A general system for automatic biomedical image segmentation using intensity neighborhoods.
Image segmentation is important with applications to several problems in biology and medicine. While extensively researched, generally, current segmentation methods perform adequately in the applications for which they were designed, but often require extensive modifications or calibrations before being used in a different application. We describe an approach that, with few modifications, can be used in a variety of image segmentation problems. The approach is based on a supervised learning strategy that utilizes intensity neighborhoods to assign each pixel in a test image its correct class based on training data. We describe methods for modeling rotations and variations in scales as well as a subset selection for training the classifiers. We show that the performance of our approach in tissue segmentation tasks in magnetic resonance and histopathology microscopy images, as well as nuclei segmentation from fluorescence microscopy images, is similar to or better than several algorithms specifically designed for each of these applications.


Stem cell-based interventions for the prevention and treatment of germinal matrix-intraventricular haemorrhage in preterm infants.
Germinal matrix-intraventricular haemorrhage (GMH-IVH) remains a substantial issue in neonatal intensive care units worldwide. Current therapies to prevent or treat GMH-IVH are limited. Stem cell-based therapies offer a potential therapeutic approach to repair, restore, and/or regenerate injured brain tissue. These preclinical findings have now culminated in ongoing human neonatal studies. To determine the benefits and harms of stem cell-based interventions for prevention or treatment of germinal matrix-intraventricular haemorrhage (GM-IVH) in preterm infants. We used the standard search strategy of Cochrane Neonatal to search the Cochrane Central Register of Controlled Trials (CENTRAL; 2019, Issue 1), in the Cochrane Library; MEDLINE via PubMed (1966 to 7 January 2019); Embase (1980 to 7 January 2019); and the Cumulative Index to Nursing and Allied Health Literature (CINAHL) (1982 to 7 January 2019). We also searched clinical trials databases, conference proceedings, and reference lists of retrieved articles for randomised controlled trials and quasi-randomised trials. We attempted to identify randomised controlled trials, quasi-randomised controlled trials, and cluster trials comparing (1) stem cell-based interventions versus control; (2) mesenchymal stromal cells (MSCs) of type or source versus MSCs of other type or source; (3) stem cell-based interventions other than MSCs of type or source versus stem cell-based interventions other than MSCs of other type or source; or (4) MSCs versus stem cell-based interventions other than MSCs. For prevention studies, we included extremely preterm infants (less than 28 weeks' gestation), 24 hours of age or less, without ultrasound diagnosis of GM-IVH; for treatment studies, we included preterm infants (less than 37 weeks' gestation), of any postnatal age, with ultrasound diagnosis of GM-IVH. For each of the included trials, two review authors independently planned to extract data (e.g. number of participants, birth weight, gestational age, type and source of MSCs, other stem cell-based interventions) and assess the risk of bias (e.g. adequacy of randomisation, blinding, completeness of follow-up). Primary outcomes considered in this review are all-cause neonatal mortality, major neurodevelopmental disability, GM-IVH, and extension of pre-existing non-severe GM-IVH. We planned to use the GRADE approach to assess the quality of evidence. Our search strategy yielded 769 references. We did not find any completed studies for inclusion. One randomised controlled trial is currently registered and ongoing. Five phase 1 trials are described in the excluded studies. Currently no evidence is available to show the benefits or harms of stem cell-based interventions for treatment or prevention of GM-IVH in preterm infants.


Autologous blood cell transplantation versus HLA-identical sibling transplantation for acute myeloid leukemia in first complete remission: a registry study from the Center for International Blood and Marrow Transplantation Research.
The optimal post-remission treatment for acute myeloid leukemia in first complete remission remains uncertain. Previous comparisons of autologous versus allogeneic hematopoietic cell transplantation noted higher relapse, but lower treatment-related mortality though using bone marrow grafts, with treatment-related mortality of 12-20%. Recognizing lower treatment-related mortality using autologous peripheral blood grafts, in an analysis of registry data from the Center for International Blood and Transplant Research, we compared treatment-related mortality, relapse, leukemia-free survival, and overall survival for patients with acute myeloid leukemia in first complete remission (median ages 36-44, range 19-60) receiving myeloablative HLA-matched sibling donor grafts (bone marrow, n=475 or peripheral blood, n=428) versus autologous peripheral blood (n=230). The 5-year cumulative incidence of treatment-related mortality was 19% (95% confidence interval, 16-23%), 20% (17-24%) and 8% (5-12%) for allogeneic bone marrow, allogeneic peripheral blood and autologous peripheral blood stem cell transplant recipients, respectively. The corresponding figures for 5-year cumulative incidence of relapse were 20% (17-24%), 26% (21-30%) and 45% (38-52%), respectively. At 5 years, leukemia-free survival and overall survival rates were similar: allogeneic bone marrow 61% (56-65%) and 64% (59-68%); allogeneic peripheral blood 54% (49-59%) and 59% (54-64%); autologous peripheral blood 47% (40-54%) and 54% (47-60%); P=0.13 and P=0.19, respectively. In multivariate analysis the incidence of treatment-related mortality was lower after autologous peripheral blood transplantation than after allogeneic bone marrow/peripheral blood transplants [relative risk 0.37 (0.20-0.69); P=0.001], but treatment failure (death or relapse) after autologous peripheral blood was significantly more likely [relative risk 1.32 (1.06-1.64); P=0.011]. The 5-year overall survival, however, was similar in patients who received autologous peripheral blood (n=230) [relative risk 1.23 (0.98-1.55); P=0.071] or allogeneic bone marrow/peripheral blood (n=903). In the absence of an HLA-matched sibling donor, autologous peripheral blood may provide acceptable alternative post-remission therapy for patients with acute myeloid leukemia in first complete remission.


Targeting Channels and Transporters in Protozoan Parasite Infections.
Infectious diseases caused by pathogenic protozoa are among the most significant causes of death in humans. Therapeutic options are scarce and massively challenged by the emergence of resistant parasite strains. Many of the current anti-parasite drugs target soluble enzymes, generate unspecific oxidative stress, or act by an unresolved mechanism within the parasite. In recent years, collections of drug-like compounds derived from large-scale phenotypic screenings, such as the malaria or pathogen box, have been made available to researchers free of charge boosting the identification of novel promising targets. Remarkably, several of the compound hits have been found to inhibit membrane proteins at the periphery of the parasites, i.e., channels and transporters for ions and metabolites. In this review, we will focus on the progress made on targeting channels and transporters at different levels and the potential for use against infections with apicomplexan parasites mainly Plasmodium spp. (malaria) and Toxoplasma gondii (toxoplasmosis), with kinetoplastids Trypanosoma brucei (sleeping sickness), Trypanosoma cruzi (Chagas disease), and Leishmania ssp. (leishmaniasis), and the amoeba Entamoeba histolytica (amoebiasis).


Effect of Prunus Mahaleb L. Seed Extract on Ethylene glycol- and Ammonium Chloride-Induced Urolithiasis in BALB/c Mice.
Kidney stone disease can be quite painful, recurrent, and affects many people. Despite advances in drug therapy, there is still a need to find effective drugs with fewer complications for long-term treatment of kidney stones and to prevent its recurrence. The present study aimed to evaluate the effect of Prunus Mahaleb L. seed extract on ethylene glycol- and ammonium chloride-induced urolithiasis in BALB/c mice. The Prunus Mahaleb L. seeds were collected in Mashhad (Iran) in June 2017. Urolithiasis was induced in male BALB/c mice by adding ethylene glycol (EG) 0.75% (v/v) and ammonium chloride (AC) 2% (w/v) to their drinking water for 21 consecutive days. A total of 72 animals were randomly divided into six groups of twelve animals each. Group 1 received purified water as control; group 2 received EG+AC in drinking water; groups 3-5 received the extracts by gavage in dosages of 100, 300, 500 mg/kg body weight, respectively; and group 6 received 888 mg/kg Sankol by gavage. Note that urolithiasis was induced in groups 3-6 in the same manner as in group 2. The data were analyzed using GraphPad Prism Software (version 5.01). The group receiving Prunus Mahaleb L. extract in a 500 mg/kg dose responded better to the treatment and less damage to the kidney tissue was observed. The serum parameters remarkably decreased in the calculi-induced animals. Besides, the acute toxicity test showed that the use of the extract was safe in animals. The results showed that the use of Prunus Mahaleb L. extract effectively prevented the formation of kidney stones.


Carbon nanotube scaffolds instruct human dendritic cells: modulating immune responses by contacts at the nanoscale.
Nanomaterials interact with cells and modify their function and biology. Manufacturing this ability can provide tissue-engineering scaffolds with nanostructures able to influence tissue growth and performance. Carbon nanotube compatibility with biomolecules motivated ongoing interest in the development of biosensors and devices including such materials. More recently, carbon nanotubes have been applied in several areas of nerve tissue engineering to study cell behavior or to instruct the growth and organization of neural networks. To gather further knowledge on the true potential of future constructs, in particular to assess their immune-modulatory action, we evaluate carbon nanotubes interactions with human dendritic cells (DCs). DCs are professional antigen-presenting cells and their behavior can predict immune responses triggered by adhesion-dependent signaling. Here, we incorporate DC cultures to carbon nanotubes and we show by phenotype, microscopy, and transcriptional analysis that in vitro differentiated and activated DCs show when interfaced to carbon nanotubes a lower immunogenic profile.


The effects of functional fiber on postprandial glycemia, energy intake, satiety, palatability and gastrointestinal wellbeing: a randomized crossover trial.
Fiber intakes in developed countries are generally below those recommended by relevant authorities. Given that many people consume fiber-depleted refined-grain products, adding functional fiber will help to increase fiber intakes. The objective of the study was to determine metabolic and sensory effects of adding fiber to bread. A double-blind pair of randomized crossover trials with a two-week washout in which two fiber-containing breads were compared with control bread. The functional fiber (fruit fiber and FibreMax) was added to yield 10 g fiber per serve (two slices). Eighty participants (n = 37 fruit fiber and n = 43 FibreMax) consumed one serve of bread (fiber or control) followed three hours later by a pasta meal consumed ad libitum. Outcome measures included glycemia, satiety, palatability, gastrointestinal wellbeing, visual appeal and subsequent energy intake of the pasta meal. Multivariate regression was undertaken to test for differences between treatment and control for blood glucose, satiety, and cumulative energy intake. Satiety responses were also compared by splitting the data into an immediate response after eating (0-30 min) and a return to hunger analysis (30-180 min). A Wilcoxon sign rank test was used for the first component (0-30 min) and Wilcoxon matched-pairs signed-rank test for the second component (30-180 min). Between treatment differences for gastrointestinal wellbeing were tested using Pearson's chi-square test or Fisher's exact test. Consumption of the fruit fiber bread reduced postprandial glycemia by 35% (95% CI 13 to 51; P = 0.004) and cumulative energy intake by 368 kJ (95% CI 163 to 531; P = 0.001). There was little influence on satiety and the bread was rated as having poor taste and smell whilst generating feelings of nausea in some participants. FibreMax enriched bread reduced glycemia by 43% (95% CI 17 to 61; P = 0.004) without influence on energy intake or satiety. Apart from a lower visual appeal, the FibreMax bread was palatable. Neither bread caused gastrointestinal discomfort related to flatulence or bloating. Enriching bread with 10 g of functional fiber per serve is feasible although reformulation is needed to create not only an acceptable bread, but a desirable product.


Separate upstream and convergent downstream pathways of G-protein- and phorbol ester-mediated Ca2+ sensitization of myosin light chain phosphorylation in smooth muscle.
The effect of phorbol ester-induced down-regulation of protein kinase C (PKC) on diacylglycerol (sn-1,2-dioctanoylglycerol, diC8)- and G-protein-coupled Ca2+ sensitization and on the relationship between phosphorylation of the regulatory myosin light chains (MLC20) and force during Ca2+ sensitization were investigated in rabbit portal vein (PV), femoral artery (FA) and ileum smooth muscle. The effects of phorbol dibutyrate (PDBu), guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and agonists on the membrane versus cytosolic distribution of PKC isoenzymes were also determined. Down-regulation of PKC abolished Ca2+ sensitization of force and the accompanying increases in MLC20 phosphorylation induced by PDBu, as well as Ca2+ sensitization of force by diC8, but not that by GTP[S], aluminum fluoride (AIF4-) or agonists (phenylephrine, endothelin or carbachol). Down-regulation also inhibited the PDBu-, but not the GTP[S]-induced increase in force under Ca(2+)-free conditions. In ileum, PDBu translocated PKCs alpha, beta 1, beta 2, epsilon and theta to the membrane fraction, and GTP[S] caused a small translocation of PKC-epsilon. Carbachol- and GTP[S]-induced Ca2+ sensitization remained unaffected in down-regulated ileum in which no cytosolic PKC-epsilon was detectable. We conclude that, although both phorbol ester-induced and G-protein-coupled Ca2+ sensitization of force are mediated by increased MLC20 phosphorylation, it is likely that PKCs alpha, beta 1, beta 2, epsilon and theta do not play an essential role in, although they may contribute to, the G-protein-coupled mechanism.


Pharmacogenomics and chemical library screens reveal a novel SCFSKP2 inhibitor that overcomes Bortezomib resistance in multiple myeloma.
While clinical benefit of the proteasome inhibitor (PI) bortezomib (BTZ) for multiple myeloma (MM) patients remains unchallenged, dose-limiting toxicities and drug resistance limit the long-term utility. The E3 ubiquitin ligase Skp1-Cullin-1-Skp2 (SCFSkp2) promotes proteasomal degradation of the cell cycle inhibitor p27 to enhance tumor growth. Increased SKP2 expression and reduced p27 levels are frequent in human cancers and are associated with therapeutic resistance. SCFSkp2 activity is increased by the Cullin-1-binding protein Commd1 and the Skp2-binding protein Cks1B. Here we observed higher CUL1, COMMD1 and SKP2 mRNA levels in CD138+ cells isolated from BTZ-resistant MM patients. Higher CUL1, COMMD1, SKP2 and CKS1B mRNA levels in patient CD138+ cells correlated with decreased progression-free and overall survival. Genetic knockdown of CUL1, COMMD1 or SKP2 disrupted the SCFSkp2 complex, stabilized p27 and increased the number of annexin-V-positive cells after BTZ treatment. Chemical library screens identified a novel compound, designated DT204, that reduced Skp2 binding to Cullin-1 and Commd1, and synergistically enhanced BTZ-induced apoptosis. DT204 co-treatment with BTZ overcame drug resistance and reduced the in vivo growth of myeloma tumors in murine models with survival benefit. Taken together, the results provide proof of concept for rationally designed drug combinations that incorporate SCFSkp2 inhibitors to treat BTZ resistant disease.


Charge transfer between acenes and PbS nanocrystals.
Organic-inorganic hybrid heterojunctions have potential as the basis for future photovoltaic devices. Herein, we report the results of investigations exploring the possibility of using pentacene and tetracene as photoelectron donors in conjunction with PbS nanocrystals (PbS-NCs). Photoinduced charge transfer was probed using external quantum efficiency measurements on acene:PbS-NC hybrid photovoltaic devices in conjunction with photoluminescence studies of the corresponding bilayer films. It is shown that photoelectron transfer from pentacene to the PbS-NCs is inefficient as compared to that between tetracene and PbS-NCs. The latter case can be rationalized in terms of the energy level alignment at the heterojunction assuming a common vacuum level. However, in the case of pentacene:PbS-NC junctions an interfacial energy level shift must be considered in order to explain the observations.


Repetitive transcranial magnetic stimulation for mal de debarquement syndrome.
Mal de debarquement syndrome (MdDS) is a chronic disorder of imbalance characterized by a feeling of rocking and swaying. The disorder starts after prolonged exposure to passive motion such as from a boat or plane. All medical treatment is palliative and symptoms that persist beyond 6 months show low likelihood of remission. This pilot study explored the feasibility and tolerability of repetitive transcranial magnetic stimulation (rTMS) as potential treatment for MdDS. Ten subjects (8 women) with persistent MdDS lasting from 10 to 91 months were given 1 session each of 4 counterbalanced protocols: left 10 Hz (high frequency), left 1 Hz (low frequency), right 10 Hz, and right 1 Hz rTMS over the dorsolateral prefrontal cortex (DLPFC). Reduction of rocking sensation reported on a visual analogue scale. 1) Right-handers improved most with 10-Hz stimulation over the left DLPFC while left-handers improved most with 10 Hz stimulation over the right DLPFC; 2) low-frequency DLPFC stimulation was associated with symptom worsening in some subjects; 3) duration of symptoms was negatively correlated with treatment response; 4) rTMS was well tolerated in MdDS subjects, showing similar rates of headache (10 of 40 sessions) as for other studies; and 5) fatigue occurred after 6 sessions usually with low-frequency stimulation. rTMS was well tolerated in subjects with MdDS with promising short-term symptom improvement. Future studies of rTMS in MdDS may consider sequential days of stimulation, longer post-rTMS observation periods, formal measurement of post-TMS fatigue, and randomization with a sham condition.


NF-κB transcriptional activity is modulated by FK506-binding proteins FKBP51 and FKBP52: a role for peptidyl-prolyl isomerase activity.
Hsp90 binding immunophilins FKBP51 and FKBP52 modulate steroid receptor trafficking and hormone-dependent biological responses. With the purpose to expand this model to other nuclear factors that are also subject to nuclear-cytoplasmic shuttling, we analyzed whether these immunophilins modulate NF-κB signaling. It is demonstrated that FKBP51 impairs both the nuclear translocation rate of NF-κB and its transcriptional activity. The inhibitory action of FKBP51 requires neither the peptidylprolyl-isomerase activity of the immunophilin nor its association with Hsp90. The TPR domain of FKBP51 is essential. On the other hand, FKBP52 favors the nuclear retention time of RelA, its association to a DNA consensus binding sequence, and NF-κB transcriptional activity, the latter effect being strongly dependent on the peptidylprolyl-isomerase activity and also on the TPR domain of FKBP52, but its interaction with Hsp90 is not required. In unstimulated cells, FKBP51 forms endogenous complexes with cytoplasmic RelA. Upon cell stimulation with phorbol ester, the NF-κB soluble complex exchanges FKBP51 for FKBP52, and the NF-κB biological effect is triggered. Importantly, FKBP52 is functionally recruited to the promoter region of NF-κB target genes, whereas FKBP51 is released. Competition assays demonstrated that both immunophilins antagonize one another, and binding assays with purified proteins suggest that the association of RelA and immunophilins could be direct. These observations suggest that the biological action of NF-κB in different cell types could be positively regulated by a high FKBP52/FKBP51 expression ratio by favoring NF-κB nuclear retention, recruitment to the promoter regions of target genes, and transcriptional activity.


How Adverse Outcome Pathways Can Aid the Development and Use of Computational Prediction Models for Regulatory Toxicology.
Efforts are underway to transform regulatory toxicology and chemical safety assessment from a largely empirical science based on direct observation of apical toxicity outcomes in whole organism toxicity tests to a predictive one in which outcomes and risk are inferred from accumulated mechanistic understanding. The adverse outcome pathway (AOP) framework provides a systematic approach for organizing knowledge that may support such inference. Likewise, computational models of biological systems at various scales provide another means and platform to integrate current biological understanding to facilitate inference and extrapolation. We argue that the systematic organization of knowledge into AOP frameworks can inform and help direct the design and development of computational prediction models that can further enhance the utility of mechanistic and in silico data for chemical safety assessment. This concept was explored as part of a workshop on AOP-Informed Predictive Modeling Approaches for Regulatory Toxicology held September 24-25, 2015. Examples of AOP-informed model development and its application to the assessment of chemicals for skin sensitization and multiple modes of endocrine disruption are provided. The role of problem formulation, not only as a critical phase of risk assessment, but also as guide for both AOP and complementary model development is described. Finally, a proposal for actively engaging the modeling community in AOP-informed computational model development is made. The contents serve as a vision for how AOPs can be leveraged to facilitate development of computational prediction models needed to support the next generation of chemical safety assessment.


Co-occurring mental disorders among criminal offenders.
Many convicted offenders suffer from major mental disorders. These offenders commit crimes with great frequency. They do not receive mental health care and are often found in isolation cells of correctional facilities. The present investigation examined lifetime multiple disorders, measured by the DIS, among a representative sample of male penitentiary inmates. Pure forms of the major mental disorders were rare. All possible combinations of the major disorders were found to exist. No patterns of groupings of disorders were evident.


Prediction model for cardiovascular events or all-cause mortality in incident dialysis patients.
Some variables including age, comorbidity of diabetes, and so on at dialysis initiation are associated with patient prognosis. Cardiovascular (CV) events are a major cause of death, and adequate models that predict prognosis in dialysis patients are warranted. Therefore, we created models using some variables at dialysis initiation. We used a database of 1,520 consecutive dialysis patients (median age, 70 years; 492 women [32.4%]) from a multicenter prospective cohort study. We established the primary endpoint as a composite of the incidence of first CV events or all-cause death. A multivariable Cox proportional hazard regression model was used to construct a model. We considered a complex and a simple model. We used area under the receiver operating characteristic curve (AUROC) to assess and compare the predictive performances of the prediction models and evaluated the improvement in discrimination using the complex model versus the simple model using net reclassification improvement (NRI). We then assessed integrated discrimination improvement (IDI) to evaluate improvements in average sensitivity and specificity. Of 392 deaths, 152 were CV-related. Totally, 506 CV events occurred during the follow-up period (median 1,285 days). Finally, 692 patients reached the primary endpoint. Baseline data were set at dialysis initiation. AUROC for the primary endpoint was 0.737 (95% confidence interval [CI], 0.712-0.761) in the simple model and 0.765 (95% CI, 0.741-0.788) in the complex model. There were significant intergroup differences in NRI (0.44; 95% CI, 0.34-0.53; p < 0.001) and IDI (0.02; 95% CI, 0.02-0.03; p < 0.001). We prepared a Shiny R application for each model to automatically calculate the predicted occurrence probability (https://statacademy.shinyapps.io/App_inaguma_20190717/). The complex model made more accurate predictions than the simple model. However, the intergroup difference was not significant. Hence, the simple model was more useful than the complex model. The tool was useful in a real-world clinical setting because it required only routinely available variables. Moreover, we emphasized that the tool could predict the incidence of CV events or all-cause mortality for individual patients. In the future, we must confirm its external validity in other prospective cohorts.


Mitochondrial state determines functionally divergent stem cell population in planaria.
Mitochondrial state changes were shown to be critical for stem cell function. However, variation in the mitochondrial content in stem cells and the implication, if any, on differentiation is poorly understood. Here, using cellular and molecular studies, we show that the planarian pluripotent stem cells (PSCs) have low mitochondrial mass compared with their progenitors. Transplantation experiments provided functional validation that neoblasts with low mitochondrial mass are the true PSCs. Further, the mitochondrial mass correlated with OxPhos and inhibiting the transition to OxPhos dependent metabolism in cultured cells resulted in higher PSCs. In summary, we show that low mitochondrial mass is a hallmark of PSCs in planaria and provide a mechanism to isolate live, functionally active, PSCs from different cell cycle stages (G0/G1 and S, G2/M). Our study demonstrates that the change in mitochondrial metabolism, a feature of PSCs is conserved in planaria and highlights its role in organismal regeneration.


Rattlesnake venom shock in the rat: development of a method.
A model in Wistar rats (n = 30, 279-345 g) was developed to study circulatory, respiratory, metabolic, and lethal effects of an intravenous infusion (30 min; 1.25, 1.5, 1.75, and 2.0 mg/kg) of rattlesnake (Crotalus viridis helleri) venom. Venom produced perfusion failure with lactacidemia, hemoconcentration, hypoproteinemia, and death. The severity of poisoning was proportional to the quantity of venom given and to the elevation in lactic acid and hematocrit. Hemorrhages in the diaphragm, intercostal muscles, and intestine were observed at necropsy. In a separate test, rats (n = 12, 311-355 g) received an infusion of 1.5 mg/kg of venom or physiological salt solution. Blood volume was measured 30 min after the end of infusion in both groups with radioiodinated (125I) human serum albumin (RIHSA) and 51Cr-labeled rat red cells. Venom produced a significant reduction in total blood volume index (35%, P less than 0.001), plasma volume index (46%, P less than 0.001), and red cell mass indec (22% P less than 0.005). The slope of the RIHSA-disappearance curve of animals that received venom was more than twice that of the control group. We conclude that perfusion failure following rattlesnake envenomation is associated with hypovolemia due to increases in vascular permeabiltiy and hemorrhage.


The myogenic factor Myf5 supports efficient skeletal muscle regeneration by enabling transient myoblast amplification.
The myogenic factor Myf5 defines the onset of myogenesis in mammals during development. Mice lacking both Myf5 and MyoD fail to form myoblasts and are characterized by a complete absence of skeletal muscle at birth. To investigate the function of Myf5 in adult skeletal muscle, we generated Myf5 and mdx compound mutants, which are characterized by constant regeneration. Double mutant mice show an increase of dystrophic changes in the musculature, although these mice were viable and the degree of myopathy was modest. Myf5 mutant muscles show a small decrease in the number of muscle satellite cells, which was within the range of physiological variations. We also observed a significant delay in the regeneration of Myf5 deficient skeletal muscles after injury. Interestingly, Myf5 deficient skeletal muscles were able to even out this flaw during the course of regeneration, generating intact muscles 4 weeks after injury. Although we did not detect a striking reduction of MyoD positive activated myoblasts or of Myf5-LacZ positive cells in regenerating muscles, a clear decrease in the proliferation rate of satellite cell-derived myoblasts was apparent in satellite cell-derived cultures. The reduction of the proliferation rate of Myf5 mutant myoblasts was also reflected by a delayed transition from proliferation to differentiation, resulting in a reduced number of myotube nuclei after 6 and 7 days of culture. We reason that Myf5 supports efficient skeletal muscle regeneration by enabling transient myoblast amplification. Disclosure of potential conflicts of interest is found at the end of this article.


The effect of N-ethylmaleimide on the Na(+)-dependent nucleoside transporter (N3) in rabbit choroid plexus.
The effect of the irreversible sulfhydryl-modifying agent, N-ethylmaleimide (NEM), on the transport of purine and pyrimidine nucleosides in choroid plexus was examined. [3H]thymidine and [3H]guanosine were used as model compounds to determine the effect of NEM on the Na(+)-dependent nucleoside transporter (N3) in rabbit choroid plexus tissue slices that were ATP-depleted with 2,4 dinitrophenol. Thymidine uptake in choroid plexus tissue slices preincubated with NEM was irreversibly inhibited in a concentration- (IC50 = 0.18 mM) and time-dependent fashion. NEM treatment also reduced the Na(+)-dependent uptake of other purine and pyrimidine nucleosides to a similar extent. In addition, an amine-selective modifying reagent, phenylisothiocyanate, had no effect on Na(+)-dependent thymidine uptake. Treatment of choroid plexus tissue slices with other sulfhydryl-modifying agents, including 4,4-dithiodipyridine, a reagent specific for cysteine residues, reduced the Na(+)-dependent uptake of thymidine. Preincubation of choroid plexus slices with NEM (2.5 mM) increased the Km of guanosine (control 64.5 +/- 5.7 microM; treated 120 +/- 23 microM) whereas the Vmax was unaffected (control 4.7 +/- 1 nmol/g/sec; treated 4.03 +/- 0.26 nmol/g/sec). These data suggest that covalent modification of sulfhydryl groups reduces the Na(+)-dependent uptake of nucleosides in choroid plexus slices by decreasing the affinity of nucleosides for the transporter.


Design and evaluation of Bacteroides DNA probes for the specific detection of human fecal pollution.
Because Bacteroides spp. are obligate anaerobes that dominate the human fecal flora, and because some species may live only in the human intestine, these bacteria might be useful to distinguish human from nonhuman sources of fecal pollution. To test this hypothesis, PCR primers specific for 16S rRNA gene sequences of Bacteroides distasonis, B. thetaiotaomicron, and B. vulgatus were designed. Hybridization with species-specific internal probes was used to detect the intended PCR products. Extracts from 66 known Bacteroides strains, representing 10 related species, were used to confirm the specificity of these PCR-hybridization assays. To test for specificity in feces, procedures were developed to prepare DNA of sufficient purity for PCR. Extracts of feces from 9 humans and 70 nonhumans (cats, dogs, cattle, hogs, horses, sheep, goats, and chickens) were each analyzed with and without an internal positive control to verify that PCR amplification was not inhibited by substances in the extract. In addition, serial dilutions from each extract that tested positive were assayed to estimate the relative abundance of target Bacteroides spp. in the sample. Depending on the primer-probe set used, either 78 or 67% of the human fecal extracts tested had high levels of target DNA. On the other hand, only 7 to 11% of the nonhuman extracts tested had similarly high levels of target DNA. An additional 12 to 20% of the nonhuman extracts had levels of target DNA that were 100- to 1,000-fold lower than those found in humans.(ABSTRACT TRUNCATED AT 250 WORDS)


Chronic effects of chlorothiazide on reabsorption by the proximal tubule of the rat.
1. The renal response to a low-sodium diet alone and a low-sodium diet plus the daily oral administration of chlorothiazide was examined in rats. Sodium restriction resulted in a decrease in sodium excretion until day 4, after which it remained constant. The administration of chlorothiazide resulted in an initial natriuresis. By day 6, however, the natriuresis had abated and thereafter sodium excretion remained the same as that of the low sodium group. 2. After the animals were in balance on their respective regimens, clearance and micropuncture studies were performed. The glomerular filtration rate was lower in the chlorothiazide-treated rats than in control rats and/or in the low-sodium group. End proximal tubule TF/Pinulin ratios were higher in the diuretic-treated animals than in control rats. TF/Pinulin ratios in low-sodium animals were lower than in the diuretic-animals but higher than in control rats. 3. These studies demonstrate that the escape from the chronic effects of chlorothiazide is due to a decrease in the glomerular filtration rate and to an increase in fractional reabsorption in the proximal tubule, resulting in a reduction in delivery of filtrate to the cortical diluting segment where chlorothiazide exerts its major inhibitory effect.


Blood-nerve barrier dysfunction contributes to the generation of neuropathic pain and allows targeting of injured nerves for pain relief.
The blood-nerve barrier (BNB) is a selectively permeable barrier that creates an immunologically and biochemically privileged space for peripheral axons and supporting cells. The breakdown of the BNB allows access of blood-borne (hematogenous) cells and molecules to the endoneurium to engage in the local inflammatory cascade. This process was examined in a mouse model of trauma-associated neuropathic pain. The impact of nerve injury-triggered opening of the BNB in the development of chronic pain behavior was investigated. Partial ligation of the sciatic nerve led to a long-lasting disruption of the BNB distal to the site of injury. Vascular endothelial growth factor (VEGF) was expressed by resident macrophages after nerve injury. Intraneural injection of VEGF decreased mechanical thresholds while opening the BNB. Serum from nerve-injured or lipopolysaccharide-treated animals elicited mechanical allodynia in naive animals, when allowed to bypass the BNB by intraneural injection. Intraneural injection of fibrinogen, a clotting protein in plasma that was found to deposit in the nerve after nerve injury, also produced a decrease in mechanical thresholds when introduced into naive nerves. These results demonstrate that blood-borne molecules may play a role in the generation of neuropathic pain, suggesting that pain may be driven from infection or injury, at a distance from the nervous system. Furthermore, the breakdown of the BNB in neuropathic conditions was exploited to permit the entry of analgesic molecules that typically cannot pass the BNB, such as ProToxin-II, a BNB-impermeable Nav1.7 inhibitor. Therapeutics utilizing this mechanism could have selective access to injured nerves over healthy tissues.


Artificial Microbial Arenas: Materials for Observing and Manipulating Microbial Consortia.
From the smallest ecological niche to global scale, communities of microbial life present a major factor in system regulation and stability. As long as laboratory studies remain restricted to single or few species assemblies, however, very little is known about the interaction patterns and exogenous factors controlling the dynamics of natural microbial communities. In combination with microfluidic technologies, progress in the manufacture of functional and stimuli-responsive materials makes artificial microbial arenas accessible. As habitats for natural or multispecies synthetic consortia, they are expected to not only enable detailed investigations, but also the training and the directed evolution of microbial communities in states of balance and disturbance, or under the effects of modulated stimuli and spontaneous response triggers. Here, a perspective on how materials research will play an essential role in generating answers to the most pertinent questions of microbial engineering is presented, and the concept of adaptive microbial arenas and possibilities for their construction from particulate microniches to 3D habitats is introduced. Materials as active and tunable components at the interface of living and nonliving matter offer exciting opportunities in this field. Beyond forming the physical horizon for microbial cultivates, they will enable dedicated intervention, training, and observation of microbial consortia.


Thyroid hormone transport by the human monocarboxylate transporter 8 and its rate-limiting role in intracellular metabolism.
Cellular entry of thyroid hormone is mediated by plasma membrane transporters. We have identified rat monocarboxylate transporter 8 (MCT8) as an active and specific thyroid hormone transporter. The MCT8 gene is located on the X-chromosome. The physiological relevance of MCT8 has been demonstrated by the identification of hemizygous mutations in this gene in males with severe psychomotor retardation and elevated serum T(3) levels. We have characterized human (h) MCT8 by analysis of iodothyronine uptake and metabolism in cell lines transiently transfected with hMCT8 cDNA alone or together with cDNA coding for iodothyronine deiodinase D1, D2, or D3. MCT8 mRNA was detected by RT-PCR in a number of human cell lines as well as in COS1 cells but was low to undetectable in other cell lines, including JEG3 cells. MCT8 protein was not detected in nontransfected cell lines tested by immunoblotting using a polyclonal C-terminal hMCT8 antibody but was detectable in transfected cells at the expected size (61 kDa). Transfection of COS1 and JEG3 cells with hMCT8 cDNA resulted in 2- to 3-fold increases in uptake of T(3) and T(4) but little or no increase in rT(3) or 3,3'-diiodothyronine (3,3'-T(2)) uptake. MCT8 expression produced large increases in T(4) metabolism by cotransfected D2 or D3, T(3) metabolism by D3, rT(3) metabolism by D1 or D2, and 3,3'-T(2) metabolism by D3. Affinity labeling of hMCT8 protein was observed after incubation of intact transfected cells with N-bromoacetyl-[(125)I]T(3). hMCT8 also facilitated affinity labeling of cotransfected D1 by bromoacetyl-T(3). Our findings indicate that hMCT8 mediates plasma membrane transport of iodothyronines, thus increasing their intracellular availability.


Surfactant prophylaxis and retinopathy of prematurity.
The use of exogenous surfactant has led to a decrease in mortality of low-birth-weight infants. In some instances, use of this drug has also led to a reduction in neonatal morbidity, such as respiratory distress syndrome. This study was undertaken to determine whether prophylactic surfactant therapy has an effect on the incidence and severity of retinopathy of prematurity. The authors reviewed the findings of sequential prospective ophthalmologic examinations performed in two neonatal intensive care units that participated in randomized trials of prophylactic exogenous mammalian surfactant. There were 43 patients in the control group (no prophylactic surfactant) and 43 patients in the prophylactic surfactant group. These groups did not differ on the basis of birth weight, gestational age, race, or gender. The incidence and severity of retinopathy of prematurity did not vary between the control and surfactant-treated infants. Only birth weight appeared to be significantly related to the incidence and severity of retinopathy of prematurity. These results suggest that the widespread use of prophylactic surfactant therapy will not change the incidence of retinopathy of prematurity in extremely low-birth-weight infants. However, the absolute number of affected patients will likely increase because of the decrease in mortality of extremely low-birth-weight patients, the patients most at risk for retinopathy of prematurity.


Controlled trial of long-term oral calcium supplementation in essential hypertension.
A randomized, double-blind, placebo-controlled crossover trial of oral calcium supplementation was carried out in 18 patients with uncomplicated essential hypertension. After 15 weeks of oral calcium supplementation, 1 g/day, of the patients' habitual diet, the only blood pressure change (compared with the results of placebo treatment) was in the average standing systolic blood pressure, which was significantly reduced (-8.6 mm Hg; p less than 0.01). The 24-hour urinary calcium excretion and the total serum calcium concentration increased significantly during calcium supplementation (p less than 0.05), indicating good compliance with the treatment. The individual blood pressure changes with high calcium intake were found to be inversely related to basal 24-hour urinary calcium excretion (r = -0.69, p less than 0.001 for standing systolic pressure; r = -0.55, p less than 0.002 for standing diastolic pressure). This correlation was independent of age, basal blood pressure, serum calcium concentration, basal 24-hour urinary sodium excretion, and body weight changes during the trial. In particular, a subgroup of six patients, who had a basal 24-hour urinary calcium excretion higher than the mean + 2 SD of a reference healthy population previously described, showed a substantial average blood pressure fall at variance with the other patients in the study. These results do not support the usefulness of an oral calcium supplement in the majority of subjects with mild essential hypertension; however, they suggest that a group of patients with a previously reported abnormality of calcium metabolism may be responsive to this therapeutic measure.


The effects of blunt trauma and cataract surgery on corneal endothelial cell density.
This study was designed to investigate the effects of trauma and cataract surgery on corneal endothelial cell density (ECD) in patients with a traumatic cataract due to blunt trauma without globe laceration. In this prospective study, 31 subjects with traumatic cataract (traumatic cataract group) and 30 subjects with a senile cataract (control group) were enrolled. The subjects with traumatic cataract were subdivided into two groups: uncomplicated surgery subgroup (n = 19) in which subjects underwent standard phacoemulsification with intraocular lens implantation and complicated surgery subgroup (n = 12) in which subjects underwent cataract surgery other than standard phacoemulsification. The ECD of the traumatic cataract group and the control group was compared preoperatively and at 3 months or later postoperatively. A P value less than 0.05 was considered statistically significant. The ECD in the eyes with traumatic cataract was 13.1% lower than that for healthy eyes preoperatively (P = 0.043). Postsurgical ECD decreased by 16.7% in complicated surgery subgroup and 11.9% in uncomplicated surgery subgroup (P = 0.049) after 3 months postoperatively. The ECD decreased by 10.8% in the control group (P = 0.489). Patients with cataracts due to blunt trauma had a decreased endothelial cell count, which was significantly aggravated by cataract surgery. The loss of corneal endothelium cells due to surgery depends on the surgical approach.


Current Status of Familial LCAT Deficiency in Japan.
Lecithin cholesterol acyltransferase (LCAT) is a lipid-modification enzyme that catalyzes the transfer of the acyl chain from the second position of lecithin to the hydroxyl group of cholesterol (FC) on plasma lipoproteins to form cholesteryl acylester and lysolecithin. Familial LCAT deficiency is an intractable autosomal recessive disorder caused by inherited dysfunction of the LCAT enzyme. The disease appears in two different phenotypes depending on the position of the gene mutation: familial LCAT deficiency (FLD, OMIM 245900) that lacks esterification activity on both HDL and ApoB-containing lipoproteins, and fish-eye disease (FED, OMIM 136120) that lacks activity only on HDL. Impaired metabolism of cholesterol and phospholipids due to LCAT dysfunction results in abnormal concentrations, composition and morphology of plasma lipoproteins and further causes ectopic lipid accumulation and/or abnormal lipid composition in certain tissues/cells, and serious dysfunction and complications in certain organs. Marked reduction of plasma HDL-cholesterol (HDL-C) and corneal opacity are common clinical manifestations of FLD and FED. FLD is also accompanied by anemia, proteinuria and progressive renal failure that eventually requires hemodialysis. Replacement therapy with the LCAT enzyme should prevent progression of serious complications, particularly renal dysfunction and corneal opacity. A clinical research project aiming at gene/cell therapy is currently underway.


Eliciting antibodies in chickens to human dimeric IgA. Removal of factors from human colostrum depressing anti IgA antibody production.
Contrary to expectation chickens did not readily elicit antibodies to IgA dimers when untreated human colostrum was used as antigen. When colostrum was fractionated by means of a column of 8% granulated agarose equilibrated with 10mM phosphate buffer, pH 7.2, a major and a minor fraction were obtained. The major or "1st fraction" consisted of two components with sedimentation coefficients of 10.9 S and 14.1 S, respectively. The minor or "2nd fraction" consisted of components of S values ranging from 2 to 6 and small amounts of 10.9 and 14.1 S material. When chickens were immunized with the "1st fraction" antibodies to dimeric IgA were produced. When the "1st and 2nd fractions" of the column were remixed and used for immunization of chickens, the immune response was as poor as when the chickens were injected with untreated colostrum. An immuno-depressing agent in colostrum was indicated. When rabbits were immunized with clarified human colostrum, antibodies against five antigens were elicited, one of the antigens being dimeric IgA. The immuno-depressing agent is therefore not universal. The purified agent suppressed antibody formation in chickens against the haemocyanin of Jasus lalandii. The "activity" is therefore not specific for IgA and the remaining four antigens in human colostrum. The purified component is a glyco-protein with a hexose content in excess of 10%. The derivatized sugars prepared from it were shown by gas liquid chromatography to be an equimolar mixture of galactose, mannose and fucose. The molecular weight (Mr) of the purified component was found to be 72,000 by sedimentation and diffusion and 80,000 by SDS page using Mr reference standards. The properties of the immuno-suppressor strongly suggest that it is the secretory piece of dimeric IgA.


Effects of tumor necrosis factor and interleukin-1 on the constriction induced by angiotensin II in rat aorta.
To better understand the different steps in the changes occurring in vascular reactivity during sepsis, we studied the effects of a short exposure to tumor necrosis factor (TNF) and interleukin-1 (IL-1) on the contraction in response to angiotensin II (ANG II). The contraction elicited by ANG II was studied by using standard isometric tension techniques in aortic rings exposed for 1 h to 25 ng/ml TNF or to 5 or 20 ng/ml IL-1. This contraction was not significantly changed by TNF but was 109 +/- 23 and 190 +/- 38% greater than in control rings after 5 and 20 ng/ml IL-1, respectively. Because the contraction induced by ANG II is modulated by the simultaneous release of prostaglandins, we tested the hypothesis that IL-1 interferes with this modulation. We found that the IL-1-induced increase in contraction in response to ANG II was completely inhibited by 10(-5) M of the cyclooxygenase inhibitor indomethacin and also by 10(-5) M of the prostaglandin H2/thromboxane A2-receptor antagonist SQ-29548. Note, however, that in rings exposed to IL-1 the contraction in response to the thromboxane A2-receptor agonist U-46619 was not significantly different from the contraction in unexposed rings. Furthermore, no loss was observed in either the vasodilator response to 10(-9)-10(-4) M of the endothelium-dependent-receptor agonist acetylcholine or in the receptor-independent contraction induced by 60 mM K+. We conclude that short exposure to IL-1, but not to TNF, produces a specific increase in the vasoconstrictor response to ANG II via mechanisms mediated by prostaglandin H2/thromboxane A2. This increase might result from an IL-1-induced shift in favor of constrictor prostanoids in the balance of the dilator/constrictor prostanoids, the release of which is associated with stimulation by ANG II.


Applied Mycology Can Contribute to Sustainable Rural Livelihoods: Building upon China's Matsutake Management Initiatives.
Matsutake mushrooms are an important part of rural livelihoods and forest ecosystems across large parts of China, as well as elsewhere in East Asia, Northern Europe and North America. Mushroom harvesters have developed sophisticated understandings of matsutake ecology and production, and are applying this knowledge in various innovative management strategies. At the same time, Chinese government agencies and scientists are promoting matsutake-based livelihoods to support development and conservation goals. We collaborated with matsutake harvesters in one Yunnan community to carry out a systematic experiment on a popular shiro-level management technique: covering matsutake shiros with either plastic or leaf litter. Our experimental results suggest that although leaf litter coverings are superior to plastic coverings, shiros that are left uncovered may produce the highest yields. Complementing our experimental work is a multi-sited household survey of existing matsutake management practices across Yunnan, which shows that a high proportion of harvesters are already engaged in a broad range of potentially beneficial management strategies. Though both findings highlight limitations of previous initiatives led by government and research actors in China, this existing body of work is an important foundation and opportunity for developing applied mycology in the region. In and beyond China, working with communities to develop site-specific management strategies through rigorous and participatory scientific inquiry can provide salient benefits for both scientists and resource users.


Gallic Acid Is the Major Active Component of Cortex Moutan in Inhibiting Immune Maturation of Human Monocyte-Derived Dendritic Cells.
Atopic dermatitis (AD) is a widely prevalent and chronically relapsing inflammatory skin disease. Penta Herbs Formula (PHF) is efficacious in improving the quality of life and reducing topical corticosteroid used in children with AD and one of the active herbs it contains is Cortex Moutan. Recent studies showed that altered functions of dendritic cells (DC) were observed in atopic individuals, suggesting that DC might play a major role in the generation and maintenance of inflammation by their production of pro-inflammatory cytokines. Hence, the aims of the present study were to identify the major active component(s) of Cortex Moutan, which might inhibit DC functions and to investigate their possible interactions with conventional corticosteroid on inhibiting the development of DC from monocytes. Monocyte-derived dendritic cells (moDC) culture model coupled with the high-speed counter-current chromatography (HSCCC), high pressure liquid chromatography (HPLC) and Liquid Chromatography-Mass Spectrometry (LCMS) analyses were used. Gallic acid was the major active component from Cortex Moutan which could dose dependently inhibit interleukin (IL)-12 p40 and the functional cluster of differentiation (CD) surface markers CD40, CD80, CD83 and CD86 expression from cytokine cocktail-activated moDC. Gallic acid could also lower the concentration of hydrocortisone required to inhibit the activation of DC.


Study of the dynamics of poly(ethylene oxide) by combining molecular dynamic simulations and neutron scattering experiments.
We performed quasielastic neutron scattering experiments and atomistic molecular dynamics simulations on a poly(ethylene oxide) (PEO) homopolymer system above the melting point. The excellent agreement found between both sets of data, together with a successful comparison with literature diffraction results, validates the condensed-phase optimized molecular potentials for atomistic simulation studies (COMPASS) force field used to produce our dynamic runs and gives support to their further analysis. This provided direct information on magnitudes which are not accessible from experiments such as the radial probability distribution functions of specific atoms at different times and their moments. The results of our simulations on the H-motions and different experiments indicate that in the high-temperature range investigated the dynamics is Rouse-like for Q-values below approximately 0.6 A(-1). We then addressed the single chain dynamic structure factor with the simulations. A mode analysis, not possible directly experimentally, reveals the limits of applicability of the Rouse model to PEO. We discuss the possible origins for the observed deviations.


Prevalence of workplace violence against healthcare workers: a systematic review and meta-analysis.
We aim to quantitatively synthesise available epidemiological evidence on the prevalence rates of workplace violence (WPV) by patients and visitors against healthcare workers. We systematically searched PubMed, Embase and Web of Science from their inception to October 2018, as well as the reference lists of all included studies. Two authors independently assessed studies for inclusion. Data were double-extracted and discrepancies were resolved by discussion. The overall percentage of healthcare worker encounters resulting in the experience of WPV was estimated using random-effects meta-analysis. The heterogeneity was assessed using the I2 statistic. Differences by study-level characteristics were estimated using subgroup analysis and meta-regression. We included 253 eligible studies (with a total of 331 544 participants). Of these participants, 61.9% (95% CI 56.1% to 67.6%) reported exposure to any form of WPV, 42.5% (95% CI 38.9% to 46.0%) reported exposure to non-physical violence, and 24.4% (95% CI 22.4% to 26.4%) reported experiencing physical violence in the past year. Verbal abuse (57.6%; 95% CI 51.8% to 63.4%) was the most common form of non-physical violence, followed by threats (33.2%; 95% CI 27.5% to 38.9%) and sexual harassment (12.4%; 95% CI 10.6% to 14.2%). The proportion of WPV exposure differed greatly across countries, study location, practice settings, work schedules and occupation. In this systematic review, the prevalence of WPV against healthcare workers is high, especially in Asian and North American countries, psychiatric and emergency department settings, and among nurses and physicians. There is a need for governments, policymakers and health institutions to take actions to address WPV towards healthcare professionals globally.


Adherence of Candida albicans to bladder mucosa: development and application of a tissue explant assay.
In order to study the interactions between Candida species and uroepithelial tissue, a tissue explant assay was developed using bladder mucosa harvested from New Zealand white rabbits. Blastoconidia of Candida albicans, Candida tropicalis and Candida glabrata attached to the uroepithelial tissue in similar quantities. However, there was significantly more adherence to the uroepithelium by pre-germinated C. albicans compared with C. albicans blastoconidia. Furthermore, the amount of uroepithelial tissue injury was directly related to the length of exposure of the tissue to Candida. Thus, this tissue explant assay may provide a useful method for investigating properties related to fungal adherence to transitional uroepithelium and organism-mediated tissue injury.


[Myositis-specific autoantibodies and their clinical significance].
The idiopathic inflammatory myopathies polymyositis (PM) and dermatomyositis (DM) have historically been defined using broad clinical and pathological criteria. These conditions affect both adults and children with clinical features including muscle weakness, skin disease, internal organ involvement, and an association with cancer in adults. Using a clinical-serological approach, PM and DM can be defined into more homogeneous subsets. In recent years, myositis-specific autoantibodies (MSAs) have been better characterized and include autoantibodies to the signal-recognition particle and aminoacyl tRNA-synthetase enzymes. In addition, clinically significant novel autoantibodies -anti-p155/p140, anti-CADM-140, and others- have been described in the disease spectrum. MSAs are directed against cytoplasmic or nuclear components involved in key regulatory intracellular processes including protein synthesis, translocation, and transcription. The striking association between unique serological profiles and distinct clinical phenotypes suggests that target autoantigens may play a role in disease induction and propagation. This review reports recent progress in myositis-specific autoantigens, particularly their clinical significance.


Characterization of human anti-porcine "natural antibodies" recovered from ex vivo perfused hearts--predominance of IgM and IgG2.
Hyperacute rejection is a major obstacle to successful transplantation of vascularized xenogeneic organs and is believed to be mediated at least in part by performed xenoreactive "natural antibodies" (NAb). In this study, human NAb that could be involved in hyperacute rejection of pig heart xenografts were identified and characterized using an ex vivo model in which pig hearts were perfused with whole blood from individual human or pig donors. This ex vivo perfusion model allows for the continuous monitoring of physiologic parameters of cardiac function as well as sequential sampling of tissue and blood. Pig hearts perfused with allogeneic pig blood maintained normal function for at least 4 hr, whereas those perfused with xenogeneic AB+ human blood never achieved normal function and rejected completely after 30 min. In three separate experiments involving different human blood donors and pig hearts, sequential samples of perfused blood revealed a progressive depletion of anti-porcine NAb. Samples of all three rejected cardiac xenografts were homogenized, and the specifically bound human anti-porcine antibodies were eluted with citric acid. The eluted antibodies were enriched approximately 50-120-fold for anti-porcine reactivity compared with serum from the corresponding donor. Eluates contained NAb of predominantly IgM and IgG2 isotypes. Immunofluorescence histology confirmed the deposition of IgM and IgG2 but not other IgG subclasses in the rejected pig hearts. Since IgG2 utilized predominantly in response to bacterial polysaccharide antigens, our findings are consistent with the possibility that some NAb arise via crossreactivity with microbial antigens and are predominantly directed against carbohydrate rather than protein antigens.


Effect of tilmicosin on chemotactic, phagocytic, and bactericidal activities of bovine and porcine alveolar macrophages.
To evaluate chemotactic, phagocytic, and bactericidal activities of bovine and porcine alveolar macrophages (AM) exposed to tilmicosin. 12 healthy calves and 12 healthy pigs. Lungs were obtained immediately after euthanasia; AM were collected by means of bronchoalveolar lavage and density gradient centrifugation. Chemotactic activity was evaluated by exposing AM to lipopolysaccharide or macrophage inhibitory peptide during incubation with tilmicosin. Phagocytic activity was evaluated by incubating AM with tilmicosin for 24 hours and then with tilmicosin-resistant Salmonella serotype Typhimurium. Bactericidal activity was evaluated by incubating AM with tilmicosin (0, 10, or 20 microg/ml for bovine AM; 0 or 10 microg/ml or 10 microg/ml but washed free of tilmicosin for porcine AM) and then with Mannheimia haemolytica (bovine AM) or with Actinobacillus pleuropneumoniae or Pasteurella multocida (porcine AM). Tilmicosin had no significant effects on chemotactic or phagocytic activities of bovine or porcine AM. The time-course of bactericidal activity was best described by polynomial equations. Time to cessation of bacterial growth and area under the time versus bacterial number curve were significantly affected by incubation of AM with tilmicosin. Results show that bactericidal activity of bovine and porcine AM was enhanced by tilmicosin, but not in proportion to the reported ability of AM to concentrate tilmicosin intracellularly. With or without exposure to tilmicosin, the time-course of bactericidal activity of bovine AM against M haemolytica and of porcine AM against A pleuropneumoniae or P multocida was too complex to be reduced to a simple linear equation.


Grip strength in children with chronic kidney disease.
The relationship between muscle strength and chronic kidney disease (CKD) in children is unknown. This study aims to quantify the association between grip strength (GS) and kidney function and to explore factors associated with grip strength in children and adolescents with CKD. We included 411 children (699 GS assessments) of the Chronic Kidney Disease in Children (CKiD) study. They were matched by age, sex, and height to a healthy control from the National Health and Nutrition Examination Survey to quantify the relationship between GS and CKD. Linear mixed models were used to identify factors associated with GS among CKD patients. Median GS z-score was - 0.72 (IQR - 1.39, 0.11) among CKD patients with CKD stages 2 through 5 having significantly lower GS than CKD stage 1. Compared with healthy controls, CKiD participants had a decreased GS z-score (- 0.53 SD lower, 95% CI - 0.67 to - 0.39) independent of race/ethnicity and body mass index. Factors associated with reduced GS included longer duration of CKD, pre-pubertal status, delayed puberty, neuropsychiatric comorbidities, need of feeding support, need for alkali therapy, and hemoglobin level. Decreased GS was also associated with both a lower frequency and intensity of physical activity. CKD is associated with impaired muscle strength in children independent of growth retardation and BMI. Exposure to CKD for a prolonged time is associated with impaired muscle strength. Potential mediators of the impact of CKD on muscle strength include growth retardation, acidosis, poor nutritional status, and low physical activity. Additional studies are needed to assess the efficacy of interventions targeted at these risk factors.


Tissue residue approach for chemical mixtures.
At the SETAC Pellston Workshop "The Tissue Residues Approach for Toxicity Assessment," held in June 2007, we discussed mixture toxicology in terms of the tissue residue approach (TRA). This article reviews the literature related to the TRA for mixtures of chemicals and recommends a practical, tiered approach that can be implemented in regulatory or risk assessment applications. As with the toxicity of individual chemicals, addressing mixture toxicity by means of the TRA has a number of significant advantages. Early work provided a theoretical basis and experimental data to support the use of TRA for mixtures; later work provided a field-based validation of the integration. However, subsequent development has been hindered by the lack of mixture toxicity data expressed in tissue or preferably target-site concentrations. We recommend a framework for addressing the toxicology of mixtures that integrates the TRA and mixture toxicology in a 3-tier approach. Tier I uses concentration addition (CA) to estimate the toxicity of mixtures regardless of the mechanism of action of the components. However, the common approach that uses a bioaccumulation factor (BAF) to predict TR from the exposure-water concentration for organics must be modified slightly for metals because, unlike organics, the BAF for a metal changes as 1) the aqueous exposure concentration changes, and 2) the concentration of other metals changes. In addition, total tissue residues of a metal are not a good predictor of toxicity, because some organisms store high concentrations of metals internally in detoxified forms. In tier I, if the combination of measured concentrations in the mixture exceeds that predicted to produce adverse effects or above-reference levels, it is necessary to proceed to tier II. Tier II is a mixed model that employs CA and independent action to estimate mixture toxicity. Tiers I and II estimate the toxicity of mixtures to individual species. In tier III, the TRA is integrated with the multisubstance potentially affected fraction (ms-PAF) method to derive TR levels that are protective of a selected percentage of species in aquatic communities (e.g., hazardous concentration for 5% of the species [HC5]).


Nutrition considerations in special environments for aquatic sports.
Elite athletes who compete in aquatic sports face the constant challenge of arduous training and competition schedules in difficult and changing environmental conditions. The huge range of water temperatures to which swimmers and other aquatic athletes are often exposed (16-31 °C for open-water swimming), coupled with altered aquatic thermoregulatory responses as compared with terrestrial athletes, can challenge the health, safety, and performance of these athletes. Other environmental concerns include air and water pollution, altitude, and jetlag and travel fatigue. However, these challenging environments provide the potential for several nutritional interventions that can mitigate the negative effects and enhance adaptation and performance. These interventions include providing adequate hydration and carbohydrate and iron intake while at altitude; optimizing body composition and fluid and carbohydrate intake when training or competing in varying water temperatures; and maximizing fluid and food hygiene when traveling. There is also emerging information on nutritional interventions to manage jetlag and travel fatigue, such as the timing of food intake and the strategic use of caffeine or melatonin. Aquatic athletes often undertake their major global competitions where accommodations feature cafeteria-style buffet eating. These environments can often lead to inappropriate choices in the type and quantity of food intake, which is of particular concern to divers and synchronized swimmers who compete in physique-specific sports, as well as swimmers who have a vastly reduced energy expenditure during their taper. Taken together, planned nutrition and hydration interventions can have a favorable impact on aquatic athletes facing varying environmental challenges.


[Role of the critical period in sex and brain differentiation: learning from dioxin-induced disorders in next generations].
The sexual differentiation of animal fetuses and infants needs stimuli by sex steroids, which are produced in their own gonads, during a short window ('critical period') of pre- and post-natal periods. Our laboratory has conducted a series of studies focusing on the damage to next generations by dioxins. When pregnant rats are exposed to a prototype of dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 1 μg/kg), sexual immaturity such as defects in copulation behavior as well as growth retardation emerges in their pups. We have provided evidence that such disorders are evoked, if not all, from a transient reduction in the gonadal synthesis of sex steroids in fetuses/infants during the critical period. Our studies also revealed that TCDD initially reduces the pituitary expression of luteinizing hormone (LH) to exert the effect on steroidogenesis. Several mechanisms seem to be involved in a TCDD-induced reduction in LH expression. For example, a change in epigenetic regulation in the pituitary and impaired energy production in the hypothalamus are suggested to contribute to the above reduction. Current our study has demonstrated that a transient reduction in the pituitary-gonad axis fixes the lowered expression of hypothalamic gonadotropin-releasing hormone, resulting in defects in sexual behavior. Through these topics, we discuss the role of the critical period in differentiation and development.


Global and regional associations of smaller cerebral gray and white matter volumes with gait in older people.
Gait impairments increase with advancing age and can lead to falls and loss of independence. Brain atrophy also occurs in older age and may contribute to gait decline. We aimed to investigate global and regional relationships of cerebral gray and white matter volumes with gait speed, and its determinants step length and cadence, in older people. In a population-based study, participants aged >60 years without Parkinson's disease or brain infarcts underwent magnetic resonance imaging and gait measurements using a computerized walkway. Linear regression was used to study associations of total gray and white matter volumes with gait, adjusting for each other, age, sex, height and white matter hyperintensity volume. Other covariates considered in analyses included weight and vascular disease history. Voxel-based morphometry was used to study regional relationships of gray and white matter with gait. There were 305 participants, mean age 71.4 (6.9) years, 54% male, mean gait speed 1.16 (0.22) m/s. Smaller total gray matter volume was independently associated with poorer gait speed (p = 0.001) and step length (p<0.001), but not cadence. Smaller volumes of cortical and subcortical gray matter in bilateral regions important for motor control, vision, perception and memory were independently associated with slower gait speed and shorter steps. No global or regional associations were observed between white matter volume and gait independent of gray matter volume, white matter hyperintensity volume and other covariates. Smaller gray matter volume in bilaterally distributed brain networks serving motor control was associated with slower gait speed and step length, but not cadence.


Genome-wide correlation analysis suggests different roles of CRISPR-Cas systems in the acquisition of antibiotic resistance genes in diverse species.
CRISPR-Cas systems are widespread in bacterial and archaeal genomes, and in their canonical role in phage defence they confer a fitness advantage. However, CRISPR-Cas may also hinder the uptake of potentially beneficial genes. This is particularly true under antibiotic selection, where preventing the uptake of antibiotic resistance genes could be detrimental. Newly discovered features within these evolutionary dynamics are anti-CRISPR genes, which inhibit specific CRISPR-Cas systems. We hypothesized that selection for antibiotic resistance might have resulted in an accumulation of anti-CRISPR genes in genomes that harbour CRISPR-Cas systems and horizontally acquired antibiotic resistance genes. To assess that question, we analysed correlations between the CRISPR-Cas, anti-CRISPR and antibiotic resistance gene content of 104 947 reference genomes, including 5677 different species. In most species, the presence of CRISPR-Cas systems did not correlate with the presence of antibiotic resistance genes. However, in some clinically important species, we observed either a positive or negative correlation of CRISPR-Cas with antibiotic resistance genes. Anti-CRISPR genes were common enough in four species to be analysed. In Pseudomonas aeruginosa, the presence of anti-CRISPRs was associated with antibiotic resistance genes. This analysis indicates that the role of CRISPR-Cas and anti-CRISPRs in the spread of antibiotic resistance is likely to be very different in particular pathogenic species and clinical environments. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.


DSM-5 full syndrome, other specified, and unspecified eating disorders in Australian adolescents: prevalence and clinical significance.
Little information is available on the prevalence of Diagnostic and Statistical Manual of Mental Disorders (DSM)-5 eating disorders in adolescence, and eating disorders remain unique in the DSM for not systematically including a criterion for clinical significance. This study aimed to provide the first prevalence report of the full suite of DSM-5 eating disorders in adolescence, and to examine the impact of applying a criterion for clinical significance. In total, 5191 (participation rate: 70%) Australian adolescents completed a survey measuring 1-month prevalence of eating disorder symptoms for all criterial, 'other specified' and unspecified eating disorders, as well as health-related quality of life and psychological distress. The point prevalence of any eating disorder was 22.2% (12.8% in boys, 32.9% in girls), and 'other specified' disorders (11.2%) were more common than full criterial disorders (6.2%). Probable bulimia nervosa and binge eating disorder, but not anorexia nervosa (AN), were more likely to be experienced by older adolescents. Most disorders were associated with an increased odds for being at a higher weight. The prevalence of eating disorders was reduced by 40% (to 13.6%) when a criterion for clinical significance was applied. Eating disorders, particularly 'other specified' syndromes, are common in adolescence, and are experienced across age, weight, socioeconomic and migrant status. The merit of adding a criterion for clinical significance to the eating disorders, similar to other DSM-5 disorders, warrants consideration. At the least, screening tools should measure distress and impairment associated with eating disorder symptoms in order to capture adolescents in greatest need for intervention.


Silk nanoparticles: from inert supports to bioactive natural carriers for drug delivery.
Silk proteins have been studied and employed for the production of drug delivery (nano)systems. They show excellent biocompatibility, controllable biodegradability and non-immunogenicity and, if needed, their properties can be modulated by blending with other polymers. Silk fibroin (SF), which forms the inner core of silk, is a (bio)material officially recognized by the Food and Drug Administration for human applications. Conversely, the potential of silk sericin (SS), which forms the external shell of silk, could still be considered under evaluation. At the best of our knowledge, nanoparticles based on silk sericin "alone" cannot be produced, due to its physicochemical instability influenced by extreme pH, high water solubility and temperature; for these reasons, it almost always needs to be combined with other polymers for the development of drug delivery systems. In this review, we focused on silk proteins as bioactive natural carriers, since they show not only optimal features as inert excipients, but also remarkable intrinsic biological activities. SF has anti-inflammatory properties, while SS presents antioxidant, anti-tyrosine, anti-aging, anti-elastase and anti-bacterial features. Here, we give an overview on SF or SS silk-based nanosystems, with particular attention on the production techniques.


On the enzymatic incorporation of an imidazole nucleotide into DNA.
The expansion of the genetic alphabet with an additional, artificial base pair is of high relevance for numerous applications in synthetic biology. The enzymatic construction of metal base pairs is an alluring strategy that would ensure orthogonality to canonical nucleic acids. So far, very little is known on the enzymatic fabrication of metal base pairs. Here, we report on the synthesis and the enzymatic incorporation of an imidazole nucleotide into DNA. The imidazole nucleotide dIm is known to form highly stable dIm-Ag+-dIm artificial base pairs that cause minimal structural perturbation of DNA duplexes and was considered to be an ideal candidate for the enzymatic construction of metal base pairs. We demonstrate that dImTP is incorporated with high efficiency and selectivity opposite a templating dIm nucleotide by the Kf exo-. The presence of Mn2+, and to a smaller extent Ag+, enhances the efficiency of this polymerization reaction, however, without being strictly required. In addition, multiple incorporation events could be observed, albeit with modest efficiency. We demonstrate that the dIm-Mn+-dIm cannot be constructed by DNA polymerases and suggest that parameters other than stability of a metal base pair and its impact on the structure of DNA duplexes govern the enzymatic formation of artificial metal base pairs.


Ischemic colitis due to obstruction of mesenteric and splenic veins: a case report.
Ischemic injury to the bowel is a well known disease entity that has a wide spectrum of pathological and clinical findings. A sudden drop in the colonic blood supply is essential to its development. We encountered a 41-year-old male patient, who presented with abdominal pain and bloody diarrhea. A colonoscopy showed markedly edematous mucosa with tortuous dilatation of the veins and a deep ulceration at the rectosigmoid junction. On an abdominal computed tomography (CT) scan and CT angiography, the mesenteric and splenic veins were absent with numerous venous collaterals for drainage. The patient gradually responded to oral aminosalicylate therapy, and was in remission after nine months. In most cases, non-occlusive ischemic injury is caused by idiopathic form and occlusive ischemia is caused by abnormalities of arteries and acute venous thrombosis. However, chronic venous insufficiency due to obstruction of macrovascular mesenteric vein rarely causes ischemia of the bowel. This report describes the first case of ischemic colitis caused by obstruction of the mesenteric and splenic veins.


Subcellular dynamics of T cell immunological synapses and kinapses in lymph nodes.
In vitro studies have revealed that T cell activation occurs during the formation of either dynamic or stable interactions with antigen-presenting cells (APC), and the respective cell junctions have been referred to as immunological kinapses and synapses. However, the relevance and molecular dynamics of kinapses and synapses remain to be established in vivo. Using two-photon imaging, we tracked the distribution of LAT-EGFP molecules during antigen recognition by activated CD4(+) T cells in lymph nodes. At steady state, LAT-EGFP molecules were preferentially found at the uropod of rapidly migrating T cells. In contrast to naïve T cells that fully stopped upon systemic antigen delivery, recently activated T cells decelerated and formed kinapses, characterized by continuous extension of membrane protrusions and by the absence of persistent LAT-EGFP clustering. On the other hand, activated CD4(+) T cells formed stable immunological synapses with antigen-loaded B cells and displayed sustained accumulation of LAT-EGFP fluorescence at the contact zone. Our results show that the state of T cell activation and the type of APC largely influence T cell-APC contact dynamics in lymph nodes. Furthermore, we provide a dynamic look at immunological kinapses and synapses in lymph nodes and suggest the existence of distinct patterns of LAT redistribution during antigen recognition.


A RSK(y) relationship with promiscuous PKA.
Where, when, and with "whom" do molecules interact? Such relations in space and time are key concepts that currently engage investigators of cellular signaling processes. The notion of compartmentalized signaling grew out of studies of adenosine 3',5'-monophosphate (cAMP) signaling processes, and this area continues to generate exciting new paradigms. Distinct clouds of cAMP are formed and shaped within cells by tethered cAMP phosphodiesterases (PDEs). AKAPosomes, formed from distinct subpopulations of cAMP-dependent protein kinase (PKA) tethered to anchoring proteins (AKAPs) together with specific substrate molecules, interpret these gradients to generate individualized responses. PKA activity is also regulated by the interaction of other proteins with the regulatory (R) or catalytic (C) subunits of PKA, and a mechanism has been uncovered in which ribosomal S6 kinase (RSK1) interacts with either PKA subunit, depending on whether RSK1 has been phosphorylated and activated by extracellular signal-regulated kinase (ERK). Thus, inactive RSK1 binds the RI subunit of PKA to sensitize it to activation, whereas activated RSK1 binds the C subunit to desensitize PKA to cAMP activation. Cross-talk between the key cAMP and ERK signaling pathways provides a mechanism that, along with distinct mechanisms of both positive and negative attenuation provided by Raf and PDE4 isoforms, can be tailored on a cell type-specific basis.


Material Needs of Emergency Department Patients: A Systematic Review.
Interest in social determinants of health (SDOH) has expanded in recent years, driven by a recognition that such factors may influence health outcomes, services use, and health care costs. One subset of SDOH is material needs such as housing and food. We conducted a systematic review of the literature on material needs among emergency department (ED) patients in the United States. We followed PRISMA guidelines for systematic review methodology. With the assistance of a research librarian, four databases were searched for studies examining material needs among ED patients. Two reviewers independently screened titles, abstracts, and full text to identify eligible articles. Information was abstracted systematically from eligible articles. Forty-three articles were eligible for inclusion. There was heterogeneity in study methods; single-center, cross-sectional studies were most common. Specific material needs examined included homelessness, poverty, housing insecurity, housing quality, food insecurity, unemployment, difficulty paying for health care, and difficulty affording basic expenses. Studies overwhelmingly supported the notion that ED patients have a high prevalence of a number of material needs. Despite some limitations in the individual studies examined in this review, the plurality of prior research confirms that the ED serves a vulnerable population with high rates of material needs. Future research is needed to better understand the role these needs play for ED patients and how to best address them.


Step time variability and pelvis acceleration patterns of younger and older adults: effects of footwear and surface conditions.
This study investigated the effects of various shoe features and surfaces on step time variability and pelvis accelerations (RMS) during walking in six younger and 22 older adults. Participants walked at a self-selected speed in five shoe conditions (standard, elevated heel, soft sole, hard sole, and high collar) on two surfaces: level and irregular. Results showed an age-related reduction in step time variability on the irregular surface and increased medio-lateral (ML) acceleration RMS in older people walking on the irregular versus the level surface. When wearing the elevated heel shoes, both young and old participants displayed significant reductions in pelvis ML accelerations, suggesting a compensatory strategy to counteract for lateral instability resulting from heel elevation. Overall, the findings demonstrate that older people are less able than younger ones to meet the stepping adaptability required for negotiating an irregular surface and, as a possible consequence, have more difficulty maintaining frontal plane stability on such a surface.


How post-translational modifications influence amyloid formation: a systematic study of phosphorylation and glycosylation in model peptides.
A reciprocal relationship between phosphorylation and O-glycosylation has been reported for many cellular processes and human diseases. The accumulated evidence points to the significant role these post-translational modifications play in aggregation and fibril formation. Simplified peptide model systems provide a means for investigating the molecular changes associated with protein aggregation. In this study, by using an amyloid-forming model peptide, we show that phosphorylation and glycosylation can affect folding and aggregation kinetics differently. Incorporation of phosphoserines, regardless of their quantity and position, turned out to be most efficient in preventing amyloid formation, whereas O-glycosylation has a more subtle effect. The introduction of a single beta-galactose does not change the folding behavior of the model peptide, but does alter the aggregation kinetics in a site-specific manner. The presence of multiple galactose residues has an effect similar to that of phosphorylation.


Histopathologic and immunohistochemical characterization of rash to human epidermal growth factor receptor 1 (HER1) and HER1/2 inhibitors in cancer patients.
Human epidermal growth factor receptor (HER) 1 and HER 1/2 inhibitors have shown benefit against a wide range of solid tumors. However, their use is associated with rash in 40% to 90% of patients, which impacts quality of life and interrupts antineoplastic therapy. The pathologic characteristics of affected skin remain unclear, precluding development of rational therapies. The aim of this study was to evaluate differences in histologic and immunohistochemical alterations in rash caused by lapatinib, a dual HER1/2 inhibitor (HER1/2i), and the single HER1 inhibitors (HER1i) cetuximab, erlotinib, and panitumumab. For each of the four drugs, skin biopsies were collected and analyzed from 8 patients with rash (n = 32). Blinded independent histologic analysis and automated measurement of 17 skin biomarkers involved in proliferation, differentiation, and inflammation were conducted. Increased expression of pAKT and decreased dermal K16 and p27 for HER1/2i when compared with each of the HER1i were observed. In addition, decreased epidermal atrophy and follicular neutrophilic infiltrate were evidenced in the skin of patients on HER1/2i when compared with HER1i. We found a lower inhibition of epidermal kinetics and decreased inflammation in HER1/2i-induced rash. These findings underscore differences in skin toxicity as related to specificity of HER blockade, concordant with clinical tolerability and decreased severity of skin toxicity seen with the HER1/2i lapatinib compared with the HER1 inhibitors cetuximab, erlotinib, and panitumumab.


Helper cell-independent cytotoxic clones in man.
We report here a class of helper cell-independent cytotoxic T cell (HITc) clones in man that can proliferate in response to antigenic stimulation as well as mediate cytotoxicity. HITc appear to be rare among clones derived from primary in vitro allosensitized culture, but constitute the majority of clones derived from cells sensitized to autologous Epstein-Barr virus-transformed lymphoblastoid cell lines. The implications of the derivation and function of HITc clones are discussed.


Spectral analysis of waveguide tapered microfiber with an ultrathin metal coating.
This work demonstrates the feasibility of a novel dispersion engineered ultrathin metal film coated on a tapered fiber with a thickness of around 10nm. To our knowledge, the dispersion characteristics of the proposed device induced by such an extremely thin metal film are described here for the first time. Experimental and simulation results indicate that the metal thin film has unique dispersion properties and intrinsic optical characteristics of strong absorption and high reflection in the near infrared light of a wavelength range of 1.25 approximately 1.65 microm, making the material and waveguide dispersions of tapered-fibers more tailorable. In addition to the ability to flatten the slope of the fundamental-mode cutoff of the transmission spectrum, the dispersion profile is heavily influenced when the ultrathin metal film is coated around the proposed tapered fibers. The optical characteristics of the spectral response caused by the ultrathin film on tapered microfibers are also investigated and analyzed.


The development of a cisgenic apple plant.
Cisgenesis represents a step toward a new generation of GM crops. The lack of selectable genes (e.g. antibiotic or herbicide resistance) in the final product and the fact that the inserted gene(s) derive from organisms sexually compatible with the target crop should rise less environmental concerns and increase consumer's acceptance. Here we report the generation of a cisgenic apple plant by inserting the endogenous apple scab resistance gene HcrVf2 under the control of its own regulatory sequences into the scab susceptible apple cultivar Gala. A previously developed method based on Agrobacterium-mediated transformation combined with a positive and negative selection system and a chemically inducible recombination machinery allowed the generation of apple cv. Gala carrying the scab resistance gene HcrVf2 under its native regulatory sequences and no foreign genes. Three cisgenic lines were chosen for detailed investigation and were shown to carry a single T-DNA insertion and express the target gene HcrVf2. This is the first report of the generation of a true cisgenic plant.


Dose-adjustment study of tamoxifen based on CYP2D6 genotypes in Japanese breast cancer patients.
CYP2D6 is a key enzyme responsible for the metabolism of tamoxifen to active metabolites, endoxifen, and 4-hydroxytamoxifen. The breast cancer patients who are heterozygous and homozygous for decreased-function and null alleles of CYP2D6 showed lower plasma concentrations of endoxifen and 4-hydroxytamoxifen compared to patients with homozygous-wild-type allele, resulting in worse clinical outcome in tamoxifen therapy. We recruited 98 Japanese breast cancer patients, who had been taking 20 mg of tamoxifen daily as adjuvant setting. For the patients who have one or no normal allele of CYP2D6, dosages of tamoxifen were increased to 30 and 40 mg/day, respectively. The plasma concentrations of tamoxifen and its metabolites were measured at 8 weeks after dose-adjustment using liquid chromatography-tandem mass spectrometry. Association between tamoxifen dose and the incidence of adverse events during the tamoxifen treatment was investigated. In the patients with CYP2D6*1/*10 and CYP2D6*10/*10, the mean plasma endoxifen levels after dose increase were 1.4- and 1.7-fold higher, respectively, than those before the increase (P < 0.001). These plasma concentrations of endoxifen achieved similar level of those in the CYP2D6*1/*1 patients receiving 20 mg/day of tamoxifen. Plasma 4-hydroxytamoxifen concentrations in the patients with CYP2D6*1/*10 and CYP2D6*10/*10 were also significantly increased to the similar levels of the CYP2D6*1/*1 patients according to the increasing tamoxifen dosages (P < 0.001). The incidence of adverse events was not significantly different between before and after dose adjustment. This study provides the evidence that dose adjustment is useful for the patients carrying CYP2D6*10 allele to maintain the effective endoxifen level.


A simple method for large-scale generation of dopamine neurons from human embryonic stem cells.
Dopamine (DA) neurons derived from human embryonic stem cells (hESCs) are potentially valuable in drug screening and as a possible source of donor tissue for transplantation in Parkinson's disease. However, existing culture protocols that promote the differentiation of DA neurons from hESCs are complex, involving multiple steps and having unreliable results between cultures. Here we report a simple and highly reproducible culture protocol that induces expandable DA neuron progenitors from hESCs in attached cultures. We found that the hESC-derived neuronal progenitors retain their full capacity to generate DA neurons after repeated passaging in the presence of basic fibroblast growth factor (bFGF) and medium conditioned with PA6 stromal cells. Using immunocytochemistry and RT-PCR, we found that the differentiated DA neurons exhibit a midbrain phenotype and express, e.g., Aldh1a, Ptx3, Nurr1, and Lmx1a. Using HPLC, we monitored their production of DA. We then demonstrated that the expanded progenitors are possible to cryopreserve without loosing the dopaminergic phenotype. With our protocol, we obtained large and homogeneous populations of dopaminergic progenitors and neurons. We conclude that our protocol can be used to generate human DA neurons suitable for the study of disease mechanisms, toxicology, drug screening, and intracerebral transplantation.


Effectiveness of Short Training in Resuscitative Endovascular Balloon Occlusion of the Aorta (REBOA) by Emergency Physicians: The Applied Course for Internal Aortic Clamping on Field Mission.
Resuscitative endovascular balloon occlusion of the aorta (REBOA) is a technique that uses internal clamping of the aorta to control abdominal, pelvic, or junctional bleeding. We created a course to train military physicians in both civilian prehospital use and battlefield use. To determine the effectiveness of this training, we conducted REBOA training for French military emergency physicians. We trained 15 military physicians, organizing the training as follows: a half-day of theoretical training, a half-day of training on mannequins, a half-day on human corpses, and a half-day on a living pig. The primary endpoint was the success rate after training. We defined success as the balloon being inflated in zone 1 of a PryTime mannequin. The secondary endpoints were the progression of each trainee during the training, the difference between the median completion duration before and after training, the median post-training duration, and the median duration for the placement of the sheath introducer before and after training. Fourteen of the physicians (93%) correctly placed the balloon in the mannequin at the end of the training period. During the training, the success rate increased from 73% to 93% (p = .33). The median time for REBOA after training was only 222 seconds (interquartile range [IQR] 194-278), significantly faster than before training (330 seconds, IQR 260-360.5; p = .0033). We also found significantly faster sheath introducer placement (148 seconds, IQR 126-203 versus 145 seconds, IQR 115.5 - 192.5; p = .426). The training can be performed successfully and paves the way for the use of REBOA by emergency physicians in austere conditions.


The Japan Flavour and Fragrance Materials Association's (JFFMA) safety assessment of acetal food flavouring substances uniquely used in Japan.
Using the procedure devised by the Joint FAO/WHO Expert Committee on Food Additives (JECFA), we performed safety evaluations on five acetal flavouring substances uniquely used in Japan: acetaldehyde 2,3-butanediol acetal, acetoin dimethyl acetal, hexanal dibutyl acetal, hexanal glyceryl acetal and 4-methyl-2-pentanone propyleneglycol acetal. As no genotoxicity study data were available in the literature, all five substances had no chemical structural alerts predicting genotoxicity. Using Cramer's classification, acetoin dimethyl acetal and hexanal dibutyl acetal were categorised as class I, and acetaldehyde 2,3-butanediol acetal, hexanal glyceryl acetal and 4-methyl-2-pentanone propyleneglycol acetal as class III. The estimated daily intakes for all five substances were within the range of 1.45-6.53 µg/person/day using the method of maximised survey-derived intake based on the annual production data in Japan from 2001, 2005, 2008 and 2010, and 156-720 µg/person/day using the single-portion exposure technique (SPET), based on the average use levels in standard portion sizes of flavoured foods. The daily intakes of the two class I substances were below the threshold of toxicological concern (TTC) - 1800 μg/person/day. The daily intakes of the three class III substances exceeded the TTC (90 μg/person/day). Two of these, acetaldehyde 2,3-butanediol acetal and hexanal glyceryl acetal, were expected to be metabolised into endogenous products after ingestion. For 4-methyl-2-pentanone propyleneglycol acetal, one of its metabolites was not expected to be metabolised into endogenous products. However, its daily intake level, based on the estimated intake calculated by the SPET method, was about 1/15 000th of the no observed effect level. It was thus concluded that all five substances raised no safety concerns when used for flavouring foods at the currently estimated intake levels. While no information on in vitro and in vivo toxicity for all five substances was available, their metabolites were judged as raising no safety concerns at the current levels of intake.


Sex, hormones and neuroeffector mechanisms.
Incidence and rate of cardiovascular disease differ between men and women across the life span. Although hypertension is more prominent in men than women, there is a group of vasomotor disorders [i.e. Raynaud's disease, postural orthostatic tachycardia syndrome and vasomotor symptoms (hot flashes) of menopause and migraine] with a female predominance. Both sex and hormones interact to modulate neuroeffector mechanisms including integrated regulation of the Sry gene and direct effect of sex steroid hormones on synthesis, release and disposition of monoamine neurotransmitters, and distribution and sensitivity of their receptors in brain areas associated with autonomic control. The interaction of the sex chromosomes and steroids also modulates these effector tissues, that is, the heart, vascular smooth muscle and endothelium. Although involvement of central serotonergic centres has been studied in regard to mood disorders such as depression, their contribution to cardiovascular risk is gaining attention. Studies are needed to further evaluate how hormonal treatments and drugs used to modulate adrenergic and serotonergic activity affect progression and risk for cardiovascular disease in men and women.