Reductions in co-contraction following neuromuscular re-education in people with knee osteoarthritis.
Both increased knee muscle co-contraction and alterations in central pain processing have been suggested to play a role in knee osteoarthritis pain. However, current interventions do not target either of these mechanisms. The Alexander Technique provides neuromuscular re-education and may also influence anticipation of pain. This study therefore sought to investigate the potential clinical effectiveness of the AT intervention in the management of knee osteoarthritis and also to identify a possible mechanism of action. A cohort of 21 participants with confirmed knee osteoarthritis were given 20 lessons of instruction in the Alexander Technique. In addition to clinical outcomes EMG data, quantifying knee muscle co-contraction and EEG data, characterising brain activity during anticipation of pain, were collected. All data were compared between baseline and post-intervention time points with a further 15-month clinical follow up. In addition, biomechanical data were collected from a healthy control group and compared with the data from the osteoarthritis subjects. Following AT instruction the mean WOMAC pain score reduced by 56 % from 9.6 to 4.2 (P < 0.01) and this reduction was maintained at 15 month follow up. There was a clear decrease in medial co-contraction at the end of the intervention, towards the levels observed in the healthy control group, both during a pre-contact phase of gait (p < 0.05) and during early stance (p < 0.01). However, no changes in pain-anticipatory brain activity were observed. Interestingly, decreases in WOMAC pain were associated with reductions in medial co-contraction during the pre-contact phase of gait. This is the first study to investigate the potential effectiveness of an intervention aimed at increasing awareness of muscle behaviour in the clinical management of knee osteoarthritis. These data suggest a complex relationship between muscle contraction, joint loading and pain and support the idea that excessive muscle co-contraction may be a maladaptive response in this patient group. Furthermore, these data provide evidence that, if the activation of certain muscles can be reduced during gait, this may lead to positive long-term clinical outcomes. This finding challenges clinical management models of knee osteoarthritis which focus primarily on muscle strengthening. ISRCTN74086288 , 4th January 2016, retrospectively registered.


Associations of High-Density Lipoprotein Particle and High-Density Lipoprotein Cholesterol With Alcohol Intake, Smoking, and Body Mass Index - The INTERLIPID Study.
Recently, high-density lipoprotein particles (HDL-P) have been found to be more strongly inversely associated with coronary artery disease (CAD) risk than their counterpart, HDL cholesterol (HDL-C). Given that lifestyle is among the first targets in CAD prevention, we compared the associations of HDL-P and HDL-C with selected lifestyle factors. Methods and Results: We examined 789 Japanese participants of the INTERLIPID Study: men (n=386) and women (n=403) aged 40-59 years in 1996-1998. Participants treated for dyslipidemias were excluded. Lifestyle factors included alcohol intake, smoking amount, and body mass index (BMI). Multivariable linear regression was used for cross-sectional analyses of these factors with HDL-P, HDL-C, HDL-P size subclasses (small, medium and large) and mean HDL-P size. In men, higher alcohol intake was associated with higher HDL-P and higher HDL-C. The associations of alcohol, however, were strongest with HDL-P. A higher smoking amount tended to be associated with lower HDL-P and HDL-C. In contrast, BMI was not associated with HDL-P, but was strongly inversely associated with HDL-C. While alcohol intake favored larger mean HDL-P size, smoking and BMI favored a lipid profile with smaller HDL-P subclasses and overall smaller mean HDL-P size. Similar, but generally weaker results were observed in women. Although both HDL-P and HDL-C are parameters of HDL, they have different associations with alcohol, smoking and BMI.


Polynucleotide phosphorylase activity may be modulated by metabolites in Escherichia coli.
RNA turnover is an essential element of cellular homeostasis and response to environmental change. Whether the ribonucleases that mediate RNA turnover can respond to cellular metabolic status is an unresolved question. Here we present evidence that the Krebs cycle metabolite citrate affects the activity of Escherichia coli polynucleotide phosphorylase (PNPase) and, conversely, that cellular metabolism is affected widely by PNPase activity. An E. coli strain that requires PNPase for viability has suppressed growth in the presence of increased citrate concentration. Transcriptome analysis reveals a PNPase-mediated response to citrate, and PNPase deletion broadly impacts on the metabolome. In vitro, citrate directly binds and modulates PNPase activity, as predicted by crystallographic data. Binding of metal-chelated citrate in the active site at physiological concentrations appears to inhibit enzyme activity. However, metal-free citrate is bound at a vestigial active site, where it stimulates PNPase activity. Mutagenesis data confirmed a potential role of this vestigial site as an allosteric binding pocket that recognizes metal-free citrate. Collectively, these findings suggest that RNA degradative pathways communicate with central metabolism. This communication appears to be part of a feedback network that may contribute to global regulation of metabolism and cellular energy efficiency.


Shorter Versus Longer Shift Durations to Mitigate Fatigue and Fatigue-Related Risks in Emergency Medical Services Personnel and Related Shift Workers: A Systematic Review.
This study comprehensively reviewed the literature on the impact of shorter versus longer shifts on critical and important outcomes for Emergency Medical Services (EMS) personnel and related shift worker groups. Six databases (e.g., PubMed/MEDLINE) were searched, including one website. This search was guided by a research question developed by an expert panel a priori and registered with the PROSPERO database of systematic reviews (2016:CRD42016040099). The critical outcomes of interest were patient safety and personnel safety. The important outcomes of interest were personnel performance, acute fatigue, sleep and sleep quality, retention/turnover, long-term health, burnout/stress, and cost to system. Screeners worked independently and full-text articles were assessed for relevance. Data abstracted from the retained literature were categorized as favorable, unfavorable, mixed/inconclusive, or no impact toward the shorter shift duration. This research characterized the evidence as very low, low, moderate, or high quality according to the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) methodology. The searched yielded n = 21,674 records. Of the 480 full-text articles reviewed, 100 reported comparisons of outcomes of interest by shift duration. We identified 24 different shift duration comparisons, most commonly 8 hours versus 12 hours. No one study reported findings for all 9 outcomes. Two studies reported findings linked to both critical outcomes of patient and personnel safety, 34 reported findings for one of two critical outcomes, and 64 did not report findings for critical outcomes. Fifteen studies were grouped to compare shifts <24 hours versus shifts ≥24 hours. None of the findings for the critical outcomes of patient and personnel safety were categorized as unfavorable toward shorter duration shifts (<24 hours). Nine studies were favorable toward shifts <24 hours for at least one of the 7 important outcomes, while findings from one study were categorized as unfavorable. Evidence quality was low or very low. The quality of existing evidence on the impact of shift duration on fatigue and fatigue-related risks is low or very low. Despite these limitations, this systematic review suggests that for outcomes considered critical or important to EMS personnel, shifts <24 hours in duration are more favorable than shifts ≥24 hours.


Coronal microleakage of five materials used to create an intracoronal seal in endodontically treated teeth.
The purpose of this study was to quantitatively compare the sealing effectiveness of five restorative materials that were used to create an intracoronal double seal. Fifty-two extracted mandibular molars were randomly divided into five groups of 10 teeth, and one positive and one negative control tooth. The crowns were removed and the pulpal floor and canal orifices were sealed with 3 mm of one of the following materials: Amalgabond, C&B Metabond, One-Step Dentin Adhesive with AEliteflo composite, One-Step with Palfique composite, or intermediate restorative material (IRM). Each tooth was affixed to a fluid filtration device and the seal was evaluated at 0, 1, 7, 30, and 90 days. The results showed a significant (p = 0.0001) difference in leakage between the materials. At 7 days, IRM, AEliteflo, and Palfique leaked significantly more than Amalgabond or C&B Metabond. Amalgabond consistently produced the best seal of all the materials throughout the duration of the study.


Genetic instability in human ovarian cancer cell lines.
We have analyzed the stability of microsatellites in cell lines derived from human ovarian cancers and found that 5 out of 10 of the ovarian tumor cell lines are genetically unstable at the majority of the loci analyzed. In clones and subclones derived serially from one of these cell lines (2774; serous cystadenocarcinoma), a very high proportion of microsatellites distributed in many different regions of the genome change their size in a mercurial fashion. We conclude that genomic instability in ovarian tumors is a dynamic and ongoing process whose high frequency may have been previously underestimated by PCR-based allelotyping of bulk tumor tissue. We have identified the source of the genetic instability in one ovarian tumor as a point mutation (R524P) in the human mismatch-repair gene MSH2 (Salmonella MutS homologue), which has recently been shown to be involved in hereditary nonpolyposis colorectal cancer. Patient 2774 was a 38-year-old heterozygote, and her normal tissue carried both mutant and wild-type alleles of the human MSH2 gene. However the wild-type allele was lost at some point early during tumorigenesis so that DNA isolated either from the patient's ovarian tumor or from the 2774 cell line carries only the mutant allele of the human MSH2 gene. The genetic instability observed in the tumor and cell line DNA, together with the germ-line mutation in a mismatch-repair gene, suggest that the MSH2 gene is involved in the onset and/or progression in a subset of ovarian cancer.


Automated analysis of brachial ultrasound image sequences: early detection of cardiovascular disease via surrogates of endothelial function.
Early detection of cardiovascular disease would allow timely institution of preventive measures. Arterial endothelium play a primary role in processes leading to the development of atherosclerotic plaque and cardiovascular disease in general. Determination of flow-mediated dilatation (FMD) of brachial arteries from B-mode ultrasound image sequences offers a noninvasive surrogate index of endothelial function. A highly automated method for analysis of brachial ultrasound image sequences is reported and its performance assessed. The method overcomes the variability of brachial ultrasound images across subjects by incorporating machine learning and quality control steps. The automated method outperformed conventional manual analysis by providing a decreased analysis bias, increased reproducibility, and improved measurement accuracy. Consequently, it decreases inter- and intraobserver as well interinstitution variability. The method has been employed in a number of population studies with thousands of subjects analyzed.


Summary of 2017 ESC guidelines on valvular heart disease, peripheral artery disease, STEMI and on dual antiplatelet therapy.
During the ESC congress in September 2017 in Barcelona, the new ESC guidelines were presented and are now available on the ESC website. The new guidelines cover management recommendations on following cardiovascular items: valvular heart disease, peripheral artery disease, ST elevation myocardial infarction (STEMI) and on dual antiplatelet therapy. The present document gives a summary of these guidelines and highlights the most important recommendations and changes in the management of these diseases. It will help to increase awareness about the new guidelines and may stimulate to consult the full document for specific items. Ultimately, the authors hope that this document will enhance implementation of new ESC guidelines in daily clinical practice.


Large scale dynamics of the Michaelis complex in Bacillus stearothermophilus lactate dehydrogenase revealed by a single-tryptophan mutant study.
Large scale dynamics within the Michaelis complex mimic of Bacillus stearothermophilus thermophilic lactate dehydrogenase, bsLDH·NADH·oxamate, were studied with site specific resolution by laser-induced temperature jump relaxation spectroscopy with a time resolution of 20 ns. NADH emission and Trp emission from the wild type and a series of single-tryptophan bsLDH mutants, with the tryptophan positions different distances from the active site, were used as reporters of evolving structure in response to the rapid change in temperature. Several distinct dynamical events were observed on the millisecond to microsecond time scale involving motion of atoms spread over the protein, some occurring concomitantly or nearly concomitantly with structural changes at the active site. This suggests that a large portion of the protein-substrate complex moves in a rather concerted fashion to bring about catalysis. The catalytically important surface loop undergoes two distinct movements, both needed for a competent enzyme. Our results also suggest that what is called "loop motion" is not just localized to the loop and active site residues. Rather, it involves the motion of atoms spread over the protein, even some quite distal from the active site. How these results bear on the catalytic mechanism of bsLDH is discussed.


The arcABC operon required for fermentative growth of Pseudomonas aeruginosa on arginine: Tn5-751-assisted cloning and localization of structural genes.
Pseudomonas aeruginosa is able to utilize L-arginine as the energy source for growth under anaerobic, nitrate-free conditions. Mutations in the chromosomal arcABC gene cluster specifying the inducible arginine deiminase pathway enzymes abolish fermentative growth on arginine. From two different arc::Tn5-751 insertion mutants of P. aeruginosa recombinant plasmids have been derived which carry a resistance marker of transposon Tn5-751 plus flanking parts of the arc region. These recombinant plasmids served to reconstruct in vitro the functional arcABC cluster on a 5.6 kb fragment, which was inserted into the broad-host-range vector pKT240. In P. aeruginosa this 5.6 kb segment complemented arcABC mutations in trans and contained the control region necessary in cis for arc enzyme induction by oxygen limitation and arginine. The results of subcloning experiments and transcriptional lacZ fusions, the polarity of transposon insertions and the effect of external promoters led to the conclusion that the structural genes arcA (for arginine deiminase), arcB (for catabolic ornithine carbamoyltransferase) and arcC (for carbamate kinase) are contiguous and transcribed in the same direction. Thus, the arcABC cluster appears to have the characteristics of an operon. In Escherichia coli the cloned arcABC genes were expressed at low, non-inducible levels; strong vector promoters enhanced arc expression up to 100-fold. This indicates that transcriptional initiation at the arc promoter(s) is poor in E. coli.


Deep iterative reconstruction for phase retrieval.
The classical phase retrieval problem is the recovery of a constrained image from the magnitude of its Fourier transform. Although there are several well-known phase retrieval algorithms, including the hybrid input-output (HIO) method, the reconstruction performance is generally sensitive to initialization and measurement noise. Recently, deep neural networks (DNNs) have been shown to provide state-of-the-art performance in solving several inverse problems such as denoising, deconvolution, and superresolution. In this work, we develop a phase retrieval algorithm that utilizes two DNNs together with the model-based HIO method. First, a DNN is trained to remove the HIO artifacts, and is used iteratively with the HIO method to improve the reconstructions. After this iterative phase, a second DNN is trained to remove the remaining artifacts. Numerical results demonstrate the effectiveness of our approach, which has little additional computational cost compared to the HIO method. Our approach not only achieves state-of-the-art reconstruction performance but also is more robust to different initialization and noise levels.


Adding a single CA 125 measurement to ultrasound imaging performed by an experienced examiner does not improve preoperative discrimination between benign and malignant adnexal masses.
To determine whether CA 125 measurement is superior to ultrasound imaging performed by an experienced examiner for discriminating between benign and malignant adnexal lesions, and to determine whether adding CA 125 to ultrasound examination improves diagnostic performance. This is a prospective multicenter study (International Ovarian Tumor Analysis (IOTA) study) conducted in nine European ultrasound centers in university hospitals. Of 1149 patients with an adnexal mass examined in the IOTA study, 83 were excluded. Of the remaining 1066 patients, 809 had CA 125 results available and were included. The patients underwent preoperative serum CA 125 measurements and transvaginal ultrasound examination by an experienced ultrasound examiner blinded to CA 125 values. The examiner classified each mass as certainly or probably benign, difficult to classify, or probably or certainly malignant. The outcome measure was the sensitivity and specificity with regard to malignancy of CA 125, ultrasound imaging and their combined use, the 'gold standard' being the histological diagnosis of the adnexal mass removed surgically within 120 days after the ultrasound examination. There were 242 (30%) malignancies. For 534 tumors judged to be certainly benign or certainly malignant by the ultrasound examiner the sensitivity and specificity of ultrasound examination and CA 125 (> or =35 U/mL indicating malignancy) were 97% vs. 86% (95% CI of difference, 4.7-17.2) and 99% vs. 79% (95% CI of difference, 15.7-24.2); for 209 tumors judged probably benign or probably malignant, sensitivity and specificity were 81% vs. 57% (95% CI of difference, 12.3-36.0) and 91% vs. 74% (95% CI of difference, 8.5-25.7); for 66 tumors that were difficult to classify, sensitivity and specificity were 57% vs. 39% (95% CI of difference, -9.7 to 41.1) and 74% vs. 67% (95% CI of difference, -14.6 to 27.7). Diagnostic performance deteriorated when CA 125 was used as a second-stage test after ultrasound examination. Specialist ultrasound examination is superior to CA 125 for preoperative discrimination between benign and malignant adnexal masses, irrespective of the diagnostic confidence of the ultrasound examiner; adding CA 125 to ultrasound does not improve diagnostic performance. Our results indicate that greater investment in education and training in gynecological ultrasound imaging would be of value.


A model framework to estimate impact and cost of genetics-based sterile insect methods for dengue vector control.
Vector-borne diseases impose enormous health and economic burdens and additional methods to control vector populations are clearly needed. The Sterile Insect Technique (SIT) has been successful against agricultural pests, but is not in large-scale use for suppressing or eliminating mosquito populations. Genetic RIDL technology (Release of Insects carrying a Dominant Lethal) is a proposed modification that involves releasing insects that are homozygous for a repressible dominant lethal genetic construct rather than being sterilized by irradiation, and could potentially overcome some technical difficulties with the conventional SIT technology. Using the arboviral disease dengue as an example, we combine vector population dynamics and epidemiological models to explore the effect of a program of RIDL releases on disease transmission. We use these to derive a preliminary estimate of the potential cost-effectiveness of vector control by applying estimates of the costs of SIT. We predict that this genetic control strategy could eliminate dengue rapidly from a human community, and at lower expense (approximately US$ 2~30 per case averted) than the direct and indirect costs of disease (mean US$ 86-190 per case of dengue). The theoretical framework has wider potential use; by appropriately adapting or replacing each component of the framework (entomological, epidemiological, vector control bio-economics and health economics), it could be applied to other vector-borne diseases or vector control strategies and extended to include other health interventions.


Depleted uranium is not toxic to rat brain endothelial (RBE4) cells.
Studies on Gulf War veterans with depleted uranium (DU) fragments embedded in their soft tissues have led to suggestions of possible DUinduced neurotoxicity. We investigated DU uptake into cultured rat brain endothelial cells (RBE4). Following the determination that DU readily enters RBE4 cells, cytotoxic effects were analyzed using assays for cell volume increase, heat shock protein 90 (Hsp90) expression, 3-[4,5-dimethylthiazol- 2-yl]-2, 5-diphenyltetrazolium bromide (MTT) reduction, and lactate dehydrogenase (LDH) activity. The results of these studies show that uptake of the U3O8 uranyl chloride form of DU into RBE4 cells is efficient, but there are little or no resulting cytotoxic effects on these cells as detected by common biomarkers. Thus, the present experimental paradigm is rather reassuring and provides no indication for overt cytotoxicity in endothelial cells exposed to DU.


Neural network models - a novel tool for predicting the efficacy of growth hormone (GH) therapy in children with short stature.
The leading method for prediction of growth hormone (GH) therapy effectiveness are multiple linear regression (MLR) models. Best of our knowledge, we are the first to apply artificial neural networks (ANN) to solve this problem. For ANN there is no necessity to assume the functions linking independent and dependent variables. The aim of study is to compare ANN and MLR models of GH therapy effectiveness. Analysis comprised the data of 245 GH-deficient children (170 boys) treated with GH up to final height (FH). Independent variables included: patients' height, pre-treatment height velocity, chronological age, bone age, gender, pubertal status, parental heights, GH peak in 2 stimulation tests, IGF-I concentration. The output variable was FH. For testing dataset, MLR model predicted FH SDS with average error (RMSE) 0.64 SD, explaining 34.3% of its variability; ANN model derived on the same pre-processed data predicted FH SDS with RMSE 0.60 SD, explaining 42.0% of its variability; ANN model derived on raw data predicted FH with RMSE 3.9 cm (0.63 SD), explaining 78.7% of its variability. ANN seem to be valuable tool in prediction of GH treatment effectiveness, especially since they can be applied to raw clinical data.


The thioacetate-ω(γ-lactam carboxamide) HDAC inhibitor ST7612AA1 as HIV-1 latency reactivation agent.
Antiretroviral therapy (ART) is unable to cure HIV infection. The ability of HIV to establish a subset of latent infected CD4(+) T cells, which remain undetectable to the immune system, becomes a major roadblock to achieve viral eradication. Histone deacetylase inhibitors (HDACi) have been shown to potently induce the reactivation of latent HIV. Here, we show that a new thiol-based HDACi, the thioacetate-ω(γ-lactam carboxamide) derivative ST7612AA1, is a potent inducer of HIV reactivation. We evaluated HIV reactivation activity of ST7612AA1 compared to panobinostat (PNB), romidepsin (RMD) and vorinostat (VOR) in cell culture models of HIV-1 latency, in latently infected primary CD4(+) T lymphocytes and in PBMCs from HIV(+) patients. ST7612AA1 potently induced HIV-1 reactivation at submicromolar concentrations with comparable potency to panobinostat or superior to vorinostat. The presence of known antiretrovirals did not affect ST7612AA1-induced reactivation and their activity was not affected by ST7612AA1. Cell proliferation and cell activation were not affected by ST7612AA1, or any other HDACi used. In conclusion, our results indicate that ST7612AA1 is a potent activator of latent HIV and that reactivation activity of ST7612AA1 is exerted without activation or proliferation of CD4(+) T cells. ST7612AA1 is a suitable candidate for further studies of HIV reactivation strategies and potential new therapies to eradicate the viral reservoirs.


Child temperaments, differential parenting, and the sibling relationships of children with autism spectrum disorder.
This study examined associations between sibling temperaments, differential parenting, and the quality of the relationships between 50 children with autism spectrum disorder (ASD) and their typically developing siblings. The temperament dimension of persistence, but not activity level or emotional intensity, was found to relate to the quality of the sibling relationship. Effects were stronger for temperaments of the typically developing siblings, but persistence levels of both groups of children interacted to predict sibling relationship quality. Persistence also was the temperament dimension associated with differential parenting, with increased levels of differential parenting occurring when siblings, and to some degree the children with ASD, were low in persistence. When siblings were dissatisfied with differential parenting, quality of the sibling relationship was compromised.


The hydroxymethylome of multiple myeloma identifies FAM72D as a 1q21 marker linked to proliferation.
Cell identity relies on the cross-talk between genetics and epigenetics and their impact on gene expression. Oxidation of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) is the first step of an active DNA demethylation process occurring mainly at enhancers and gene bodies and, as such, participates in processes governing cell identity in normal and pathological conditions. Although genetic alterations are well documented in multiple myeloma (MM), epigenetic alterations associated with this disease have not yet been thoroughly analyzed. To gain insight into the biology of MM, genome-wide 5hmC profiles were obtained and showed that regions enriched in this modified base overlap with MM enhancers and super enhancers and are close to highly expressed genes. Through the definition of a MM-specific 5hmC signature, we identified FAM72D as a poor prognostic gene located on 1q21, a region amplified in high risk myeloma. We further uncovered that FAM72D functions as part of the FOXM1 transcription factor network controlling cell proliferation and survival and we evidenced an increased sensitivity of cells expressing high levels of FOXM1 and FAM72 to epigenetic drugs targeting histone deacetylases and DNA methyltransferases.


Metachromasy: an experimental and theoretical reevaluation.
Non-chromotropic substances such as fibrin and gelatin and most tissue and cellular structures stain orthochromatically with internal dye concentrations of such metachromatic dyes as methylene blue and toluidine blue which, if in solution, would be metachromatic. Therefore, at ordinary levels of staining these substances depress the natural tendency of these dyes to change color. However, at elevated levels of dye-binding metachromasy eventually occurs. This phenomenon is explained on the basis of the distribution of dye-binding sites. In these substrates, by contrast with chromotropic substances, many binding sites are too far removed for dye interaction, consequently the interaction frequency can become high enough to produce a color change only as saturation of the available sites is approached. It is also shown that the destruction of color is a characteristic of metachromasy and that water molecules intercalated between approximated dye ions are responsible for the loss and change of color. A concept of metachromasy is proposed in which the interaction between water molecules and suitably approximated dye ions plays an essential role. The experimental studies are described against a background of the history and evolution of ideas on metachromasy. The literature is reviewed and reassessed particularly from the physicochemical viewpoint.


Cross-presentation of virus-like particles by skin-derived CD8(-) dendritic cells: a dispensable role for TAP.
Virus-like particles (VLP) induce efficient CTL responses although they do not carry any genetic information. Here, we analyzed MHC class I associated presentation of VLP-derived CTL-epitopes in vivo. After intradermal injection of VLP containing the immunodominant epitope (p33) of lymphocytic choriomeningitis virus (p33-VLP), presentation of peptide p33 in draining lymph nodes was largely restricted to CD8(-) skin-derived dendritic cells (DC). Surprisingly, and in contrast to findings with tumor cells, TAP1-deficient DC and macrophages mediated efficient cross-presentation of VLP-derived p33 in vivo and in vitro. However, the ability of TAP1-deficient DC to cross-present p33-VLP was reduced compared to wild-type DC, indicating that in DC, both TAP-dependent and TAP-independent pathways were operative. In contrast, macrophages cross-presented p33-VLP normally in the absence of TAP. The TAP-dependent pathway of cross-presentation is therefore confined to DC while both macrophages and DC harbor the TAP-independent pathway. In summary, the results show that VLP-derived epitopes are cross-presented by CD8(-) DC in vivo in a partial TAP-independent fashion and highlight important differences in the processing machinery of DC versus macrophages.


Omega-3 fatty acids in the gravid pig uterus as affected by maternal supplementation with omega-3 fatty acids.
Two experiments evaluated the ability of maternal fatty acid supplementation to alter conceptus and endometrial fatty acid composition. In Exp. 1, treatments were 1) the control, a corn-soybean meal diet; 2) flax, the control diet plus ground flax (3.75% of diet); and 3) protected fatty acids (PFA), the control plus a protected fish oil source rich in n-3 PUFA (Gromega, JBS United Inc., Sheridan, IN; 1.5% of diet). Supplements replaced equal parts of corn and soybean meal. When gilts reached 170 d of age, PG600 (PMSG and hCG, Intervet USA, Millsboro, DE) was injected to induce puberty, and dietary treatments (n = 8/treatment) were initiated. When detected in estrus, gilts were artificially inseminated. On d 40 to 43 of gestation, 7 gilts in the control treatment, 8 gilts in the PFA treatment, and 5 gilts in the flax treatment were pregnant and were slaughtered. Compared with the control treatment, the flax treatment tended to increase eicosapentaenoic acid (EPA: C20:5n-3) in fetuses (0.14 vs. 0.25 +/- 0.03 mg/g of dry tissue; P = 0.055), whereas gilts receiving PFA had more (P < 0.05) docosahexaenoic acid (DHA: C22:6n-3) in their fetuses (5.23 vs. 4.04 +/- 0.078 mg/g) compared with gilts fed the control diet. Both the flax and PFA diets increased (P < 0.05) DHA (0.60, 0.82, and 0.85 +/- 0.078 mg/g for the control, flax, and PFA diet, respectively) in the chorioallantois. In the endometrium, EPA and docosapentaenoic acid (C22:5n-3) were increased by the flax diet (P < 0.001; P < 0.05), whereas gilts receiving PFA had increased DHA (P < 0.001). The flax diet selectively increased EPA, and the PFA diet selectively increased DHA in the fetus and endometrium. In Exp. 2, gilts were fed diets containing PFA (1.5%) or a control diet beginning at approximately 170 of age (n = 13/treatment). A blood sample was collected after 30 d of treatment, and gilts were artificially inseminated when they were approximately 205 d old. Conceptus and endometrial samples were collected on d 11 to 19 of pregnancy. Plasma samples indicated that PFA increased (P < 0.005) circulating concentrations of EPA and DHA. Endometrial EPA was increased (P < 0.001) for gilts fed the PFA diet. In extraembryonic tissues, PFA more than doubled (P < 0.001) the EPA (0.13 vs. 0.32 +/- 0.013 mg/g) and DHA (0.39 vs. 0.85 +/- 0.05 mg/g). In embryonic tissue on d 19, DHA was increased (P < 0.05) by PFA (0.20 vs. 0.30 +/- 0.023 mg/g). Supplementing n-3 PUFA, beginning 30 d before breeding, affected endometrial, conceptus, and fetal fatty acid composition in early pregnancy. Dynamic day effects in fatty acid composition indicate this may be a critical period for maternal fatty acid resources to affect conceptus development and survival.


Identification and Molecular Mapping of a Gene Conferring Resistance to Pyrenophora tritici-repentis Race 3 in Tetraploid Wheat.
ABSTRACT Race 3 of the fungus Pyrenophora tritici-repentis, causal agent of tan spot, induces differential symptoms in tetraploid and hexaploid wheat, causing necrosis and chlorosis, respectively. This study was conducted to examine the genetic control of resistance to necrosis induced by P. tritici-repentis race 3 and to map resistance genes identified in tetraploid wheat (Triticum turgidum). A mapping population of recombinant inbred lines (RILs) was developed from a cross between the resistant genotype T. tur-gidum no. 283 (PI 352519) and the susceptible durum cv. Coulter. Based on the reactions of the Langdon-T. dicoccoides (LDN[DIC]) disomic substitution lines, chromosomal location of the resistance genes was determined and further molecular mapping of the resistance genes for race 3 was conducted in 80 RILs of the cross T. turgidum no. 283/Coulter. Plants were inoculated at the two-leaf stage and disease reaction was assessed 8 days after inoculation based on lesion type. Disease reaction of the LDN(DIC) lines and molecular mapping on the T. turgidum no. 283/Coulter population indicated that the gene, designated tsn2, conditioning resistance to race 3 is located on the long arm of chromosome 3B. Genetic analysis of the F(2) generation and of the F(4:5) and F(6:7) families indicated that a single recessive gene controlled resistance to necrosis induced by race 3 in the cross studied.


Reliability and Normative Data for the Dynamic Visual Acuity Test for Vestibular Screening.
The purpose of this study was to determine reliability of computerized dynamic visual acuity (DVA) testing and to determine reference values for younger and older adults. A primary function of the vestibular system is to maintain gaze stability during head motion. The DVA test quantifies gaze stabilization with the head moving versus stationary. Commercially available computerized systems allow clinicians to incorporate DVA into their assessment; however, information regarding reliability and normative values of these systems is sparse. Forty-six healthy adults, grouped by age, with normal vestibular function were recruited. Each participant completed computerized DVA testing including static visual acuity, minimum perception time, and DVA using the NeuroCom inVision System. Testing was performed by two examiners in the same session and then repeated at a follow-up session 3 to 14 days later. Intraclass correlation coefficients (ICCs) were used to determine inter-rater and test-retest reliability. ICCs for inter-rater reliability ranged from 0.323 to 0.937 and from 0.434 to 0.909 for horizontal and vertical head movements, respectively. ICCs for test-retest reliability ranged from 0.154 to 0.856 and from 0.377 to 0.9062 for horizontal and vertical head movements, respectively. Overall, raw scores (left/right DVA and up/down DVA) were more reliable than DVA loss scores. Reliability of a commercially available DVA system has poor-to-fair reliability for DVA loss scores. The use of a convergence paradigm and not incorporating the forced choice paradigm may contribute to poor reliability.


Comparative histomorphological assessment of the testes of two Clarias species from the Okavango Delta Panhandle, Botswana.
The testes of two Clarias species from the Okavango Delta Panhandle were histomorphologically assessed for differences in structure mainly because the testes varied externally in colour, with Clarias ngamensis having black testes while those of Clarias gariepinus were off-white. Although a detailed histological description of normal testes in C. gariepinus has been compiled based on laboratory experimentation, there is limited histomorphological field data available on both species which could be used as reference material in the histology and histomorphology assessment of gonads both in the laboratory and field experiments. In August 2006 and 2007 fifteen fish per species of C. ngamensis and C. gariepinus were collected from the Shakawe Panhandle in the Okavango Delta. Testicular samples excised from the fish were fixed in 10% neutral buffered formalin and later transported to the University of Johannesburg where laboratory analyses using standard histological procedures were conducted. Microscopic assessments were used to describe the testicular morphology and the reproductive developmental stages. Haematoxylin and Eosin (H&E), Periodic Acid Schiff (PAS), Perl's Prussian Blue and Gordon and Sweets Silver Stain Solution were used to enhance finer cellular detail and the morphology of various structures present in the catfish testes. Results emanating from this study (a) form a histomorphological baseline set of data from an undisturbed wetland system for comparison between normal morphology and anomalies within the catfish species, (b) contribute to the limited database of the normal histomorphology of gonads in Southern African freshwater fish species and (c) expand the academic knowledge and skills required for the conservation of wetland ecosystems in Southern Africa.


Pharmacokinetics and clinical efficacy of lorazepam in children with severe malaria and convulsions.
To investigate the pharmacokinetics and clinical efficacy of intravenous (i.v.) and intramuscular (i.m.) lorazepam (LZP) in children with severe malaria and convulsions. Twenty-six children with severe malaria and convulsions lasting > or =5 min were studied. Fifteen children were given a single dose (0.1 mg kg(-1)) of i.v. LZP and 11 received a similar i.m. dose. Blood samples were collected over 72 h for determination of plasma LZP concentrations. Plasma LZP concentration-time data were fitted using compartmental models. Median [95% confidence interval (CI)] LZP concentrations of 65.1 ng ml(-1) (50.2, 107.0) and 41.4 ng ml(-1) (22.0, 103.0) were attained within median (95% CI) times of 30 min (10, 40) and 25 min (20, 60) following i.v. and i.m. administration, respectively. Concentrations were maintained above the reported therapeutic concentration (30 ng ml(-1)) for at least 8 h after dosing via either route. The relative bioavailability of i.m. LZP was 89%. A single dose of LZP was effective for rapid termination of convulsions in all children and prevention of seizure recurrence for >72 h in 11 of 15 children (73%, i.v.) and 10 of 11 children (91%, i.m), without any clinically apparent respiratory depression or hypotension. Three children (12%) died. Administration of LZP (0.1 mg kg(-1)) resulted in rapid achievement of plasma LZP concentrations within the reported effective therapeutic range without significant cardiorespiratory effects. I.m administration of LZP may be more practical in rural healthcare facilities in Africa, where venous access may not be feasible.


Multiple sexual partnerships and their correlates among Facebook users in Swaziland: an online cross-sectional study.
Social networking sites (SNSs) have been suggested to facilitate risky sexual activities. However, it is unknown and of concern how SNSs such as Facebook shape risky sexual activities in developing settings such as Swaziland, the country hardest hit by HIV and AIDS. We conducted an online cross-sectional study in 2012 to explore the prevalence of multiple sexual partnerships (MSPs) and their correlates among Facebook users in Swaziland. The response rate was 44.1% (N = 882); relatively, an equal proportion of men 82.7% (341/414) and 82.9% (388/468) women had ever had sex. Of those sexually active, 44.9% of men and 30.7% of women reported having sex with someone they met on Facebook. Approximately half of the participants (61.6% men, 41.0% women and 50.6% total) reported MSPs over the past 12 months. Multiple logistic regression analysis revealed that time spent on Facebook, "finding it easier to initiate a romantic conversation on Facebook" and having had sex with someone met on Facebook were significantly associated with having MSPs (adjusted odds ratio = 1.6-3.8). The potential impact of risky sexual behaviour among Facebook users should be appropriately addressed particularly in high HIV-prevalent settings like Swaziland.


A Robust strategy enabling addressable porous 3D carbon-based functional nanomaterials in miniaturized systems.
3D-porous carbon nanomaterials and their hybrids are ideal materials for energy storage and conversion, biomedical research, and wearable sensors, yet today's fabrication methods are too complicated and inefficient to implement into miniaturized systems. Instead, it is shown here that 3D-carbon nanofibrous electrodes of various designs, shapes and sizes, on flexible substrates, under ambient conditions and without complicated equipment and procedures can simply be "written" via a one-step laser-induced carbonization on electrospun nanofibers. Analytical functionalities are realized as full control over native polymer chemistry doping of the polymer (e.g. with metals) is provided. Similarly, being able to control mat morphology and its impact on the electroanalytical performance was studied. Ultimately, optimized writing conditions were harnessed for superior (bio)analytical sensing of important biomarkers (NADH, dopamine). The new procedure hence paves the way for future controlled studies on this 3D nanomaterial, for a multitude of functionalization and design possibilities, and for mass production capabilities necessary for their application in the real world.


eggNOG 4.5: a hierarchical orthology framework with improved functional annotations for eukaryotic, prokaryotic and viral sequences.
eggNOG is a public resource that provides Orthologous Groups (OGs) of proteins at different taxonomic levels, each with integrated and summarized functional annotations. Developments since the latest public release include changes to the algorithm for creating OGs across taxonomic levels, making nested groups hierarchically consistent. This allows for a better propagation of functional terms across nested OGs and led to the novel annotation of 95 890 previously uncharacterized OGs, increasing overall annotation coverage from 67% to 72%. The functional annotations of OGs have been expanded to also provide Gene Ontology terms, KEGG pathways and SMART/Pfam domains for each group. Moreover, eggNOG now provides pairwise orthology relationships within OGs based on analysis of phylogenetic trees. We have also incorporated a framework for quickly mapping novel sequences to OGs based on precomputed HMM profiles. Finally, eggNOG version 4.5 incorporates a novel data set spanning 2605 viral OGs, covering 5228 proteins from 352 viral proteomes. All data are accessible for bulk downloading, as a web-service, and through a completely redesigned web interface. The new access points provide faster searches and a number of new browsing and visualization capabilities, facilitating the needs of both experts and less experienced users. eggNOG v4.5 is available at http://eggnog.embl.de.


Bilateral field advantage in visual enumeration.
A number of recent studies have demonstrated superior visual processing when the information is distributed across the left and right visual fields than if the information is presented in a single hemifield (the bilateral field advantage). This effect is thought to reflect independent attentional resources in the two hemifields and the capacity of the neural responses to the left and right hemifields to process visual information in parallel. Here, we examined whether a bilateral field advantage can also be observed in a high-level visual task that requires the information from both hemifields to be combined. To this end, we used a visual enumeration task--a task that requires the assimilation of separate visual items into a single quantity--where the to-be-enumerated items were either presented in one hemifield or distributed between the two visual fields. We found that enumerating large number (>4 items), but not small number (<4 items), exhibited the bilateral field advantage: enumeration was more accurate when the visual items were split between the left and right hemifields than when they were all presented within the same hemifield. Control experiments further showed that this effect could not be attributed to a horizontal alignment advantage of the items in the visual field, or to a retinal stimulation difference between the unilateral and bilateral displays. These results suggest that a bilateral field advantage can arise when the visual task involves inter-hemispheric integration. This is in line with previous research and theory indicating that, when the visual task is attentionally demanding, parallel processing by the neural responses to the left and right hemifields can expand the capacity of visual information processing.


Different molecular and structural adaptations with eccentric and conventional strength training in elderly men and women.
Reprogramming of gene expression contributes to structural and functional adaptation of muscle tissue in response to altered use. The aim of this study was to investigate mechanisms for observed improvements in leg extension strength, gain in relative thigh muscle mass and loss of body and thigh fat content in response to eccentric and conventional strength training in elderly men (n = 14) and women (n = 14; average age of the men and women: 80.1 ± 3.7 years) by means of structural and molecular analyses. Biopsies were collected from m. vastus lateralis in the resting state before and after 12 weeks of training with two weekly resistance exercise sessions (RET) or eccentric ergometer sessions (EET). Gene expression was analyzed using custom-designed low-density PCR arrays. Muscle ultrastructure was evaluated using EM morphometry. Gain in thigh muscle mass was paralleled by an increase in muscle fiber cross-sectional area (hypertrophy) with RET but not with EET, where muscle growth is likely occurring by the addition of sarcomeres in series or by hyperplasia. The expression of transcripts encoding factors involved in muscle growth, repair and remodeling (e.g., IGF-1, HGF, MYOG, MYH3) was increased to a larger extent after EET than RET. MicroRNA 1 expression was decreased independent of the training modality, and was paralleled by an increased expression of IGF-1 representing a potential target. IGF-1 is a potent promoter of muscle growth, and its regulation by microRNA 1 may have contributed to the gain of muscle mass observed in our subjects. EET depressed genes encoding mitochondrial and metabolic transcripts. The changes of several metabolic and mitochondrial transcripts correlated significantly with changes in mitochondrial volume density. Intramyocellular lipid content was decreased after EET concomitantly with total body fat. Changes in intramyocellular lipid content correlated with changes in body fat content with both RET and EET. In the elderly, RET and EET lead to distinct molecular and structural adaptations which might contribute to the observed small quantitative differences in functional tests and body composition parameters. EET seems to be particularly convenient for the elderly with regard to improvements in body composition and strength but at the expense of reducing muscular oxidative capacity.


Role of interleukin-6 in a patient with tumor necrosis factor receptor-associated periodic syndrome: assessment of outcomes following treatment with the anti-interleukin-6 receptor monoclonal antibody tocilizumab.
In this report, we describe treatment outcomes in the first case of a patient with tumor necrosis factor receptor-associated periodic syndrome (TRAPS) treated with the anti-interleukin-6 (anti-IL-6) receptor monoclonal antibody tocilizumab. Since IL-6 levels are elevated in TRAPS, we hypothesized that tocilizumab might be effective. The patient, a 52-year-old man with lifelong TRAPS in whom treatment with etanercept and anakinra had failed, was administered tocilizumab for 6 months, and the therapeutic response was assessed by measurement of monocyte CD16 expression and cytokine levels. Following treatment, the evolving acute attack was aborted and further attacks of TRAPS were prevented. The patient did not require corticosteroids and showed significant clinical improvement in scores for pain, stiffness, and well-being. Moreover, the acute-phase response diminished significantly with treatment. Monocyte CD16 expression was reduced and the numbers of circulating CD14+CD16+ and CD14++CD16- monocytes were transiently decreased. However, cytokine levels were not reduced. This case supports the notion of a prominent role for IL-6 in mediating the inflammatory attacks in TRAPS, but blockade of IL-6 did not affect the underlying pathogenesis. These preliminary findings require confirmation.


Spatial pattern of 2009 dengue distribution in Kuala Lumpur using GIS application.
In the last few years in Malaysia, dengue fever has increased dramatically and has caused huge public health concerns. The present study aimed to establish a spatial distribution of dengue cases in the city of Kuala Lumpur using a combination of Geographic Information System (GIS) and spatial statistical tools. Collation of data from 1,618 dengue cases in 2009 was obtained from Kuala Lumpur City Hall (DBKL). These data were processed and then converted into GIS format. Information on the average monthly rainfall was also used to correlate with the distribution pattern of dengue cases. To asses the spatial distribution of dengue cases, Average Nearest Neighbor (ANN) Analysis was applied together with spatial analysis with the ESRI ArcGIS V9.3 programme. Results indicated that the distribution of dengue cases in Kuala Lumpur for the year 2009 was spatially clustered with R value less than 1 (R = 0.42; z-scores = - 4.47; p < 0.001). Nevertheless, when this pattern was further analyzed according to month by each zone within Kuala Lumpur, two distinct patterns were observed which include a clustered pattern (R value < 1) between April to June and a dispersed pattern (R value > 1) between August and November. In addition, the mean monthly rainfall has not influenced the distribution pattern of the dengue cases. Implementation of control measures is more difficult for dispersed pattern compared to clustered pattern. From this study, it was found that distribution pattern of dengue cases in Kuala Lumpur in 2009 was spatially distributed (dispersed or clustered) rather than cases occurring randomly. It was proven that by using GIS and spatial statistic tools, we can determine the spatial distribution between dengue and population. Utilization of GIS tools is vital in assisting health agencies, epidemiologist, public health officer, town planner and relevant authorities in developing efficient control measures and contingency programmes to effectively combat dengue fever.


Integrative analysis of sequencing and array genotype data for discovering disease associations with rare mutations.
In the large cohorts that have been used for genome-wide association studies (GWAS), it is prohibitively expensive to sequence all cohort members. A cost-effective strategy is to sequence subjects with extreme values of quantitative traits or those with specific diseases. By imputing the sequencing data from the GWAS data for the cohort members who are not selected for sequencing, one can dramatically increase the number of subjects with information on rare variants. However, ignoring the uncertainties of imputed rare variants in downstream association analysis will inflate the type I error when sequenced subjects are not a random subset of the GWAS subjects. In this article, we provide a valid and efficient approach to combining observed and imputed data on rare variants. We consider commonly used gene-level association tests, all of which are constructed from the score statistic for assessing the effects of individual variants on the trait of interest. We show that the score statistic based on the observed genotypes for sequenced subjects and the imputed genotypes for nonsequenced subjects is unbiased. We derive a robust variance estimator that reflects the true variability of the score statistic regardless of the sampling scheme and imputation quality, such that the corresponding association tests always have correct type I error. We demonstrate through extensive simulation studies that the proposed tests are substantially more powerful than the use of accurately imputed variants only and the use of sequencing data alone. We provide an application to the Women's Health Initiative. The relevant software is freely available.


Hypointense and hyperintense lesions on magnetic resonance imaging in secondary-progressive MS patients.
Cranial magnetic resonance imaging (MRI) is widely used to monitor disease activity in clinical trials in multiple sclerosis (MS). The purpose of this study is to examine lesion burden as determined from hypointense regions on postcontrast T1-weighted scans (or black holes), and lesion burden on conventional T2-weighted scans, from a cohort of secondary progressive MS patients who participated in a placebo-controlled, randomized, double-blind cross-over trial assessing the therapeutic efficacy of cladribine. T2 lesion volumes and black hole volumes are approximately normal distributed when log-transformed, and are highly correlated (adjusted R2 = 0.63). Changes in clinical scores could be predicted with a reasonable degree of precision from baseline scores and changes in T2 lesion volumes (adjusted R2 values 0.52-0.7). Stratification schemes for clinical trials should include the acute proportion of the disease (enhancing T1 lesions), degree of permanent damage (black holes), and T2 lesion volume.


Predicting 24-hour urinary sodium excretion in Afro-Caribbean Barbadians by comparing urine sodium excretion over different durations versus spot collection.
Urinary sodium excretion is used as an assessment tool for salt intake and salt handling. Even though cumbersome, the most reliable and readily used method in clinical and epidemiological studies is the 24-hour urine collection. This study investigates other appropriate means ofpredicting 24-hour urinary sodium excretion in a sample of Afro-Caribbeans in Barbados by assessing the correlation of actual and estimated urinary sodium excretion between a 24-hour urine collection sample, 12-hour (AM and PM), and spot (AM and PM) urine collections. A convenient sample of 30 healthy participants of Afro-Caribbean origin between the ages of 21 and 55 years was recruited for the study. The 24-hour urine samples and anthropometric data were collected as documented in the study's standard clinical procedure. A 24-hour urine sample was collected as two separate 12-hour AM and PM samples. In addition, two spot samples (AM and PM) were taken during each 12-hour sample collection period. Analysis of the urinary sodium and creatinine was done with a Roche/Hitachi Modular System (Roche Diagnostics, IN, USA). SPSS version 19 was used to analyse the data to make inferences. Thirty Afro-Caribbean subjects participated in this study: 16 females and 14 males. The average age and body mass index (BMI) were 38 +/- 17 years and 25.32 +/- 5.98 kg/m2, respectively. The greatest correlation of the estimated 24-hour sodium excretion to the measured 24-hour sodium excretion was observed in the 12-hour PM sample (Pearson's correlation, r = 0.786, p < 0.001) followed by the 12-hour AM sample (Pearson's correlation, r = 0.774, p < 0.001). The PM spot sample showed a weaker, but still statistically significant correlation to the 24-hour timed sample (Pearson 's correlation, r = 0.404, p < 0.045). The AM spot sample showed a very weak and insignificant correlation (Pearson 's correlation, r = 0.05, p = 0.807) to the 24-hour timed sample. Similarly to the whole sample, the gender analysis demonstrated that estimated 24-hour sodium excretion in the female's 12-hour PM sample had the greatest correlation (r = 0.819, p < 0.001) to the measured 24-hour sodium excretion, followed by the 12-hour AM (r = 0.793, p = 0.001) and the PM spot samples (r = 0.741, p = 0.02). The correlation between variables is weaker in males compared to the females. Overall, this study shows a clear correlation between the estimated 24-hour sodium excretion from the 12-hour timed PM sample and the measured 24-hour sodium excretion. Such findings support the thought of using other alternatives to determine sodium excretion, in view of replacing the cumbersome 24-hour urinary collection with a smaller timed sample. Nonetheless, a more robust and randomized population sample as well as a method to correct for high creatinine variability is required to further enhance the significance of the obtained results.


Subclinical Thyroid Dysfunction and Frailty Among Older Men.
Both subclinical thyroid dysfunction and frailty are common among older individuals, but data on the relationship between these 2 conditions are conflicting. The purpose of this study was to assess the cross-sectional and prospective associations between subclinical thyroid dysfunction and frailty and the 5 frailty subdomains (sarcopenia, weakness, slowness, exhaustion, and low activity). The Osteoporotic Fractures in Men Study is a prospective cohort study. Men older than 65 years (n = 1455) were classified into 3 groups of thyroid status: subclinical hyperthyroidism (n = 26, 1.8%), subclinical hypothyroidism (n = 102, 7.0%), and euthyroidism (n = 1327, 91.2%). Frailty was defined using a slightly modified Cardiovascular Health Study Index: men with 3 or more criteria were considered frail, men with 1 to 2 criteria were considered intermediately frail, and men with no criteria were considered robust. We assessed the cross-sectional relationship between baseline thyroid function and the 3 categories of frailty status (robust/intermediate/frail) as well as the prospective association between baseline thyroid function and subsequent frailty status and mortality after a 5-year follow-up. At baseline, compared with euthyroid participants, men with subclinical hyperthyroidism had an increased likelihood of greater frailty status (adjusted odds ratio, 2.48; 95% confidence interval, 1.15-5.34), particularly among men aged <74 years at baseline (odds ratio for frailty, 3.63; 95% confidence interval, 1.21-10.88). After 5 years of follow-up, baseline subclinical hypothyroidism and hyperthyroidism were not consistently associated with overall frailty status or frailty components. Among community-dwelling older men, subclinical hyperthyroidism, but not subclinical hypothyroidism, is associated with increased odds of prevalent but not incident frailty.


DNA double-strand break repair pathways, chromosomal rearrangements and cancer.
Chromosomal rearrangements, which can lead to oncogene activation and tumour suppressor loss, are a hallmark of cancer cells. Such outcomes can result from both the repair and misrepair of DNA ends, which arise from a variety of lesions including DNA double strand breaks (DSBs), collapsed replication forks and dysfunctional telomeres. Here we review the mechanisms by which non-homologous end joining (NHEJ) and homologous recombination (HR) repair pathways can both promote chromosomal rearrangements and also suppress them in response to such lesions, in accordance with their increasingly recognised tumour suppressor function. Further, we consider how chromosomal rearrangements, together with a modular approach towards understanding their etiology, may be exploited for cancer therapy.


Effects of negative pressures on epithelial tight junctions and migration in wound healing.
Negative-pressure wound therapy has recently gained popularity in chronic wound care. This study attempted to explore effects of different negative pressures on epithelial migration in the wound-healing process. The electric cell-substrate impedance sensing (ECIS) technique was used to create a 5 x 10(-4) cm(2) wound in the Madin-Darby canine kidney (MDCK) and human keratinocyte (HaCaT) cells. The wounded cells were cultured in a negative pressure incubator at ambient pressure (AP) and negative pressures of 75 mmHg (NP(75)), 125 mmHg (NP(125)), and 175 mmHg (NP(175)). The effective time (ET), complete wound healing time (T(max)), healing rate (R(heal)), cell diameter, and wound area over time at different pressures were evaluated. Traditional wound-healing assays were prepared for fluorescent staining of cells viability, cell junction proteins, including ZO-1 and E-cadherin, and actins. Amount of cell junction proteins at AP and NP(125) was also quantified. In MDCK cells, the ET (1.25 +/- 0.27 h), T(max) (1.76 +/- 0.32 h), and R(heal) (2.94 +/- 0.62 x 10(-4) cm(2)/h) at NP(125) were significantly (P < 0.01) different from those at three other pressure conditions. In HaCaT cells, the T(max) (7.34 +/- 0.29 h) and R(heal) (6.82 +/- 0.26 x 10(-5) cm(2)/h) at NP(125) were significantly (P < 0.01) different from those at NP(75). Prominent cell migration features were identified in cells at the specific negative pressure. Cell migration activities at different pressures can be documented with the real-time wound-healing measurement system. Negative pressure of 125 mmHg can help disassemble the cell junction to enhance epithelial migration and subsequently result in quick wound closure.


Pharmacovigilance of biopharmaceuticals: challenges remain.
Biopharmaceuticals are important treatment options for a variety of chronic and sometimes life-threatening diseases. Compared with the traditional small molecule drugs, biopharmaceuticals have specific characteristics, which might also influence their safety profile. They have, for example, a complex production process, limited predictability of preclinical to clinical data, a high potential for immunogenicity, and adverse events can often be related to an exaggerated pharmacology. The limited predictability of preclinical to clinical data and the known limitations of randomized controlled trials results in limited knowledge of the safety profile of biopharmaceuticals at the point of their approval, underlining the need for pharmacovigilance. Due to their specific characteristics, pharmacovigilance activities required for biopharmaceuticals might differ from those required for small molecules. This review discusses characteristics and potential challenges with the pharmacovigilance and risk management of biopharmaceuticals as compared with small molecules, and proposes remedies for some of the emerging problems. Spontaneous reporting of adverse drug reactions (ADRs) is important in the detection of new, rare and/or serious ADRs. However, causality assessment remains complicated because of concomitant diseases or drugs. This is particularly the case with biopharmaceuticals, as they are often indicated to treat severe and/or life-threatening diseases in patients who often have other diseases and are treated with concomitant medication. Proactive risk management has been implemented in the EU by the obligatory submission of an EU risk management plan (EU-RMP). In this, the (potential) risks should be described and pharmacovigilance activities proposed. Pharmacovigilance activities can be either routine or additional (post-authorization safety studies [PASS]) activities. During safety assessment, stakeholders are encouraged to use knowledge obtained with biopharmaceuticals with a comparable pharmacology. PASS of biopharmaceuticals with a comparable pharmacology may therefore be used to complement each other. Since biopharmaceuticals are often used in a specialized hospital setting, it is expected that large population-based databases will contain limited information on biopharmaceuticals. Registries have therefore been shown to be an important tool to obtain pharmacovigilance data. Since small changes in the production and purification process might alter the safety profile, activities to improve traceability of the specific biopharmaceutical responsible for the ADR should be taken into account. Key messages in safety management of biopharmaceuticals remain: be prepared for the unexpected, be aware of confounding by disease (severity) and maintain exposure ascertainment/traceability throughout the logistical chain.


Medium-term variability of blood pressure and potential underdiagnosis of hypertension in patients with previous transient ischemic attack or minor stroke.
Blood pressure (BP) is a major risk factor for stroke. However, the variability of systolic and diastolic BP (SBP and DBP) means that single measurements do not provide a reliable measure of usual BP. Although 24-hour ambulatory BP monitoring can correct for the effects of short-term variation, there is also important medium-term variability. The extent of medium-term variability in BP is most marked in patients with a previous transient ischemic attack (TIA) or stroke. We studied the potential impact of this variability on the likely recognition of hypertension. We analyzed multiple repeated measurements of BP in 3 large cohorts with a TIA or minor stroke: the UK-TIA trial (n=2098), the Dutch TIA trial (n=2953), and the European Carotid Surgery Trial (ECST; n=2646). Regression dilution ratios and coefficients of variation were calculated for SBP and DBP from baseline and repeated measurements during the subsequent 12 months. Categorization based on single baseline measurements was also compared with categorization based on the subsequent "usual" BP. The correlation between measurements of BP at baseline and 3 to 5 months later was poor (R(2) from 0.17 to 0.31 for SBP and from 0.10 to 0.20 for DBP). Categorization of patients by baseline values resulted in substantial misclassification in relation to usual BP. For example, of patients with an SBP <140 mm Hg at baseline, the percentage with a usual SBP >or=140 mm Hg was 31.6% in the UK-TIA trial, 48.2% in the Dutch TIA trial, and 57.7% in the ECST. At least 3 consecutive measurements of SBP <120 mm Hg were required to be >90% certain that subsequent usual SBP would not be >or=140 mm Hg. Given the greater medium-term variability of BP in patients with a previous TIA or stroke than in the general population, single measurements of "normal" or "low" BP will substantially underestimate the true prevalence of hypertension.


Mast cell TLR2 signaling is crucial for effective killing of Francisella tularensis.
TLR signaling is critical for early host defense against pathogens, but the contributions of mast cell TLR-mediated mechanisms and subsequent effector functions during pulmonary infection are largely unknown. We have previously demonstrated that mast cells, through the production of IL-4, effectively control Francisella tularensis replication. In this study, the highly human virulent strain of F. tularensis SCHU S4 and the live vaccine strain were used to investigate the contribution of mast cell/TLR regulation of Francisella. Mast cells required TLR2 for effective bacterial killing, regulation of the hydrolytic enzyme cathepsin L, and for coordination and trafficking of MHC class II and lysosomal-associated membrane protein 2. Infected TLR2(-/-) mast cells, in contrast to wild-type and TLR4(-/-) cells, lacked detectable IL-4 and displayed increased cell death with a 2-3 log increase of F. tularensis replication, but could be rescued with rIL-4 treatment. Importantly, MHC class II and lysosomal-associated membrane protein 2 localization with labeled F. tularensis in the lungs was greater in wild-type than in TLR2(-/-) mice. These results provide evidence for the important effector contribution of mast cells and TLR2-mediated signaling on early innate processes in the lung following pulmonary F. tularensis infection and provide additional insight into possible mechanisms by which intracellular pathogens modulate respiratory immune defenses.


Homogeneous and heterogeneous noncovalent dimers of formaldehyde and thioformaldehyde: structures, energetics, and vibrational frequencies.
This work provides the first characterization of five stationary points of the homogeneous thioformaldehyde dimer, (CH2S)2, and seven stationary points of the heterogeneous formaldehyde/thioformaldehyde dimer, CH2O/CH2S, with correlated ab initio electronic structure methods. Full geometry optimizations and corresponding harmonic vibrational frequencies were computed with second-order Møller-Plesset perturbation theory (MP2) and 13 different density functionals in conjunction with triple-ζ basis sets augmented with diffuse and multiple sets of polarization functions. The MP2 results indicate that the three stationary points of (CH2S)2 and four of CH2O/CH2S are minima, in contrast to two stationary points of the formaldehyde dimer, (CH2O)2. Single-point energies were also computed using the explicitly correlated MP2-F12 and CCSD(T)-F12 methods and basis sets as large as heavy-aug-cc-pVTZ. The (CH2O)2 and CH2O/CH2S MP2 and MP2-F12 binding energies deviated from the CCSD(T)-F12 binding energies by no more than 0.2 and 0.4 kcal mol(-1), respectively. The (CH2O)2 and CH2O/CH2S global minimum is the same at every level of theory. However, the MP2 methods overbind (CH2S)2 by as much as 1.1 kcal mol(-1), effectively altering the energetic ordering of the thioformaldehyde dimer minima relative to the CCSD(T)-F12 energies. The CCSD(T)-F12 binding energies of the (CH2O)2 and CH2O/CH2S stationary points are quite similar, with the former ranging from around -2.4 to -4.6 kcal mol(-1) and the latter from about -1.1 to -4.4 kcal mol(-1). Corresponding (CH2S)2 stationary points have appreciably smaller CCSD(T)-F12 binding energies ranging from ca. -1.1 to -3.4 kcal mol(-1). The vibrational frequency shifts upon dimerization are also reported for each minimum on the MP2 potential energy surfaces.


Measurement of both cyclic [3H]AMP and cyclic [3H]GMP in cultured vascular smooth muscle cells labeled with [3H]hypoxanthine: use in studies of cardiovascular drugs.
We describe a method for prelabeling cultured vascular smooth muscle cells that permits rapid and accurate measurements of changes in the amounts of cyclic AMP and of cyclic GMP in 5 x 10(5) cells. This procedure utilizes [3H]hypoxanthine to radiolabel both the adenine and guanine nucleotide pools and simple column chromatographic steps to isolate and separate the 3H-labeled cyclic nucleotides. The application of the method to studies of the actions of cardiovascular drugs on vascular smooth muscle cells is illustrated by measurements of the effects of isoproterenol, nitroprusside, and inhibitors of cyclic AMP phosphodiesterases on the cyclic nucleotide levels in these cells. If required, the mass amounts of cyclic AMP and cyclic GMP present could be determined by measurement of the specific radioactivities of the precursor [3H]ATP and [3H]GTP, respectively. The cyclic nucleotide values calculated by the latter method were almost identical to those obtained with larger numbers of cells using commercially available radioimmunoassays, thus validating the prelabeling assays. The method described should be applicable to any type of cultured cell that can utilize [3H]hypoxanthine to replenish its ATP and GTP pools.


Dectin-1 is a major beta-glucan receptor on macrophages.
Zymosan is a beta-glucan- and mannan-rich particle that is widely used as a cellular activator for examining the numerous responses effected by phagocytes. The macrophage mannose receptor (MR) and complement receptor 3 (CR3) have historically been considered the major macrophage lectins involved in the nonopsonic recognition of these yeast-derived particles. Using specific carbohydrate inhibitors, we show that a beta-glucan receptor, but not the MR, is a predominant receptor involved in this process. Furthermore, nonopsonic zymosan binding was unaffected by genetic CD11b deficiency or a blocking monoclonal antibody (mAb) against CR3, demonstrating that CR3 was not the beta-glucan receptor mediating this activity. To address the role of the recently described beta-glucan receptor, Dectin-1, we generated a novel anti-Dectin-1 mAb, 2A11. Using this mAb, we show here that Dectin-1 was almost exclusively responsible for the beta-glucan-dependent, nonopsonic recognition of zymosan by primary macro-phages. These findings define Dectin-1 as the leukocyte beta-glucan receptor, first described over 50 years ago, and resolves the long-standing controversy regarding the identity of this important molecule. Furthermore, these results identify Dectin-1 as a new target for examining the immunomodulatory properties of beta-glucans for therapeutic drug design.


Oxidative stress and antioxidant status in neonates born to pre-eclamptic mother.
Pre-eclampsia is a significant health problem and is the leading cause of maternal and perinatal mortality and morbidity. Low birth weight and prematurity are very common in pre-eclamptic mothers. Pre-eclampsia is associated with oxidative stress in the maternal circulation. To observe the effect of pre-eclampsia on neonates, this study was designed to explore oxidative stress and anti-oxidant status in the fetal circulation in pre-eclampsia. For this purpose, we collected cord bloods during delivery from Bangabandhu Sheikh Mujib Medical University. Twenty samples were collected from uncomplicated (normotensive) mothers and 15 samples were collected from pre-eclamptic mothers (maternal age matched). Thiobarbituric acid reactive substances (TBARS), lipid hydroperoxide, protein carbonyl value, lipid profile, total anti-oxidant status (TAS), vitamin C, serum total protein and albumin were measured. It was observed that TBARS and lipid hydroperoxide were significantly (P < 0.001) increased, protein carbonyl content were also significantly (P < 0.001) increased but total anti-oxidant status (P < 0.001) and vitamin C level were significantly (P < 0.05) decreased in cord blood from pre-eclamptic mother compared to control group. Cholesterol, TG, LDL level was elevated and HDL were lowered in cord blood in pre-eclamptic group compared to normotensive group. In pre-eclamptic group, cord blood total protein, albumin and globulin level were significantly decreased compared to control group. As pre-eclampsia is associated with increased oxidative stress and decreased anti-oxidant status, the results of these investigations suggest that oxidative stress and antioxidant status are altered towards proatherogenic level in cord blood of pre-eclamptic women which may ultimately be responsible for different complications of newborn babies of pre-eclamptic mothers.


The Jak2V617F oncogene associated with myeloproliferative diseases requires a functional FERM domain for transformation and for expression of the Myc and Pim proto-oncogenes.
The V617F activating point mutation in Jak2 is associated with a proportion of myeloproliferative disorders. In normal hematopoietic cells, Jak2 signals only when associated with a growth factor receptor, such as the erythropoietin receptor (EpoR). We sought to identify the molecular requirements for activation of Jak2V617F by introducing a point mutation in the FERM domain (Y114A), required for receptor binding. Whereas BaF3.EpoR cells are readily transformed by Jak2V617F to Epo independence, we found that the addition of the FERM domain mutation blocked transformation and the induction of reactive oxygen species. Further, while cells expressing Jak2V617F had constitutive activation of STAT5, cells expressing Jak2V617F/Y114A did not, suggesting that signaling is defective at a very proximal level. In addition, expression of the Myc and Pim proto-oncogenes by Jak2V617F was found to be FERM domain dependent. An inducible constitutively active STAT5 mutant expressed in BaF3 cells was sufficient to induce Myc and Pim. Finally, the FERM domain in Jak2V617F was also required for abnormal hematopoiesis in transduced primary murine fetal liver cells. Overall, our results suggest that constitutive activation of Jak2 requires an intact FERM domain for a transforming phenotype, and is necessary for activation of the major target of Jak2, STAT5.


Expression of NPAT, a novel substrate of cyclin E-CDK2, promotes S-phase entry.
To understand the mechanisms by which CDKs regulate cell cycle progression, it is necessary to identify and characterize the physiological substrates of these kinases. We have developed a screening method to identify novel CDK substrates. One of the cDNAs identified in the screen is identical to the recently isolated NPAT gene. Here we show that NPAT associates with cyclin E-CDK2 in vivo and can be phosphorylated by this CDK. The protein level of NPAT peaks at the G1/S boundary. Overexpression of NPAT accelerates S-phase entry, and this effect is enhanced by coexpression of cyclin E-CDK2. These results suggest that NPAT is a substrate of cyclin E-CDK2 and plays a role in S-phase entry.


Genomic and proteomic analyses of Prdm5 reveal interactions with insulator binding proteins in embryonic stem cells.
PRDM proteins belong to the SET domain protein family, which is involved in the regulation of gene expression. Although few PRDM members possess histone methyltransferase activity, the molecular mechanisms by which the other members exert transcriptional regulation remain to be delineated. In this study, we find that Prdm5 is highly expressed in mouse embryonic stem (mES) cells and exploit this cellular system to characterize molecular functions of Prdm5. By combining proteomics and next-generation sequencing technologies, we identify Prdm5 interaction partners and genomic occupancy. We demonstrate that although Prdm5 is dispensable for mES cell maintenance, it directly targets genomic regions involved in early embryonic development and affects the expression of a subset of developmental regulators during cell differentiation. Importantly, Prdm5 interacts with Ctcf, cohesin, and TFIIIC and cooccupies genomic loci. In summary, our data indicate how Prdm5 modulates transcription by interacting with factors involved in genome organization in mouse embryonic stem cells.


Substituent effects on the relaxation dynamics of furan, furfural and β-furfural: a combined theoretical and experimental approach.
For the series furan, furfural and β-furfural we investigated the effect of substituents and their positioning on the photoinduced relaxation dynamics in a combined theoretical and experimental approach. Using time resolved photoelectron spectroscopy with a high intensity probe pulse, we can, for the first time, follow the whole deactivation process of furan through a two photon probe signal. Using the extended 2-electron 2-orbital model [Nenov et al., J. Chem. Phys., 2011, 135, 034304] we explain the formation of one central conical intersection and predict the influence of the aldehyde group of the derivatives on its geometry. This, as well as the relaxation mechanisms from photoexcitation to the final outcome was investigated using a variety of theoretical methods. Complete active space self consistent field was used for on-the-fly calculations while complete active space perturbation theory and coupled cluster theory were used to accurately describe critical configurations. Experiment and theory show the relaxation dynamics of furfural and β-furfural to be slowed down, and together they disclose an additional deactivation pathway, which is attributed to the nO lonepair state introduced with the aldehyde group.


High-speed stroboscopic imaging with frequency-doubled supercontinuum.
We present a supercontinuum (SC) light source designed for stroboscopic white light interferometry. The compact, cost-effective SC source is built from off-the-shelf optical components and operates both in the visible and near-IR at arbitrary repetition rates in the 10 kHz-1 MHz frequency range. We estimate that our source allows performing dynamic white-light interferometric characterization of rapidly oscillating objects up to several tens of megahertz. Its current potential is demonstrated by capturing the movement of a microelectromechanical system oscillating at 2.16 MHz with sub-100 nm accuracy.


The orisome: structure and function.
During the cell division cycle of all bacteria, DNA-protein complexes termed orisomes trigger the onset of chromosome duplication. Orisome assembly is both staged and stringently regulated to ensure that DNA synthesis begins at a precise time and only once at each origin per cycle. Orisomes comprise multiple copies of the initiator protein DnaA, which oligomerizes after interacting with specifically positioned recognition sites in the unique chromosomal replication origin, oriC. Since DnaA is highly conserved, it is logical to expect that all bacterial orisomes will share fundamental attributes. Indeed, although mechanistic details remain to be determined, all bacterial orisomes are capable of unwinding oriC DNA and assisting with loading of DNA helicase onto the single-strands. However, comparative analysis of oriCs reveals that the arrangement and number of DnaA recognition sites is surprisingly variable among bacterial types, suggesting there are many paths to produce functional orisome complexes. Fundamental questions exist about why these different paths exist and which features of orisomes must be shared among diverse bacterial types. In this review we present the current understanding of orisome assembly and function in Escherichia coli and compare the replication origins among the related members of the Gammaproteobacteria. From this information we propose that the diversity in orisome assembly reflects both the requirement to regulate the conformation of origin DNA as well as to provide an appropriate cell cycle timing mechanism that reflects the lifestyle of the bacteria. We suggest that identification of shared steps in orisome assembly may reveal particularly good targets for new antibiotics.


Cell-free plasma human immunodeficiency virus type 1 titer assessed by culture and immunocapture-reverse transcription-polymerase chain reaction.
The relationship between plasma human immunodeficiency virus type 1 (HIV-1) infectious titer, determined by quantitative fivefold end-point dilution culture, and the detection of genomic HIV-1 RNA by immunocapture-cDNA-polymerase chain reaction was determined. The optimal plasma specimen collection and storage conditions for the use of such virologic markers for clinical trials were also determined. The variabilities in the measurement of infectious HIVLAI titer associated with intra- and interdonor peripheral blood mononuclear cells were 1.2 and 0.86 log10 50% tissue culture infective doses (TCID50)/ml (95% confidence interval range), respectively. Plasma HIV-1 titers did not change significantly after storing whole blood for 6 h either at 4 degrees C or ambient temperature or plasma for a median of 267 days (range, 259 to 482) at -70 degrees C. The detection of genomic HIV-1 RNA encapsulated in viral particles was very consistent, reproducible, and unaffected by either heparin or acid citrate or by multiple freeze-thawing. The HIV-1 RNA titers also appeared to generally correlate with the biologic titer obtained by the microculture assay. The consistency in infectious HIV-1 titer was evaluated by using 27 unfrozen plasma specimens collected from five subjects over 1 to 9 days. The median change in HIV-1 titer relative to baseline was -0.5 log10 TCID50/ml (interquartile range, -1.03 to 0.175 log10). In contrast, no significant change in HIV-1 RNA for the same frozen plasma specimens was noted. As such, immunocapture-cDNA-polymerase chain reaction may be a useful measure of plasma viremia for studying the natural history of HIV disease and assessing response to therapy.


Distribution of low-molecular lipophilic extractives beneath the surface of air- and kiln-dried Scots pine sapwood boards.
During industrial wood drying, extractives migrate towards the wood surfaces and make the material more susceptible to photo/biodegradation. The present work provides information about the distribution, quantity and nature of lipophilic substances beneath the surface in air- and kiln-dried Scots pine (Pinus sylvestris L.) sapwood boards. Samples were taken from knot-free sapwood surfaces and the composition of lipophilic extractives, phenols and low-molecular fatty/resin acids layers at different nominal depths below the surface was studied gravimetrically, by UV-spectrometry and by gas chromatography-mass spectrometry (GC-MS). The concentration of total extractives was significantly higher in kiln-dried than in air-dried samples and was higher close to the surface than in the layers beneath. The scatter in the values for the lipophilic extractives was high in both drying types, being highest for linoleic acid and slightly lower for palmitic, oleic and stearic acids. The amount of fatty acids was low in kiln-dried boards, probably due to a stronger degradation due to the high temperature employed. The most abundant resin acid was dehydroabietic acid followed by pimaric, isopimaric, and abietic acids in both drying types. It is concluded that during kiln-drying a migration front is created at a depth of 0.25 mm with a thickness of about 0.5 mm.


Protective role of 1,25(OH)2 vitamin D3 in the mucosal injury and epithelial barrier disruption in DSS-induced acute colitis in mice.
Intestinal hyper-permeability plays a critical role in the etiopathogenesis of inflammatory bowel disease (IBD) by affecting the penetration of pathogens, toxic compounds and macromolecules. 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the active form of vitamin D, has been shown to be an important regulator of IBD and recent epidemiology suggests that patients with IBD have an impaired vitamin D status. The purpose of this study is to investigate the possible protective effects of 1,25(OH)2D3 on mucosal injury and epithelial barrier disruption on dextran sulfate sodium (DSS)-induced acute colitis model. We used DSS-induced acute colitis model to investigate the protective effects of 1,25(OH)2D3 on mucosal injury and epithelial barrier integrity. Severity of colitis was evaluated by disease activity index (DAI), body weight (BW) change, colon length, histology, myeloperoxidase (MPO) activity, and proinflammatory cytokine production including tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). In vitro the protective role of 1,25(OH)2D3 was assessed by incubating Caco-2 cells with or without DSS and measuring transepithelial electrical resistance (TEER) and fluorescein isothiocyanate dextran (FITC-D). The intestinal permeability was analyzed by FITC-D, bacterial translocation and measurement of lipopolysaccharide (LPS). Ultrastructural features of the colon tissue and Caco-2 cell monolayer were observed by electron microscopy. Expressions of tight junction (TJ) proteins in the colon mucosa and Caco-2 cells were detected by immunohistochemistry, immunofluorescence, Western blot and real-time fluorescent quantitative PCR, respectively. DSS-induced acute colitis model was characterized by a reduced BW, AUC of BW, serum calcium, higher DAI, AUC of DAI, shortened colon length, elevated MPO activity, worsened histologic inflammation, increased mononuclear cell numbers in mesenteric lymph nodes (MLNs) and colonic lamina propria (LP), and enhanced proteins and mRNA levels of TNF-α and IFN-γ. 1,25(OH)2D3 markedly increased expressions of TJ proteins and mRNA and decreased the FITC-D permeability and the level of LPS. Furthermore, 1,25(OH)2D3 abrogated bacterial translocation to MLNs and ameliorated ultrastructural features of the colon epithelium by scanning electron microscopy (SEM). In vitro, 1,25(OH)2D3 increased TEER, TJ proteins and mRNA expressions, decreased the FITC-D permeability, and preserved structural integrity of the TJ in Caco-2 cells. 1,25(OH)2D3 may play a protective role in mucosal barrier homeostasis by maintaining the integrity of junction complexes and in healing capacity of the colon epithelium. 1,25(OH)2D3 may represent an attractive and novel therapeutic agent for the adjuvant therapy of IBD.


Guard Cell Salicylic Acid Signaling Is Integrated into Abscisic Acid Signaling via the Ca2+/CPK-Dependent Pathway.
The phenolic hormone salicylic acid (SA) induces stomatal closure. It has been suggested that SA signaling is integrated with abscisic acid (ABA) signaling in guard cells, but the integration mechanism remains unclear. The Ca2+-independent protein kinase Open Stomata1 (OST1) and Ca2+-dependent protein kinases (CPKs) are key for ABA-induced activation of the slow-type anion channel SLAC1 and stomatal closure. Here, we show that SA-induced stomatal closure and SA activation of slow-type anion channel are impaired in the CPK disruption mutant cpk3-2 cpk6-1 but not in the OST1 disruption mutant ost1-3 We also found that the key phosphorylation sites of SLAC1 in ABA signaling, serine-59 and serine-120, also are important for SA signaling. Chemiluminescence-based detection of superoxide anion revealed that SA did not require CPK3 and CPK6 for the induction of reactive oxygen species production. Taken together, our results suggest that SA activates peroxidase-mediated reactive oxygen species signal that is integrated into Ca2+/CPK-dependent ABA signaling branch but not the OST1-dependent signaling branch in Arabidopsis (Arabidopsis thaliana) guard cells.


The Inhibition of Ocean Acidification on the Formation of Oyster Calcified Shell by Regulating the Expression of Cgchs1 and Cgchit4.
The biosynthesis of a calcified shell is critical for the development of oyster larvae. This process can be severely inhibited by CO2-induced ocean acidification, causing mass mortality of oyster larvae. However, the underlying molecular mechanism of such process has not been well explored until now. In the present study, a homolog of chitin synthase (named as Cgchs1) and a homolog of chitinase (named as Cgchit4) were identified from the Pacific oyster Crassostrea gigas. The cDNA sequences of Cgchs1 and Cgchit4 were of 813 bp and 2118 bp, encoding a putative polypeptide of 271 amino acids and 706 amino acids, respectively. There were a Chitin_synth_2 domain and a Glyco_18 domain in the inferred amino acid sequences of Cgchs1 and Cgchit4, respectively. Both Cgchs1 and Cgchit4 shared high sequence identity with their homologs in vertebrates. In addition, when oyster larvae were exposed to acidification treatment (pH 7.4), their shell biosynthesis process was seriously restrained. The expression level of Cgchs1 mRNA was significantly suppressed while that of Cgchit4 was dramatically activated upon acidification treatment. Cgchs1 and Cgchit4 are critical enzymes for chitin metabolism, and such changes in their mRNA expression could result in the decrease of chitin content in oyster larvae's shells, which might lead to the failure of shell formation. Therefore, results in the present study suggested that acidified seawater might inhibit the formation of oyster calcified shell by suppressing the biosynthesis of chitin.


Pine bark extract (Pycnogenol®) suppresses cigarette smoke-induced fibrotic response via transforming growth factor-β1/Smad family member 2/3 signaling.
Chronic obstructive pulmonary diseases (COPD) is an important disease featured as intense inflammation, protease imbalance, and air flow limitation and mainly induced by cigarette smoke (CS). In present study, we explored the effects of Pycnogenol® (PYC, pine bark extract) on pulmonary fibrosis caused by CS+lipopolysaccharide (LPS) exposure. Mice were treated with LPS intranasally on day 12 and 26, followed by CS exposure for 1 h/day (8 cigarettes per day) for 4 weeks. One hour before CS exposure, 10 and 20 mg/kg of PYC were administered by oral gavage for 4 weeks. PYC effectively reduced the number of inflammatory cells and proinflammatory mediators caused by CS+LPS exposure in bronchoalveolar lavage fluid. PYC inhibited the collagen deposition on lung tissue caused by CS+LPS exposure, as evidenced by Masson's trichrome stain. Furthermore, transforming growth factor-β1 (TGF-β1) expression and Smad family member 2/3 (Smad 2/3) phosphorylation were effectively suppressed by PYC treatment. PYC markedly reduced the collagen deposition caused by CS+LPS exposure, which was closely involved in TGF-β1/Smad 2/3 signaling, which is associated with pulmonary fibrotic change. These findings suggest that treatment with PYC could be a therapeutic strategy for controlling COPD progression.


The differential effects of chronic imipramine or citalopram administration on physiological and behavioral outcomes in naïve mice.
Tricyclics and selective serotonin reuptake inhibitors (SSRIs) are probably the most widely employed reference antidepressants in animal studies on depression. Using imipramine and citalopram, we sought to assess which drug would be more appropriate as pharmacological reference in paradigms of depression in C57BL6N mice by measuring their effect on liquid consumption, home cage activity, body weight and long-term memory in naïve animals treated with each compound at generally used dose of 15 mg/kg/day. Continuous logging of home cage movement, weekly monitoring of vertical activity in a novel cage, and body weight was recorded during four-week treatment period and for four weeks after discontinuation of the antidepressant; sucrose preference was evaluated at weekly intervals during drug administration. A novel object recognition memory test was performed in mice treated the antidepressants for two weeks. Compared to control, imipramine-treated mice displayed increased sucrose and water intake, as well as enhanced home-cage and novelty exploration activities, and reduced body weight. Imipramine also impaired learning in the object recognition task, but citalopram diminished object exploration sufficiently to invalidate the test. Citalopram-treated animals demonstrated no changes in a sucrose test and had elevated body mass. Thus basic physiological and behavioral outcomes in naïve mice were significantly altered by the chronic administration of imipramine and, to a lesser extent, citalopram. As altered variables are crucial for the evaluation of antidepressant-like effects in mice, our data suggest that, at commonly used doses, both drugs must be applied in mouse models of depression with caution.


Variables associated with communicative participation in people with multiple sclerosis: a regression analysis.
To explore variables associated with self-reported communicative participation in a sample (n = 498) of community-dwelling adults with multiple sclerosis (MS). A battery of questionnaires was administered online or on paper per participant preference. Data were analyzed using multiple linear backward stepwise regression. The dependent variable was an item response theory score of communicative participation measured by a subset of items from the Communicative Participation Item Bank asking respondents to rate how much their health condition interfered with participation in real-life speech communication situations. Thirteen independent variables were included in the model as self-reported symptoms: problems thinking, slurred speech, vision loss, pain, mobility, depression, fatigue, perceived social support, age, education level, employment status, gender, and MS duration. Fatigue, slurred speech, depression, problems thinking, employment status, and social support were significantly associated with communicative participation, accounting for 48.7% of the variance. Communicative participation is significantly associated with multiple variables, only some of which reflect communication disorders. If the goal of intervention is to improve communicative participation, intervention may need to extend beyond traditional speech-language pathology boundaries to include other health symptoms as well as personal, social, and physical environments.


Sumoylation by Ubc9 regulates the stem cell compartment and structure and function of the intestinal epithelium in mice.
Small ubiquitin-like modifiers (SUMOs) are attached to other proteins to regulate their function (sumoylation). We investigated the role of Ubc9, which covalently attaches SUMOs to proteins, in the gastrointestinal tract of adult mice. We investigated the effects of decreased sumoylation in adult mammals by generating mice with an inducible knockout (by injection of 4-hydroxytamoxifen) of the E2 enzyme Ubc9 (Ubc9fl/-/ROSA26-CreERT2 mice). We analyzed the phenotypes using a range of histologic techniques. Loss of Ubc9 from adult mice primarily affected the small intestine. Ubc9fl/-/ROSA26-CreERT2 mice died within 6 days of 4-hydroxytamoxifen injection, losing 20% or less of their body weight and developing severe diarrhea on the second day after injection. Surprisingly, other epithelial tissues appeared to be unaffected at that stage. Decreased sumoylation led to the depletion of the intestinal proliferative compartment and to the rapid disappearance of stem cells. Sumoylation was required to separate the proliferative and differentiated compartments from the crypt and control differentiation and function of the secretory lineage. Sumoylation was required for nucleus positioning and polarized organization of actin in the enterocytes. Loss of sumoylation caused detachment of the enterocytes from the basal lamina, as observed in tissue fragility diseases. We identified the intermediate filament keratin 8 as a SUMO substrate in epithelial cells. Sumoylation maintains intestinal stem cells and the architecture, mechanical stability, and function of the intestinal epithelium of mice.