Arterial and venous anatomies of the deep inferior epigastric perforator and superficial inferior epigastric artery flaps.
This study uses three- and four-dimensional computed tomographic angiography and venography to evaluate the microvascular anatomy and perfusion of the deep inferior epigastric artery perforator (DIEP) and superficial inferior epigastric artery (SIEA) flaps. Ten DIEP flaps harvested from fresh cadavers and two abdominoplasty specimens were used. Studies of the largest single perforators from the medial and lateral rows were performed. Injections of the vena comitans of the perforators and of the superficial inferior epigastric veins were performed to evaluate venous drainage. Zone IV was not perfused following injection of a lateral row perforator, whereas injection of a medial row perforator consistently resulted in perfusion of zone IV. Image analysis revealed the presence of large-diameter linking vessels at the level of the subdermal plexus between the perforators of the medial row, whereas lateral row perforators predominantly perfused the lateral aspect of the flap and perfused the medial row perforators by means of recurrent flow through the subdermal plexus. Medial row perforators perfused the flap in a central elliptical pattern, whereas lateral row perforators predominantly perfused the ipsilateral portion of the flap. The SIEA vessel branches were seen to course to the subdermal plexus and then to perfuse ipsilateral perforators through the subdermal plexus. Venous drainage occurred by means of the superficial inferior epigastric veins and the venae comitantes of the perforators. This study demonstrates that flow through medial and lateral row perforators occurs in physiologically stereotyped patterns. A medial row perforator should be selected if zone IV perfusion is required. The SIEA was consistently seen to only perfuse a hemiflap.


Network reorganization of dynamic covalent polymer gels with exchangeable diarylbibenzofuranone at ambient temperature.
Reversible bonds and interactions have been utilized to build stimuli-responsive and reorganizable polymer networks that show recyclability, plasticity, and self-healing. In addition, reorganization of polymer gels at ambient temperature, such as room or body temperature, is expected to lead to several biomedical applications. Although these stimuli-responsive properties originate from the reorganization of the polymer networks, not such microscopic structural changes but instead only macroscopic properties have been the focus of previous work. In the present work, the reorganization of gel networks with diarylbibenzofuranone (DABBF)-based dynamic covalent linkages in response to the ambient temperature was systematically investigated from the perspective of both macroscopic and microscopic changes. The gels continued to swell in suitable solvents above room temperature but attained equilibrium swelling in nonsolvents or below room temperature because of the equilibrium of DABBF linkages, as supported by electron paramagnetic resonance measurements. Small-angle X-ray scattering measurements revealed the mesh sizes of the gels to be expanded and the network structures reorganized under control at ambient temperature.


Development of a Semiselective Medium for Detection of Nalanthamala psidii, Causal Agent of Wilt of Guava.
Guava wilt, caused by Nalanthamala psidii, has become an important disease of guava (Psidium guajava) in Taiwan since the 1970s. This study was conducted to develop a semiselective medium for detecting N. psidii in soil and in tissues of diseased guava trees. Among 9 carbon and 21 nitrogen compounds tested in a modified Czapek-Dox medium, the most effective carbon and nitrogen sources for mycelial growth of N. psidii were sucrose and glycine, respectively. Among eight fungicides tested, iprodione at 5 μg ml-1 and azoxystrobin at 1 μg ml-1 were the most effective fungicides for detection of N. psidii in artificially infested soil or in naturally infected guava debris. Based on the requirement for carbon and nitrogen sources and response to fungicides, a semiselective medium designated as modified sucrose-glycine semiselective medium (mSGSSM) was developed for isolation of N. psidii, using the modified Czapek-Dox medium containing 3% sucrose, 0.3% glycine, iprodione at 5 μg ml-1, azoxystrobin at 1 μg ml-1, streptomycin at 200 μg ml-1, and neomycin at 200 μg ml-1. Colonies of N. psidii on mSGSSM at 30°C for 5 to 10 days were white to orange with sparse aerial hyphae. N. psidii was detected more accurately and efficiently on mSGSSM than on other media, including potato dextrose agar, modified Nash-Snyder medium, and modified Czapek-Dox medium. This semiselective medium is effective in detection of N. psidii from various parts of diseased guava trees and in soil; therefore, it would be a useful medium for etiological, ecological, and epidemiological studies of guava wilt.


Predicting shockwave lithotripsy outcome for urolithiasis using clinical and stone computed tomography texture analysis variables.
We aimed to develop and evaluate a statistical model, which included known pre-treatment factors and new computed tomography texture analysis (CTTA) variables, for its ability to predict the likelihood of a successful outcome after extracorporeal shockwave lithotripsy (SWL) treatment for renal and ureteric stones. Up to half of patients undergoing SWL may fail treatment. Better prediction of which cases will likely succeed SWL will help patients to make an informed decision on the most effective treatment modality for their stone. 19 pre-treatment factors for SWL success, including 6 CTTA variables, were collected from 459 SWL cases at a single centre. Univariate and multivariable analyses were performed by independent statisticians to predict the probability of a stone free (both with and without residual fragments) outcome after SWL. A multivariable model had an overall accuracy of 66% on Receiver Operator Curve (ROC) analysis to predict for successful SWL outcome. The variables most frequently chosen for the model were those which represented stone size. Although previous studies have suggested SWL can be reliably predicted using pre-treatment factors and that analysis of CT stone images may improve outcome prediction, the results from this study have not produced a useful model for SWL outcome prediction.


Combining Unfolding Reversibility Studies and Molecular Dynamics Simulations to Select Aggregation-Resistant Antibodies.
The efficient development of new therapeutic antibodies relies on developability assessment with biophysical and computational methods to find molecules with drug-like properties such as resistance to aggregation. Despite the many novel approaches to select well-behaved proteins, antibody aggregation during storage is still challenging to predict. For this reason, there is a high demand for methods that can identify aggregation-resistant antibodies. Here, we show that three straightforward techniques can select the aggregation-resistant antibodies from a dataset with 13 molecules. The ReFOLD assay provided information about the ability of the antibodies to refold to monomers after unfolding with chemical denaturants. Modulated scanning fluorimetry (MSF) yielded the temperatures that start causing irreversible unfolding of the proteins. Aggregation was the main reason for poor unfolding reversibility in both ReFOLD and MSF experiments. We therefore performed temperature ramps in molecular dynamics (MD) simulations to obtain partially unfolded antibody domains in silico and used CamSol to assess their aggregation potential. We compared the information from ReFOLD, MSF, and MD to size-exclusion chromatography (SEC) data that shows whether the antibodies aggregated during storage at 4, 25, and 40 °C. Contrary to the aggregation-prone molecules, the antibodies that were resistant to aggregation during storage at 40 °C shared three common features: (i) higher tendency to refold to monomers after unfolding with chemical denaturants, (ii) higher onset temperature of nonreversible unfolding, and (iii) unfolding of regions containing aggregation-prone sequences at higher temperatures in MD simulations.


Implementing resilience engineering for healthcare quality improvement using the CARE model: a feasibility study protocol.
Resilience engineering (RE) is an emerging perspective on safety in complex adaptive systems that emphasises how outcomes emerge from the complexity of the clinical environment. Complexity creates the need for flexible adaptation to achieve outcomes. RE focuses on understanding the nature of adaptations, learning from success and increasing adaptive capacity. Although the philosophy is clear, progress in applying the ideas to quality improvement has been slow. The aim of this study is to test the feasibility of translating RE concepts into practical methods to improve quality by designing, implementing and evaluating interventions based on RE theory. The CARE model operationalises the key concepts and their relationships to guide the empirical investigation. The settings are the Emergency Department and the Older Person's Unit in a large London teaching hospital. Phases 1 and 2 of our work, leading to the development of interventions to improve the quality of care, are described in this paper. Ethical approval has been granted for these phases. Phase 1 will use ethnographic methods, including observation of work practices and interviews with staff, to understand adaptations and outcomes. The findings will be used to collaboratively design, with clinical staff in interactive design workshops, interventions to improve the quality of care. The evaluation phase will be designed and submitted for ethical approval when the outcomes of phases 1 and 2 are known. Study outcomes will be knowledge about the feasibility of applying RE to improve quality, the development of RE theory and a validated model of resilience in clinical work which can be used to guide other applications. Tools, methods and practical guidance for practitioners will also be produced, as well as specific knowledge of the potential effectiveness of the implemented interventions in emergency and older people's care. Further studies to test the application of RE at a larger scale will be required, including studies of other healthcare settings, organisational contexts and different interventions.


FluKB: A Knowledge-Based System for Influenza Vaccine Target Discovery and Analysis of the Immunological Properties of Influenza Viruses.
FluKB is a knowledge-based system focusing on data and analytical tools for influenza vaccine discovery. The main goal of FluKB is to provide access to curated influenza sequence and epitope data and enhance the analysis of influenza sequence diversity and the analysis of targets of immune responses. FluKB consists of more than 400,000 influenza protein sequences, known epitope data (357 verified T-cell epitopes, 685 HLA binders, and 16 naturally processed MHC ligands), and a collection of 28 influenza antibodies and their structurally defined B-cell epitopes. FluKB was built using a modular framework allowing the implementation of analytical workflows and includes standard search tools, such as keyword search and sequence similarity queries, as well as advanced tools for the analysis of sequence variability. The advanced analytical tools for vaccine discovery include visual mapping of T- and B-cell vaccine targets and assessment of neutralizing antibody coverage. FluKB supports the discovery of vaccine targets and the analysis of viral diversity and its implications for vaccine discovery as well as potential T-cell breadth and antibody cross neutralization involving multiple strains. FluKB is representation of a new generation of databases that integrates data, analytical tools, and analytical workflows that enable comprehensive analysis and automatic generation of analysis reports.


Genetic Variants Influencing Joint Damage in Mexican Americans and European Americans With Rheumatoid Arthritis.
Joint destruction in rheumatoid arthritis (RA) is heritable, but knowledge on specific genetic determinants of joint damage in RA is limited. We have used the Immunochip array to examine whether genetic variants influence variation in joint damage in a cohort of Mexican Americans (MA) and European Americans (EA) with RA. We studied 720 MA and 424 EA patients with RA. Joint damage was quantified using a radiograph of both hands and wrists, scored using Sharp's technique. We conducted association analyses with the transformed Sharp score and the Immunochip single nucleotide polymorphism (SNP) data using PLINK. In MAs, 15 SNPs from chromosomes 1, 5, 9, 17 and 22 associated with joint damage yielded strong p-values (p < 1 × 10(-4) ). The strongest association with joint damage was observed with rs7216796, an intronic SNP located in the MAP3K14 gene, on chromosome 17 (β ± SE = -0.25 ± 0.05, p = 6.23 × 10(-6) ). In EAs, 28 SNPs from chromosomes 1, 4, 6, 9, and 21 showed associations with joint damage (p-value < 1 × 10(-4) ). The best association was observed on chromosome 9 with rs59902911 (β ± SE = 0.86 ± 0.17, p = 1.01 × 10(-6) ), a synonymous SNP within the CARD9 gene. We also observed suggestive evidence for some loci influencing joint damage in MAs and EAs. We identified two novel independent loci (MAP3K14 and CARD9) strongly associated with joint damage in MAs and EAs and a few shared loci showing suggestive evidence for association.


Patterns of Benign Prostate Hyperplasia Based on Magnetic Resonance Imaging Are Correlated With Lower Urinary Tract Symptoms and Continence in Men Undergoing a Robot-assisted Radical Prostatectomy for Prostate Cancer.
To investigate the association between benign prostatic hyperplasia (BPH) patterns, classified by magnetic resonance imaging (MRI), with lower urinary tract symptoms (LUTS) or continence, preoperatively and after robot-assisted laparoscopic radical prostatectomy (RARP). This retrospective study included 49 prostate cancer patients, with prostate size >47 cm3, who underwent an endorectal MRI followed by RARP. Five BPH patterns were identified according to Wasserman, and additional prostate measurements were recorded. LUTS were assessed using the International Prostate Symptom Score and the PR25-LUTS-Questionnaire score. Continence was assessed using the International Consultation of Incontinence Questionnaire-Short Form. BPH pattern 3 (44.9%) was identified most common, followed by pattern 5 (26.6%), 1 (24.5%), and 2 and 4 (both 2%). BPH patterns were significant predictors of preoperative LUTS, with pedunculated with bilateral transition zone (TZ) and retrourethral enlargement (pattern 5) causing more severe symptoms compared with bilateral TZ and retrourethral enlargement (pattern 3) and bilateral TZ enlargement (pattern 1), whereas pattern 3 was additionally associated with more voiding symptoms compared with pattern 1. None of the BPH patterns was predictive of postoperative LUTS and continence. Independent predictors of continence at 12 months were lower preoperative PR25-LUTS score (P = .022) and longer membranous urethral length (P = .025). MRI is useful for classifying patients in BPH patterns which are strongly associated with preoperative LUTS. However, BPH patterns did not predict remnant LUTS or postoperative incontinence. Postoperative continence status was only associated with preoperative LUTS and membranous urethra length.


International Federation of gynecology and obstetrics staging classification for cancer of the ovary, fallopian tube, and peritoneum: estimation of survival in patients with node-positive epithelial ovarian cancer.
The objective of this study was to determine the survival of patients with node-positive epithelial ovarian cancer according to the 2014 International Federation of Gynecology and Obstetrics (FIGO) staging system. We performed a retrospective chart review. Data from all consecutive patients with node-positive epithelial ovarian cancer (stages IIIC and IV) who underwent cytoreductive surgery at the Mayo Clinic from 1996 to 2000 were reassessed to evaluate the prognostic significance of the new FIGO stages. Multivariate Cox regression was performed, and Kaplan-Meier survival curves constructed. The distribution of the restaged patients was as follows: IIIA1, 23 patients (IIIA1i, 9 patients; and IIIA1ii, 14 patients); IIIA2, 3 patients; IIIB, 4; IIIC, 67 patients; IVA, 4 patients; and IVB, 15 patients. In the univariate analysis, the relative risk for positive nodes greater than 10 mm on the longer axis was 2.57 and 3.00 for patients with microscopic peritoneal disease, compared with patients with microscopic positive nodes. However, the difference was not statistically significant. Moreover, the univariate analyses revealed statistically significant differences for 2014 FIGO stages (IIIA, IIIB, IIIC, and IVA-B), anatomical sites of peritoneal metastases, and disease staged at IIIC because of the presence of omental metastases. Multivariate analysis showed that survival was higher in patients restaged to IIIA-B than in those restaged to IIIC and IV (hazard ratios, 2.75 and 3.16, respectively; P = 0.002). The hazard ratio for patients with abdominal peritoneal metastases was 2.76 compared with patients with pelvic peritoneal metastases (P = 0.001). The current 2014 FIGO staging system for ovarian cancer successfully correlates survival, anatomical location of peritoneal metastases, and extra-abdominal lymph node metastases.


Dihydrocodeine for detoxification and maintenance treatment in individuals with opiate use disorders.
Medical treatment and detoxification from opiate disorders includes oral administration of opioid agonists. Dihydrocodeine (DHC) substitution treatment is typically low threshold and therefore has the capacity to reach wider groups of opiate users. Decisions to prescribe DHC to patients with less severe opiate disorders centre on its perceived safety, reduced toxicity, shorter half-life and more rapid onset of action, and potential retention of patients. This review set out to investigate the effects of DHC in comparison to other pharmaceutical opioids and placebos in the detoxification and substitution of individuals with opiate use disorders. To investigate the effectiveness of DHC in reducing illicit opiate use and other health-related outcomes among adults compared to other drugs or placebos used for detoxification or substitution therapy. In February 2019 we searched Cochrane Drugs and Alcohol's Specialised Register, CENTRAL, PubMed, Embase and Web of Science. We also searched for ongoing and unpublished studies via ClinicalTrials.gov, the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP) and Trialsjournal.com. All searches included non-English language literature. We handsearched references of topic-related systematic reviews and the included studies. We included randomised controlled trials that evaluated the effect of DHC for detoxification and maintenance substitution therapy for adolescent (aged 15 years and older) and adult illicit opiate users. The primary outcomes were abstinence from illicit opiate use following detoxification or maintenance therapy measured by self-report or urinalysis. The secondary outcomes were treatment retention and other health and behaviour outcomes. We followed the standard methodological procedures that are outlined by Cochrane. This includes the GRADE approach to appraise the quality of evidence. We included three trials (in five articles) with 385 opiate-using participants that measured outcomes at different follow-up periods in this review. Two studies with 150 individuals compared DHC with buprenorphine for detoxification, and one study with 235 participants compared DHC to methadone for maintenance substitution therapy. We downgraded the quality of evidence mainly due to risk of bias and imprecision. For the two studies that compared DHC to buprenorphine, we found low-quality evidence of no significant difference between DHC and buprenorphine for detoxification at six-month follow-up (risk ratio (RR) 0.59, 95% confidence interval (CI) 0.25 to 1.39; P = 0.23) in the meta-analysis for the primary outcome of abstinence from illicit opiates. Similarly, low-quality evidence indicated no difference for treatment retention (RR 1.29, 95% CI 0.99 to 1.68; P = 0.06). In the single trial that compared DHC to methadone for maintenance substitution therapy, the evidence was also of low quality, and there may be no difference in effects between DHC and methadone for reported abstinence from illicit opiates (mean difference (MD) -0.01, 95% CI -0.31 to 0.29). For treatment retention at six months' follow-up in this single trial, the RR calculated with an intention-to-treat analysis also indicated that there may be no difference between DHC and methadone (RR 1.04, 95% CI 0.94 to 1.16). The studies that compared DHC to buprenorphine reported no serious adverse events, while the DHC versus methadone study reported one death due to methadone overdose. We found low-quality evidence that DHC may be no more effective than other commonly used pharmacological interventions in reducing illicit opiate use. It is therefore premature to make any conclusive statements about the effectiveness of DHC, and it is suggested that further high-quality studies are conducted, especially in low- to middle-income countries.


Epidermal growth factor receptor ligands in murine models for erythropoietic protoporphyria: potential novel players in the progression of liver injury.
Activation of the epidermal growth factor receptor (EGFR) plays an important role in liver regeneration and resistance to acute injury. However its chronic activation participates in the progression of liver disease, including fibrogenesis and malignant transformation. Hepatobiliary disease represents a constant feature in the clinically relevant Fechm1pas/Fechm1pas genetic model of erythropoietic protoporphyria (EPP). Similarly, chronic administration of griseofulvin to mice induces pathological changes similar to those found in patients with EPP-associated liver injury. We investigated the hepatic expression of the EGFR and its seven most relevant ligands in Fechm1pas/Fechm1pas mice bred in three different backgrounds, and in griseofulvin-induced protoporphyria. We observed that the expression of amphiregulin, betacellulin and epiregulin was significantly increased in young EPP mice when compared to aged-matched controls in all genetic backgrounds. The expression of these ligands was also tested in older (11 months) BALB/cJ EPP mice, and it was found to remain induced, while that of the EGFR was downregulated. Griseofulvin feeding also increased the expression of amphiregulin, betacellulin and epiregulin. Interestingly, protoporphyrin accumulation in cultured hepatic AML-12 cells readily elicited the expression of these three EGFR ligands. Our findings suggest that protoporphyrin could directly induce the hepatic expression of EGFR ligands, and that their chronic upregulation might participate in the pathogenesis of EPP-associated liver disease.


Vesicular Trans-Cell Wall Transport in Fungi: A Mechanism for the Delivery of Virulence-Associated Macromolecules?
Fungal cells are encaged in rigid, complex cell walls. Until recently, there was remarkably little information regarding the trans-fungal cell wall transfer of intracellular macromolecules to the extracellular space. Recently, several studies have begun to elucidate the mechanisms that fungal cells utilize to secrete a wide variety of macromolecules through the cell wall. The combined use of transmission electron microscopy, serology, biochemistry, proteomics and lipidomics have revealed that the fungal pathogens Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida parapsilosis and Sporothrix schenckii, as well as the model yeast Saccharomyces cerevisiae, each produces extracellular vesicles that carry lipids, proteins, polysaccharides and pigment-like structures of unquestionable biological significance. Compositional analysis of the C. neoformans and H. capsulatum extracellular vesicles suggests that they may function as 'virulence bags', with the potential to modulate the host-pathogen interaction in favor of the fungus. The cellular origin of the extracellular vesicles remains unknown, but morphological and biochemical features indicate that they are similar to the well-described mammalian exosomes.


Dental pulp regeneration in paediatric dentistry: the role of stem cells.
In the last decade, tissue engineering has undergone enormous expansion in the fields of regenerative medicine and dentistry. In response to mechanical and chemical injuries, the dentine-pulp complex possesses a regenerative ability due to a speci?c population of mesenchymal stem cells, which have been widely described in literature. The postnatal dental pulp also contains progenitor stem cells that participate in dental regeneration. Knowledge of the regenerative potential of these cells is important in terms of understanding and applying their biological and chemical characteristics. Through the isolation and exploration of the regenerative abilities of these stem cells, new therapeutic possibilities may be possible, opening the way for improving paediatric endodontic therapy.


Endothelial nitric oxide synthase gene is associated with vessel stenosis in Korean population.
Nitric oxide (NO) produced by endothelial NO synthase (eNOS) mediates endothelium-dependent vasodilation and antithrombotic action. Controversial results regarding the association of eNOS gene polymorphisms with vessel stenosis have been reported. Age- and sex-matched 932 individuals (656 subjects having 1-, 2-, and 3-stenosed vessels and 276 controls without stenosis) living in Seoul and surrounding suburbs were selected. A GT missense mutation in exon 7 (894GT) was screened using PCR-restriction fragment length polymorphism analysis. The genotypes of a 27-bp insertion/deletion in intron 4 (eNOS4b/a) and a TC mutation in promoter region of -786 (-786TC) were determined by the banding pattern on gel electrophoresis and a commercially available minisequencing protocol (SNaPshot), respectively. The eNOS4a allele was highly linked to the -786C allele (r=0.93, P<0.0001) while there was no linkage between eNOS4a allele and 894T allele or between 894T allele and -786C allele. Furthermore, 894T allele, but not eNOS4a (-786C) allele, was associated with the presence, but not the number, of stenosed vessels (odds ratio=1.57 for dominant effect of the T allele, P<0.05, and 1.49 for additive effect, P<0.05). Multiple logistic regression analysis revealed that 894T allele and hypertension were predictive independent risk factors for the presence of vessel stenosis. Our data suggest that eNOS gene polymorphisms may play an important role in the pathogenesis of vessel stenosis in Korean population.


Origin of 1/f noise in hydration dynamics on lipid membrane surfaces.
Water molecules on lipid membrane surfaces are known to contribute to membrane stability by connecting lipid molecules and acting as a water bridge. Although water structures and diffusivities near the membrane surfaces have been extensively studied, hydration dynamics on the surfaces has remained an open question. Here we investigate residence time statistics of water molecules on the surface of lipid membranes using all-atom molecular dynamics simulations. We show that hydration dynamics on the lipid membranes exhibits 1/f noise. Constructing a dichotomous process for the hydration dynamics, we find that residence times in each state follow a power-law with exponential cutoff and that the process can be regarded as a correlated renewal process where interoccurrence times are correlated. The results imply that the origin of the 1/f noise in hydration dynamics on the membrane surfaces is a combination of a power-law distribution with cutoff of interoccurrence times of switching events and a long-term correlation between the interoccurrence times. These results suggest that the 1/f noise attributed to the correlated renewal process may contribute to the stability of the hydration layers and lipid membranes.


Effect of computer-generated tailored feedback on glycemic control in people with diabetes in the community: a randomized controlled trial.
It is unknown whether computer-generated, patient-tailored feedback leads to improvements in glycemic control in people with type 2 diabetes. We recruited people with type 2 diabetes aged ≥ 40 years with a glycated hemoglobin (A1C) ≥ 7%, living in Hamilton, Canada, who were enrolled in a community-based program (Diabetes Hamilton) that provided regular evidence-based information and listings of community resources designed to facilitate diabetes self-management. After completing a questionnaire, participants were randomly allocated to either receive or not receive periodic computer-generated, evidence-based feedback on the basis of their questionnaire responses and designed to facilitate improved glycemic control and diabetes self-management. The primary outcome was a change in A1C after 1 year. A total of 465 participants (50% women, mean age 62 years, and mean A1C 7.83%) were randomly assigned, and 12-month A1C values were available in 96% of all participants, at which time the A1C level had decreased by an absolute amount of 0.24 and 0.15% in the intervention and control groups, respectively. The difference in A1C reduction for the intervention versus control group was 0.09% (95% CI -0.08 to 0.26; P = 0.3). No between-group differences in measures of quality of life, diabetes self-management behaviors, or clinical outcomes were observed. Providing computer-generated tailored feedback to registrants of a generic, community-based program that supports diabetes self-management does not lead to lower A1C levels or a better quality of life than participation in the community-based program (augmented by periodic A1C testing) alone.


Sequential events in thymocyte differentiation and thymus regeneration revealed by a combination of bromodeoxyuridine DNA labeling and antimitotic drug treatment.
The proliferative status of thymocyte cell subsets in vivo was assessed by observing the effect of two antimitotic drugs, hydroxyurea (HU) and demecolcine. Both drugs had the greatest effect on the double-positive (DP) subset followed by the L3T4+ single-positive (SP) subset. However, the decrease in the latter type was delayed by several days, showing that their precursors rather than the cells themselves were killed by HU. Double-negative (DN) cells were less affected, indicating that they contain a resting subset and that they are renewed by emigration rather than by autonomous in situ proliferation. After drug treatment all cycling cells were eliminated from the thymus but, as shown by in vivo and in vitro bromodeoxyuridine incorporation, new cells rapidly reentered in cycle, starting from DN cells and Lyt-2+ SP cells and followed by DP cells. Lyt-2+ SP cycling cells represent an intermediary stage between DN and DP cells, and they are very transient. Injection of HU 24 h after in vivo BrdUrd labeling eliminated most labeled DN cells, but did not prevent the emergence of L3T4+ SP-labeled cells on day 3 as observed in control thymuses. These results suggest that these L3T4+ SP cells are generated from DP cells in the absence of proliferation. Cycling cells in the regenerating thymus were first located at the corticomedullary junction and then in the subcapsular region, suggesting a reverse migration process to that observed after cessation of proliferation. A model is proposed to summarize these sequential events.


Construction of a baculovirus-silkworm multigene expression system and its application on producing virus-like particles.
A new baculovirus-silkworm multigene expression system named Bombyx mori MultiBac is developed and described here, by which multiple expression cassettes can be introduced into the Bombyx mori nuclear polyhedrosis virus (BmNPV) genome efficiently. The system consists of three donor vectors (pCTdual, pRADM and pUCDMIG) and an invasive diaminopimelate (DAP) auxotrophic recipient E. coli containing BmNPV-Bacmid (BmBacmid) with a homologous recombination region, an attTn7 site and a loxp site. Two genes carried by pCTdual are firstly inserted into BmBacmid by homologous recombination, while the other eight genes in pRADM and pUCDMIG are introduced into BmBacmid through Tn7 transposition and cre-loxp recombination. Then the invasive and DAP auxotrophic E. coli carrying recombinant BmBacmid is directly injected into silkworm for expressing heterologous genes in larvae or pupae. Three structural genes of rotavirus and three fluorescent genes have been simultaneously expressed in silkworm larvae using our new system, resulting in the formation of virus-like particles (VLPs) of rotavirus and the color change of larvae. The VLPs were purified from hemolymph by ultracentrifugation using CsCl gradients, with a yield of 12.7 µg per larva. For the great capacity of foreign genes and the low cost of feeding silkworm, this high efficient BmMultiBac expression system provides a suitable platform to produce VLPs or protein complexes.


Myocardial contraction and hyaluronic acid mechanotransduction in epithelial-to-mesenchymal transformation of endocardial cells.
Epithelial-to-mesenchymal transition (EMT) of endocardial cells is a critical initial step in the formation of heart valves. The collagen gel in vitro model has provided significant information on the role of growth factors regulating EMT but has not permitted investigation of mechanical factors. Therefore we sought to develop a system to probe the effects of mechanical inputs on endocardial EMT by incorporating hyaluronic acid (HA), the primary component of endocardial cushions in developing heart valves, into the gel assay. This was achieved using a combination collagen and crosslinkable methacrylated HA hydrogel (Coll-MeHA). Avian atrioventricular canal explants on Coll-MeHA gels showed increased numbers of transformed cells. Analysis of the mechanical properties of Coll-MeHA gels shows that stiffness does not directly affect EMT. Hydrogel deformation from the beating myocardium of explants directly led to higher levels of regional gel deformation and larger average strain magnitudes associated with invaded cells on Coll-MeHA gels. Inhibition of this contraction reduced EMT on all gel types, although to a lesser extent on Coll-MeHA gels. Using the system we have developed, which permits the manipulation of mechanical factors, we have demonstrated that active mechanical forces play a role in the regulation of endocardial EMT.


Transcriptome-Wide Annotation of m5C RNA Modifications Using Machine Learning.
The emergence of epitranscriptome opened a new chapter in gene regulation. 5-methylcytosine (m5C), as an important post-transcriptional modification, has been identified to be involved in a variety of biological processes such as subcellular localization and translational fidelity. Though high-throughput experimental technologies have been developed and applied to profile m5C modifications under certain conditions, transcriptome-wide studies of m5C modifications are still hindered by the dynamic and reversible nature of m5C and the lack of computational prediction methods. In this study, we introduced PEA-m5C, a machine learning-based m5C predictor trained with features extracted from the flanking sequence of m5C modifications. PEA-m5C yielded an average AUC (area under the receiver operating characteristic) of 0.939 in 10-fold cross-validation experiments based on known Arabidopsis m5C modifications. A rigorous independent testing showed that PEA-m5C (Accuracy [Acc] = 0.835, Matthews correlation coefficient [MCC] = 0.688) is remarkably superior to the recently developed m5C predictor iRNAm5C-PseDNC (Acc = 0.665, MCC = 0.332). PEA-m5C has been applied to predict candidate m5C modifications in annotated Arabidopsis transcripts. Further analysis of these m5C candidates showed that 4nt downstream of the translational start site is the most frequently methylated position. PEA-m5C is freely available to academic users at: https://github.com/cma2015/PEA-m5C.


Acute cardiac injury events ≤30 days after laboratory-confirmed influenza virus infection among U.S. veterans, 2010-2012.
Cardiac injury is a known potential complication of influenza infection. Because U.S. veterans cared for at the U.S. Department of Veterans Affairs are older and have more cardiovascular disease (CVD) risk factors than the general U.S. population, veterans are at risk for cardiac complications of influenza infection. We investigated biomarkers of cardiac injury characteristics and associated cardiac events among veterans who received cardiac biomarker testing ≤30 days after laboratory-confirmed influenza virus infection. Laboratory-confirmed influenza cases among veterans cared for at U.S. Department of Veterans Affairs' facilities for October 2010-December 2012 were identified using electronic medical records (EMRs). Influenza confirmation was based on respiratory specimen viral culture or antigen or nucleic acid detection. Acute cardiac injury (ACI) was defined as an elevated cardiac biomarker (troponin I or creatinine kinase isoenzyme MB) >99 % of the upper reference limit occurring ≤30 days after influenza specimen collection. EMRs were reviewed for demographics, CVD history and risk factors, and ACI-associated cardiac events. Among 38,197 patients with influenza testing results, 4,469 (12 %) had a positive result; 600 of those patients had cardiac biomarker testing performed ≤30 days after influenza testing, and 143 (24 %) had one or more elevated cardiac biomarkers. Among these 143, median age was 73 years (range 44-98 years), and 98 (69 %) were non-Hispanic white. All patients had one or more CVD risk factors, and 98 (69 %) had a history of CVD. Eighty-six percent of ACI-associated events occurred within 3 days of influenza specimen collection date. Seventy patients (49 %) had documented or probable acute myocardial infarction, 8 (6 %) acute congestive heart failure, 6 (4 %) myocarditis, and 4 (3 %) atrial fibrillation. Eleven (8 %) had non-cardiac explanations for elevated cardiac biomarkers, and 44 (31 %) had no documented explanation. Sixty-eight (48 %) patients had received influenza vaccination during the related influenza season. Among veterans with laboratory-confirmed influenza infection and cardiac biomarker testing ≤30 days after influenza testing, approximately 25 % had evidence of ACI, the majority within 3 days. Approximately half were myocardial infarctions. Our findings emphasize the importance of considering ACI associated with influenza infection among patients at high risk, including this older population with prevalent CVD risk factors.


Functional Status and Well-Being in People with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Compared with People with Multiple Sclerosis and Healthy Controls.
People with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) continue to struggle to have their condition recognised as disabling in the face of public and professional prejudice and discrimination. The aim of this study was to compare the functional status and well-being of people with well-characterised ME/CFS with people with multiple sclerosis (PWMS), as well as healthy controls (HCs). In this cross-sectional study, we used data collected as part of the UK ME/CFS Biobank to compare actual participant scores from the Medical Outcomes Survey Short Form-36 v2™ (SF-36v2™) between groups, as a proxy for impact of disability, and from a bespoke questionnaire seeking data on employment and income. People with ME/CFS scored significantly lower than PWMS or HCs in almost all SF-36v2™ areas. Prominent were lower scores for people with ME/CFS in the Physical Component Summary and Role Physical and Social Function domains, while the smallest differences were seen in the Mental Health domain. Responses to the bespoke questionnaire indicated that people with ME/CFS in this study work fewer hours and have lower incomes compared with people in the other two groups. Using SF-36v2™ scores as a proxy, people with ME/CFS were measurably more disabled than PWMS or HCs in this study population. Furthermore, employment and income data are consistent with loss of functional status. These findings should encourage the health community to recognise the disabling effects of ME/CFS, to advocate for the needs of people with ME/CFS, and to investigate strategies to address the cost of the disease to both individuals and society.


CNTF receptor subunit α as a marker for glioma tumor-initiating cells and tumor grade: laboratory investigation.
Tumor-initiating cells are uniquely resilient to current treatment modalities and play an important role in tumor resistance and recurrence. The lack of specific tumor-initiating cell markers to identify and target these cells presents a major obstacle to effective directed therapy. To identify tumor-initiating cell markers in primary brain tumors, the authors compared the proteomes of glioma tumor-initiating cells to their differentiated progeny using a novel, nongel/shotgun-based, multidimensional liquid-chromatography protein separation technique. An in vivo xenograft model was used to demonstrate the tumorigenic and stem cell properties of these cells. Western blot and immunofluorescence analyses were used to confirm findings of upregulated ciliary neurotrophic factor receptor subunit-α (CNTFRα) in undifferentiated tumor-initiating cells and gliomas of increasing tumor grade. Sequencing of the CNTFRα coding regions was performed for mutation analysis. Finally, antibody-dependent cell-mediated cytotoxicity was used to establish the role of CNTFRα as a potential immunotherapeutic target. Ciliary neurotrophic factor receptor subunit-α expression was increased in tumor-initiating cells and was decreased in the cells' differentiated progeny, and expression levels increased with glioma grade. Mutations of CNTFRα are not common in gliomas. Functional studies using CNTF treatment in glioma tumor-initiating cells showed induction of differentiation through the CNTFRα pathway. Treatment with anti-CNTFRα antibody resulted in increased antibody-dependent cell-mediated cytotoxicity in CNTFRα expressing DAOY cells but not in cell lines that lack CNTFRα. These data indicate that CNTFRα plays a role in the formation or maintenance of tumor-initiating cells in gliomas, is a marker that correlates with histological grade, may underlie treatment resistance in some cases, and is a potential therapeutic target.


Deoxyspergualin in relapsing and refractory Wegener's granulomatosis.
Conventional therapy of Wegener's granulomatosis with cyclophosphamide and corticosteroids is limited by incomplete remissions and a high relapse rate. The efficacy and safety of an alternative immunosuppressive drug, deoxyspergualin, was evaluated in patients with relapsing or refractory disease. A prospective, international, multicentre, single-limb, open-label study. Entry required active Wegener's granulomatosis with a Birmingham vasculitis activity score (BVAS) > or =4 and previous therapy with cyclophosphamide or methotrexate. Immunosuppressive drugs were withdrawn at entry and prednisolone doses adjusted according to clinical status. Deoxyspergualin, 0.5 mg/kg per day, was self-administered by subcutaneous injection in six cycles of 21 days with a 7-day washout between cycles. Cycles were stopped early for white blood count less than 4000 cells/mm(3). The primary endpoint was complete remission (BVAS 0 for at least 2 months) or partial remission (BVAS <50% of entry score). After the sixth cycle azathioprine was commenced and follow-up continued for 6 months. 42/44 patients (95%) achieved at least partial remission and 20/44 (45%) achieved complete remission. BVAS fell from 12 (4-25), median (range) at baseline to 2 (0-14) at the end of the study (p<0.001). Prednisolone doses were reduced from 20 to 8 mg/day (p<0.001). Relapses occurred in 18 (43%) patients after a median of 170 (44-316) days after achieving remission. Severe or life-threatening (> or = grade 3) treatment-related adverse events occurred in 24 (53%) patients mostly due to leucopaenias. Deoxyspergualin achieved a high rate of disease remission and permitted prednisolone reduction in refractory or relapsing Wegener's granulomatosis. Adverse events were common but rarely led to treatment discontinuation.


Regulation of group II metabotropic glutamate receptors by G protein-coupled receptor kinases: mGlu2 receptors are resistant to homologous desensitization.
We examined the regulation of mGlu2 and mGlu3 metabotropic glutamate receptor signaling prompted by the emerging role of these receptor subtypes as therapeutic targets for psychiatric disorders, such as anxiety and schizophrenia. In transfected human embryonic kidney 293 cells, G-protein-coupled receptor kinase (GRK) 2 and GRK3 fully desensitized the agonist-dependent inhibition of cAMP formation mediated by mGlu3 receptors. In contrast, GRK2 or other GRKs did not desensitize the cAMP response to mGlu2 receptor activation. Desensitization of mGlu3 receptors by GRK2 required an intact kinase activity, as shown by the use of the kinase-dead mutant GRK2-K220R or the recombinant GRK2 C-terminal domain. Overexpression of beta-arrestin1 also desensitized mGlu3 receptors and did not affect the cAMP signaling mediated by mGlu2 receptors. The difference in the regulation of mGlu2 and mGlu3 receptors was signal-dependent because GRK2 desensitized the activation of the mitogen-activated protein kinase pathway mediated by both mGlu2 and mGlu3 receptors. In vivo studies confirmed the resistance of mGlu2 receptor-mediated cAMP signaling to homologous desensitization. Wild-type, mGlu2(-/-), or mGlu3(-/-) mice were treated intraperitoneally with saline or the mixed mGlu2/3 receptor agonist (-)-2-oxa-4-aminobicyclo[3.1.0]-exhane-4,6-dicarboxylic acid (LY379268; 1 mg/kg) once daily for 7 days. Inhibition of forskolin-stimulated cAMP formation by LY379268 was measured in cortical slices prepared 24 h after the last injection. Agonist pretreatment fully desensitized the cAMP response in wild-type and mGlu2(-/-) mice but had no effect in mGlu3(-/-) mice, in which LY379268 could only activate the mGlu2 receptor. We predict the lack of tolerance when mixed mGlu2/3 receptor agonists or selective mGlu2 enhancers are used continually in patients.


COVID-19 (Novel Coronavirus 2019) - recent trends.
The World Health Organization (WHO) has issued a warning that, although the 2019 novel coronavirus (COVID-19) from Wuhan City (China), is not pandemic, it should be contained to prevent the global spread. The COVID-19 virus was known earlier as 2019-nCoV. As of 12 February 2020, WHO reported 45,171 cases and 1115 deaths related to COVID-19. COVID-19 is similar to Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) virus in its pathogenicity, clinical spectrum, and epidemiology. Comparison of the genome sequences of COVID-19, SARS-CoV, and Middle East Respiratory Syndrome coronavirus (MERS-CoV) showed that COVID-19 has a better sequence identity with SARS-CoV compared to MERS CoV. However, the amino acid sequence of COVID-19 differs from other coronaviruses specifically in the regions of 1ab polyprotein and surface glycoprotein or S-protein. Although several animals have been speculated to be a reservoir for COVID-19, no animal reservoir has been already confirmed. COVID-19 causes COVID-19 disease that has similar symptoms as SARS-CoV. Studies suggest that the human receptor for COVID-19 may be angiotensin-converting enzyme 2 (ACE2) receptor similar to that of SARS-CoV. The nucleocapsid (N) protein of COVID-19 has nearly 90% amino acid sequence identity with SARS-CoV. The N protein antibodies of SARS-CoV may cross react with COVID-19 but may not provide cross-immunity. In a similar fashion to SARS-CoV, the N protein of COVID-19 may play an important role in suppressing the RNA interference (RNAi) to overcome the host defense. This mini-review aims at investigating the most recent trend of COVID-19.


Metamorphic changes in the intestine of three species of lampreys.
Measurements have been made of those changes which lead to increases in the surface area of the intestine during the metamorphosis of three species of lampreys. Although the intestine of the Southern Hemisphere lamprey, Geotria australis, increases in length by 1.13 times and in diameter by 1.12 times, the main factor influencing the 5.71 times increase in surface area is the development of longitudinal folds. The contribution of the typhlosole to the internal perimeter of the intestine is less in most life cycle stages of G. australis than in Lampetra spp. The changes in the various intestinal measurements of the nonparasitic species L. planeri parallel those of the presumed ancestral parasitic species, L. fluviatilis, during the first six stages of metamorphosis. However, the longitudinal folds, but not the typhlosole, subsequently start regressing in L. planeri just after the time when the rate of gonadal development increases markedly. An account is also given of the pattern of fold formation and the development of the typhlosolar vein in G. australis.


Two-dimensional electrophoretic analysis of radio-frequency radiation-exposed MCF7 breast cancer cells.
Although many in vitro studies have previously been conducted to elucidate the biological effects of radio frequency (RF) radiation over the past decades, the existence and nature of any effects is still inconclusive. In an effort to further elucidate this question, we have monitored changes in protein expression profiles in RF-exposed MCF7 human breast cancer cells using two-dimensional gel electrophoresis. MCF7 cells were exposed to 849 MHz RF radiation for 1 h per day for three consecutive days at specific absorption rates (SARs) of either 2 W/Kg or 10 W/kg. During exposure, the temperature in the exposure chamber was kept in an isothermal condition. Twenty-four hours after the final RF exposure, the protein lysates from MCF cells were prepared and two-dimensional electrophoretic analyses were conducted. The protein expression profiles of the MCF cells were not significantly altered as the result of RF exposure. None of the protein spots on the two-dimensional electrophoretic gels showed reproducible changes in three independent experiments. To determine effect of RF radiation on protein expression profiles more clearly, three spots showing altered expression without reproducibility were identified using electrospray ionization tandem mass spectrometry analysis and their expressions were examined with RT-PCR and Western blot assays. There was no alteration in their mRNA and protein levels. As we were unable to observe any significant and reproducible changes in the protein expression profiles of the RF radiation-exposed MCF7 cells using high throughput and non-high throughput techniques, it seems unlikely that RF exposure modulates the protein expression profile.


Thinking about changing mobility practices: how a social practice approach can help.
Policy efforts directed at encouraging physical activity have had minimal success to date. Drawing on Bourdieu's theory of practice, we suggest that a social practice framing might provide useful ways of thinking about why and how some practices do and could change. This article takes three case studies of transformations in mobility practices to explore conditions of possibility for change, using a secondary analysis of qualitative data from studies on cycling in London and fell running in the English Lake District. Three modes of transformation: unthinkable, thwarted and resisted, are rooted in differential interrelationships of field, habitus and doxa in these contrasting cases. We suggest that the notion of tacit, practical knowledge is more useful to understanding why change is thinkable or unthinkable than participants' reasoned accounts of their practice; that where new social fields are available that are congruent with habitus, change is possible and that where field and habitus are tightly aligned, the conditions of possibility for change are reduced. Efforts directed at changing practice might usefully focus not on behaviour or environments but on identifying the social fields in which mobility practices are likely to be malleable. The sociology of public health needs to focus less on health behaviour and more on social practice.


Exploring the dog-human relationship by combining fMRI, eye-tracking and behavioural measures.
Behavioural studies revealed that the dog-human relationship resembles the human mother-child bond, but the underlying mechanisms remain unclear. Here, we report the results of a multi-method approach combining fMRI (N = 17), eye-tracking (N = 15), and behavioural preference tests (N = 24) to explore the engagement of an attachment-like system in dogs seeing human faces. We presented morph videos of the caregiver, a familiar person, and a stranger showing either happy or angry facial expressions. Regardless of emotion, viewing the caregiver activated brain regions associated with emotion and attachment processing in humans. In contrast, the stranger elicited activation mainly in brain regions related to visual and motor processing, and the familiar person relatively weak activations overall. While the majority of happy stimuli led to increased activation of the caudate nucleus associated with reward processing, angry stimuli led to activations in limbic regions. Both the eye-tracking and preference test data supported the superior role of the caregiver's face and were in line with the findings from the fMRI experiment. While preliminary, these findings indicate that cutting across different levels, from brain to behaviour, can provide novel and converging insights into the engagement of the putative attachment system when dogs interact with humans.


Venous limb gangrene and fatal hemorrhage: adverse consequences of HIT "overdiagnosis" in a patient with antiphospholipid syndrome.
This unfortunate patient case highlights the problems with "overdiagnosis" of HIT. Despite "positive" tests for HIT antibodies, the low pretest probability for HIT and the known propensity of patients with APS to yield false-positive HIT antibody results suggests that the patient did not have a true diagnosis of HIT. Moreover, the early administration of warfarin and the choice of argatroban for parenteral anticoagulation when monitoring was hindered by a prolonged baseline aPTT likely play a key factor in the progression of UE DVT to VLG. Ironically, the problems of anticoagulant monitoring posed by the prolonged baseline aPTT likely contributed to the subsequent overanticoagulation and fatal pulmonary hemorrhage. With benefit of hindsight, avoiding the temptation to test for HIT in a low pretest probability situation, and treatment with either heparin using anti-factor Xa monitoring or with non-aPTT-monitored therapy such as LMWH or fondaparinux would likely have resulted in a more favorable clinical course.


Over-the-counter availability of antimicrobial agents, self-medication and patterns of resistance in Karachi, Pakistan.
To determine whether the free availability of antimicrobial agents leads to misuse through self-medication, a house-to-house semi-structured interview was held in three different socio-economic areas of Karachi, Pakistan. Of the 2348 households visited, 1342 (57%) participated; this included 9209 individuals. Three hundred and twenty-two (3.5%) had used one or more antimicrobial in the previous 4 weeks, equivalent to 43 agents per 1000 persons per month. The most frequently used agents were amoxycillin (16.7%), co-trimoxazole (15.7%), erythromycin (10.9%), ampicillin/cloxacillin (Ampiclox, 9.1%) and metronidazole (4.5%). Of these, 91.4% were prescribed by a physician, 2.3% were advised by a chemist and 6.3% were used as self-medication. Self-medication increased with socio-economic status. High levels of resistance were found to ampicillin, co-trimoxazole, chloramphenicol and erythromycin. If these high resistance levels are related to the high frequency of antimicrobial use, over-the-counter availability cannot be held responsible. Education of the medical profession seems to be the single most important tool to control misuse of antimicrobial agents. Innovative approaches for continuous medical education are urgently needed.


In search of the in vivo identity of mesenchymal stem cells.
In spite of the advances in the knowledge of adult stem cells (ASCs) during the past few years, their natural activities in vivo are still poorly understood. Mesenchymal stem cells (MSCs), one of the most promising types of ASCs for cell-based therapies, are defined mainly by functional assays using cultured cells. Defining MSCs in vitro adds complexity to their study because the artificial conditions may introduce experimental artifacts. Inserting these results in the context of the organism is difficult because the exact location and functions of MSCs in vivo remain elusive; the identification of the MSC niche is necessary to validate results obtained in vitro and to further the knowledge of the physiological functions of this ASC. Here we show an analysis of the evidence suggesting a perivascular location for MSCs, correlating these cells with pericytes, and present a model in which the perivascular zone is the MSC niche in vivo, where local cues coordinate the transition to progenitor and mature cell phenotypes. This model proposes that MSCs stabilize blood vessels and contribute to tissue and immune system homeostasis under physiological conditions and assume a more active role in the repair of focal tissue injury. The establishment of the perivascular compartment as the MSC niche provides a basis for the rational design of additional in vivo therapeutic approaches. This view connects the MSC to the immune and vascular systems, emphasizing its role as a physiological integrator and its importance in tissue repair/regeneration.


Identification of a novel post-hydrolytic state in CFTR gating.
Adenosine triphosphate (ATP)-binding cassette (ABC) transporters, ubiquitous proteins found in all kingdoms of life, catalyze substrates translocation across biological membranes using the free energy of ATP hydrolysis. Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of this superfamily in that it functions as an ATP-gated chloride channel. Despite difference in function, recent studies suggest that the CFTR chloride channel and the exporter members of the ABC protein family may share an evolutionary origin. Although ABC exporters harness the free energy of ATP hydrolysis to fuel a transport cycle, for CFTR, ATP-induced dimerization of its nucleotide-binding domains (NBDs) and subsequent hydrolysis-triggered dimer separation are proposed to be coupled, respectively, to the opening and closing of the gate in its transmembrane domains. In this study, by using nonhydrolyzable ATP analogues, such as pyrophosphate or adenylyl-imidodiphosphate as baits, we captured a short-lived state (state X), which distinguishes itself from the previously identified long-lived C2 closed state by its fast response to these nonhydrolyzable ligands. As state X is caught during the decay phase of channel closing upon washout of the ligand ATP but before the channel sojourns to the C2 closed state, it likely emerges after the bound ATP in the catalysis-competent site has been hydrolyzed and the hydrolytic products have been released. Thus, this newly identified post-hydrolytic state may share a similar conformation of NBDs as the C2 closed state (i.e., a partially separated NBD and a vacated ATP-binding pocket). The significance of this novel state in understanding the structural basis of CFTR gating is discussed.


Development of a novel bag-mediated filtration system for environmental recovery of poliovirus.
Poliovirus (PV) is on the verge of global eradication. Due to asymptomatic shedding, eradication certification requires environmental and clinical surveillance. Current environmental surveillance methods involve collection and processing of 400-mL to 1-L grab samples by a two-phase separation method, where sample volume limits detection sensitivity. Filtration of larger sample volumes facilitates increased detection sensitivity. This study describes development of a pumpless in-field filtration system for poliovirus recovery from environmental waters. Recovery of PV types 1, 2, and 3 were compared for glass wool, ViroCap, and NanoCeram (PV1 only) filters. Seeded experiments were performed using 10(5) plaque forming units of PV inoculated into 10-L volumes of secondary effluent, surface water, or a 50:50 mixture of each at pH 7.0. Filter eluates were plated onto buffalo green monkey kidney cells for virus enumeration by plaque assay. Across all water types, recovery from glass wool filters for PV1, PV2, and PV3 averaged 17%, 28%, and 6%, respectively. Recovery from ViroCaps for PV1, PV2, and PV3 averaged 44%, 70%, and 81%, respectively. 10-L samples of moderate turbidity water were processed through ViroCap filters in less than 30 minutes using a pumpless, bag-mediated filtration system. Bag-mediated filtration offers a simple, compact, and efficient method for enhanced environmental PV surveillance.


Rapid time course of action potentials in spines and remote dendrites of mouse visual cortex neurons.
Axonally initiated action potentials back-propagate into spiny dendrites of central mammalian neurons and thereby regulate plasticity at excitatory synapses on individual spines as well as linear and supralinear integration of synaptic inputs along dendritic branches. Thus, the electrical behaviour of individual dendritic spines and terminal dendritic branches is critical for the integrative function of nerve cells. The actual dynamics of action potentials in spines and terminal branches, however, are not entirely clear, mostly because electrode recording from such small structures is not feasible. Additionally, the available membrane potential imaging techniques are limited in their sensitivity and require substantial signal averaging for the detection of electrical events at the spatial scale of individual spines. We made a critical improvement in the voltage-sensitive dye imaging technique to achieve multisite recordings of backpropagating action potentials from individual dendritic spines at a high frame rate. With this approach, we obtained direct evidence that in layer 5 pyramidal neurons from the visual cortex of juvenile mice, the rapid time course of somatic action potentials is preserved throughout all cellular compartments, including dendritic spines and terminal branches of basal and apical dendrites. The rapid time course of the action potential in spines may be a critical determinant for the precise regulation of spike timing-dependent synaptic plasticity within a narrow time window.


From ideology to logistics: the organizational aspects of syringe exchange in a period of institutional consolidation.
The initial period in the establishment of syringe exchange projects is often characterized by overt conflict: between community AIDS activists, on the one hand, and public officials and political leaders who remain ideologically opposed to the introduction of measures perceived as condoning illicit drug use. In this context, professionals concerned with legitimating the new institutions of syringe exchange may sometimes neglect aspects of their everyday logistics and social organization, obscuring the important choices which have to be made to carry these initiatives forward. In particular, the contrast between formally-constituted institutions-the "storefront" or "community-based" syringe exchange programs (SEPs)-and the model of low-threshold syringe availability through pharmacies, vending machines, and user networks, is here presented not as an either/or choice but rather as a pair of complementary strategies which respond to diverse needs and target different populations. The advantages and disadvantages of each particular approach make it likely that maximum effectiveness will be achieved through a combination of every possible form of needle distribution, each tailored to specific and cultural circumstances. The case is here examined in the light of the experience of the SEPs in New York City, from their clandestine origins in 1990 through their first years of official functioning in 1992-1996.


Spatiotemporal Variation in Environmental Vibrio cholerae in an Estuary in Southern Coastal Ecuador.
Cholera emergence is strongly linked to local environmental and ecological context. The 1991-2004 pandemic emerged in Perú and spread north into Ecuador's El Oro province, making this a key site for potential re-emergence. Machala, El Oro, is a port city of 250,000 inhabitants, near the Peruvian border. Many livelihoods depend on the estuarine system, from fishing for subsistence and trade, to domestic water use. In 2014, we conducted biweekly sampling for 10 months in five estuarine locations, across a gradient of human use, and ranging from inland to ocean. We measured water-specific environmental variables implicated in cholera growth and persistence: pH, temperature, salinity, and algal concentration, and evaluated samples in five months for pathogenic and non-pathogenic Vibrio cholerae, by polymerase chain reaction (PCR). We found environmental persistence of pandemic strains O1 and O139, but no evidence for toxigenic strains. Vibrio cholerae presence was coupled to algal and salinity concentration, and sites exhibited considerable seasonal and spatial heterogeneity. This study indicates that environmental conditions in Machala are optimal for cholera re-emergence, with risk peaking during September, and higher risk near urban periphery low-income communities. This highlights a need for surveillance of this coupled cholera-estuarine system to anticipate potential future cholera outbreaks.


Maitotoxin-4, a Novel MTX Analog Produced by Gambierdiscus excentricus.
Maitotoxins (MTXs) are among the most potent toxins known. These toxins are produced by epi-benthic dinoflagellates of the genera Gambierdiscus and Fukuyoa and may play a role in causing the symptoms associated with Ciguatera Fish Poisoning. A recent survey revealed that, of the species tested, the newly described species from the Canary Islands, G. excentricus, is one of the most maitotoxic. The goal of the present study was to characterize MTX-related compounds produced by this species. Initially, lysates of cells from two Canary Island G. excentricus strains VGO791 and VGO792 were partially purified by (i) liquid-liquid partitioning between dichloromethane and aqueous methanol followed by (ii) size-exclusion chromatography. Fractions from chromatographic separation were screened for MTX toxicity using both the neuroblastoma neuro-2a (N2a) cytotoxicity and Ca2+ flux functional assays. Fractions containing MTX activity were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) to pinpoint potential MTX analogs. Subsequent non-targeted HRMS analysis permitted the identification of a novel MTX analog, maitotoxin-4 (MTX4, accurate mono-isotopic mass of 3292.4860 Da, as free acid form) in the most toxic fractions. HRMS/MS spectra of MTX4 as well as of MTX are presented. In addition, crude methanolic extracts of five other strains of G. excentricus and 37 other strains representing one Fukuyoa species and ten species, one ribotype and one undetermined strain/species of Gambierdiscus were screened for the presence of MTXs using low resolution tandem mass spectrometry (LRMS/MS). This targeted analysis indicated the original maitotoxin (MTX) was only present in one strain (G. australes S080911_1). Putative maitotoxin-2 (p-MTX2) and maitotoxin-3 (p-MTX3) were identified in several other species, but confirmation was not possible because of the lack of reference material. Maitotoxin-4 was detected in all seven strains of G. excentricus examined, independently of their origin (Brazil, Canary Islands and Caribbean), and not detected in any other species. MTX4 may therefore serve as a biomarker for the highly toxic G. excentricus in the Atlantic area.


Decreased expression levels of rat liver glutathione S-transferase A2 and albumin during the acute phase response are mediated by HNF1 (hepatic nuclear factor 1) and IL6DEX-NP.
The acute phase response is characterized by positive and negative regulation of many liver proteins including GSTs (glutathione S-transferases) and albumin. The expression of albumin and some GSTs are dependent on HNF1 (hepatic nuclear factor 1). Interleukin 6 plus dexamethasone induce a nuclear protein (IL6DEX-NP) in rat hepatocytes in vitro that binds to a promoter element adjacent to the HNF1 site of rGSTA2 and decreases its expression. We determined how HNF1 and IL6DEX-NP regulate rGSTA2 and albumin expression in rats during the acute phase response after LPS (lipopolysaccharide) treatment. Expression of rGSTA2 and albumin mRNA decreased 3 h after LPS treatment and remained low for 48 h. Transcription rates showed a similar pattern but albumin transcription was less affected. HNF1 and IL6DEX-NP binding to the rGSTA2 promoter was present in control livers but was absent at 3 and 6 h after LPS. By 12 h, HNF1 and IL6DEX-NP binding to the rGSTA2 promoter reappeared and increased to above normal at 48 h. The patterns of HNF1 and IL6DEX-NP binding to the albumin promoter were similar. Affinity of IL6DEX-NP for the albumin promoter was less than that for the rGSTA2 promoter and changes in the transcription rates were consistent with the difference. Early decreases in rGSTA2 and albumin during the acute phase response are due to decreased binding of HNF1. Later persistent decreases in transcriptional rate of rGSTA2 and to a lesser extent albumin are due to increased IL6DEX-NP binding. IL6DEX-NP appears to be an important negative regulator of gene expression in vitro and in vivo.


Efficient frequency-domain sample selection for recovering limited-support images.
An image whose region of support is smaller than its bounding rectangle can, in principle, be reconstructed from a subset of the Nyquist samples. However, determining such a sampling set that gives a stable reconstruction is a difficult and computationally intensive problem. An algorithm is developed for determining periodic nonuniform sampling patterns that is orders of magnitude faster than existing algorithms. The speedup is achieved by using a sequential selection algorithm and heuristic metrics for the quality of sampling sets that are fast to compute, as opposed to the more rigorous linear algebraic metrics that have been used previously. Simulations show that the sampling sets determined using the new algorithm give image reconstructions that are of accuracy comparable with those determined by other slower algorithms.


Annexin A2/TLR2/MYD88 pathway induces arginase 1 expression in tumor-associated neutrophils.
Myeloid lineage cells suppress T cell viability through arginine depletion via arginase 1 (ARG1). Despite numerous studies exploring the mechanisms by which ARG1 perturbs lymphocyte function, the cellular populations responsible for its generation and release remain poorly understood. Here, we showed that neutrophil lineage cells and not monocytes or macrophages expressed ARG1 in human non-small cell lung cancer (NSCLC). Importantly, we showed that approximately 40% of tumor-associated neutrophils (TANs) actively transcribed ARG1 mRNA. To determine the mechanism by which ARG1 mRNA is induced in TANs, we utilized FPLC followed by MS/MS to screen tumor-derived factors capable of inducing ARG1 mRNA expression in neutrophils. These studies identified ANXA2 as the major driver of ARG1 mRNA expression in TANs. Mechanistically, ANXA2 signaled through the TLR2/MYD88 axis in neutrophils to induce ARG1 mRNA expression. The current study describes what we believe to be a novel mechanism by which ARG1 mRNA expression is regulated in neutrophils in cancer and highlights the central role that neutrophil lineage cells play in the suppression of tumor-infiltrating lymphocytes.


Modelling seasonal dynamics, population stability, and pest control in Aedes japonicus japonicus (Diptera: Culicidae).
The invasive temperate mosquito Aedes japonicus japonicus is a potential vector for various infectious diseases and therefore a target of vector control measures. Even though established in Germany, it is unclear whether the species has already reached its full distribution potential. The possible range of the species, its annual population dynamics, the success of vector control measures and future expansions due to climate change still remain poorly understood. While numerous studies on occurrence have been conducted, they used mainly presence data from relatively few locations. In contrast, we used experimental life history data to model the dynamics of a continuous stage-structured population to infer potential seasonal densities and ask whether stable populations are likely to establish over a period of more than one year. In addition, we used climate change models to infer future ranges. Finally, we evaluated the effectiveness of various stage-specific vector control measures. Aedes j. japonicus has already established stable populations in the southwest and west of Germany. Our models predict a spread of Ae. j. japonicus beyond the currently observed range, but likely not much further eastwards under current climatic conditions. Climate change models, however, will expand this range substantially and higher annual densities can be expected. Applying vector control measures to oviposition, survival of eggs, larvae or adults showed that application of adulticides for 30 days between late spring and early autumn, while ambient temperatures are above 9 °C, can reduce population density by 75%. Continuous application of larvicide showed similar results in population reduction. Most importantly, we showed that with the consequent application of a mixed strategy, it should be possible to significantly reduce or even extinguish existing populations with reasonable effort. Our study provides valuable insights into the mechanisms concerning the establishment of stable populations in invasive species. In order to minimise the hazard to public health, we recommend vector control measures to be applied in 'high risk areas' which are predicted to allow establishment of stable populations to establish.


Stress, lifestyle, and quality of life in midlife and older Australian women: results from the Stress and the Health of Women Study.
Chronic psychological stress may pose a serious threat to health, although the mechanisms are not fully understood. This study examines the impact of stress on modifiable lifestyle factors, depressive symptoms, health-related quality of life (HRQOL) and chronic illness in older Australian women. Cross-sectional data were collected from a random sample of 181 older adults aged 60 to 70 years from rural and urban areas of South-East Queensland, Australia. We used structural equation modelling to examine associations between stress, modifiable lifestyle factors, HRQOL, and chronic illness. Parameter estimates show that older women who reported life stressors where they felt helpless and feared for their life (high-magnitude stressors) also reported higher body mass index (p = .03) and more chronic illness (p < .01). In contrast, duration of exposure to life stressors was associated with higher depressive symptom scores (Center for Epidemiologic Studies Depression Scale; p = .02) and sleep disturbance scores (p < .01). Our findings support the link between traumatic personal histories (exposure to high-magnitude stressors) and unhealthy lifestyle factors. Findings highlight the need for more research on how stress reduction, a healthy lifestyle, and positive coping strategies can be used to reduce the effects of high-magnitude stress on HRQOL and chronic illness.


The validation of an in vitro colonic motility assay as a biomarker for gastrointestinal adverse drug reactions.
Motility-related gastrointestinal adverse drug reactions (GADRs), such as constipation and diarrhea, are some of the most frequently reported adverse events associated with the clinical development of new chemical entities, and for marketed drugs. However, biomarkers capable of detecting such GADRs are lacking. Here, we describe an in vitro assay developed to detect and quantify changes in intestinal motility as a surrogate biomarker for constipation/diarrhea-type GADRs. In vitro recordings of intraluminal pressure were used to monitor the presence of colonic peristaltic motor complexes (CPMCs) in mouse colonic segments. CPMC frequency, contractile and total mechanical activity were assessed. To validate the assay, two experimental protocols were conducted. Initially, five drugs with known gastrointestinal effects were tested to determine optimal parameters describing excitation and inhibition as markers for disturbances in colonic motility. This was followed by a "blinded" evaluation of nine drugs associated with or without clinically identified constipation/diarrhea-type GADRs. Concentration-response relationships were determined for these drugs and the effects were compared with their maximal free therapeutic plasma concentration in humans. The assay detected stimulatory and inhibitory responses, likely correlating to the occurrence of diarrhea or constipation. Concentration-related effects were identified and potential mechanisms of action were inferred for several drugs. Based on the results from the fourteen drugs asssessed, the sensitivity of the assay was calculated at 90%, with a specificity of 75% and predictive capacity of 86%. These results support the potential use of this assay in screening for motility-related GADRs during early discovery phase, safety pharmacology assessment.


Effects of pseudophosphorylation mutants on the structural dynamics of smooth muscle myosin regulatory light chain.
We have performed 50 independent molecular dynamics (MD) simulations to determine the effect of pseudophosphorylation mutants on the structural dynamics of smooth muscle myosin (SMM) regulatory light chain (RLC). We previously showed that the N-terminal phosphorylation domain of RLC simultaneously populates two structural states in equilibrium, closed and open, and that phosphorylation at S19 induces a modest shift toward the open state, which is sufficient to activate smooth muscle. However, it remains unknown why pseudophosphorylation mutants poorly mimic phosphorylation-induced activation of SMM. We performed MD simulations of unphosphorylated, phosphorylated, and three pseudophosphorylated RLC mutants: S19E, T18D/S19D and T18E/S19E. We found that the S19E mutation does not shift the equilibrium toward the open state, indicating that simple charge replacement at position S19 does not mimic the activating effect of phosphorylation, providing a structural explanation for previously published functional data. In contrast, mutants T18D/S19D and T18E/S19E shift the equilibrium toward the open structure and partially activate in vitro motility, further supporting the model that an increase in the mol fraction of the open state is coupled to SMM motility. Structural analyses of the doubly-charged pseudophosphorylation mutants suggest that alterations in an interdomain salt bridge between residues R4 and D100 results in impaired signal transmission from RLC to the catalytic domain of SMM, which explains the low ATPase activity of these mutants. Our results demonstrate that phosphorylation produces a unique structural balance in the RLC. These observations have important implications for our understanding of the structural aspects of activation and force potentiation in smooth and striated muscle.


Short-term complications after bilateral internal mammary artery grafting--a retrospective study.
To analyze our experience of conducting Bilateral Internal Mammary Artery (BIMA) grafting, using both pedicled and free grafts for coronary revascularization, assessing the safety and efficacy of the procedure and comparing any differences observed between the two techniques of harvesting the grafts. A retrospective cross sectional study was conducted in July 2010 in which all 48 patients undergoing bilateral internal mammary artery grafting for coronary bypass surgery at the Aga Khan University Hospital from 1996 to 2010 were reviewed and evaluated. The main outcome measures were perioperative mortality and early morbidity with particular reference to cerebrovascular accidents, sternal wound complications and re-opening for bleeding. Chi-square test was used to elicit any meaningful associations between patient characteristics and post-operative outcomes. Of the 48 patients undergoing bilateral IMA grafting, 46 were males and 2 females. The mean age was 48 +/- 9.4 years. Dyslipidaemia and smoking were the most common identifiable risk factors for coronary vascular disease. Following the 40 elective and 8 emergent surgeries, there was one hospital death. The most common post-operative complications were arrhythmias and wound infection. Diabetes and obesity were not observed to be associated with an increased risk of developing surgical site infection. Our study suggests that the short-term efficacy and the safety profile of bilateral IMA grafting for coronary revascularization is clinically acceptable. Long-term follow-up studies should be done to assess the cardiac event-free survival of these individuals evaluating its applicability as a routine for coronary artery bypass grafting in Pakistan.


Ovarian hyperstimulation syndrome: current views on pathophysiology, risk factors, prevention, and management.
To summarize current views on the pathophysiology, risk factors, prevention, clinical features, and management of Ovarian Hyperstimulation Syndrome (OHSS). Literature review OHSS is a condition characterized by increased capillary permeability, and experimental evidence has identified a provocative link to pathologic vasoactive cytokine actions. Although the ultimate physiologic mechanism of OHSS is not yet known, there are well-known risk factors that must be considered during the administration of medications to treat infertility. Clinical features are consequences of third-spaced intravascular fluid, and OHSS may become life-threatening secondary to thromboembolism or compromised pulmonary or cardiovascular function. Cornerstones of prevention have historically included cycle cancellation, coasting, decreased dosing of human chorionic gonadotropin (hCG) trigger, use of an agonist trigger, and cryopreservation of all embryos. Newer methods of prevention include the administration of a dopamine agonist medication. Management options for OHSS include outpatient transvaginal paracentesis, outpatient transabdominal paracentesis, and inpatient hospitalization with or without paracentesis. OHSS continues to be a serious complication of assisted reproductive therapy (ART), with no universally agreed upon best method of prevention. Coasting and cryopreservation of all embryos are the most commonly used approaches in the literature, but cycle cancellation is the only method that can completely prevent the development of OHSS. Dopamine agonists are currently being investigated to both prevent and improve the clinical course in OHSS. Recent publications suggest that outpatient paracentesis both prevents the need for inpatient hospitalization and is a cost-effective strategy.


Microarray-based pncA genotyping of pyrazinamide-resistant strains of Mycobacterium tuberculosis.
Drug-resistant Mycobacterium tuberculosis poses a significant threat to the treatment of tuberculosis (TB). The current susceptibility testing for the first-line TB drug pyrazinamide (PZA) is not only time-consuming but also difficult, due to the requirement for acid pH for drug activity. Predominantly, resistance to PZA in M. tuberculosis is caused by mutations in the pncA gene, and the detection of pncA mutations can be an indicator of PZA resistance. In this study, the use of a previously developed microarray method for the rapid detection of PZA-resistant M. tuberculosis based on identifying mutations in the pncA gene was evaluated. Microarray analysis was performed in a blind manner on 33 clinical isolates of M. tuberculosis for which the sequence of the pncA gene had not previously been determined. The results showed that all mutations in PZA-resistant strains identified by DNA sequencing could be unambiguously detected by the microarray method. It is concluded that the microarray method is a valuable tool for the rapid screening and genetic identification of potential PZA-resistant M. tuberculosis strains.


Patient attitudes to clinical trials: development of a questionnaire and results from asthma and cancer patients.
To develop a questionnaire to assess patients' views of clinical trials, and to report the results from the questionnaire in two patient groups: asthma and cancer. A 43 item questionnaire asking patients about their views to clinical trials was developed on the basis of interviews with trialists and focus groups with patients. The questionnaire was mailed to patients with a diagnosis of either asthma or cancer. A set of items was then selected, via statistical analyses, to form the core of the questionnaire. Patients with a diagnosis of cancer in one NHS Hospital Trust, and patients with a diagnosis of asthma in two NHS Hospital Trusts. Completed questionnaires were received from 353 cancer patients and 578 asthma patients. Factor analyses of the data indicated that 22 items contributed to five dimensions: 'positive beliefs', 'safety', 'information needs', 'negative expectations' and 'patient involvement'. Differences between asthma and cancer patients on these dimensions were small. A regression of these dimension scores against a variable asking if patients would be willing to take part in trials found that 'safety' and 'information needs' did not contribute significantly to the model for either asthma or cancer patients. A questionnaire has been developed for use in assessing patients' views towards clinical trials. Results from the surveys reported here suggest that patient views about the importance of trials and beliefs about the value of patient involvement are likely to be predictive of whether or not patients will agree to take part in a study.


Lobophora (Dictyotales) Species Richness, Ecology and Biogeography Across the North-Eastern Atlantic Archipelagos and Description of Two New Species1.
The brown alga Lobophora (Dictyotales, Phaeophyceae) is an important macroalga in the North-eastern Atlantic archipelagos (i.e., Macaronesia). Notably in the Canaries it can dominate benthic assemblages. While the genus has been the subject of several ecological studies in the Canaries, no study has yet been conducted to assess species-level diversity of Lobophora in Macaronesia. We reassessed the diversity of Lobophora in Macaronesia, reporting the presence of seven species (L. caboverdeana sp. nov., L. canariensis, L. dagamae sp. nov., L. delicata, L. dispersa, L. littlerorum, and L. schneideri). Lobophora spp. from Macaronesia are morphologically and ecologically distinguishable. In the Canaries, L. schneideri dominates the photophilic assemblages from the intertidal to 20-30 m depth. Lobophora dagamae sp. nov. grows in less illuminated shallow habitats, and replaces L. schneideri from 30 to ~80 m. Lobophora canariensis also has a wide vertical distribution, from the intertidal to deep waters, while L. delicata, L. dispersa and L. littlerorum grow in shallow waters. The dominance of species with an upright habit versus prostrate or crustose species may be mediated by the pressure of herbivores. Four species have an amphi-Atlantic distribution: L. littlerorum, L. canariensis, L. delicata, and L. schneideri. Lobophora schneideri and L. delicata are furthermore distributed in the Mediterranean Sea. By sampling a pivotal region in the Atlantic, this study significantly improves our knowledge of Lobophora biogeography in the Atlantic Ocean. Macaronesia constitutes a species-poor region for Lobophora where no diversification events occurred, and a region of overlap between the Greater Caribbean and the Indo-Pacific.


Three-dimensional quantum photonic elements based on single nitrogen vacancy-centres in laser-written microstructures.
To fully integrate quantum optical technology, active quantum systems must be combined with resonant microstructures and optical interconnects harvesting and routing photons in three diemsnsions (3D) on one chip. We fabricate such combined structures for the first time by using two-photon laser lithography and a photoresist containing nanodiamonds including nitrogen vacancy-centers. As an example for possible functionality, single-photon generation, collection, and transport is successfully accomplished. The single photons are efficiently collected via resonators and routed in 3D through waveguides, all on one optical chip. Our one-step fabrication scheme is easy to implement, scalable and flexible. Thus, other complex assemblies of 3D quantum optical structures are feasible as well.


Expression of voltage-gated K+ channels in insulin-producing cells. Analysis by polymerase chain reaction.
We have used the polymerase chain reaction (PCR) with primers against the S5 and S6 regions of voltage-gated K+ channels to identify 8 different specific amplification products using poly(A)+ RNA isolated from islets of Langerhans from obese hyperglycemic (ob/ob) mice and from the two insulin-producing cell lines HIT T15 and RINm5F. Sequence analysis suggests that they derive from mRNAs coding for a family of voltage-gated K+ channels; 5 of these have been recently identified in mammalian brain and 3 are novel. These hybridize in classes to different mRNAs which distribute differently to a number of tissues and cell lines including insulin-producing cells.


Replicative senescence induced by Romo1-derived reactive oxygen species.
Persistent accumulation of DNA damage induced by reactive oxygen species (ROS) is proposed to be a major contributor toward the aging process. Furthermore, an increase in age-associated ROS is strongly correlated with aging in various species, including humans. Here we showed that the enforced expression of the ROS modulator 1 (Romo1) triggered premature senescence by ROS production, and this also contributed toward induction of DNA damage. Romo1-derived ROS was found to originate in the mitochondrial electron transport chain. Romo1 expression gradually increased in proportion to population doublings of IMR-90 human fibroblasts. An increase in ROS production in these cells with high population doubling was blocked by the Romo1 knockdown using Romo1 small interfering RNA. Romo1 knockdown also inhibited the progression of replicative senescence. Based on these results, we suggest that age-related ROS levels increase, and this contributes to replicative senescence, which is directly associated with Romo1 expression.


Accumulation and Elimination of Enrofloxacin and Ciprofloxacin in Tissues of Shrimp Litopenaeus vannamei under Laboratory and Farm Conditions.
This study aimed to quantify the accumulation and elimination of Enrofloxacin (ENRO) and Ciprofloxacin (CIPRO) in cultivated Litopenaeus vannamei under controlled laboratory and farm conditions. Laboratory- and farm-raised shrimp were given feed supplemented with 200 mg/kg ENRO for 14 days, followed by a 16-day diet without antibiotics. The levels of ENRO and CIPRO were analyzed by High Performance Liquid Chromatography (HPLC). In the laboratory, ENRO concentrations in the muscle and hepatopancreas reached a maximum (C(max)) of 0.54 ± 0.26 μg/g and 3.52 ± 1.9 μg/g, respectively; C(max) values for CIPRO in the laboratory were 0.18 ± 0.13 μg/g (muscle) and 1.05 ± 0.20 μg/g (hepatopancreas). In farmed shrimp, C(max) values for ENRO were 0.36 ± 0.17 μg/g muscle and 1.60 ± 0.82 μg/g in the hepatopancreas; CIPRO C(max) values were 0.03 ± 0.02 μg/g (muscle) and 0.36 ± 0.08 μg/g (hepatopancreas). Two to fourteen days were necessary to eliminate both antibiotics from muscular tissue and four to more fourteen days for complete elimination of the antibiotics from the hepatopancreas. These results should be considered in terms of minimum concentrations necessary to inhibit Vibrio bacteria to determine whether the current use of this antibiotic is effective in controlling disease.


Development of a disease severity and responder index for psoriatic arthritis (PsA)--report of the OMERACT 10 PsA special interest group.
Work within the Group for Research and Assessment of Psoriasis and Psoriatic Arthritis (GRAPPA) to develop and validate composite disease activity measures in PsA has progressed. At the Outcome Measures in Rheumatology Clinical Trials (OMERACT) 8 meeting, a core set of domains to be assessed in randomized controlled trials (RCT) and longitudinal observational studies (LOS) of PsA was agreed upon. At OMERACT 10, work to date regarding proposed composite responder indices was presented. Five proposed composite responder definitions for PsA were reviewed and discussed including new data from the GRACE (GRAppa Composite Exercise) study. There was agreement that the work to date was promising, and that developing composite outcome measures for use in RCT and LOS was important. Further work was required, including data on followup timepoints and less common phenotypes of PsA, to ensure that all subgroups were represented within GRACE. During discussion on the concept of composite measures for PsA, based on predominant/little/no involvement in several domains (such as skin versus joints, enthesitis, dactylitis, spondyloarthritis) it was acknowledged that a simple summative score encompassing all domains of PsA would be difficult to construct psychometrically and may not be appropriate. Ideally, any composite measure should retain the ability to differentiate between activity in individual domains, such as enthesitis or skin psoriasis, so that the influence of each can be assessed independently. Further work is required within the GRACE dataset to develop an optimal composite measure for PsA. Several proposals to date have shown preliminary validity according to the OMERACT filter.


Strong photoluminescence emission from resonant Fibonacci quantum wells.
Strong photoluminescence (PL) emission from a resonant Fibonacci quantum well (FQW) is demonstrated. The maximum PL intensity in the FQW is significantly stronger than that in a periodic QW under the Bragg or anti-Bragg conditions. Moreover, the peaks of the squared electric field in the FQW are located very near each of the QWs. The optimal PL spectrum in the FQW has an asymmetrical form rather than the symmetrical one in the periodic case. The maximum PL intensity and the corresponding thickness filling factor in the FQW become greater with increasing generation order.


Caver Web 1.0: identification of tunnels and channels in proteins and analysis of ligand transport.
Caver Web 1.0 is a web server for comprehensive analysis of protein tunnels and channels, and study of the ligands' transport through these transport pathways. Caver Web is the first interactive tool allowing both the analyses within a single graphical user interface. The server is built on top of the abundantly used tunnel detection tool Caver 3.02 and CaverDock 1.0 enabling the study of the ligand transport. The program is easy-to-use as the only required inputs are a protein structure for a tunnel identification and a list of ligands for the transport analysis. The automated guidance procedures assist the users to set up the calculation in a way to obtain biologically relevant results. The identified tunnels, their properties, energy profiles and trajectories for ligands' passages can be calculated and visualized. The tool is very fast (2-20 min per job) and is applicable even for virtual screening purposes. Its simple setup and comprehensive graphical user interface make the tool accessible for a broad scientific community. The server is freely available at https://loschmidt.chemi.muni.cz/caverweb.


A continuous perfusion microplate for cell culture.
We describe a 96-well microplate with fluidically connected wells that enables the continuous fluid perfusion between wells without the need for external pumping. A single unit in such a perfusion microplate consists of three wells: a source well, a sample (cell culture) well in the middle and a waste well. Fluid perfusion is achieved using a combination of the hydrostatic pressure generated by different liquid levels in the wells and the fluid wicking through narrow strips of a cellulose membrane connecting the wells. There is an excellent correspondence between the observed perfusion flow dynamics and the flow simulations based on Darcy's Law. Hepatocytes (C3A cells) cultured for 4 days in the perfusion microplate with no media exchange in the cell culture well had the same viability as hepatocytes exposed to a daily exchange of media. EOC 20 cells that require media conditioned by LADMAC cells were shown to be equally viable in the adjacent cell culture well of the perfusion microplate with LADMAC cells cultured in the source well. Tegafur, a prodrug, when added to primary human hepatocytes in the source well, was metabolized into a cytotoxic metabolite that kills colon cancer cells (HCT 116) cultured in the adjacent cell culture well; no toxicity was observed when only medium was in the source well. These results suggest that the perfusion microplate is a useful tool for a variety of cell culture applications with benefits ranging from labor savings to enabling in vivo-like toxicity studies.


Reiterative use of the EGF receptor triggers differentiation of all cell types in the Drosophila eye.
The Drosophila eye has contributed much to our knowledge of cell differentiation. This understanding has primarily come from the study of the R7 photoreceptor; much less is known about the development of the other classes of photoreceptor or the nonneuronal cone or pigment cells. I have used a dominant-negative form of the Drosophila epidermal growth factor receptor (DER) to show that this receptor tyrosine kinase (RTK) is required for the differentiation of all these cell types, and I have also shown that DER is sufficient to trigger their development. DER is even required in R7, where it can replace Sevenless, another RTK. These results broaden our view of eye development to include the whole ommatidium and suggest that reiterative activation of DER is critical for triggering the differentiation of all cell types.