HPs. This study demonstrates the potential for time-resolved single-cell measurements to explain population dynamics from a microscopic point of view.


Influence of plant polymers on the distribution and cultivation of bacteria in the phylum Acidobacteria.
Members of the phylum Acidobacteria are among the most abundant bacteria in soil. Although they have been characterized as versatile heterotrophs, it is unclear if the types and availability of organic resources influence their distribution in soil. The potential for organic resources to select for different acidobacteria was assessed using molecular and cultivation-based approaches with agricultural and managed grassland soils in Michigan. The distribution of acidobacteria varied with the carbon content of soil: the proportion of subdivision 4 sequences was highest in agricultural soils (ca. 41%) that contained less carbon than grassland soils, where the proportions of subdivision 1, 3, 4, and 6 sequences were similar. Either readily oxidizable carbon or plant polymers were used as the sole carbon and energy source to isolate heterotrophic bacteria from these soils. Plant polymers increased the diversity of acidobacteria cultivated but decreased the total number of heterotrophs recovered compared to readily oxidizable carbon. Two phylogenetically novel Acidobacteria strains isolated on the plant polymer medium were characterized. Strains KBS 83 (subdivision 1) and KBS 96 (subdivision 3) are moderate acidophiles with pH optima of 5.0 and 6.0, respectively. Both strains grew slowly (μ = 0.01 h(-1)) and harbored either 1 (strain KBS 83) or 2 (strain KBS 96) copies of the 16S rRNA encoding gene-a genomic characteristic typical of oligotrophs. Strain KBS 83 is a microaerophile, growing optimally at 8% oxygen. These metabolic characteristics help delineate the niches that acidobacteria occupy in soil and are consistent with their widespread distribution and abundance.


Clinical evaluation of epithelioid angiomyolipoma.
Epithelioid angiomyolipomas (AML) of the kidney are malignant tumors with aggressive clinical behavior. The Authors report on some cases epithelioid AM recently diagnosed at their Institute to highlight the spectrum of clinical presentations. In all cases, renal lesions observed at computed tomography were suspicious for renal cell carcinoma (RCC). Histologically, these tumors can resemble RCC. The diagnosis of epithelioid AML was established by positive staining for melanoma and smooth muscle cell markers, and presence of perivascular epithelioid cells. Four patient presented with a renal tumor extending into the inferior vena cava to the level of the hepatic veins. Two patients developed recurrent, metastatic disease following nephrectomy. These tumors are distinguished from RCC by positive immunostaining for melanoma markers and smooth muscle cell markers. They resemble conventional RCC on imaging. Epithelioid AML, may be locally aggressive and metastasized.


Lappaol F, a novel anticancer agent isolated from plant arctium Lappa L.
In an effort to search for new cancer-fighting therapeutics, we identified a novel anticancer constituent, Lappaol F, from plant Arctium Lappa L. Lappaol F suppressed cancer cell growth in a time- and dose-dependent manner in human cancer cell lines of various tissue types. We found that Lappaol F induced G(1) and G(2) cell-cycle arrest, which was associated with strong induction of p21 and p27 and reduction of cyclin B1 and cyclin-dependent kinase 1 (CDK1). Depletion of p21 via genetic knockout or short hairpin RNA (shRNA) approaches significantly abrogated Lappaol F-mediated G(2) arrest and CDK1 and cyclin B1 suppression. These results suggest that p21 seems to play a crucial role in Lappaol F-mediated regulation of CDK1 and cyclin B1 and G(2) arrest. Lappaol F-mediated p21 induction was found to occur at the mRNA level and involved p21 promoter activation. Lappaol F was also found to induce cell death in several cancer cell lines and to activate caspases. In contrast with its strong growth inhibitory effects on tumor cells, Lappaol F had minimal cytotoxic effects on nontumorigenic epithelial cells tested. Importantly, our data also demonstrate that Lappaol F exhibited strong growth inhibition of xenograft tumors in nude mice. Lappaol F was well tolerated in treated animals without significant toxicity. Taken together, our results, for the first time, demonstrate that Lappaol F exhibits antitumor activity in vitro and in vivo and has strong potential to be developed as an anticancer therapeutic.


Protonation/reduction dynamics at the [4Fe-4S] cluster of the hydrogen-forming cofactor in [FeFe]-hydrogenases.
The [FeFe]-hydrogenases of bacteria and algae are the most efficient hydrogen conversion catalysts in nature. Their active-site cofactor (H-cluster) comprises a [4Fe-4S] cluster linked to a unique diiron site that binds three carbon monoxide (CO) and two cyanide (CN-) ligands. Understanding microbial hydrogen conversion requires elucidation of the interplay of proton and electron transfer events at the H-cluster. We performed real-time spectroscopy on [FeFe]-hydrogenase protein films under controlled variation of atmospheric gas composition, sample pH, and reductant concentration. Attenuated total reflection Fourier-transform infrared spectroscopy was used to monitor shifts of the CO/CN- vibrational bands in response to redox and protonation changes. Three different [FeFe]-hydrogenases and several protein and cofactor variants were compared, including element and isotopic exchange studies. A protonated equivalent (HoxH) of the oxidized state (Hox) was found, which preferentially accumulated at acidic pH and under reducing conditions. We show that the one-electron reduced state Hred' represents an intrinsically protonated species. Interestingly, the formation of HoxH and Hred' was independent of the established proton pathway to the diiron site. Quantum chemical calculations of the respective CO/CN- infrared band patterns favored a cysteine ligand of the [4Fe-4S] cluster as the protonation site in HoxH and Hred'. We propose that proton-coupled electron transfer facilitates reduction of the [4Fe-4S] cluster and prevents premature formation of a hydride at the catalytic diiron site. Our findings imply that protonation events both at the [4Fe-4S] cluster and at the diiron site of the H-cluster are important in the hydrogen conversion reaction of [FeFe]-hydrogenases.


Mathematical models of cytotoxic effects in endpoint tumor cell line assays: critical assessment of the application of a single parametric value as a standard criterion to quantify the dose-response effects and new unexplored proposal formats.
The development of convenient tools for describing and quantifying the effects of standard and novel therapeutic agents is essential for the research community, to perform more precise evaluations. Although mathematical models and quantification criteria have been exchanged in the last decade between different fields of study, there are relevant methodologies that lack proper mathematical descriptions and standard criteria to quantify their responses. Therefore, part of the relevant information that can be drawn from the experimental results obtained and the quantification of its statistical reliability are lost. Despite its relevance, there is not a standard form for the in vitro endpoint tumor cell lines' assays (TCLA) that enables the evaluation of the cytotoxic dose-response effects of anti-tumor drugs. The analysis of all the specific problems associated with the diverse nature of the available TCLA used is unfeasible. However, since most TCLA share the main objectives and similar operative requirements, we have chosen the sulforhodamine B (SRB) colorimetric assay for cytotoxicity screening of tumor cell lines as an experimental case study. In this work, the common biological and practical non-linear dose-response mathematical models are tested against experimental data and, following several statistical analyses, the model based on the Weibull distribution was confirmed as the convenient approximation to test the cytotoxic effectiveness of anti-tumor compounds. Then, the advantages and disadvantages of all the different parametric criteria derived from the model, which enable the quantification of the dose-response drug-effects, are extensively discussed. Therefore, model and standard criteria for easily performing the comparisons between different compounds are established. The advantages include a simple application, provision of parametric estimations that characterize the response as standard criteria, economization of experimental effort and enabling rigorous comparisons among the effects of different compounds and experimental approaches. In all experimental data fitted, the calculated parameters were always statistically significant, the equations proved to be consistent and the correlation coefficient of determination was, in most of the cases, higher than 0.98.


DNA methylation changes facilitated evolution of genes derived from Mutator-like transposable elements.
Mutator-like transposable elements, a class of DNA transposons, exist pervasively in both prokaryotic and eukaryotic genomes, with more than 10,000 copies identified in the rice genome. These elements can capture ectopic genomic sequences that lead to the formation of new gene structures. Here, based on whole-genome comparative analyses, we comprehensively investigated processes and mechanisms of the evolution of putative genes derived from Mutator-like transposable elements in ten Oryza species and the outgroup Leersia perieri, bridging ~20 million years of evolutionary history. Our analysis identified thousands of putative genes in each of the Oryza species, a large proportion of which have evidence of expression and contain chimeric structures. Consistent with previous reports, we observe that the putative Mutator-like transposable element-derived genes are generally GC-rich and mainly derive from GC-rich parental sequences. Furthermore, we determine that Mutator-like transposable elements capture parental sequences preferentially from genomic regions with low methylation levels and high recombination rates. We explicitly show that methylation levels in the internal and terminated inverted repeat regions of these elements, which might be directed by the 24-nucleotide small RNA-mediated pathway, are different and change dynamically over evolutionary time. Lastly, we demonstrate that putative genes derived from Mutator-like transposable elements tend to be expressed in mature pollen, which have undergone de-methylation programming, thereby providing a permissive expression environment for newly formed/transposable element-derived genes. Our results suggest that DNA methylation may be a primary mechanism to facilitate the origination, survival, and regulation of genes derived from Mutator-like transposable elements, thus contributing to the evolution of gene innovation and novelty in plant genomes.


Sequence-based differentiation of strains in the Mycobacterium avium complex.
The complete 16S-23S rDNA internal transcribed spacer (ITS) was sequenced in 35 reference strains of the Mycobacterium avium complex. Twelve distinct ITS sequences were obtained, each of which defined a "sequevar"; a sequevar consists of the strain or strains which have a particular sequence. ITS sequences were identified which corresponded to M. avium (16 strains, four ITS sequevars) and Mycobacterium intracellulare (12 strains, one ITS sequevars). The other seven M. avium complex strains had ITS sequences which varied greatly from those of M. avium and M. intracellulare and from each other. The 16S-23S rDNA ITS was much more variable than 16S rDNA, which is widely used for genus and species identification. Phylogenetic trees based on the ITS were compatible with those based on 16S rDNA but were more detailed and had longer branches. The results of ITS sequencing were consistent with the results of hybridization with M. avium and M. intracellulare probes (Gen-Probe) for 30 of 31 strains tested. Serologic testing correlated poorly with ITS sequencing. Strains with the same sequence were different serovars, and those of the same serovar had different sequences. Sequencing of the 16S-23S rDNA ITS should be useful for species and strain differentiation for a wide variety of bacteria and should be applicable to studies of epidemiology, diagnosis, virulence, and taxonomy.


Selecting high priority quality measures for breast cancer quality improvement.
Although many quality measures have been created, there is no consensus regarding which are the most important. We sought to develop a simple, explicit strategy for prioritizing breast cancer quality measures based on their potential to highlight areas where quality improvement efforts could most impact a population. Using performance data for 9019 breast cancer patients treated at 10 National Comprehensive Cancer Network institutions, we assessed concordance relative to 30 reliable, valid breast cancer process-based treatment measures. We identified 4 attributes that indicated there was room for improvement and characterized the extent of burden imposed by failing to follow each measure: number of nonconcordant patients, concordance across all institutions, highest concordance at any 1 institution, and magnitude of benefit associated with concordant care. For each measure, we used data from the concordance analyses to derive the first 3 attributes and surveyed expert breast cancer physicians to estimate the fourth. A simple algorithm incorporated these attributes and produced a final score for each measure; these scores were used to rank the measures. We successfully prioritized quality measures using explicit, objective methods and actual performance data. The number of nonconcordant patients had the greatest influence on the rankings. The highest-ranking measures recommended chemotherapy and hormone therapy for hormone-receptor positive tumors and radiation therapy after breast-conserving surgery. This simple, explicit approach is a significant departure from methods used previously, and effectively identifies breast cancer quality measures that have broad clinical relevance. Systematically prioritizing quality measures could increase the efficiency and efficacy of quality improvement efforts and substantially improve outcomes.


Mammalian cell defence mechanisms against the cytotoxicity of NaYF4:(Er,Yb,Gd) nanoparticles.
Water-soluble upconversion nanoparticles (UCNPs), based on polyvinylpyrrolidone (PVP)-coated NaYF4:Er3+,Yb3+,Gd3+, with various concentrations of Gd3+ ions and relatively high upconversion efficiencies, were synthesized. The internalization and cytotoxicity of the thus obtained UCNPs were evaluated in three cell lines (HeLa, HEK293 and astrocytes). No cytotoxicity was observed even at concentrations of UCNPs up to 50 μg ml-1. The fate of the UCNPs within the cells was studied by examining their upconversion emission spectra with confocal microscopy and confirming these observations with transmission electron microscopy. It was found that the cellular uptake of the UCNPs occurred primarily by clathrin-mediated endocytosis, whereas they were secreted from the cells via lysosomal exocytosis. The results of this study, focused on the mechanisms of the cellular uptake, localization and secretion of UCNPs, demonstrate, for the first time, the co-localization of UCNPs within discrete cell organelles.


Effects of Panax ginseng, consumed with and without glucose, on blood glucose levels and cognitive performance during sustained 'mentally demanding' tasks.
Single doses of the traditional herbal treatment Panax ginseng have recently been shown to lower blood glucose levels and elicit cognitive improvements in healthy, overnight-fasted volunteers. The specific mechanisms responsible for these effects are not known. However, cognitive improvements may be related to the glycaemic properties of Panax ginseng. Using a double-blind, placebo-controlled, balanced-crossover design, 27 healthy young adults completed a 10 minute "cognitive demand" test battery at baseline. They then consumed capsules containing either ginseng (extract G115) or a placebo and 30 minutes later a drink containing glucose or placebo. A further 30 minutes later (i.e. 60 minutes post-baseline/capsules) they completed the "cognitive demand" battery six times in immediate succession. Depending on the condition to which the participant was allocated on that particular day, the combination of capsules/drink treatments corresponded to a dose of: 0mg G115/0 mg glucose (placebo); 200mg G115/0 mg glucose (ginseng); 0 mg G115/25 g glucose (glucose) or 200 mg G115/25 g glucose (ginseng/glucose combination). The 10 minute "cognitive demand" battery comprised a Serial Threes subtraction task (2 min); a Serial Sevens subtraction task (2 min); a Rapid Visual Information Processing task (5 min); and a "mental fatigue" visual analogue scale. Blood glucose levels were measured prior to the day's treatment, and before and after the post-dose completions of the battery. The results showed that both Panax ginseng and glucose enhanced performance of a mental arithmetic task and ameliorated the increase in subjective feelings of mental fatigue experienced by participants during the later stages of the sustained, cognitively demanding task performance. Accuracy of performing the Rapid Visual Information Processing task (RVIP) was also improved following the glucose load. There was no evidence of a synergistic relationship between Panax ginseng and exogenous glucose ingestion on any cognitive outcome measure. Panax ginseng caused a reduction in blood glucose levels 1 hour following consumption when ingested without glucose. These results confirm that Panax ginseng may possess glucoregulatory properties and can enhance cognitive performance.


Prospective study of the relationship between coffee and tea with colorectal cancer risk: the PLCO Cancer Screening Trial.
Coffee and tea are commonly consumed and carry potential anticancer components that could reduce the risk of colorectal cancer; however, their relationships with colorectal cancer risk remain inconsistent. A prospective analysis was carried out to examine the relationships of coffee and tea intake with colorectal cancer risk in 57,398 men and women in the intervention arm of the National Cancer Institute-Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial, a national screening study that limits differential detection biases. Coffee and tea intakes were assessed by food frequency questionnaire. Six hundred and eighty-one incident colorectal cancer cases were ascertained during a median follow-up of 11.4 years. Greater coffee intake was not associated with risk of colorectal cancer (relative risk (RR)=1.08, 95% confidence interval (CI)=0.79-1.48, Ptrend=0.23). Stratifying by cancer site (Pheterogeneity=0.48) or stage (Pheterogeneity=0.83) did not alter the relationship. Associations remained unchanged in subsets of participants for either caffeinated or decaffeinated coffee or when stratifying by several colorectal cancer risk factors. Similarly, greater tea intake was not associated with colorectal cancer risk overall (RR=0.77, 95% CI=0.55-1.09, Ptrend=0.17) or by cancer site (Pheterogeneity=0.14) or stage (Pheterogeneity=0.60). These associations were not modified by several colorectal cancer risk factors. The findings of this study do not provide evidence to suggest that drinking coffee or tea is beneficial in protecting against colorectal cancer.


Novel pink bollworm resistance to the Bt toxin Cry 1Ac: effects on mating, oviposition, larval development and survival.
Bt cotton plants are genetically engineered to produce insecticidal toxins from the Bacillus thuringiensis (Bt) Berliner (Bacillales: Bacillaceae) bacterium and target key lepidopteran pests. In all previous strains of pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) selected in the laboratory for resistance to insecticidal Cry1Ac toxin using an artificial diet containing the toxin, resistance to Cry1Ac and to Bt cotton is linked to three cadherin alleles (r1, r2, and r3). In contrast, the BG(4) pink bollworm strain was selected for resistance to Bt cotton by feeding larvae for four days in each of 42 generations on bolls of 'NuCOTN33B(R)' that expressed Cry1Ac toxin. After additional selection for eleven generations on Cry1Ac-incorporated diet, the susceptibility to Cry1Ac, fecundity, egg viability, and mating of this strain (Bt4R) was compared with the unselected Cry1Ac-susceptible parent strain. Some larvae of the Bt4R strain survived on diet containing >or= 10 microg Cry1Ac per milliliter artificial diet, but none survived on transgenic cotton bolls. In contrast to strains selected exclusively on Cry1Ac diet, some survival of progeny of reciprocal moth crosses of Bt4R resistant and Bt-susceptible strains occurred on Cry1Ac-treated diet, suggesting differences in levels of dominance. The Bt4R resistant strain does not have the r1, r2, or r3 mutant cadherin genes as do all previous strains of pink bollworm selected on Cry1Ac-treated artificial diet. The combined results suggest a mechanism of resistance to Cry1Ac that is different from previously described cadherin mutations.


Whole genome sequencing and rare variant analysis in essential tremor families.
Essential tremor (ET) is one of the most common movement disorders. The etiology of ET remains largely unexplained. Whole genome sequencing (WGS) is likely to be of value in understanding a large proportion of ET with Mendelian and complex disease inheritance patterns. In ET families with Mendelian inheritance patterns, WGS may lead to gene identification where WES analysis failed to identify the causative single nucleotide variant (SNV) or indel due to incomplete coverage of the entire coding region of the genome, in addition to accurate detection of larger structural variants (SVs) and copy number variants (CNVs). Alternatively, in ET families with complex disease inheritance patterns with gene x gene and gene x environment interactions enrichment of functional rare coding and non-coding variants may explain the heritability of ET. We performed WGS in eight ET families (n = 40 individuals) enrolled in the Family Study of Essential Tremor. The analysis included filtering WGS data based on allele frequency in population databases, rare SNV and indel classification and association testing using the Mixed-Model Kernel Based Adaptive Cluster (MM-KBAC) test. A separate analysis of rare SV and CNVs segregating within ET families was also performed. Prioritization of candidate genes identified within families was performed using phenolyzer. WGS analysis identified candidate genes for ET in 5/8 (62.5%) of the families analyzed. WES analysis in a subset of these families in our previously published study failed to identify candidate genes. In one family, we identified a deleterious and damaging variant (c.1367G>A, p.(Arg456Gln)) in the candidate gene, CACNA1G, which encodes the pore forming subunit of T-type Ca(2+) channels, CaV3.1, and is expressed in various motor pathways and has been previously implicated in neuronal autorhythmicity and ET. Other candidate genes identified include SLIT3 which encodes an axon guidance molecule and in three families, phenolyzer prioritized genes that are associated with hereditary neuropathies (family A, KARS, family B, KIF5A and family F, NTRK1). Functional studies of CACNA1G and SLIT3 suggest a role for these genes in ET disease pathogenesis.


Therapeutic drug monitoring in dried blood spots using liquid microjunction surface sampling and high resolution mass spectrometry.
Dried blood spots (DBS) are a versatile and stable tool for direct clinical blood analysis. Ambient high-resolution mass spectrometry is emerging as a method of choice for their quantitative analysis, for instance in therapeutic drug monitoring. Here, we coupled liquid microjunction surface sampling technology, a so-called Flowprobe, with an Orbitrap mass spectrometer and demonstrated the utility of this set-up for direct quantification of multiple drugs in DBS on filter paper. A three-layer set-up that we had introduced earlier enabled introduction of internal standards into DBS. We furthermore took an established point-of-care test system a step further and analyzed disposable test fields for blood glucose monitoring also for Flowprobe-based acetaminophen screening without additional sample preparation. Using as little as 2 μL blood, the method had an LOD of 1 μg mL(-1) (coefficient of variation of ≤15%) and acetaminophen recoveries of 82 to 119% for blinded samples, as assessed by LC-MS/MS. Half an hour after ingestions of a single 1000 mg acetaminophen dose, indistinguishable drug levels were measured in three healthy volunteers by LC-MS/MS and Flowprobe-Orbitrap MS analysis of DBS. Flowprobe analysis of DBS was 6- to 100-times more sensitive than corresponding desorption electrospray ionization MS analysis for four drugs. For instance, the LOD for salicylic acid analysis was 0.07 ng mL(-1) with Flowprobe measurement. Furthermore, we showed that multi-component analysis of five different substances, which may mimic polypharmacy in diabetes patients, in one blood sample for screening purposes was feasible. Taken together, our study suggests that microjunction surface sampling of DBS on filter paper and disposable point-of-care test fields may be developed into routine methods for near-patient multi-compound therapeutic drug monitoring that may advance blood screening analysis for patients with polypharmacy.


Effect of intensive versus standard lipid-lowering treatment with atorvastatin on the progression of calcified coronary atherosclerosis over 12 months: a multicenter, randomized, double-blind trial.
Recent clinical trials have suggested that intensive versus standard lipid-lowering therapy provides for additional benefit. Electron-beam computed tomography provides the opportunity to quantify the progression of coronary artery calcification (CAC) in serial measurements. In a multicenter, randomized, double-blind trial, 471 patients (age 61+/-8 years) who had no history of coronary artery disease and no evidence of high-grade coronary stenoses (>50% diameter reduction) were randomized if they had > or =2 cardiovascular risk factors and moderate calcified coronary atherosclerosis as evidenced by a CAC score > or =30. Patients were assigned to receive 80 mg or 10 mg of atorvastatin per day over 12 months. Progression of CAC volume scores could be analyzed in 366 patients. After pretreatment with 10 mg of atorvastatin for 4 weeks, 12 months of study medication reduced LDL cholesterol from 106+/-22 to 87+/-33 mg/dL in the group randomized to receive 80 mg of atorvastatin (P<0.001), whereas levels remained stable in the group randomized to receive 10 mg (108+/-23 at baseline, 109+/-28 mg/dL at the end of the study, P=NS). The mean progression of CAC volume scores, corrected for the baseline CAC volume score, was 27% (95% CI 20.8% to 33.1%) in the 80-mg atorvastatin group and 25% (95% CI 19.1% to 30.8%) in the 10-mg atorvastatin group (P=0.65). CAC progression showed no relationship with on-treatment LDL cholesterol levels. We did not observe a relationship between on-treatment LDL cholesterol levels and the progression of calcified coronary atherosclerosis. Over a period of 12 months, intensive atorvastatin therapy was unable to attenuate CAC progression compared with standard atorvastatin therapy. The possibility remains that the time window was too short to demonstrate an effect.


Seasonal variation in weight-for-age in a pediatric emergency room.
The authors assess seasonal variations in the prevalence of low weight-for-age among young children visiting the pediatric emergency room of a city hospital. We analyzed data on 11,118 children ages 6 to 24 months who visited the Boston City Hospital Pediatric Emergency Department between July 1989 and June 1992. Medical diagnoses were documented on a randomly selected subsample of 1,569 children. In addition, a questionnaire about food insecurity was administered to a convenience subsample of 269 families with children under 3 years of age. The percentage of children visiting the emergency room with weight-for-age below the fifth percentile was significantly higher for the three months following the coldest months than for the remaining months of the year, controlling for year of measurement. In the subsample, gastrointestinal illness was correlated with both season of measurement and weight-for-age, but the seasonal effect remained for the entire sample after controlling for dehydration. The questionnaire data suggested a relationship between economic stress and food insecurity that might help explain the seasonal effect. Families who were without heat or who were threatened with utility turnoff in the previous winter were twice as likely as other families to report that their children were hungry or at risk for hunger. Winter and early spring constituted periods of increased nutritional risk in the in this sample of predominantly low-income children, probably because of the increased caloric associated with cold stress and infections. Further research is needed to assess whether decreased caloric availability due to high heating costs, a "heat or eat" effect, also contributes to this phenomenon.


Effects of the tubulin-colchicine complex on microtubule dynamic instability.
The effects of the tubulin-colchicine complex (Tu-Col) on the dynamic behavior of microtubules have been examined under steady-state conditions in vitro. The addition of Tu-Col to tubulin microtubules at steady state results in only partial microtubule disassembly. Nevertheless, both the rate and the extent of tubulin exchange into microtubules are markedly suppressed by concentrations of Tu-Col which are low relative to the total amount of free tubulin. In addition, the time-dependent changes in microtubule length distribution, characteristic of dynamic instability, are suppressed by the addition of Tu-Col. Examination by video-enhanced dark-field microscopy of individual microtubules in the presence of Tu-Col shows that the principal effect of this complex is to reduce the growth rate at both ends of the microtubule. We have used computer simulation to rationalize the action of Tu-Col in terms of its effects on the experimentally observable parameters, namely, the rates of growth and shortening and the mean lifetimes of growth and shortening, which provide an empirical description of the dynamic behavior of microtubules. The results have been interpreted within the framework of the lateral cap formulation for microtubule dynamic instability [Martin, S. R., Schilstra, M. J., & Bayley, P. M. (1993) Biophys. J. 65, 578-596]. The simplest model mechanism requires only that Tu-Col binds to the microtubule end and inhibits further addition reactions in either the 5-start or the 8-start direction of the microtubule lattice. Monte Carlo simulations show that Tu-Col can, in this way, cause major suppression of the dynamic transitions of microtubules without inducing bulk microtubule disassembly. This type of mechanism could be important for the regulation of microtubule dynamics in vivo.


Accumulation of mutant lamin A causes progressive changes in nuclear architecture in Hutchinson-Gilford progeria syndrome.
Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder, commonly caused by a point mutation in the lamin A gene that results in a protein lacking 50 aa near the C terminus, denoted LADelta50. Here we show by light and electron microscopy that HGPS is associated with significant changes in nuclear shape, including lobulation of the nuclear envelope, thickening of the nuclear lamina, loss of peripheral heterochromatin, and clustering of nuclear pores. These structural defects worsen as HGPS cells age in culture, and their severity correlates with an apparent increase in LADelta50. Introduction of LADelta50 into normal cells by transfection or protein injection induces the same changes. We hypothesize that these alterations in nuclear structure are due to a concentration-dependent dominant-negative effect of LADelta50, leading to the disruption of lamin-related functions ranging from the maintenance of nuclear shape to regulation of gene expression and DNA replication.


Structure of the PH domain from Bruton's tyrosine kinase in complex with inositol 1,3,4,5-tetrakisphosphate.
The activity of Bruton's tyrosine kinase (Btk) is important for the maturation of B cells. A variety of point mutations in this enzyme result in a severe human immunodeficiency known as X-linked agammaglobulinemia (XLA). Btk contains a pleckstrin-homology (PH) domain that specifically binds phosphatidylinositol 3,4,5-trisphosphate and, hence, responds to signalling via phosphatidylinositol 3-kinase. Point mutations in the PH domain might abolish membrane binding, preventing signalling via Btk. We have determined the crystal structures of the wild-type PH domain and a gain-of-function mutant E41K in complex with D-myo-inositol 1,3,4,5-tetra-kisphosphate (Ins (1,3,4,5)P4). The inositol Ins (1,3,4,5)P4 binds to a site that is similar to the inositol 1,4,5-trisphosphate binding site in the PH domain of phospholipase C-delta. A second Ins (1,3,4,5)P4 molecule is associated with the domain of the E41K mutant, suggesting a mechanism for its constitutive interaction with membrane. The affinities of Ins (1,3,4,5)P4 to the wild type (Kd = 40 nM), and several XLA-causing mutants have been measured using isothermal titration calorimetry. Our data provide an explanation for the specificity and high affinity of the interaction with phosphatidylinositol 3,4,5-trisphosphate and lead to a classification of the XLA mutations that reside in the Btk PH domain. Mis-sense mutations that do not simply destabilize the PH fold either directly affect the interaction with the phosphates of the lipid head group or change electrostatic properties of the lipid-binding site. One point mutation (Q127H) cannot be explained by these facts, suggesting that the PH domain of Btk carries an additional function such as interaction with a Galpha protein.


Folding pathway mediated by an intramolecular chaperone. The inhibitory and chaperone functions of the subtilisin propeptide are not obligatorily linked.
The subtilisin propeptide functions as an intramolecular chaperone (IMC) that facilitates correct folding of the catalytic domain while acting like a competitive inhibitor of proteolytic activity. Upon completion of folding, subtilisin initiates IMC degradation to complete precursor maturation. Existing data suggest that the chaperone and inhibitory functions of the subtilisin IMC domain are interdependent during folding. Based on x-ray structure of the IMC-subtilisin complex, we introduce a point mutation (E112A) to disrupt three hydrogen bonds that stabilize the interface between the protease and its IMC domain. This mutation within subtilisin does not alter the folding kinetics but dramatically slows down autoprocessing of the IMC domain. Inhibition of E112A-subtilisin activity by the IMC added in trans is 35-fold weaker than wild-type subtilisin. Although the IMC domain displays substantial loss of inhibitory function, its ability to chaperone E112A-subtilisin folding remains intact. Our results show that (i) the chaperone activity of the IMC domain is not obligatorily linked with its ability to bind with and inhibit active subtilisin; (ii) degradation and not autoprocessing of the IMC domain is the rate-limiting step in precursor maturation; and (iii) the Glu(112) residue within the IMC-subtilisin interface is not crucial for initiating folding but is important in maintaining the IMC structure capable of binding subtilisin.


Incidence of Guillain-Barré syndrome in Chile: a population-based study.
The Guillain-Barré syndrome (GBS) incidence rate (IR) varies between 0.16 and 3.00 cases per 100,000 inhabitants. Little data exist on the epidemiology of GBS in Latin American countries. Our objective was to describe GBS epidemiology based on a national database in a Latin American country and to contribute to the global map of GBS epidemiology. This was a retrospective study that included all reported GBS cases in Chile between 2001 and 2012. Gender, age, seasonal occurrence, and geographical distribution were analyzed. A total of 4,158 GBS cases were identified from 19,513,655 registries. The mean age was 37 ± 24 years, and 59% of patients were male (male to female ratio of 1.5 : 1). Gender IR was 2.53/100,000 for males and 1.68/100,000 for females. The overall standardized IR was 2.1/100,000, although this varied between 1.61/100,000 (2001) and 2.35/100,000 (2010). The seasonal distribution was as follows: autumn 22%; winter 25%; spring 27%; and summer 26%. The geographical IR were as follows: far North 1.49/100,000; North 1.94/100,000; Central 1.97/100,000; South 3.18/100,000; and far South 2.78/100,000. The reported IR of GBS in Chile was similar to other studies based on national databases. In Chile, IR was greater in men and in the south.


Immunophenotypic analysis of peripheral T-cell neoplasms. A multiparameter flow cytometric approach.
We retrospectively reviewed multiparameter flow cytometric analyses in 50 peripheral T-cell neoplasms (PTCNs). Results were interpreted within the context of a large cohort of nonneoplastic T-cell populations. All PTCN diagnoses were confirmed with morphologic and/or molecular analysis. Aberrant populations were defined as discrete immunophenotypic clusters exhibiting loss of or increased or diminished expression of T-cell antigens relative to internal immunophenotypically normal T-cell populations. An antigenic pattern was considered abnormal if it exceeded ranges for T-cell subsets in specific anatomic sites or was not normally encountered. Forty-six of 50 and 41 of 50 demonstrated 1 or more and 2 or more aberrations, respectively. The most common abnormally expressed antigen was CD3, followed by CD7, CD5, and CD2. Except for CD7, abnormally dim or bright antigen expression was more common than deletion. Only 3 cases were abnormal solely based on expansion of an otherwise immunophenotypically normal population; the remainder had patterns of antigen expression not seen in nonneoplastic populations. These data indicate that most PTCNs are aberrant by multiparameter flow analysis. However, results must be interpreted within the context of thorough knowledge of the immunophenotypic spectrum of nonneoplastic T cells.


Effect of particulate aggregation in aquatic environments on the beam attenuation and its utility as a proxy for particulate mass.
Marine aggregates, agglomerations of particles and dissolved materials, are an important particulate pool in aquatic environments, but their optical properties are not well understood. To improve understanding of the optical properties of aggregates, two related studies are presented. In the first, an in situ manipulation experiment is described, in which beam attenuation of undisturbed and sheared suspensions are compared. Results show that in the sheared treatment bulk particle size decreases and beam attenuation increases, consistent with the hypothesis that a significant fraction of mass in suspension is contained in fragile aggregates. Interestingly, the magnitude of increase in beam attenuation is less than expected if the aggregates are modeled as solid spheres. Motivated by this result, a second study is presented, in which marine aggregates are modeled to assess how the beam attenuation of aggregates differs from that of their constituent particles and from solid particles of the same mass. The model used is based on that of Latimer [Appl. Opt. 24, 3231 (1985)] and mass specific attenuation is compared with that based on homogeneous and solid particles, the standard model for aquatic particles. In the modeling we use recent research relating size and solid fraction of aquatic aggregates. In contrast with Mie theory, this model provides a rather size-insensitive mass specific attenuation for most relevant sizes. This insensitivity is consistent with the observations that mass specific beam-attenuation of marine particles is in the range 0.2-0.6m(2)/gr despite large variability in size distribution and composition across varied aquatic environments.


Jugular venous reflux could influence cerebral blood flow: a transcranial Doppler study.
Studies have found significant associations between jugular venous reflux (JVR) and neurological disorders. However, there still lacks evidences that JVR could influence cerebral circulation. The aim of the present study is trying to provide evidences that the retrogradely-transmitted venous pressure of JVR could reach cerebral venous system and has an influence on cerebral blood flow (CBF). We recruited 50 volunteers. Only 42 subjects' data (55.81 ± 19.96, 23 - 85 years; 9 women) were analyzed due to poor temporal windows in eight subjects. JVR was determined by color-coded Duplex sonography. Transcranial Doppler study was used to examine the CBF changes during Valsalva maneuver (VM) in each subject. All JVRs were detected during VM. We divided subjects into people with right JVR (n=12), left JVR (n=13) and no JVR (n=21) and four had bilateral JVR. There was a more decrease in CBF during and immediately after VM in right-JVR group than no-JVR group, though the baseline characteristics and arterial blood pressure changes were similar. There were no demographic and hemodynamic differences between left-JVR group and no-JVR group. We are the first to provide evidences that right JVR during VM could influence CBF. However, whether left JVR with or without right JVR may have similar effect on CBF deserves further study. The definite mechanism underlying this finding needs further studies.


E-cigarettes and smoking cessation in real-world and clinical settings: a systematic review and meta-analysis.
Smokers increasingly use e-cigarettes for many reasons, including attempts to quit combustible cigarettes and to use nicotine where smoking is prohibited. We aimed to assess the association between e-cigarette use and cigarette smoking cessation among adult cigarette smokers, irrespective of their motivation for using e-cigarettes. PubMed and Web of Science were searched between April 27, 2015, and June 17, 2015. Data extracted included study location, design, population, definition and prevalence of e-cigarette use, comparison group (if applicable), cigarette consumption, level of nicotine dependence, other confounders, definition of quitting smoking, and odds of quitting smoking. The primary endpoint was cigarette smoking cessation. Odds of smoking cessation among smokers using e-cigarettes compared with smokers not using e-cigarettes were assessed using a random effects meta-analysis. A modification of the ACROBAT-NRSI tool and the Cochrane Risk of Bias Tool were used to assess bias. This meta-analysis is registered with PROSPERO (number CRD42015020382). 38 studies (of 577 studies identified) were included in the systematic review; all 20 studies with control groups (15 cohort studies, three cross-sectional studies, and two clinical trials) were included in random effects meta-analysis and sensitivity analyses. Odds of quitting cigarettes were 28% lower in those who used e-cigarettes compared with those who did not use e-cigarettes (odds ratio [OR] 0·72, 95% CI 0·57-0·91). Association of e-cigarette use with quitting did not significantly differ among studies of all smokers using e-cigarettes (irrespective of interest in quitting cigarettes) compared with studies of only smokers interested in cigarette cessation (OR 0·63, 95% CI 0·45-0·86 vs 0·86, 0·60-1·23; p=0·94). Other study characteristics (design, population, comparison group, control variables, time of exposure assessment, biochemical verification of abstinence, and definition of e-cigarette use) were also not associated with the overall effect size (p≥0·77 in all cases). As currently being used, e-cigarettes are associated with significantly less quitting among smokers. National Institutes of Health, National Cancer Institute, FDA Center for Tobacco Products.


Photolytic generation of benzhydryl cations and radicals from quaternary phosphonium salts: how highly reactive carbocations survive their first nanoseconds.
UV irradiation (266 or 280 nm) of benzhydryl triarylphosphonium salts Ar(2)CH-PAr(3)(+)X(-) yields benzhydryl cations Ar(2)CH(+) and/or benzhydryl radicals Ar(2)CH(•). The efficiency and mechanism of the photo-cleavage were studied by nanosecond laser flash photolysis and by ultrafast spectroscopy with a state-of-the-art femtosecond transient spectrometer. The influences of the photo-electrofuge (Ar(2)CH(+)), the photo-nucleofuge (PPh(3) or P(p-Cl-C(6)H(4))(3)), the counterion (X(-) = BF(4)(-), SbF(6)(-), Cl(-), or Br(-)), and the solvent (CH(2)Cl(2) or CH(3)CN) were investigated. Photogeneration of carbocations from Ar(2)CH-PAr(3)(+)BF(4)(-) or -SbF(6)(-) is considerably more efficient than from typical neutral precursors (e.g., benzhydryl chlorides or bromides). The photochemistry of phosphonium salts is controlled by the degree of ion pairing, which depends on the solvent and the concentration of the phosphonium salts. High yields of carbocations are obtained by photolyses of phosphonium salts with complex counterions (X(-) = BF(4)(-) or SbF(6)(-)), while photolyses of phosphonium halides Ar(2)CH-PPh(3)(+)X(-) (X(-) = Cl(-) or Br(-)) in CH(2)Cl(2) yield benzhydryl radicals Ar(2)CH(•) due to photo-electron transfer in the excited phosphonium halide ion pair. At low concentrations in CH(3)CN, the precursor salts are mostly unpaired, and the photo-cleavage mechanism is independent of the nature of the counter-anions. Dichloromethane is better suited for generating the more reactive benzhydryl cations than the more polar and more nucleophilic solvents CH(3)CN or CF(3)CH(2)OH. Efficient photo-generation of the most reactive benzhydryl cations (3,5-F(2)-C(6)H(3))(2)CH(+) and (4-(CF(3))-C(6)H(4))(2)CH(+) was only achieved using the photo-leaving group P(p-Cl-C(6)H(4))(3) and the counter-anion SbF(6)(-) in CH(2)Cl(2). The lifetimes of the photogenerated benzhydryl cations depend greatly on the decay mechanisms, which can be reactions with the solvent, with the photo-leaving group PAr(3), or with the counter-anion X(-) of the precursor salt. However, the nature of the photo-leaving group and the counterion of the precursor phosphonium salt do not affect the rates of the reactions of the obtained benzhydryl cations toward added nucleophiles. The method presented in this work allows us to generate a wide range of donor- and acceptor-substituted benzhydryl cations Ar(2)CH(+) for the purpose of studying their electrophilic reactivities.


[Evaluation of segmentary contractility in Chagas' disease by using the integral of the myocardial velocity gradient (myocardial strain) obtained through tissue Doppler echocardiography].
To quantify the percentage of contractility of different myocardial segments in patients with Chagas' disease by measuring myocardial strain and to assess the differences in the radial and longitudinal ventricular contractile function in the undetermined and dilated forms of chronic chagasic cardiomyopathy as compared with those in a group of healthy individuals. The study comprised 39 individuals [20 (51.3%) of the male sex] divided into the following 4 groups: 1) Nl: 17 (43.6%) healthy individuals; 2) Und: 7 (17.9%) patients with the undetermined form of Chagas' disease; 3) C1: 7 (17.9%) patients with the chronic form of Chagas' disease with ejection fraction < 50%; and 4) C2: 8 (20.5%) patients with the chronic form of Chagas' disease with ejection fraction > 50%. After performing baseline echocardiography, Doppler tissue images were recorded to measure myocardial strain in different segments on longitudinal and transversal parasternal, and apical 2- and 4-chamber views. The percentage of contractility in the different myocardial segments, both the radial and longitudinal components, is greater in healthy individuals than in patients with the chronic form of Chagas' disease, and in those with the undetermined form of the disease as compared with that of chronic chagasic patients with EF < 50%. Left ventricular radial contractility is greater than left ventricular longitudinal contractility in all groups (Nl, Und, and Chronic). The data presented allow us to propose a progressive character of myocardial impairment in patients with Chagas' disease.


Evolutionary dynamics of interlinked public goods traits: an experimental study of siderophore production in Pseudomonas aeruginosa.
Public goods cooperation is common in microbes, and there is much interest in understanding how such traits evolve. Research in recent years has identified several important factors that shape the evolutionary dynamics of such systems, yet few studies have investigated scenarios involving interactions between multiple public goods. Here, we offer general predictions about the evolutionary trajectories of two public goods traits having positive, negative or neutral regulatory influence on one another's expression, and we report on a test of some of our predictions in the context of Pseudomonas aeruginosa's production of two interlinked iron-scavenging siderophores. First, we confirmed that both pyoverdine and pyochelin siderophores do operate as public goods under appropriate environmental conditions. We then tracked their production in lines experimentally evolved under different iron-limitation regimes known to favour different siderophore expression profiles. Under strong iron limitation, where pyoverdine represses pyochelin, we saw a decline in pyoverdine and a concomitant increase in pyochelin - consistent with expansion of pyoverdine-defective cheats derepressed for pyochelin. Under moderate iron limitation, pyochelin declined - again consistent with an expected cheat invasion scenario - but there was no concomitant shift in pyoverdine because cross-suppression between the traits is unidirectional only. Alternating exposure to strong and moderate iron limitation caused qualitatively similar though lesser shifts compared to the constant-environment regimes. Our results confirm that the regulatory interconnections between public goods traits can significantly modulate the course of evolution, yet also suggest how we can start to predict the impacts such complexities will have on phenotypic divergence and community stability.


Viewing child pornography: prevalence and correlates in a representative community sample of young Swedish men.
Most research on child pornography use has been based on selected clinical or criminal justice samples; risk factors for child pornography use in the general population remain largely unexplored. In this study, we examined prevalence, risk factors, and correlates of viewing depictions of adult-child sex in a population-representative sample of 1,978 young Swedish men (17-20 years, Mdn = 18 years, overall response rate, 77 %). In an anonymous, school-based survey, participants self-reported sexual coercion experiences, attitudes and beliefs about sex, perceived peer attitudes, and sexual interests and behaviors; including pornography use, sexual interest in children, and sexually coercive behavior. A total of 84 (4.2 %) young men reported they had ever viewed child pornography. Most theory-based variables were moderately and significantly associated with child pornography viewing and were consistent with models of sexual offending implicating both antisociality and sexual deviance. In multivariate logistic regression analysis, 7 of 15 tested factors independently predicted child pornography viewing and explained 42 % of the variance: ever had sex with a male, likely to have sex with a child aged 12-14, likely to have sex with a child 12 or less, perception of children as seductive, having friends who have watched child pornography, frequent pornography use, and ever viewed violent pornography. From these, a 6-item Child Pornography Correlates Scale was constructed and then cross-validated in a similar but independent Norwegian sample.


Designing green oxidation catalysts for purifying environmental waters.
We describe the synthesis, characterization, aqueous behavior, and catalytic activity of a new generation of Fe(III)-TAML (tetraamido macrocycle ligand) activators of peroxides (2), variants of [Fe{(OC)(2)(o,o'-NC(6)H(4)NCO)(2)CMe(2)}(OH(2))(-)] (2d), which have been designed to be especially suitable for purifying water of recalcitrant oxidizable pollutants. Activation of H(2)O(2) by 2 (k(I)) as a function of pH was analyzed via kinetic studies of Orange II bleaching. This was compared with the known behavior of the first generation of Fe(III)-TAMLs (1). Novel reactivity features impact the potential for oxidant activation for water purification by 2d and its aromatic ring-substituted dinitro (2e) and tetrachloro (2f) derivatives. Thus, the maximum activity for 2e occurs at pH 9, the closest yet to the EPA guidelines for drinking water (6.5-8.5), allowing 2e to rapidly activate H(2)O(2) at pH 7.7. In water, 2e has two axial water ligands with pK(a)'s of 8.4 and 10.0 (25 degrees C). The former is the lowest for all Fe(III)-TAMLs developed to date and is key to 2e's exceptional catalytic activity in neutral and slightly basic solutions. Below pH 7, 2d was found to be quite sensitive to demetalation in phosphate buffers. This was overcome by iterative design to give 2e (hydrolysis rate 2d > 100 x 2e). Mechanistic studies highlight 2e's increased stability by establishing that to demetalate 2e at a comparable rate to which H(2)PO(4)(-) demetalates 2d, H(3)PO(4) is required. A critical criterion for green catalysts for water purification is the avoidance of endocrine disruptors, which can impair aquatic life. Fe(III)-TAMLs do not alter transcription mediated by mammalian thyroid, androgen, or estrogen hormone receptors, suggesting that 2 do not bind to the receptors and reducing concerns that the catalysts might have endocrine disrupting activity.


Does metabosensitive afferent fibers activity differ from slow- and fast-twitch muscles?
This study was designed to investigate the metabosensitive afferent response evoked by electrically induced fatigue (EIF), lactic acid (LA) and potassium chloride (KCl) in three muscle types. We recorded the activity of groups III-IV afferents originating from soleus, gastrocnemius and tibialis anterior muscles. Our data showed a same pattern of response in the three muscles after chemical injections, i.e., a bell curve with maximal discharge rate at 1 mM for LA injections and a linear relationship between KCl concentrations and the afferent discharge rate. Furthermore, a stronger response was recorded after EIF in the gastrocnemius muscle compared to the two other muscles. The change in afferent discharge after 1 mM LA injection was higher for the gastrocnemius muscle compared to the response obtained with the corresponding concentration applied in the two other muscles, whereas changes to KCl injections did not dramatically differ between the three muscles. We conclude that anatomical (mass, phenotype, vascularization, receptor and afferent density…) and functional (flexor vs. extensor) differences between muscles could explain the amplitude of these responses.


Androgen-primed chemotherapy-experimental confirmation of efficacy.
A current hypothesis suggests that androgen administration prior to chemotherapy (androgen priming) may potentiate tumor cytotoxicity in prostate cancer. The Dunning R3327G rat prostatic tumor model was used to test this concept experimentally. Control groups without priming included (1) intact untreated, (2) castrate alone and (3) castrate+ chemotherapy (cyclophosphamide, 30 mg/kg/day for 2 days with repeat cycle in 25 days- CTX). Two experimental groups received androgens, one before and one after chemotherapy. Treatment effect was monitored by quantitating tumor volume and animal survival. Control groups receiving castration and chemotherapy had a retardation of tumor growth and a prolongation of survival when compared to untreated animals. Androgen priming before but not after chemotherapy enhanced the degree of tumor suppression. With the androgen-priming protocol, all androgen-primed tumors had regressed, 3/6 tumors had disappeared and 3 were only palpable. At the same time point, tumors in all the other groups were actively growing and had volumes greater than the initial values (P less than 0.01). Median survival was significantly prolonged in primed animals 191 vs 40 days for untreated animals and 150 days for the nonprimed castration + chemotherapy animals (P less than 0.02). These findings have been repeated with several replicate experiments. These observations confirm the hypothesis that androgen priming can potentiate chemotherapy in an experimental system.


Effects of lipid composition and solution conditions on the mechanical properties of membrane vesicles.
The mechanical properties of cell-sized giant unilamellar liposomes were studied by manipulating polystyrene beads encapsulated within the liposomes using double-beam laser tweezers. Mechanical forces were applied to the liposomes from within by moving the beads away from each other, which caused the liposomes to elongate. Subsequently, a tubular membrane projection was generated in the tip at either end of the liposome, or the bead moved out from the laser trap. The force required for liposome transformation reached maximum strength just before formation of the projection or the moving out of the bead. By employing this manipulation system, we investigated the effects of membrane lipid compositions and environment solutions on the mechanical properties. With increasing content of acidic phospholipids, such as phosphatidylglycerol or phosphatidic acid, a larger strength of force was required for the liposome transformation. Liposomes prepared with a synthetic dimyristoylphosphatidylcholine, which has uniform hydrocarbon chains, were transformed easily compared with liposomes prepared using natural phosphatidylcholine. Surprisingly, bovine serum albumin or fetuin (soluble proteins that do not bind to membranes) decreased liposomal membrane rigidity, whereas the same concentration of sucrose showed no particular effect. These results show that the mechanical properties of liposomes depend on their lipid composition and environment.


Diffuse Lewy body disease manifesting as corticobasal syndrome: A rare form of Lewy body disease.
To describe clinical and pathologic characteristics of diffuse Lewy body disease (DLBD) manifesting as corticobasal syndrome (CBS). In 523 autopsy-confirmed cases of DLBD, we identified 11 patients diagnosed with CBS. For comparison, we studied 22 DLBD brains with antemortem presentation of dementia with Lewy bodies (DLB). Given previous studies suggesting the importance of pathology in peri-Rolandic cortices in CBS, we used digital pathology to count Lewy bodies and to quantify intracytoplasmic and neuritic α-synuclein and phospho-tau burden in the motor cortex. DLBD patients with antemortem features of CBS were significantly younger at disease onset and less likely to have REM sleep behavior disorder than DLBD cases who met clinical criteria for DLB during life. Patients with DLBD manifesting as CBS had more Lewy bodies in the motor cortex than DLBD manifesting as clinically probable DLB. Three cases had concomitant progressive supranuclear palsy and 4 cases had concomitant Alzheimer disease as probable correlates of CBS. The neuropathology underlying CBS is heterogeneous, including corticobasal degeneration, Alzheimer disease, and progressive supranuclear palsy. This study suggests that atypical variants of Lewy body disease with severe peri-Rolandic Lewy-related pathology can present clinically as CBS. Patients with DLBD who present as CBS tend to have an earlier age at onset and are less likely to have clinical features of DLB, such as dream enactment behavior during sleep, visual hallucinations, and levodopa-responsive parkinsonism. Future studies with biofluid or molecular imaging biomarkers for α-synuclein will permit better recognition of this uncommon pathologic substrate of CBS.


Mass spectrometry in the characterization of cereal seed proteins.
In less then a decade, applications of matrix-assisted laser desorption/ionisation (MALDI) and electrospray ionisation (ESI) mass spectrometry to the investigation of prolamins have rapidly evolved from measurements of the molecular mass of isolated proteins to a proteomic approach attempting to characterise the complete protein pattern in the seed. Mass spectrometry is currently making significant contributions to the understanding of the composition and structure of the gluten proteins and, in turn, to the elucidation of structure-function relationships. Results obtained using mass spectrometry, including determination of the molecular masses of prolamins, direct verification of gene-derived sequences, determination of the number of cysteine residues and localisation of disulphide bonds, investigation of the gluten toxicity for celiac patients, qualitative and quantitative determination of gliadins in food and determination of the protein pattern and its modification during seed maturation by proteomic approaches, are summarised here, to illustrate current trends and individuate possible future perspectives.


Transformation of chicken cells by the gene encoding the catalytic subunit of PI 3-kinase.
The avian sarcoma virus 16 (ASV 16) is a retrovirus that induces hemangiosarcomas in chickens. Analysis of the ASV 16 genome revealed that it encodes an oncogene that is derived from the cellular gene for the catalytic subunit of phosphoinositide 3-kinase (PI 3-kinase). The gene is referred to as v-p3k, and like its cellular counterpart c-p3k, it is a potent transforming gene in cultured chicken embryo fibroblasts (CEFs). The products of the viral and cellular p3k genes have PI 3-kinase activity. CEFs transformed with either gene showed elevated levels of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate and activation of Akt kinase.


Short faces, big tongues: developmental origin of the human chin.
During the course of human evolution, the retraction of the face underneath the braincase, and closer to the cervical column, has reduced the horizontal dimension of the vocal tract. By contrast, the relative size of the tongue has not been reduced, implying a rearrangement of the space at the back of the vocal tract to allow breathing and swallowing. This may have left a morphological signature such as a chin (mental prominence) that can potentially be interpreted in Homo. Long considered an autopomorphic trait of Homo sapiens, various extinct hominins show different forms of mental prominence. These features may be the evolutionary by-product of equivalent developmental constraints correlated with an enlarged tongue. In order to investigate developmental mechanisms related to this hypothesis, we compare modern 34 human infants against 8 chimpanzee fetuses, whom development of the mandibular symphysis passes through similar stages. The study sets out to test that the shared ontogenetic shape changes of the symphysis observed in both species are driven by the same factor--space restriction at the back of the vocal tract and the associated arrangement of the tongue and hyoid bone. We apply geometric morphometric methods to extensive three-dimensional anatomical landmarks and semilandmarks configuration, capturing the geometry of the cervico-craniofacial complex including the hyoid bone, tongue muscle and the mandible. We demonstrate that in both species, the forward displacement of the mental region derives from the arrangement of the tongue and hyoid bone, in order to cope with the relative horizontal narrowing of the oral cavity. Because humans and chimpanzees share this pattern of developmental integration, the different forms of mental prominence seen in some extinct hominids likely originate from equivalent ontogenetic constraints. Variations in this process could account for similar morphologies.


Acute inflammation initiates the regenerative response in the adult zebrafish brain.
The zebrafish regenerates its brain after injury and hence is a useful model organism to study the mechanisms enabling regenerative neurogenesis, which is poorly manifested in mammals. Yet the signaling mechanisms initiating such a regenerative response in fish are unknown. Using cerebroventricular microinjection of immunogenic particles and immunosuppression assays, we showed that inflammation is required and sufficient for enhancing the proliferation of neural progenitors and subsequent neurogenesis by activating injury-induced molecular programs that can be observed after traumatic brain injury. We also identified cysteinyl leukotriene signaling as an essential component of inflammation in the regenerative process of the adult zebrafish brain. Thus, our results demonstrate that in zebrafish, in contrast to mammals, inflammation is a positive regulator of neuronal regeneration in the central nervous system.


Internet health information seeking behavior and antiretroviral adherence in persons living with HIV/AIDS.
Abstract While the Internet has the potential to educate persons living with HIV/AIDS (PLWHA), websites may contain inaccurate information and increase the risk of nonadherence with antiretroviral therapy (ART). The objectives of our study were to determine the extent to which PLWHA engage in Internet health information seeking behavior (IHISB) and to determine whether IHISB is associated with ART adherence. We conducted a survey of adult, English-speaking HIV-infected patients at four HIV outpatient clinic sites in the United States (Baltimore, Maryland; Detroit, Michigan; New York, and Portland, Oregon) between December 2004 and January 2006. We assessed IHISB by asking participants how much information they had received from the Internet since acquiring HIV. The main outcome was patient-reported ART adherence over the past three days. Data were available on IHISB for 433 patients, 334 of whom were on ART therapy. Patients had a mean age of 45 (standard error [SE] 0.45) years and were mostly male (66%), African American (58%), and had attained a high school degree (73%). Most (55%) reported no IHISB, 18% reported some, and 27% reported "a fair amount" or "a great deal." Patients who reported higher versus lower levels of IHISB were significantly younger, had achieved a higher level of education, and had higher medication self-efficacy. In unadjusted analyses, higher IHISB was associated with ART adherence (odds ratio [OR], 2.96, 95% confidence interval [CI] 1.27-6.94). This association persisted after adjustment for age, gender, race, education, clinic site, and medication self-efficacy (adjusted odds ratio [AOR] 2.76, 95% CI 1.11-6.87). Our findings indicate that IHISB is positively associated with ART adherence even after controlling for potentially confounding variables. Future studies should investigate the ways in which Internet health information may promote medication adherence among PLWHA.


Exposure to Mono-n-Butyl Phthalate in Women with Endometriosis and Its Association with the Biological Effects on Human Granulosa Cells.
To study the association between urinary phthalate metabolite levels, endometriosis, and their effects on human granulosa cells, we recruited patients who underwent laparoscopy to confirm endometriosis (n = 123) and control patients (n = 78). Liquid chromatography-tandem mass spectrometry was used to measure the following five urinary phthalate metabolites: mono-n-butyl phthalate (MnBP), mono(2-ethylhexyl) phthalate, monobenzyl phthalate, mono(2-ethyl-5-oxo-hexyl) phthalate, and mono(2-ethyl-5-hydroxyhexyl) phthalate. Urinary MnBP levels were higher in patients with endometriosis than in controls after multivariable logistic regression including the number of deliveries, body mass index, and use of medicine as covariables. MnBP correlates with other phthalate metabolites. Previous studies found that endometriosis was a detrimental condition for granulosa cells. In our study, we observed whether MnBP affected granulosa cells. MnBP treatment altered the gene expression of BIRC5, BUB1B, CDC20, cyclin B1, IL-1β, TNF-α, inhibin-B, StAR, and P450ssc and attenuated the ratio of the mitochondrial membrane potential in human granulosa cells. Moreover, MnBP decreased the expression of the anti-Mullerian hormone. These findings suggest that MnBP concentration is associated with endometriosis and may affect the health and steroidogenesis of human granulosa cells.


Plasma proteins as biomarkers of the aging process.
This study was designed to characterize the rat serum proteins as biomarkers of the normal aging process. Crossed immunoelectrophoresis or electroimmunodiffusion quantitation of proteins was performed in rats aged 6, 12, 24, and 30 mo. Selection of healthy animals was based on confrontation of crossed immunoelectrophoresis patterns with those of experimentally inflamed young adults and with individual anatomopathological data. Convergence of inflammatory patterns and severe histological lesions was the exclusion criterion. Senescence-induced decrease was demonstrated for eight proteins [negative senescence reactants (SRs-)] and increase for six proteins [positive SRs (SRs+)]. Most SRs belonged to the class of proteins responsive to acute inflammation [acute phase reactants (APRs)]. One SR+, the thyroxine-binding globulin, a high-affinity thyroid hormone binder, emerged as a particularly reliable senescence biomarker, showing the highest aging-related variation (8-fold increase from 6 to 30 mo) and not belonging to the APR class. Chronic treatment with perindopril, an angiotensin I-converting enzyme inhibitor used in heart and renal disease therapy, significantly enhanced thyroxine-binding capacity, possibly by preventing age-related alterations of serum lipids. Serum protein patterns prove valuable both as indexes for selecting aging animals free from superimposed pathologies and as parameters of senescence-induced changes in protein biosynthesis.


Spectral signatures of hydrated proton vibrations in water clusters.
The ease with which the pH of water is measured obscures the fact that there is presently no clear molecular description for the hydrated proton. The mid-infrared spectrum of bulk aqueous acid, for example, is too diffuse to establish the roles of the putative Eigen (H3O+) and Zundel (H5O2+) ion cores. To expose the local environment of the excess charge, we report how the vibrational spectrum of protonated water clusters evolves in the size range from 2 to 11 water molecules. Signature bands indicating embedded Eigen or Zundel limiting forms are observed in all of the spectra with the exception of the three- and five-membered clusters. These unique species display bands appearing at intermediate energies, reflecting asymmetric solvation of the core ion. Taken together, the data reveal the pronounced spectral impact of subtle changes in the hydration environment.


SUMO modification of Huntingtin and Huntington's disease pathology.
Huntington's disease (HD) is characterized by the accumulation of a pathogenic protein, Huntingtin (Htt), that contains an abnormal polyglutamine expansion. Here, we report that a pathogenic fragment of Htt (Httex1p) can be modified either by small ubiquitin-like modifier (SUMO)-1 or by ubiquitin on identical lysine residues. In cultured cells, SUMOylation stabilizes Httex1p, reduces its ability to form aggregates, and promotes its capacity to repress transcription. In a Drosophila model of HD, SUMOylation of Httex1p exacerbates neurodegeneration, whereas ubiquitination of Httex1p abrogates neurodegeneration. Lysine mutations that prevent both SUMOylation and ubiquitination of Httex1p reduce HD pathology, indicating that the contribution of SUMOylation to HD pathology extends beyond preventing Htt ubiquitination and degradation.


X-ray and Cryo-electron Microscopy Structures of Monalysin Pore-forming Toxin Reveal Multimerization of the Pro-form.
β-Barrel pore-forming toxins (β-PFT), a large family of bacterial toxins, are generally secreted as water-soluble monomers and can form oligomeric pores in membranes following proteolytic cleavage and interaction with cell surface receptors. Monalysin has been recently identified as a β-PFT that contributes to the virulence of Pseudomonas entomophila against Drosophila. It is secreted as a pro-protein that becomes active upon cleavage. Here we report the crystal and cryo-electron microscopy structure of the pro-form of Monalysin as well as the crystal structures of the cleaved form and of an inactive mutant lacking the membrane-spanning region. The overall structure of Monalysin displays an elongated shape, which resembles those of β-pore-forming toxins, such as Aerolysin, but is devoid of a receptor-binding domain. X-ray crystallography, cryo-electron microscopy, and light-scattering studies show that pro-Monalysin forms a stable doughnut-like 18-mer complex composed of two disk-shaped nonamers held together by N-terminal swapping of the pro-peptides. This observation is in contrast with the monomeric pro-form of the other β-PFTs that are receptor-dependent for membrane interaction. The membrane-spanning region of pro-Monalysin is fully buried in the center of the doughnut, suggesting that upon cleavage of pro-peptides, the two disk-shaped nonamers can, and have to, dissociate to leave the transmembrane segments free to deploy and lead to pore formation. In contrast with other toxins, the delivery of 18 subunits at once, nearby the cell surface, may be used to bypass the requirement of receptor-dependent concentration to reach the threshold for oligomerization into the pore-forming complex.


Cells on hierarchically-structured platforms hosting functionalized nanoparticles.
In this work, we report on a novel approach to develop hierarchically-structured cell culture platforms incorporating functionalized gold nanoparticles (AuNPs). In particular, the hierarchical substrates comprise primary pseudo-periodic arrays of silicon microcones combined with a secondary nanoscale pattern of homogeneously deposited AuNPs terminated with bio-functional moieties. AuNPs with various functionalities (i.e. oligopeptides, small molecules and oligomers) were successfully attached onto the microstructures. Experiments with PC12 cells on hierarchical substrates incorporating AuNPs carrying the RGD peptide showed an impressive growth and NGF-induced differentiation of the PC12 cells, compared to that on the NP-free, bare, micropatterned substrates. The exploitation of the developed methodology for the binding of AuNPs as carriers of specific bio-functional moieties onto micropatterned culture substrates for cell biology studies is envisaged.


The late effects of mantle field radiotherapy: the information and support needs of women survivors of Hodgkin's disease.
To improve understanding of the information and support needs of women at risk from breast cancer after earlier treatment with mantle field radio therapy for Hodgkin's disease. A multiple case study approach presenting the detailed accounts of three participants has been used to represent the issues raised by a wider sub-set of 15 Hodgkin's disease survivors. These women were participants in a larger qualitative study of 50 long term survivors of adolescent cancer. They were interviewed in depth either face to face or via the telephone. Data were analysed using methods of data reduction, display and conclusion drawing, three case studies were then purposively selected to illustrate key themes in the wider data set. Discovering the risk of late effect breast cancer, particularly via the media, caused shock. Reliable information was difficult to locate and the support needs of the women were not met by medical services. The impact of the new risk was exacerbated by the earlier cancer experience. Information on the risk of late effects to cancer survivors regarded as trustworthy is not easy to access. When the risk of a treatment related malignancy is identified there must be a reliable way of informing both those at risk and primary health care physicians who then need to take into account the cancer history of the patient and its ongoing effect on their emotional vulnerability.


2011 update on pancreas transplantation: comprehensive trend analysis of 25,000 cases followed up over the course of twenty-four years at the International Pancreas Transplant Registry (IPTR).
This study aimed to analyze the outcome of pancreas and pancreas-kidney transplantations based on the comprehensive follow-up data reported to the International Pancreas Transplant Registry (IPTR). As of December 2010, more than 35,000 pancreas transplantations have been reported to the IPTR: more than 24,000 transplantations in the US and more than 12,000 outside the US. Cases with follow-up information until March 2011 were included in the analysis. Pancreas transplantations in diabetic patients were divided into 3 categories: those performed simultaneously with a kidney (SPK) (75%), those given after a previous kidney transplantation (PAK) (18%), and pancreas transplantation alone (PTA) (7%). The total number of pancreas transplantations steadily increased until 2004 but has since declined. The largest decrease was seen in PAK, which decreased by 50% from 2004 through 2010. Comparatively, the number of SPK decreased by 7% during this time. Era analysis of US transplantations between 1987 and 2010 showed changes in recipient and donor characteristics. Recipient age at transplantation increased significantly as well as transplantations in type 2 diabetes patients. The trend over time was towards tighter donor criteria. There was a concentration on younger donors, preferable trauma victims, with short preservation time. Surgical techniques for the drainage of the pancreatic duct changed over time, too. Now enteric drainage is the predominantly used technique in combination with systemic drainage of the venous effluent of the pancreas graft. Immunosuppressive protocols developed towards antibody induction therapy with tacrolimus and MMF as maintenance therapy. The rate of transplantations with steroid avoidance increased over time in all 3 categories. These changes have led to improved patient and graft survival. Patient survival now reaches over 95% at one year post-transplant and over 83% after 5 years. The best graft survival was found in SPK with 86% pancreas and 93% kidney graft function at one year. PAK pancreas graft function reached 80%, and PTA pancreas graft function reached 78% at one year. In all 3 categories, early technical graft loss rates decreased significantly to 8-9%. Likewise, the 1-year immunological graft loss rate also decreased: in SPK, the immunological 1-year graft loss rate was 1.8%, in PAK 3.7%, and in PTA 6.0%. Patient survival and graft function improved significantly over the course of 24 years of pancreas transplantation in all 3 categories. With further reduction in surgical complications and improvements in immunosuppressive protocols, pancreas transplantation offers excellent outcomes for patients with labile diabetes.


Symmetrical arrangement of positively charged residues around the 5-fold axes of SAT type foot-and-mouth disease virus enhances cell culture of field viruses.
Field isolates of foot-and-mouth disease viruses (FMDVs) utilize integrin-mediated cell entry but many, including Southern African Territories (SAT) viruses, are difficult to adapt to BHK-21 cells, thus hampering large-scale propagation of vaccine antigen. However, FMDVs acquire the ability to bind to cell surface heparan sulphate proteoglycans, following serial cytolytic infections in cell culture, likely by the selection of rapidly replicating FMDV variants. In this study, fourteen SAT1 and SAT2 viruses, serially passaged in BHK-21 cells, were virulent in CHO-K1 cells and displayed enhanced affinity for heparan, as opposed to their low-passage counterparts. Comparative sequence analysis revealed the fixation of positively charged residues clustered close to the icosahedral 5-fold axes of the virus, at amino acid positions 83-85 in the βD-βE loop and 110-112 in the βF-βG loop of VP1 upon adaptation to cultured cells. Molecular docking simulations confirmed enhanced binding of heparan sulphate to a model of the adapted SAT1 virus, with the region around VP1 arginine 112 contributing the most to binding. Using this information, eight chimeric field strain mutant viruses were constructed with additional positive charges in repeated clusters on the virion surface. Five of these bound heparan sulphate with expanded cell tropism, which should facilitate large-scale propagation. However, only positively charged residues at position 110-112 of VP1 enhanced infectivity of BHK-21 cells. The symmetrical arrangement of even a single amino acid residue in the FMD virion is a powerful strategy enabling the virus to generate novel receptor binding and alternative host-cell interactions.