Transient demyelination increases the efficiency of retrograde AAV transduction.
Adeno-associated virus (AAV) is capable of mediating retrograde viral transduction of central and peripheral neurons. This occurs at a relatively low efficiency, which we previously found to be dependent upon capsid serotype. We sought to augment retrograde transduction by providing increased axonal access to peripherally delivered AAV. Others have described utilizing full transection of peripheral nerves to mediate retrograde viral transduction of motor neurons. Here, we examined the ability of a transient demyelinating event to modulate levels of retrograde AAV transduction. Transient demyelination does not cause lasting functional deficits. Ethidium bromide (EtBr)-induced transient demyelination of the sciatic nerve resulted in significant elevation of retrograde transduction of both motor and sensory neurons. Retrograde transduction levels of motor neurons and heavily myelinated, large-diameter sensory neurons increased at least sixfold following peripheral delivery of self-complementary AAV serotype 1 (scAAV1) and serotype 2 (scAAV2), when preceded by demyelination. These findings identify a means of significantly enhancing retrograde vector transport for use in experimental paradigms requiring either retrograde neuronal identification and gene expression, or translational treatment paradigms.


Genetics and pathogenesis of inflammatory bowel disease.
Recent advances have provided substantial insight into the maintenance of mucosal immunity and the pathogenesis of inflammatory bowel disease. Cellular programs responsible for intestinal homeostasis use diverse intracellular and intercellular networks to promote immune tolerance, inflammation or epithelial restitution. Complex interfaces integrate local host and microbial signals to activate appropriate effector programs selectively and even drive plasticity between these programs. In addition, genetic studies and mouse models have emphasized the role of genetic predispositions and how they affect interactions with microbial and environmental factors, leading to pro-colitogenic perturbations of the host-commensal relationship.


Communication: double-hybrid functionals from adiabatic-connection: the QIDH model.
A new approach stemming from the adiabatic-connection (AC) formalism is proposed to derive parameter-free double-hybrid (DH) exchange-correlation functionals. It is based on a quadratic form that models the integrand of the coupling parameter, whose components are chosen to satisfy several well-known limiting conditions. Its integration leads to DHs containing a single parameter controlling the amount of exact exchange, which is determined by requiring it to depend on the weight of the MP2 correlation contribution. Two new parameter-free DHs functionals are derived in this way, by incorporating the non-empirical PBE and TPSS functionals in the underlying expression. Their extensive testing using the GMTKN30 benchmark indicates that they are in competition with state-of-the-art DHs, yet providing much better self-interaction errors and opening a new avenue towards the design of accurate double-hybrid exchange-correlation functionals departing from the AC integrand.


Twenty year comparison of a Bjork-Shiley mechanical heart valve with porcine bioprostheses.
To compare survival and outcome in patients receiving a mechanical or bioprosthetic heart valve prosthesis. Randomised prospective trial. Tertiary cardiac centre. Between 1975 and 1979, patients were randomised to receive either a Bjork-Shiley or a porcine prostheses. The mitral valve was replaced in 261 patients, the aortic in 211, and both valves in 61 patients. Follow up now averages 20 years. Death, reoperation, bleeding, embolism, and endocarditis. After 20 years there was no difference in survival (Bjork-Shiley v porcine prosthesis (mean (SEM)): 25.0 (2.7)% v 22.6 (2.7)%, log rank test p = 0.39). Reoperation for valve failure was undertaken in 91 patients with porcine prostheses and in 22 with Bjork-Shiley prostheses. An analysis combining death and reoperation as end points confirmed that Bjork-Shiley patients had improved survival with the original prosthesis intact (23.5 (2.6)% v 6.7 (1.6)%, log rank test p < 0.0001); this difference became apparent after 8-10 years in patients undergoing mitral valve replacement, and after 12-14 years in those undergoing aortic valve replacement. Major bleeding was more common in Bjork-Shiley patients (40.7 (5.4)% v 27.9 (8.4)% after 20 years, p = 0.008), but there was no significant difference in major embolism or endocarditis. Survival with an intact valve is better among patients with the Bjork-Shiley spherical tilting disc prosthesis than with a porcine prosthesis but there is an attendant increased risk of bleeding.


CORRELATION OF STRUCTURAL AND FUNCTIONAL OUTCOME MEASURES IN A PHASE ONE TRIAL OF CILIARY NEUROTROPHIC FACTOR IN TYPE 2 IDIOPATHIC MACULAR TELANGIECTASIA.
Macular telangiectasia Type 2 is a bilateral, progressive, potentially blinding retinal disease characterized by both vascular and neurodegenerative signs. Both the area of the break in the ellipsoid zone seen in "en face" optical coherence tomographic (OCT) images and microperimetric focal retinal sensitivity loss have been proposed as potential measures of progression in macular telangiectasia. The authors aimed to assess the characteristics and interrelationship of these structural and functional disease markers from the data collected in a phase one clinical trial of ciliary neurotrophic factor in macular telangiectasia. Orthogonal topographic (en face) maps of the ellipsoid zone were generated from Heidelberg Spectralis OCT volume scans (15 × 10° area, 30-μm B-scan intervals) or Zeiss Cirrus HD-OCT 4000 512 × 128 cube scans. Mesopic microperimetry was performed on CenterVue MAIA perimeters, using a Goldmann III stimulus in a custom test grid. Structural and functional data were analyzed by two methods: by calculating aggregate loss and by simple thresholding. The alignment quality of structural and functional data was also evaluated. Overall, the break area showed a good correlation with aggregate sensitivity loss (ρ = 0.834, P < 0.0001, 95% confidence interval 0.716-0.906) but also with the number of test points below a threshold value (e.g., <20 dB: ρ = 0.843, P < 0.0001, 95% confidence interval 0.755-0.902). Significant misalignment of the MAIA test grid was apparent in 13/48 visits of 7/14 eyes. The authors found a good correlation between ellipsoid zone break area and function loss. En face OCT mapping of the ellipsoid zone appears to demonstrate structural change before mesopic microperimetry can detect a focal loss of retinal sensitivity. Thresholding offers a quick alternative to calculating aggregate sensitivity loss.


Medial Meniscus Scaffold Implantation in Combination with Concentrated Bone Marrow Aspirate Injection: Minimum 3-Year Follow-up.
The objective of this study is to show the short-term clinical and radiological outcomes of concentrated bone marrow aspirate (CBMA) injection administered in combination with medial meniscus scaffold implantation. Twenty-three patients who received intra-articular CBMA injection in combination with polyurethane-based medial meniscus scaffold implantation were evaluated within the scope of this study. The International Knee Documentation Committee (IKDC) questionnaire and the Knee injury and Osteoarthritis Outcome Score (KOOS) were used to evaluate the results, and the visual analog scale was used to assess the pain scores. Magnetic resonance imaging (MRI) scans were obtained in the preoperative period and at postoperative months 1, 12, 24, and 36 to assess the scaffold position as well as chondral degeneration/damage in a comparative manner. MRI assessment was performed by using the modified Outerbridge scale for cartilage and the Genovese scoring system for the meniscal implant. Twenty-three patients who were included in the study were evaluated for a mean follow-up period of 38.3 months. Patients exhibited statistically significant improvement according to all scoring data from the preoperative period until the follow-up period. The mean postoperative extrusion at year 3 was 2.39 mm (distribution 2.30-2.56 mm). There was no significant difference in the distribution of the degree of chondral damage between the preoperative and 3-year follow-up periods. Four patients did not show any improvement nor had lower scores according to the assessment. Medial meniscus scaffold implantation combined with intra-articular CBMA injection resulted in a significant improvement in all functions and pain scores as well as a statistically significant clinical improvement in IKDC and KOOS values in the short-term follow-up. The Level of evidence for this study is IV.


Spatial and Temporal Mapping of Breast Cancer Lung Metastases Identify TREM2 Macrophages as Regulators of the Metastatic Boundary.
Cancer mortality primarily stems from metastatic recurrence, emphasizing the urgent need for developing effective metastasis-targeted immunotherapies. To better understand the cellular and molecular events shaping metastatic niches, we used a spontaneous breast cancer lung metastasis model to create a single-cell atlas spanning different metastatic stages and regions. We found that premetastatic lungs are infiltrated by inflammatory neutrophils and monocytes, followed by the accumulation of suppressive macrophages with the emergence of metastases. Spatial profiling revealed that metastasis-associated immune cells were present in the metastasis core, with the exception of TREM2+ regulatory macrophages uniquely enriched at the metastatic invasive margin, consistent across both murine models and human patient samples. These regulatory macrophages (Mreg) contribute to the formation of an immune-suppressive niche, cloaking tumor cells from immune surveillance. Our study provides a compendium of immune cell dynamics across metastatic stages and niches, informing the development of metastasis-targeting immunotherapies. Temporal and spatial single-cell analysis of metastasis stages revealed new players in modulating immune surveillance and suppression. Our study highlights distinct populations of TREM2 macrophages as modulators of the microenvironment in metastasis, and as the key immune determinant defining metastatic niches, pointing to myeloid checkpoints to improve therapeutic strategies. This article is featured in Selected Articles from This Issue, p. 2489.


Do antibody responses to the influenza vaccine persist year-round in the elderly? A systematic review and meta-analysis.
The influenza vaccine is less immunogenic in older than younger adults, and the duration of protection is unclear. Determining if protection persists beyond a typical seasonal epidemic is important for climates where influenza virus activity is year-round. A systematic review protocol was developed and registered with PROSPERO [CRD42015023847]. Electronic databases were searched systematically for studies reporting haemagglutination-inhibition (HI) titres 180-360days following vaccination with inactivated trivalent seasonal influenza vaccine, in adults aged ⩾65years. Geometric mean titre (GMT) and seroprotection (HI titre ⩾1:40) at each time point was extracted. A Bayesian model was developed of titre trajectories from pre-vaccination to Day 360. In the meta-analysis, studies were aggregated using a random-effects model to compare pre-vaccination with post-vaccination HI titres at Day 21-42 ('seroconversion'), Day 180 and Day 360. Potential sources of bias were systematically assessed, and heterogeneity explored. 2864 articles were identified in the literature search, of which nineteen met study inclusion/exclusion criteria. Sixteen studies contained analysable data from 2565 subjects. In the Bayesian model, the proportion of subjects seroprotected increased from 41-51% pre-vaccination to 75-78% at seroconversion. Seroprotection subsequently fell below 60% for all serotypes by Day 360: A/H1 42% (95% CI 38-46), A/H3 59% (54-63), B 47% (42-52). The Bayesian model of GMT trajectories revealed a similar pattern. By Day 360, titres were similar to pre-vaccination levels. In the meta-analysis, no significant difference in proportion of subjects seroprotected, 0 (-0.11, 0.11) or in log2GMT 0.30 (-0.02, 0.63) was identified by Day 360 compared with pre-vaccination. The quality of this evidence was limited to moderate on account of significant participant dropout. The review found consistent evidence that HI antibody responses following influenza vaccination do not reliably persist year-round in older adults. Alternative vaccination strategies could provide clinical benefits in regions where year-round protection is important.


Recognition of host proteins by Helicobacter cysteine-rich protein C.
Tetratricopeptide- and sel1-like repeat (SLR) proteins modulate various cellular activities, ranging from transcription regulation to cell-fate control. Helicobacter cysteine-rich proteins (Hcp) consist of several SLRs that are cross-linked by disulfide bridges and have been implicated in host/pathogen interactions. Using pull-down proteomics, several human proteins including Nek9, Hsp90, and Hsc71 have been identified as putative human interaction partners for HcpC. The interaction between the NimA-like protein kinase Nek9 and HcpC has been validated by ELISA and surface plasmon resonance. Recombinant Nek9 is recognized by HcpC with a dissociation constant in the lower micromolar range. This interaction is formed either directly between Nek9 and HcpC or via the formation of a complex with Hsc71. The HcpC homologue HcpA possesses no affinity for Nek9, suggesting that the reported interaction is rather specific for HcpC. These results are consistent with previous observations where Nek9 was targeted upon bacterial or viral invasion. However, further experiments will be required to show that the reported interactions also occur in vivo.


Cognitive flexibility and religious disbelief.
Cognitive flexibility is operationalized in the neuropsychological literature as the ability to shift between modes of thinking and adapt to novel or changing environments. Religious belief systems consist of strict rules and rituals that offer adherents certainty, consistency, and stability. Consequently, we hypothesized that religious adherence and practice of repetitive religious rituals may be related to the persistence versus flexibility of one's cognition. The present study investigated the extent to which tendencies towards cognitive flexibility versus persistence are related to three facets of religious life: religious affiliation, religious practice, and religious upbringing. In a large sample (N = 744), we found that religious disbelief was related to cognitive flexibility across three independent behavioural measures: the Wisconsin Card Sorting Test, Remote Associates Test, and Alternative Uses Test. Furthermore, lower frequency of religious service attendance was related to cognitive flexibility. When analysing participants' religious upbringing in relation to their current religious affiliation, it was manifest that current affiliation was more influential than religious upbringing in all the measured facets of cognitive flexibility. The findings indicate that religious affiliation and engagement may shape and be shaped by cognitive control styles towards flexibility versus persistence, highlighting the tight links between flexibility of thought and religious ideologies.


A computational study of Tat-CDK9-Cyclin binding dynamics and its implication in transcription-dependent HIV latency.
HIV is a virus that attacks the T cells. HIV may either actively replicate or become latent within host cells for years. Since HIV uses its own protein Tat to hijack the host CDK9-Cyclin complex for transcription, Tat is implicated in transcription-dependent HIV latency. To quantify the impact of Tat binding, we propose a computational framework to probe the dynamics of the CDK9-Cyclin interface and the ATP pocket reorganization upon binding by different Tat mutants. Specifically, we focus on mutations at three Tat residues P10, W11, and N12 that are known to interact directly with CDK9 based on the crystal structure of the Tat-CDK9-Cyclin complex. Our molecular dynamics simulations show that the CDK9-Cyclin interface becomes slightly weaker for P10S and W11R mutants but tighter for the K12N mutant. Furthermore, the side chain orientation of residue K48 in the ATP pocket of CDK9 is similar to the inactive state in P10S and W11R simulations, but similar to the active state in K12N simulations. These are consistent with some existing but puzzling observations of latency for these mutants. This framework may hence help gain a better understanding of the role of Tat in the transcription-dependent HIV latency establishment.


Glass-clad single-crystal germanium optical fiber.
Long lengths (250 meters) of a flexible 150 microm diameter glass-clad optical fiber containing a 15 microm diameter crystalline and phase-pure germanium core was fabricated using conventional optical fiber draw techniques. X-ray diffraction and spontaneous Raman scattering measurements showed the core to be very highly crystalline germanium with no observed secondary phases. Elemental analysis confirmed a very well-defined core-clad interface with a step-profile in composition and nominally 4 weight-percent oxygen having diffused into the germanium core from the glass cladding. For this proof-of-concept fiber, polycrystalline n-type germanium of unknown dopant concentration was used. The measured infrared transparency of the starting material was poor and, as a likely outcome, the attenuation of the resultant fiber was too high to be measured. However, the larger Raman cross-section, infrared and terahertz transparency of germanium over silicon should make these fibers of significant value for fiber-based mid- to long-wave infrared and terahertz waveguides and Raman-shifted infrared light sources once high-purity, high-resistivity germanium is employed.


Immunotherapy for prostate cancer enters its golden age.
In the United States, prostate cancer is the most frequent malignancy in men and ranks second in terms of mortality. Although recurrent or metastatic disease can be managed initially with androgen ablation, most patients eventually develop castration-resistant disease within a number of years, for which conventional treatments (eg, chemotherapy) provide only modest benefits. In the last few years, immunotherapy has emerged as an exciting therapeutic modality for advanced prostate cancer, and this field is evolving rapidly. Encouragingly, the US Food and Drug Administration (FDA) has recently approved two novel immunotherapy agents for patients with advanced cancer: the antigen presenting cell-based product sipuleucel-T and the anti-CTLA4 (cytotoxic T-lymphocyte antigen 4) antibody ipilimumab, based on improvements in overall survival in patients with castration-resistant prostate cancer and metastatic melanoma, respectively. Currently, a number of trials are investigating the role of various immunological approaches for the treatment of prostate cancer, many of them with early indications of success. As immunotherapy for prostate cancer enters its golden age, the challenge of the future will be to design rational combinations of immunotherapy agents with each other or with other standard prostate cancer treatments in an effort to improve patient outcomes further.


Phospholipids act as secondary receptor during the entry of the enveloped, double-stranded RNA bacteriophage phi6.
Bacteriophage phi6 is the type member of the family Cystoviridae and infects Gram-negative Pseudomonas syringae cells. The virion consists of a protein-rich lipid envelope enclosing a nucleocapsid. The nucleocapsid covers the icosahedral polymerase complex that encloses the double-stranded RNA genome. Here, we demonstrate that nucleocapsid surface protein P8 is the single nucleocapsid component interacting with the cytoplasmic membrane. This interaction takes place between P8 and phospholipid. Based on this and previous studies, we propose a model where the periplasmic nucleocapsid interacts with the phospholipid head groups and, when the membrane voltage exceeds the threshold of 110 mV, this interaction drives the nucleocapsid through the cytoplasmic membrane, resulting in an intracellular vesicle containing the nucleocapsid.


Contact cooling of the skin.
Skin precooling can be used to reduce epidermal thermal damage in laser procedures (such as hair removal) where the target structures are located up to several millimetres below the skin surface. We have developed and experimentally verified a computational model that describes contact precooling of a multilayered skin structure prior to laser irradiation. The skin surface is assumed to be brought into thermal contact with a cold plate made of material with a high thermal conductivity. The approximate analytical solution for the skin temperature is obtained by considering the plate as a local heat sink. The time evolution of temperature (in both the skin and the plate) is simulated numerically to yield the optimal cooling parameters. To experimentally verify the numerical results of the model, we performed direct measurements of skin temperature for contact cooling with a sapphire plate held at several different temperatures in the range +10 to -30 degrees C.


Embryo maternal dialogue: From pregnancy recognition to proliferation control.
Embryo-maternal dialogue starts shortly after fertilization and is exerted through both local and systemic signaling. We have discovered specific embryo derived pre-implantation factors (PIF), novel peptides that are secreted already at the two cell stage and which modulate cellular immunity. In the fallopian tube the embryo, a partial allograft, is tolerated by the mother. Embryo derived peripheral signaling (PIF) is also detected prior to implantation in maternal sera. This signal may also help prime the endometrium to facilitate implantation. Upon implantation, embryo-endometrial communication becomes direct and highly amplified. When the immune privilege appears to be secured, embryogenesis proper initiates. This requires proliferation/differentiation to be tightly controlled. Knowledge of proliferation promoters is ample while the factors involved in its control remain less understood. We have identified a class of novel proteins/peptides, developmental proteins (DPs), that are present in the embryo before a mature immune system has developed. Their role is to create a balance between pro and antiproliferative forces, to promote normal proliferation while controlling abnormal cell proliferation (i.e. due to carcinogens, toxins, viruses, and ionic radiation). DPs, may also redirect growth towards functionality through differentiation. DPs appear to act through a specific receptor negating growth factors action through promotion of tumor suppressors and inhibition of tumor promoters at 2 minutes, blocking DNA synthesis at ~24 hours, and promoting apoptosis at ~48 hours. When an embryo becomes incompatible with life, DPs may lead to growth arrest, PIF-like compounds decline, the immune system to be restored and the pregnancy is rejected. Final identification and use of PIF and DPs is likely to help both managing early pregnancy disorders and aid in treatment of proliferative disorders due to cancer and viral infection.


A systematic review of the effectiveness of strategies to improve health care provider performance in low- and middle-income countries: Methods and descriptive results.
Health care provider (HCP) performance in low- and middle-income countries (LMICs) is often inadequate. The Health Care Provider Performance Review (HCPPR) is a comprehensive systematic review of the effectiveness and cost of strategies to improve HCP performance in LMICs. We present the HCPPR's methods, describe methodological and contextual attributes of included studies, and examine time trends of study attributes. The HCPPR includes studies from LMICs that quantitatively evaluated any strategy to improve HCP performance for any health condition, with no language restrictions. Eligible study designs were controlled trials and interrupted time series. In 2006, we searched 15 databases for published studies; in 2008 and 2010, we completed searches of 30 document inventories for unpublished studies. Data from eligible reports were double-abstracted and entered into a database, which is publicly available. The primary outcome measure was the strategy's effect size. We assessed time trends with logistic, Poisson, and negative binomial regression modeling. We were unable to register with PROSPERO (International Prospective Register of Systematic Reviews) because the protocol was developed prior to the PROSPERO launch. We screened 105,299 citations and included 824 reports from 499 studies of 161 intervention strategies. Most strategies had multiple components and were tested by only one study each. Studies were from 79 countries and had diverse methodologies, geographic settings, HCP types, work environments, and health conditions. Training, supervision, and patient and community supports were the most commonly evaluated strategy components. Only 33.6% of studies had a low or moderate risk of bias. From 1958-2003, the number of studies per year and study quality increased significantly over time, as did the proportion of studies from low-income countries. Only 36.3% of studies reported information on strategy cost or cost-effectiveness. Studies have reported on the efficacy of many strategies to improve HCP performance in LMICs. However, most studies have important methodological limitations. The HCPPR is a publicly accessible resource for decision-makers, researchers, and others interested in improving HCP performance.


Three dehalogenases and physiological restraints in the biodegradation of haloalkanes by Arthrobacter sp. strain HA1.
Arthrobacter sp. strain HA1 utilizes 18 C2-to-C8 1-haloalkanes for growth and synthesizes an inducible 1-bromoalkane debrominase of unknown physiological function (R. Scholtz, T. Leisinger, F. Suter, and A.M. Cook, J. Bacteriol. 169:5016-5021, 1987) in addition to an inducible 1-chlorohexane halidohydrolase which dehalogenates some 50 substrates, including alpha, omega-dihaloalkanes. alpha, omega-Dihaloalkanes were utilized by cultures of strain HA1 under certain conditions only. C9 and C8 homologs prevented growth. At suitable concentrations, C7-to-C5 homologs could serve as sole sources of carbon and energy for growth. C4 and C3 homologs could be utilized only in the presence of a second substrate (e.g., butanol), and the C2 homolog was not degraded. Kinetics of growth and substrate utilization indicated that cells of strain HA1 growing in butanol-salts medium could be used to test whether compounds induced the 1-chlorohexane halidohydrolase. No gratuitous induction of synthesis of the enzyme was observed. Many enzyme substrates (e.g., bromobenzene) did not induce synthesis of the enzyme, though the enzyme sequence to degrade the product (phenol) was present. Some inducers (e.g., bromomethane) were enzyme substrates but not growth substrates. In an attempt to find a physiological role for the 1-bromoalkane debrominase, we observed that several long-chain haloaliphatic compounds (greater than C9; e.g., 1-bromohexadecane and 1-chlorohexadecane) were utilized for growth and that induced cells could dehalogenate several 1-haloalkanes (at least C4 to C16). The dehalogenation of the long-chain compounds could not be assayed in the cell extract, so we presume that a third haloalkane dehalogenase was present.(ABSTRACT TRUNCATED AT 250 WORDS)


Permissive roles of hematopoietin and cytokine tyrosine kinase receptors in early T-cell development.
Although several cytokines have been demonstrated to be critical regulators of development of multiple blood cell lineages, it remains disputed to what degree they act through instructive or permissive mechanisms. Signaling through the FMS-like tyrosine kinase 3 (FLT3) receptor and the hematopoietin IL-7 receptor alpha (IL-7Ralpha) has been demonstrated to be of critical importance for sustained thymopoiesis. Signaling triggered by IL-7 and thymic stromal lymphopoietin (TSLP) is dependent on IL-7Ralpha, and both ligands have been implicated in T-cell development. However, we demonstrate that, whereas thymopoiesis is abolished in adult mice doubly deficient in IL-7 and FLT3 ligand (FLT3L), TSLP does not play a key role in IL-7-independent or FLT3L-independent T lymphopoiesis. Furthermore, whereas previous studies implicated that the role of other cytokine tyrosine kinase receptors in T lymphopoiesis might not involve permissive actions, we demonstrate that ectopic expression of BCL2 is sufficient not only to partially correct the T-cell phenotype of Flt3l(-/-) mice but also to rescue the virtually complete loss of all discernable stages of early T lymphopoiesis in Flt3l(-/-)Il7r(-/-) mice. These findings implicate a permissive role of cytokine receptors of the hematopoietin and tyrosine kinase families in early T lymphopoiesis.


ZEB1 enhances transendothelial migration and represses the epithelial phenotype of prostate cancer cells.
Metastatic colonization involves cancer cell lodgment or adherence in the microvasculature and subsequent migration of those cells across the endothelium into a secondary organ site. To study this process further, we analyzed transendothelial migration of human PC-3 prostate cancer cells in vitro. We isolated a subpopulation of cells, TEM4-18, that crossed an endothelial barrier more efficiently, but surprisingly, were less invasive than parental PC-3 cells in other contexts in vitro. Importantly, TEM4-18 cells were more aggressive than PC-3 cells in a murine metastatic colonization model. Microarray and FACS analysis of these cells showed that the expression of many genes previously associated with leukocyte trafficking and cancer cell extravasation were either unchanged or down-regulated. Instead, TEM4-18 cells exhibited characteristic molecular markers of an epithelial-to-mesenchymal transition (EMT), including frank loss of E-cadherin expression and up-regulation of the E-cadherin repressor ZEB1. Silencing ZEB1 in TEM4-18 cells resulted in increased E-cadherin and reduced transendothelial migration. TEM4-18 cells also express N-cadherin, which was found to be necessary, but not sufficient for increased transendothelial migration. Our results extend the role of EMT in metastasis to transendothelial migration and implicate ZEB1 and N-cadherin in this process in prostate cancer cells.


What is the financial incentive to immigrate? An analysis of salary disparities between health workers working in the Caribbean and popular destination countries.
The continuous migration of Human Resources for Health (HRH) compromises the quality of health services in the developing supplying countries. The ability to increase earnings potentially serves as a strong motivator for HRH to migrate abroad. This study adds to limited available literature on HRH salaries within the Caribbean region and establishes the wage gap between selected Caribbean and popular destination countries. Salaries are reported for registered nurses, medical doctors and specialists. Within these cadres, experience is incorporated at three different levels. Earnings are compared using purchasing power parity (PPP) exchange rates allowing for cost of living adjusted salary differentials, awarded to different levels of work experience for the chosen health cadres in the selected Caribbean countries (Jamaica, Dominica, St Lucia and Grenada) and the three destination countries (United States, United Kingdom and Canada). Registered nurses in the destination countries, across all experience levels, have greater spending power compared to their Caribbean counterparts. Recently qualified registered nurses earn substantially more in the UK (86.4%), US (214.2%) and Canada (182.5% more). The highest PPP salary ($) gap amongst more experienced nurses (5-10 years) is found within the US, with a gap of 163.9%. PPP salary gaps amongst medical doctors were pronounced, with experienced cadres (10-20 years of experience) in the US earning 316.3% more than their Caribbean counterparts, whilst UK doctors (183.5%) and Canadian doctors (251.3%) also earning significantly more. Large salary differentials remained for medical specialists and consultants. US specialist salaries were 540.4% higher than their Caribbean based counterparts, whilst UK and Canadian specialists earned 95.2 and 181.6% more respectively. The PPP adjusted HRH salaries in the three destination countries are superior to those of comparable HRH working in the Caribbean countries selected. The extent of the salary gaps vary according to country and the health cadre under examination, but remain considerable even for newly qualified HRH. The financial incentive to migrate for HRH trained and working in the Caribbean region remains strong, with governments having to consider earning potential abroad when formulating policies and strategies aimed at retaining health professionals.


Genomics pipelines and data integration: challenges and opportunities in the research setting.
The emergence and mass utilization of high-throughput (HT) technologies, including sequencing technologies (genomics) and mass spectrometry (proteomics, metabolomics, lipids), has allowed geneticists, biologists, and biostatisticians to bridge the gap between genotype and phenotype on a massive scale. These new technologies have brought rapid advances in our understanding of cell biology, evolutionary history, microbial environments, and are increasingly providing new insights and applications towards clinical care and personalized medicine. Areas covered: The very success of this industry also translates into daunting big data challenges for researchers and institutions that extend beyond the traditional academic focus of algorithms and tools. The main obstacles revolve around analysis provenance, data management of massive datasets, ease of use of software, interpretability and reproducibility of results. Expert commentary: The authors review the challenges associated with implementing bioinformatics best practices in a large-scale setting, and highlight the opportunity for establishing bioinformatics pipelines that incorporate data tracking and auditing, enabling greater consistency and reproducibility for basic research, translational or clinical settings.


Dietary procyanidins lower triglyceride levels signaling through the nuclear receptor small heterodimer partner.
Hypertriglyceridemia is an independent risk factor in the development of cardiovascular diseases, and we have previously reported that oral administration of a grape seed procyanidin extract (GSPE) drastically decreases plasma levels of triglycerides (TG) and apolipoprotein B (ApoB) in normolipidemic rats, with a concomitant induction in the hepatic expression of the nuclear receptor small heterodimer partner (NR0B2/SHP). Our objective in this study was to elucidate whether SHP is the mediator of the reduction of TG-rich ApoB-containing lipoproteins triggered by GSPE. We show that GSPE inhibited TG and ApoB secretion in human hepatocarcinoma HepG2 cells and had and hypotriglyceridemic effect in wild-type mouse. The TG-lowering action of GSPE was abolished in HepG2 cells transfected with a SHP-specific siRNA and in a SHP-null mouse. Moreover, in mouse liver, GSPE downregulated several lipogenic genes, including steroid response element binding protein 1c (SREBP-1c), and upregulated carnitine palmitoyltransferase-1A (CPT-1A) and apolipoprotein A5 (ApoA5), in a SHP-dependent manner. In HepG2 cells GSPE also inhibited ApoB secretion, but in a SHP-independent manner. In conclusion, SHP is a key mediator of the hypotriglyceridemic response triggered by GSPE. This novel signaling pathway of procyanidins through SHP may be relevant to explain the health effects ascribed to the regular consumption of dietary flavonoids.


The neuroinflammatory response in humans after traumatic brain injury.
Traumatic brain injury is a significant cause of morbidity and mortality worldwide. An epidemiological association between head injury and long-term cognitive decline has been described for many years and recent clinical studies have highlighted functional impairment within 12 months of a mild head injury. In addition chronic traumatic encephalopathy is a recently described condition in cases of repetitive head injury. There are shared mechanisms between traumatic brain injury and Alzheimer's disease, and it has been hypothesized that neuroinflammation, in the form of microglial activation, may be a mechanism underlying chronic neurodegenerative processes after traumatic brain injury. This study assessed the microglial reaction after head injury in a range of ages and survival periods, from <24-h survival through to 47-year survival. Immunohistochemistry for reactive microglia (CD68 and CR3/43) was performed on human autopsy brain tissue and assessed 'blind' by quantitative image analysis. Head injury cases were compared with age matched controls, and within the traumatic brain injury group cases with diffuse traumatic axonal injury were compared with cases without diffuse traumatic axonal injury. A major finding was a neuroinflammatory response that develops within the first week and persists for several months after traumatic brain injury, but has returned to control levels after several years. In cases with diffuse traumatic axonal injury the microglial reaction is particularly pronounced in the white matter. These results demonstrate that prolonged microglial activation is a feature of traumatic brain injury, but that the neuroinflammatory response returns to control levels after several years.


In vitro Antioxidant Potentials of Cyperus rotundus L. Rhizome Extracts and Their Phytochemical Analysis.
Cyperus rotundus L. (family Cyperaceae), native to India, is a multivalent medicinal plant widely used in conventional medicine. The research reports on bioactive components from C. rotundus L. are scanty. The objective of the study was to optimize the best solvent system and bioprospect the possible phytochemicals in C. rotundus L. rhizome (CRR). The phytochemicals were extracted from the rhizomes of C. rotundus L. by successive Soxhlet technique with solvents of increasing polarity. The resultant extracts were analyzed for their total flavonoid content (TFC), total phenolic content (TPC), total proanthocyanidin content (TPAC), in vitro antioxidant potential, and inhibition of lipid peroxidation. The 70% acetone extract of CRR was analyzed using gas chromatography-mass spectrometry (GC-MS) for probable phytochemicals. The TPC, TFC, and TPAC estimates ranged from 0.036 ± 0.002 to 118.924 ± 5.946 μg/mg extract, 7.196 ± 0.359 to 200.654 ± 10.032 μg/mg extract, and 13.115 ± 0.656 to 45.901 ± 2.295 μg/mg extract, respectively. The quantities of TPC, TFC, and TPAC were found to be the highest in 70% acetone extract. The 70% acetone and 70% methanol extracts revealed best radical scavenging effect. GC-MS analysis of CRR extract revealed the presence of a novel compound 1 (2)-acetyl-3 (5)-styryl-5 (3)-methylthiopyrazole. The study indicated that 70% acetone and 70% methanol extracts of CRRs can be a potential source of antioxidants. The studies suggest 70% methanol and acetone as the suitable solvents for the extraction of phytochemicalsNovel compound 1(2)-Acetyl-3(5)-styryl-5(3)-methylthiopyrazole was detected in 70% acetone extract. Abbreviations used: ACRE: Acetone C. rotundus L. rhizome extract; AlCl3: Aluminum chloride; AQRE: Aqueous C. rotundus L. rhizome extract; CE: Catechin Equivalent; CHRE: Chloroform C. rotundus L. rhizome extract; CRR: C. rotundus L. rhizome; DPPH: 2,2 diphenyl-1-picrylhydrazyl; ETRE: Ethanolic C. rotundus L. rhizome extract; EARE: Ethyl acetate C. rotundus L. rhizome extract; FRP: Ferric reducing power; GAE: Gallic acid equivalent; GC-MS: Gas chromatography-mass spectrometry; HERE: Hexane C. rotundus L. rhizome extract; MERE: Methanolic C. rotundus L. rhizome extract; PERE: Petroleum ether C. rotundus L. rhizome extract; QE: Quercetin equivalent; RNS: Reactive nitrogen species; ROS: Reactive oxygen species; TFC: Total flavonoid content; TPC: Total phenolic content; TPAC: Total proanthocyanidin content.


Continuous flow synthesis of a pharmaceutical intermediate: a computational fluid dynamics approach.
Continuous flow chemistry has the potential to greatly improve efficiency in the synthesis of active pharmaceutical ingredients (APIs); however, the optimization of these processes can be complicated by a large number of variables affecting reaction success. In this work, a screening design of experiments was used to compare computational fluid dynamics (CFD) simulations with experimental results. CFD simulations and experimental results both identified the reactor residence time and reactor temperature as the most significant factors affecting product yield for this reaction within the studied design space. A point-to-point comparison of the results showed absolute differences in product yield as low as 2.4% yield at low residence times and up to 19.1% yield at high residence times with strong correlation between predicted and experimental percent yields. CFD was found to underestimate the product yields at low residence times and overestimate at higher residence times. The correlation in predicted product yield and the agreement in identifying significant factors in reaction performance reveals the utility of CFD as a valuable tool in the design of continuous flow tube reactors with significantly reduced experimentation.


Nonclinical and pharmacokinetic assessments to evaluate the potential of tedizolid and linezolid to affect mitochondrial function.
Prolonged treatment with the oxazolidinone linezolid is associated with myelosuppression, lactic acidosis, and neuropathies, toxicities likely caused by impairment of mitochondrial protein synthesis (MPS). To evaluate the potential of the novel oxazolidinone tedizolid to cause similar side effects, nonclinical and pharmacokinetic assessments were conducted. In isolated rat heart mitochondria, tedizolid inhibited MPS more potently than did linezolid (average [± standard error of the mean] 50% inhibitory concentration [IC50] for MPS of 0.31 ± 0.02 μM versus 6.4 ± 1.2 μM). However, a rigorous 9-month rat study comparing placebo and high-dose tedizolid (resulting in steady-state area under the plasma concentration-time curve values about 8-fold greater than those with the standard therapeutic dose in humans) showed no evidence of neuropathy. Additional studies explored why prolonged, high-dose tedizolid did not cause these mitochondriopathic side effects despite potent MPS inhibition by tedizolid. Murine macrophage (J774) cell fractionation studies found no evidence of a stable association of tedizolid with eukaryotic mitochondria. Monte Carlo simulations based on population pharmacokinetic models showed that over the course of a dosing interval using standard therapeutic doses, free plasma concentrations fell below the respective MPS IC50 in 84% of tedizolid-treated patients (for a median duration of 7.94 h) and 38% of linezolid-treated patients (for a median duration of 0 h). Therapeutic doses of tedizolid, but not linezolid, may therefore allow for mitochondrial recovery during antibacterial therapy. The overall results suggest that tedizolid has less potential to cause myelosuppression and neuropathy than that of linezolid during prolonged treatment courses. This, however, remains a hypothesis that must be confirmed in clinical studies.


Polydrug Use and HIV Risk Among People Who Inject Heroin in Tijuana, Mexico: A Latent Class Analysis.
Although most people who inject drugs (PWID) in Tijuana, Mexico, primarily inject heroin, injection and non-injection use of methamphetamine and cocaine is common. We examined patterns of polydrug use among heroin injectors to inform prevention and treatment of drug use and its health and social consequences. Participants were PWID residing in Tijuana, aged ≥18 years who reported heroin injection in the past six months and were recruited through respondent-driven sampling (n = 1,025). Latent class analysis was conducted to assign individuals to classes on a probabilistic basis, using four indicators of past six-month polydrug and polyroute use: cocaine injecting, cocaine smoking or snorting, methamphetamine injecting, and methamphetamine smoking or snorting. Latent class membership was regressed onto covariates in a multinomial logistic regression. Latent class analyses testing 1, 2, 3, and 4 classes were fit, with the 3-class solution fitting best. Class 1 was defined by predominantly heroin use (50.2%, n = 515); class 2 by methamphetamine and heroin use (43.7%, n = 448), and class 3 by methamphetamine, cocaine, and heroin use (6.0%, n = 62). Bivariate and multivariate analyses indicated a group of methamphetamine and cocaine users that exhibited higher-risk sexual practices and lower heroin injecting frequency, and a group of methamphetamine users who were younger and more likely to be female. Discrete subtypes of heroin PWID were identified based on methamphetamine and cocaine use patterns. These findings have identified subtypes of heroin injectors who require more tailored interventions to reduce the health and social harms of injecting drug use.


Global pattern of trends in streamflow and water availability in a changing climate.
Water availability on the continents is important for human health, economic activity, ecosystem function and geophysical processes. Because the saturation vapour pressure of water in air is highly sensitive to temperature, perturbations in the global water cycle are expected to accompany climate warming. Regional patterns of warming-induced changes in surface hydroclimate are complex and less certain than those in temperature, however, with both regional increases and decreases expected in precipitation and runoff. Here we show that an ensemble of 12 climate models exhibits qualitative and statistically significant skill in simulating observed regional patterns of twentieth-century multidecadal changes in streamflow. These models project 10-40% increases in runoff in eastern equatorial Africa, the La Plata basin and high-latitude North America and Eurasia, and 10-30% decreases in runoff in southern Africa, southern Europe, the Middle East and mid-latitude western North America by the year 2050. Such changes in sustainable water availability would have considerable regional-scale consequences for economies as well as ecosystems.


Gamma knife surgery for vestibular schwannoma: 10-year experience of 195 cases.
The authors conducted a study to determine the optimal radiation dose for vestibular schwannoma (VS) and to examine the histopathology in cases of treatment failure for better understanding of the effects of irradiation. A retrospective study was performed of 195 patients with VS; there were 113 female and 82 male patients whose mean age was 51 years (range 11-82 years). Seventy-two patients (37%) had undergone partial or total excision of their tumor prior to gamma knife surgery (GKS). The mean tumor volume was 4.1 cm3 (range 0.04-23.1 cm3). Multiisocenter dose planning placed a prescription dose of 11 to 18.2 Gy on the 50 to 94% isodose located at the tumor margin. Clinical and magnetic resonance (MR) imaging follow-up evaluations were performed every 6 months. A loss of central enhancement was demonstrated on MR imaging in 69.5% of the patients. At the latest MR imaging assessment decreased or stable tumor volume was demonstrated in 93.6% of the patients. During a median follow-up period of 31 months resection was avoided in 96.8% of cases. Uncontrolled tumor swelling was noted in five patients at 3.5, 17, 24, 33, and 62 months after GKS, respectively. Twelve of 20 patients retained serviceable hearing. Two patients experienced a temporary facial palsy. Two patients developed a new trigeminal neuralgia. There was no treatment-related death. Histopathological examination of specimens in three cases (one at 62 months after GKS) revealed a long-lasting radiation effect on vessels inside the tumor. Radiosurgery had a long-term radiation effect on VSs for up to 5 years. A margin 12-Gy dose with homogeneous distribution is effective in preventing tumor progression, while posing no serious threat to normal cranial nerve function.


Computational evidence that frequency trajectory theory does not oppose but emerges from age-of-acquisition theory.
According to the age-of-acquisition hypothesis, words acquired early in life are processed faster and more accurately than words acquired later. Connectionist models have begun to explore the influence of the age/order of acquisition of items (and also their frequency of encounter). This study attempts to reconcile two different methodological and theoretical approaches (proposed by Lambon Ralph & Ehsan, 2006 and Zevin & Seidenberg, 2002) to age-limited learning effects. The current simulations extend the findings reported by Zevin and Seidenberg (2002) that have shown that frequency trajectories (FTs) have limited and specific effects on word-reading tasks. Using the methodological framework proposed by Lambon Ralph and Ehsan (2006), which makes it possible to compare word-reading and picture-naming tasks in connectionist networks, we were able to show that FT has a considerable influence on age-limited learning effects in a picture naming task. The findings show that when the input-output mappings are arbitrary (simulating picture naming tasks), the links formed by the network become entrenched as a result of early experience and that subsequent variations in frequency of exposure of the items have only a minor impact. In contrast, when the mappings between input-output are quasi-systematic or systematic (simulating word-reading tasks), the training of new items was generalized and resulted in the suppression of age-limited learning effects. At a theoretical level, we suggest that FT, which simultaneously takes account of time and the level of exposure across time, represents a more precise and modulated measure compared with the order of introduction of the items and may lead to innovative hypotheses in the field of age-limited learning effects.


Activation of p42erk2 MAPK and p90rsk by IL-2 occurs independently of protein kinase C.
Stimulation of the human interleukin-2 (IL-2)-dependent cell line, Kit225, with IL-2 resulted in the rapid activation of microtubule-associated protein-2 kinase (MAPK), as demonstrated by the tyrosine phosphorylation of p42erk2, the reduced mobility of MAPK in SDS-PAGE gels, and the increased ability of lysates from these cells to phosphorylate myelin basic protein. Measurements of kinase activity in immunoprecipitates of p9O ribosomal S6 kinase (p90rsk) demonstrated that stimulation of this cell line with IL-2 also resulted in the activation of p90rsk. Further, increased MAPK activity occurred following IL-2 stimulation, but not following phorbol 12-myristate 13-acetate (PMA) stimulation in cells depleted of PKC by prolonged treatment with a high concentration of PMA. These results demonstrate that IL-2 stimulation results in the activation of MAPK and p90rsk and that this activation occurs in a PKC-independent manner.


Developmental toxicity evaluation of esterified propoxylated glycerol (EPG) administered in the diet to New Zealand white rabbits.
The safety of a "core" version of esterified propoxylated glycerols (EPGs) was assessed in a developmental toxicity study in New Zealand white rabbits, Hra:(NZW)SPF. Four groups each of 18 inseminated female rabbits received diets ad libitum containing concentrations of 0%, 2.5%, 5%, and 10% EPG (w/w) with 6% corn oil (w/w). No treatment-related effects were observed in any maternal toxicity parameter, including maternal body weight and weight gain, feed consumption, or clinical signs of toxicity. There were no statistically significant treatment-related effects in gestational parameters, including pre- and post-implantation loss, litter size, sex ratio, fetal body weight, and crown-rump length. The incidences of fetal external, visceral, and skeletal malformations or variations were also comparable across groups. A no-observable-adverse-effect level (NOAEL) of 10% EPG (approximately 4.76 g/kg bw/day) for both maternal and developmental toxicity is proposed based on the results of this study.


Tumor Tissue Explant Culture of Patient-Derived Xenograft as Potential Prioritization Tool for Targeted Therapy.
Despite of remarkable progress made in the head and neck cancer (HNC) therapy, the survival rate of this metastatic disease remain low. Tailoring the appropriate therapy to patients is a major challenge and highlights the unmet need to have a good preclinical model that will predict clinical response. Hence, we developed an accurate and time efficient drug screening method of tumor ex vivo analysis (TEVA) system, which can predict patient-specific drug responses. In this study, we generated six patient derived xenografts (PDXs) which were utilized for TEVA. Briefly, PDXs were cut into 2 × 2 × 2 mm3 explants and treated with clinically relevant drugs for 24 h. Tumor cell proliferation and death were evaluated by immunohistochemistry and TEVA score was calculated. Ex vivo and in vivo drug efficacy studies were performed on four PDXs and three drugs side-by-side to explore correlation between TEVA and PDX treatment in vivo. Efficacy of drug combinations was also ventured. Optimization of the culture timings dictated 24 h to be the time frame to detect drug responses and drug penetrates 2 × 2 × 2 mm3 explants as signaling pathways were significantly altered. Tumor responses to drugs in TEVA, significantly corresponds with the drug efficacy in mice. Overall, this low cost, robust, relatively simple and efficient 3D tissue-based method, employing material from one PDX, can bypass the necessity of drug validation in immune-incompetent PDX-bearing mice. Our data provides a potential rationale for utilizing TEVA to predict tumor response to targeted and chemo therapies when multiple targets are proposed.


No Man Left Behind: Effectively Engaging Male Military Veterans in Counseling.
Ex-military men have emerged as a vulnerable subgroup for mental illness amid long-standing trends signaling men's reticence to seek professional help. Less explored is how men engage or disengage when they actually do enter helping programs. Contrasting decades of quantitative research pairing masculine ideology with low help seeking (i.e., describing the problem), this article draws on qualitative data to distill factors that help men become engaged and committed to counseling (i.e., identifying solutions). Shared is an evaluation of a treatment program with high success rates and virtually no dropouts-a unique occurrence in men's counseling. Enhanced Critical Incident Technique data suggest that helping men feel competent and free from judgment in the company of down-to-earth peers and genuine practitioners are instrumental in helping men draw benefit from counseling. While appealing to male gender roles may be critical in recruiting men to counseling, men can transition to embrace virtues (i.e., that might be shared by men and women alike) and universal human needs as counseling progresses.


A high-accuracy pseudospectral full-vectorial leaky optical waveguide mode solver with carefully implemented UPML absorbing boundary conditions.
The previously developed full-vectorial optical waveguide eigenmode solvers using pseudospectral frequency-domain (PSFD) formulations for optical waveguides with arbitrary step-index profile is further implemented with the uniaxial perfectly matched layer (UPML) absorption boundary conditions for treating leaky waveguides and calculating their complex modal effective indices. The role of the UPML reflection coefficient in achieving high-accuracy mode solution results is particularly investigated. A six-air-hole microstructured fiber is analyzed as an example to compare with published high-accuracy multipole method results for both the real and imaginary parts of the effective indices. It is shown that by setting the UPML reflection coefficient values as small as on the order of 10(-40) ∼ 10(-70), relative errors in the calculated complex effective indices can be as small as on the order of 10(-12).


Bedside-to-Bench Translational Research for Chronic Heart Failure: Creating an Agenda for Clients Who Do Not Meet Trial Enrollment Criteria.
Congestive heart failure (CHF) is a chronic condition usually without cure. Significant developments, particularly those addressing pathophysiology, mainly started at the bench. This approach has seen many clinical observations initially explored at the bench, subsequently being trialed at the bedside, and eventually translated into clinical practice. This evidence, however, has several limitations, importantly the generalizability or external validity. We now acknowledge that clinical management of CHF is more complicated than merely translating bench-to-bedside evidence in a linear fashion. This review aims to help explore this evolving area from an Australian perspective. We describe the continuation of research once core evidence is established and describe how clinician-scientist collaboration with a bedside-to-bench view can help enhance evidence translation and generalizability. We describe why an extension of the available evidence or generating new evidence is occasionally needed to address the increasingly diverse cohort of patients. Finally, we explore some of the tools used by basic scientists and clinicians to develop evidence and describe the ones we feel may be most beneficial.


Hook Breathing Facilitates SaO2 Recovery After Deep Dives in Freedivers With Slow Recovery.
To facilitate recovery from hypoxia, many freedivers use a breathing method called "hook breathing" (HB) after diving, involving an interrupted exhale to build up intrapulmonary pressure. Some divers experience a delay in recovery of arterial oxygen saturation (SaO2) after diving, interpreted as symptoms of mild pulmonary edema, and facilitated recovery may be especially important in this group to avoid hypoxic "blackout." We examined the influence of HB on recovery of SaO2 in freedivers with slow recovery (SR) and fast recovery (FR) of SaO2 after deep "free immersion" (FIM) apnea dives to 30 m depth. Twenty-two male freedivers, with a mean (SD) personal best in the discipline FIM of 57(26) m, performed two 30 m deep dives, one followed by HB and one using normal breathing (NB) during recovery, at different days and weighted order. SaO2 and heart rate (HR) were measured via pulse oximetry during recovery. The SR group (n = 5) had a faster SaO2 recovery using HB, while the FR group (n = 17) showed no difference between breathing techniques. At 105 s, the SR group reached a mean (SD) SaO2 of 95(5)% using HB, while using NB, their SaO2 was 87(5)% (p < 0.05), and 105-120 s after surfacing SaO2 was higher with HB (p < 0.05). In SR subjects, the average time needed to reach 95% SaO2 with HB was 60 s, while it was 120 s at NB (p < 0.05). HR was similar in the SR group, while it was initially elevated at HB in the FR group (p < 0.05). We conclude that HB efficiently increases SaO2 recovery in SR individuals, but not in the FR group. The proposed mechanism is that increased pulmonary pressure with HB will reverse any pulmonary edema and facilitate oxygen uptake in divers with delayed recovery.


Effect of childhood vaccination and antibiotic use on pneumococcal populations and genome-wide associations with disease among children in Nepal: an observational study.
Pneumococcal disease is a leading cause of bacterial pneumonia and invasive bacterial disease among children globally. The reason some strains of pneumococci are more likely to cause disease, and how interventions such as vaccines and antibiotics affect pneumococcal strains is poorly understood. We aimed to identify genetic regions under selective pressure and those associated with disease through the analysis of pneumococcal whole-genome sequences. Whole-genome sequencing was performed on pneumococcal isolates collected between January, 2005, and May, 2018, in Kathmandu, Nepal, which included programmatic ten-valent pneumococcal conjugate vaccine (PCV10) introduction in 2015. Isolates were from three distinct cohorts: nasopharyngeal swabs of healthy community-based children, nasopharyngeal swabs of children admitted to hospital with pneumonia, and sterile-site cultures from children admitted to hospital. Across these cohorts we examined serotype distribution, antibiotic resistance, strain distribution, and regions of recombination to determine genes that were undergoing diversifying selection. Genome-wide association studies comparing pneumonia and sterile-site isolates with healthy carriage were used to determine novel variants associated with disease. After programmatic introduction of PCV10, there was a decline in vaccine covered serotypes; however, strains that had expressed these serotypes continued to exist in the post-PCV population. We identified GPSC9 to be a strain of concern due to its high prevalence in disease, multidrug resistance, and ability to switch to an unencapsulated phenotype via insertion of virulence factor pspC into the cps locus. Antibiotic resistance loci to co-trimoxazole were found to be prevalent (pre-PCV10 78% vs post-PCV10 81%; p=0·27) and increasingly prevalent to penicillin (pre-PCV10 15% vs post-PCV10 32%; p<0·0001). Regions with multiple recombinations were identified spanning the surface protein virulence factors pspA and pspC and antibiotic targets pbpX, folA, folC, folE, and folP. Furthermore, we identified variants in lacE2 to be strongly associated with isolates from children with pneumonia and PRIP to be strongly associated with isolates from sterile sites. Our work highlights the effect of pneumococcal conjugate vaccines, antibiotics, and host-pathogen interaction in pneumococcal variation, and the pathogen's capability of adapting to these factors at both population-wide and strain-specific levels. Ongoing surveillance of disease-associated strains and further investigation of lacE2 and PRIP as serotype-independent targets for therapeutic interventions is required. Gavi, The Vaccine Alliance; WHO; Bill & Melinda Gates Foundation; Wellcome Sanger Institute; and US Centers for Disease Control and Prevention.


Development of a High-Throughput ex-Vivo Burn Wound Model Using Porcine Skin, and Its Application to Evaluate New Approaches to Control Wound Infection.
Biofilm formation in wounds is considered a major barrier to successful treatment, and has been associated with the transition of wounds to a chronic non-healing state. Here, we present a novel laboratory model of wound biofilm formation using ex-vivo porcine skin and a custom burn wound array device. The model supports high-throughput studies of biofilm formation and is compatible with a range of established methods for monitoring bacterial growth, biofilm formation, and gene expression. We demonstrate the use of this model by evaluating the potential for bacteriophage to control biofilm formation by Staphylococcus aureus, and for population density dependant expression of S. aureus virulence factors (regulated by the Accessory Gene Regulator, agr) to signal clinically relevant wound infection. Enumeration of colony forming units and metabolic activity using the XTT assay, confirmed growth of bacteria in wounds and showed a significant reduction in viable cells after phage treatment. Confocal laser scanning microscopy confirmed the growth of biofilms in wounds, and showed phage treatment could significantly reduce the formation of these communities. Evaluation of agr activity by qRT-PCR showed an increase in activity during growth in wound models for most strains. Activation of a prototype infection-responsive dressing designed to provide a visual signal of wound infection, was related to increased agr activity. In all assays, excellent reproducibility was observed between replicates using this model.


Induction of Effective Immunity against Trypanosoma cruzi.
Chagas disease, caused by Trypanosoma cruzi, is a major public health issue. Limitations in immune responses to natural T. cruzi infection usually result in parasite persistence with significant complications. A safe, effective, and reliable vaccine would reduce the threat of T. cruzi infections; however, no suitable vaccine is currently available due to a lack of understanding of the requirements for induction of fully protective immunity. We established a T. cruzi strain expressing green fluorescent protein (GFP) under the control of dihydrofolate reductase degradation domain (DDD) with a hemagglutinin (HA) tag, GFP-DDDHA, which was induced by trimethoprim-lactate (TMP-lactate), which results in the death of intracellular parasites. This attenuated strain induces very strong protection against reinfection. Using this GFP-DDDHA strain, we investigated the mechanisms underlying the protective immune response in mice. Immunization with this strain led to a response that included high levels of gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α), as well as a rapid expansion of effector and memory T cells in the spleen. More CD8+ T cells differentiate to memory cells following GFP-DDDHA infection than after infection with a wild-type (WT) strain. The GFP-DDDHA strain also provides cross-protection against another T. cruzi isolate. IFN-γ is important in mediating the protection, as IFN-γ knockout (KO) mice failed to acquire protection when infected with the GFP-DDDHA strain. Immune cells demonstrated earlier and stronger protective responses in immunized mice after reinfection with T. cruzi than those in naive mice. Adoptive transfers with several types of immune cells or with serum revealed that several branches of the immune system mediated protection. A combination of serum and natural killer cells provided the most effective protection against infection in these transfer experiments.


Size Matters: Assessing Optimum Soil Sample Size for Fungal and Bacterial Community Structure Analyses Using High Throughput Sequencing of rRNA Gene Amplicons.
We examined the effect of different soil sample sizes obtained from an agricultural field, under a single cropping system uniform in soil properties and aboveground crop responses, on bacterial and fungal community structure and microbial diversity indices. DNA extracted from soil sample sizes of 0.25, 1, 5, and 10 g using MoBIO kits and from 10 and 100 g sizes using a bead-beating method (SARDI) were used as templates for high-throughput sequencing of 16S and 28S rRNA gene amplicons for bacteria and fungi, respectively, on the Illumina MiSeq and Roche 454 platforms. Sample size significantly affected overall bacterial and fungal community structure, replicate dispersion and the number of operational taxonomic units (OTUs) retrieved. Richness, evenness and diversity were also significantly affected. The largest diversity estimates were always associated with the 10 g MoBIO extractions with a corresponding reduction in replicate dispersion. For the fungal data, smaller MoBIO extractions identified more unclassified Eukaryota incertae sedis and unclassified glomeromycota while the SARDI method retrieved more abundant OTUs containing unclassified Pleosporales and the fungal genera Alternaria and Cercophora. Overall, these findings indicate that a 10 g soil DNA extraction is most suitable for both soil bacterial and fungal communities for retrieving optimal diversity while still capturing rarer taxa in concert with decreasing replicate variation.


Antigen-pulsed CD8alpha+ dendritic cells generate an immune response after subcutaneous injection without homing to the draining lymph node.
Two subsets of murine splenic dendritic cells, derived from distinct precursors, can be distinguished by surface expression of CD8alpha homodimers. The functions of the two subsets remain controversial, although it has been suggested that the lymphoid-derived (CD8alpha+) subset induces tolerance, whereas the myeloid-derived (CD8alpha-) subset has been shown to prime naive T cells and to generate memory responses. To study their capacity to prime or tolerize naive CD4(+) T cells in vivo, purified CD8alpha+ or CD8alpha- dendritic cells were injected subcutaneously into normal mice. In contrast to CD8alpha- dendritic cells, the CD8alpha+ fraction failed to traffic to the draining lymph node and did not generate responses to intravenous peptide. However, after in vitro pulsing with peptide, strong in vivo T cell responses to purified CD8alpha+ dendritic cells could be detected. Such responses may have been initiated via transfer of peptide-major histocompatibility complex complexes to migratory host CD8alpha- dendritic cells after injection. These data suggest that correlation of T helper cell type 1 (Th1) and Th2 priming with injection of CD8alpha+ and CD8alpha- dendritic cells, respectively, may not result from direct T cell activation by lymphoid versus myeloid dendritic cells, but rather from indirect modification of the response to immunogenic CD8alpha- dendritic cells by CD8alpha+ dendritic cells.


Pesticide levels and environmental risk in aquatic environments in China--A review.
China is one of the largest producers and consumers of pesticides in the world today. Along with the widespread use of pesticides and industrialization, there is a growing concern for water quality. The present review aims to provide an overview of studies on pesticides in aquatic environments in China. The levels in the water, sediment and biota were scored according to a detailed environmental classification system based on ecotoxicological effect, which is therefore a useful tool for assessing the risk these compounds pose to the aquatic ecosystem. Our review reveals that the most studied areas in China are the most populated and the most developed economically and that the most frequently studied pesticides are DDT and HCH. We show maps of where studies have been conducted and show the ecotoxicological risk the pesticides pose in each of the matrices. Our review pinpoints the need for biota samples to assess the risk. A large fraction of the results from the studies are given an environmental classification of "very bad" based on levels in biota. In general, the risk is higher for DDT than HCH. A few food web studies have also been conducted, and we encourage further study of this important information from this region. The review reveals that many of the most important agricultural provinces (e.g., Henan, Hubei and Hunan) with the largest pesticide use have been the subject of few studies on the environmental levels of pesticides. We consider this to be a major knowledge gap for understanding the status of pesticide contamination and related risk in China. Furthermore, there is also a lack of studies in remote Chinese environments, which is also an important knowledge gap. The compounds analyzed and reported in the studies represent a serious bias because a great deal of attention is given to DDT and HCH, whereas the organophosphate insecticides dominating current use are less frequently investigated. For the future, we point to the need for an organized monitoring plan designed according to the knowledge gaps in terms of geographical distribution, compounds included, and risks.


Topical application of honey in the management of chemo/radiotherapy-induced oral mucositis: A systematic review and network meta-analysis.
Mucositis is an inflammatory response of mucosal epithelial cells to the cytotoxic effects of chemotherapy and radiation therapy. To assess the comparative efficacy of honey for patients with cancer undergoing chemo/radiotherapy-induced oral mucositis through a systematic review and network meta-analysis. A network meta-analysis was used to identify evidence from relevant randomized controlled trials (RCTs). We searched PubMed, Embase, and the Cochrane Library for publications up to November 2017. The prespecified primary efficacy outcome was the treatment effect of moderate-severe oral mucositis with honey. We performed subgroup analyses and meta-regressions according to the age group, cancer type, mucositis cause, honey type, control arm and type of assessment scale. Moreover, secondary efficacy outcomes were treatment completed, onset time of mucositis, swallowing diary, fungal colonization, bacterial colonisation and analgesic use. And, we did standardize meta-analyses using the random-effects model, later completing the random-effects network meta-analyses by different treatment/control arms. A total of 17 RCTs were eligible (22 analyses), involving 1265 patients and 13 arms. Honey treatment arm significantly increased the therapeutic effect of chemo/radiotherapy-induced moderate-severe oral mucositis (0.25, 0.14-0.46); significant efficacy was observed in a large proportion of subgroups. The meta-regression may have identified the causes of heterogeneity as the honey type (P = 0.038). Therefore, we need to perform further comparisons of difference in honey types and controls by network meta-analysis, and the results from network meta-analysis revealed that pure natural honey was superior in therapeutic effect (0.05, 0.01-0.46). For secondary outcomes, significant effect was found in decreasing onset time of mucositis (0.41, 0.08-0.73), while no increase in adverse effects was observed. The study is registered with PROSPERO (CRD42017070873). The adjuvant treatment honey is effective and safe for patients with cancer undergoing chemo/radiotherapy-induced oral mucositis, especially applied pure natural local honey can be invoked as a first-line adjuvant therapy agent.


Theoretical study of the mechanism of proton transfer in the esterase EstB from Burkholderia gladioli.
Esterase EstB from Burkholderia gladioli belongs to a novel class of esterases homologous to penicillin binding proteins, notably DD-peptidase and class C β-lactamases. It can cleave the side chain acetyl ester group from cephalosporins leaving the β-lactam ring intact, which is a feature of relevance to industrial biocatalytic applications in the production of semisynthetic cephalosporin derivatives. Due to its important role as a potential biocatalyst in industry, the significance of EstB has been greatly appreciated. However, the molecular basis for those residues involving catalysis of EstB remains elusive. By analyzing the crystal structure of EstB, we identified a conserved water molecule in active-site cavity which might mediate an intramolecular proton transfer (PT) from Lys78 to Asp186 via Tyr133. Then a combined computational approach including molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations was employed to explore this presumable PT mode in the native enzyme. A 30 ns MD simulation of the enzyme highlights the conserved H-bond network involving Lys78, Tyr133, Asp186, and the conserved water molecule in the active site. In particular, the water molecule did not exchange with bulk solvent, indicating its structural and functional relevance. The energy profile calculated by QM/MM approach displayed a notably low PT barrier (2.2 kcal/mol) and a dramatic energy difference (14.1 kcal/mol) in reactants versus immediate products, which implies that the proposed proton shuttle is concerted and energetically favorable. Our studies offer a reasonable pathway to yield a free base by assisting Lys78 deprotonation, thereby paving the way for future studies on Ser75 activation that is a critical step in catalysis by EstB, as well as biocatalyst development by rational attempts. This PT mode would also afford clues for the forthcoming investigation on acyltransferase LovD that is homologous to EstB.


Chloroplast membrane alterations in triazine-resistant Amaranthus retroflexus biotypes.
The effectiveness of diuron, atrazine, procyazine, and cyanazine were compared in controlling growth of redroot pigweed (Amaranthus retroflexus L.) in hydroponic culture. A very marked differential inhibition response was observed for atrazine between resistant and susceptible biotypes. Procyazine and cyanazine exhibited less dramatic differential responses, whereas diuron was equally effective in controlling growth in both biotypes. Photosystem II activity of chloroplasts from both triazine-resistant and triazine-susceptible biotypes was inhibited by diuron but only the chloroplasts from triazine-susceptible biotypes were inhibited significantly by atrazine. The photochemical activity of chloroplasts from triazine-resistant biotypes was partially resistant to procyazine or cyanazine inhibition. The parallel lack of diuron differential effects, partial procyazine and cyanazine differential response, and very marked atrazine differential response in both whole plant and chloroplast assays indicates that the chloroplast is the site of selective herbicide tolerance in these triazine-resistant redroot pigweed biotypes.Photosystem II photochemical properties were characterized by analysis of chlorophyll fluorescence transients in the presence or absence of herbicides. Data with susceptible chloroplasts indicated that both diuron and atrazine inhibit electron flow very near the primary electron acceptor of photosystem II. Only diuron altered the fluorescence transient in resistant chloroplasts. In untreated preparations there were marked differences in the fast phases of the fluorescence increase in resistant vs. susceptible chloroplasts; these data are interpreted as showing that the resistant plastids have an alteration in the rate of reoxidation of the primary photosystem II electron acceptor. Electrophoretic analysis of chloroplast membrane proteins of the two biotypes showed small changes in the electrophoretic mobilities of two polypeptide species. The data provide evidence for the following herbicide resistance mechanism: genetically controlled modification of the herbicide target site.


Risk of transmission associated with sharing drug injecting paraphernalia: analysis of recent hepatitis C virus (HCV) infection using cross-sectional survey data.
Sharing injecting paraphernalia (containers, filters and water) poses a risk of transmitting the hepatitis C virus (HCV). The prevalence of, and risk of HCV from, such behaviour has not been extensively reported in Europe. People who inject drugs (PWID) were recruited in cross-sectional surveys from services providing sterile injecting equipment across Scotland between 2008 and 2010. Participants completed a questionnaire and provided a blood spot for anonymous testing. Logistic regression was used to examine the association between recent HCV infection (anti-HCV negative and HCV-RNA positive) and self-reported measures of injecting equipment sharing in the 6 months preceding interview. Twelve per cent of the sample reported sharing needles/syringes, and 40% reported sharing paraphernalia in the previous 6 months. The adjusted odds ratios (AOR) for sharing needles/syringes (+/- paraphernalia), and sharing only paraphernalia in the last 6 months were 6.7 (95% CI 2.6-17.1) and 3.0 (95% CI 1.2-7.5), respectively. Among those who reported not sharing needles/syringes, sharing containers and filters were both significantly associated with recent HCV infection (AOR 3.1, 95% CI 1.3-7.8 and 3.1, 95% CI 1.3-7.5, respectively); sharing water was not. We present the first study to apply a cross-sectional approach to the analysis of the association between sharing paraphernalia and incident HCV infection and demonstrate consistent results with previous longitudinal studies. The prevalence of paraphernalia sharing in our study population is high, representing significant potential for HCV transmission.


The histone H3 lysine 9 methyltransferases G9a and GLP regulate polycomb repressive complex 2-mediated gene silencing.
G9a/GLP and Polycomb Repressive Complex 2 (PRC2) are two major epigenetic silencing machineries, which in particular methylate histone H3 on lysines 9 and 27 (H3K9 and H3K27), respectively. Although evidence of a crosstalk between H3K9 and H3K27 methylations has started to emerge, their actual interplay remains elusive. Here, we show that PRC2 and G9a/GLP interact physically and functionally. Moreover, combining different genome-wide approaches, we demonstrate that Ezh2 and G9a/GLP share an important number of common genomic targets, encoding developmental and neuronal regulators. Furthermore, we show that G9a enzymatic activity modulates PRC2 genomic recruitment to a subset of its target genes. Taken together, our findings demonstrate an unanticipated interplay between two main histone lysine methylation mechanisms, which cooperate to maintain silencing of a subset of developmental genes.


eRegistries: Electronic registries for maternal and child health.
The Global Roadmap for Health Measurement and Accountability sees integrated systems for health information as key to obtaining seamless, sustainable, and secure information exchanges at all levels of health systems. The Global Strategy for Women's, Children's and Adolescent's Health aims to achieve a continuum of quality of care with effective coverage of interventions. The WHO and World Bank recommend that countries focus on intervention coverage to monitor programs and progress for universal health coverage. Electronic health registries - eRegistries - represent integrated systems that secure a triple return on investments: First, effective single data collection for health workers to seamlessly follow individuals along the continuum of care and across disconnected cadres of care providers. Second, real-time public health surveillance and monitoring of intervention coverage, and third, feedback of information to individuals, care providers and the public for transparent accountability. This series on eRegistries presents frameworks and tools to facilitate the development and secure operation of eRegistries for maternal and child health. In this first paper of the eRegistries Series we have used WHO frameworks and taxonomy to map how eRegistries can support commonly used electronic and mobile applications to alleviate health systems constraints in maternal and child health. A web-based survey of public health officials in 64 low- and middle-income countries, and a systematic search of literature from 2005-2015, aimed to assess country capacities by the current status, quality and use of data in reproductive health registries. eRegistries can offer support for the 12 most commonly used electronic and mobile applications for health. Countries are implementing health registries in various forms, the majority in transition from paper-based data collection to electronic systems, but very few have eRegistries that can act as an integrating backbone for health information. More mature country capacity reflected by published health registry based research is emerging in settings reaching regional or national scale, increasingly with electronic solutions. 66 scientific publications were identified based on 32 registry systems in 23 countries over a period of 10 years; this reflects a challenging experience and capacity gap for delivering sustainable high quality registries. Registries are being developed and used in many high burden countries, but their potential benefits are far from realized as few countries have fully transitioned from paper-based health information to integrated electronic backbone systems. Free tools and frameworks exist to facilitate progress in health information for women and children.


Imaging appearances of musculoskeletal developmental variants in the pediatric population.
Variations in musculoskeletal development in children are commonly encountered. These variants often have a confusing appearance on imaging and may simulate pathologic conditions. However, in many instances, these normal variants have certain features that allow for confident determination of the benign nature of these entities. An awareness of the characteristic imaging features is therefore important for radiologists. In this review, we focus on 4 specific categories of variants in the development: (1) variations in the normal ossification of skeletal structures, (2) the appearance of tendinous and ligamentous insertions in the developing skeleton, (3) overlapping lines that can be confused with fractures or other pathologic conditions, and (4) variant orientation of normal bones. We review the etiology and imaging appearance of these entities and also describe methods of differentiating these benign entities from pathologic lesions. Although in certain cases, correlation with clinical parameters is needed to confidently diagnose the lesion as benign, in many cases, an appreciation of the characteristic imaging features alone would suffice and prevent a potentially costly workup.


Alpha 2 Na+,K+-ATPase silencing induces loss of inflammatory response and ouabain protection in glial cells.
Ouabain (OUA) is a cardiac glycoside that binds to Na+,K+-ATPase (NKA), a conserved membrane protein that controls cell transmembrane ionic concentrations and requires ATP hydrolysis. At nM concentrations, OUA activates signaling pathways that are not related to its typical inhibitory effect on the NKA pump. Activation of these signaling pathways protects against some types of injury of the kidneys and central nervous system. There are 4 isoforms of the alpha subunit of NKA, which are differentially distributed across tissues and may have different physiological roles. Glial cells are important regulators of injury and inflammation in the brain and express the α1 and α2 NKA isoforms. This study investigated the role of α2 NKA in OUA modulation of the neuroinflammatory response induced by lipopolysaccharide (LPS) in mouse primary glial cell cultures. LPS treatment increased lactate dehydrogenase release, while OUA did not decrease cell viability and blocked LPS-induced NF-κB activation. Silencing α2 NKA prevented ERK and NF-κB activation by LPS. α2 NKA also regulates TNF-α and IL-1β levels. The data reported here indicate a significant role of α2 NKA in regulating central LPS effects, with implications in the associated neuroinflammatory processes.


Pathogenesis-targeted, disease-modifying therapies in Parkinson disease.
Parkinson disease is an inexorably progressive neurodegenerative disorder. Multiple attempts have been made to establish therapies for Parkinson disease which provide neuroprotection or disease modification-two related, but not identical, concepts. However, to date, none of these attempts have succeeded. Many challenges exist in this field of research, including a complex multisystem disorder that includes dopaminergic and non-dopaminergic features; poorly understood and clearly multifaceted disease pathogenic mechanisms; a lack of reliable animal models; an absence of effective biomarkers of disease state, progression, and target engagement; and the confounding effects of potent symptomatic therapy. In this article, we will review previous, ongoing, and potential future trials designed to alter the progressive course of the disease from the perspective of the targeted underlying pathogenic mechanisms.


Defects in the Ferroxidase That Participates in the Reductive Iron Assimilation System Results in Hypervirulence in Botrytis Cinerea.
The plant pathogen Botrytis cinerea is responsible for gray-mold disease, which infects a wide variety of species. The outcome of this host-pathogen interaction, a result of the interplay between plant defense and fungal virulence pathways, can be modulated by various environmental factors. Among these, iron availability and acquisition play a crucial role in diverse biological functions. How B. cinerea obtains iron, an essential micronutrient, during infection is unknown. We set out to determine the role of the reductive iron assimilation (RIA) system during B. cinerea infection. This system comprises the BcFET1 ferroxidase, which belongs to the multicopper oxidase (MCO) family of proteins, and the BcFTR1 membrane-bound iron permease. Gene knockout and complementation studies revealed that, compared to the wild type, the bcfet1 mutant displays delayed conidiation, iron-dependent sclerotium production, and significantly reduced whole-cell iron content. Remarkably, this mutant exhibited a hypervirulence phenotype, whereas the bcftr1 mutant presents normal virulence and unaffected whole-cell iron levels and developmental programs. Interestingly, while in iron-starved plants wild-type B. cinerea produced slightly reduced necrotic lesions, the hypervirulence phenotype of the bcfet1 mutant is no longer observed in iron-deprived plants. This suggests that B. cinerea bcfet1 knockout mutants require plant-derived iron to achieve larger necrotic lesions, whereas in planta analyses of reactive oxygen species (ROS) revealed increased ROS levels only for infections caused by the bcfet1 mutant. These results suggest that increased ROS production, under an iron sufficiency environment, at least partly underlie the observed infection phenotype in this mutant.IMPORTANCE The plant-pathogenic fungus B. cinerea causes enormous economic losses, estimated at anywhere between $10 billion and $100 billion worldwide, under both pre- and postharvest conditions. Here, we present the characterization of a loss-of-function mutant in a component involved in iron acquisition that displays hypervirulence. While in different microbial systems iron uptake mechanisms appear to be critical to achieve full pathogenic potential, we found that the absence of the ferroxidase that is part of the reductive iron assimilation system leads to hypervirulence in this fungus. This is an unusual and rather underrepresented phenotype, which can be modulated by iron levels in the plant and provides an unexpected link between iron acquisition, reactive oxygen species (ROS) production, and pathogenesis in the Botrytis-plant interaction.


Outcomes of cataract surgery in a rural and urban south Indian population.
To assess the visual outcome after cataract surgery in a south Indian population. Population-based cross-sectional study of subjects aged 40 years or more. Three thousand nine hundred and twenty-four rural subjects from 27 contiguous villages and 3850 urban subjects from five randomly selected divisions were studied. All subjects underwent a comprehensive ophthalmic examination that included visual acuity, refraction, slit-lamp biomicroscopy, applanation tonometry, gonioscopy, and dilated retinal examination. Chi square test, t test and multivariate analysis were used. Five hundred and twenty-eight (216 males, 312 females, 781 eyes) rural subjects (13.5%, 95% confidence interval (CI) 12.4% to 14.6%) and 406 (197 males, 209 females, 604 eyes) urban subjects (10.5%, 95% CI 9.6-11.5%) had undergone cataract surgery. Outcome of cataract surgery was defined based on visual acuity. Using best-corrected visual acuity for classification, the single most important cause for visual impairment was cystoid macular edema in the aphakic group and posterior capsule opacification in the pseudophakic group. Aphakia (visual acuity of < 20/60 to <or= 20/400--odds ratio (OR) 1.8; 95% CI 1.3 to 2.6%, visual acuity of < 20/400--OR 6.2; 95% 4.0 to 9.8%), rural residence (visual acuity of < 20/60 to <or= 20/400--OR 3.2; 95% CI 2.2 to 4.5% and visual acuity of < 20/400--OR OR 3.5; 95% CI 2.3 to 5.5%) were associated with visual impairment. The urban cataract-operated population had significantly more pseudophakics ( P < 0.001), men ( P = 0.02) and literates ( P < 0.001). In the rural group the prevalence of cataract surgery (13.5% vs. 10.5%, P P < 0.001) were significantly greater. In 30% of rural and 16% of urban subjects uncorrected refraction was the cause of visual impairment. Surgery-related complications were major causes for visual acuity of < 20/60.


Molecular Regulation of Temperature-Dependent Floral Induction in Tulipa gesneriana.
The vegetative-to-reproductive phase change in tulip (Tulipa gesneriana) is promoted by increasing temperatures during spring. The warm winters of recent years interfere with this process and are calling for new adapted cultivars. A better understanding of the underlying molecular mechanisms would be of help, but unlike the model plant Arabidopsis (Arabidopsis thaliana), very little is known about floral induction in tulip. To shed light on the gene regulatory network controlling flowering in tulip, RNA sequencing was performed on meristem-enriched tissue collected under two contrasting temperature conditions, low and high. The start of reproductive development correlated with rounding of the shoot apical meristem and induction of TGSQA expression, a tulip gene with a high similarity to Arabidopsis APETALA1 Gene Ontology enrichment analysis of differentially expressed genes showed the overrepresentation of genes potentially involved in floral induction, bulb maturation, and dormancy establishment. Expression analysis revealed that TERMINAL FLOWER1 (TgTFL1) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1-like1 (TgSOC1-like1) might be repressors, whereas TgSOC1-like2 likely is an activator, of flowering. Subsequently, the flowering time-associated expression of eight potential flowering time genes was confirmed in three tulip cultivars grown in the field. Additionally, heterologous functional analyses in Arabidopsis resulted in flowering time phenotypes in line with TgTFL1 being a floral repressor and TgSOC1-like2 being a floral activator in tulip. Taken together, we have shown that long before morphological changes occur in the shoot apical meristem, the expression of floral repressors in tulip is suppressed by increased ambient temperatures, leading either directly or indirectly to the activation of potential flowering activators shortly before the commencement of the phase change.


Canadian Association of Radiologists White Paper on De-identification of Medical Imaging: Part 2, Practical Considerations.
The application of big data, radiomics, machine learning, and artificial intelligence (AI) algorithms in radiology requires access to large data sets containing personal health information. Because machine learning projects often require collaboration between different sites or data transfer to a third party, precautions are required to safeguard patient privacy. Safety measures are required to prevent inadvertent access to and transfer of identifiable information. The Canadian Association of Radiologists (CAR) is the national voice of radiology committed to promoting the highest standards in patient-centered imaging, lifelong learning, and research. The CAR has created an AI Ethical and Legal standing committee with the mandate to guide the medical imaging community in terms of best practices in data management, access to health care data, de-identification, and accountability practices. Part 2 of this article will inform CAR members on the practical aspects of medical imaging de-identification, strengths and limitations of de-identification approaches, list of de-identification software and tools available, and perspectives on future directions.


Organization and regulation of the neurotoxin genes in Clostridium botulinum and Clostridium tetani.
Botulinum and tetanus neurotoxins are structurally and functionally related 150 kDa proteins that are potent inhibitors of neuroexocytosis. Botulinum neurotoxin associates with non-toxic proteins to form complexes of various sizes. The botulinum neurotoxin and non-toxic protein genes are clustered in a DNA segment called the botulinum locus. This locus is probably located on a mobile or degenerate mobile element, which accounts for the various genomic localizations (chromosome, plasmid, phage) in different Clostridium botulinum types. The botulinum neurotoxin and non-toxic protein genes are organized in two polycistronic operons (ntnh-bont and ha operons) transcribed in opposite orientations. The gene that separates the two operons of the botulinum locus in C. botulinum A encodes a 21 kDa protein BotR/A, which is a positive regulator of the expression of the botulinum locus genes. Similarly, in Clostridium tetani, the gene located immediately upstream of the tetanus toxin gene, encodes a positive regulatory protein, TetR. BotR and TetR are possibly alternative sigma factors related to TxeR and UviA, which regulate C. difficile toxin and C. perfringens bacteriocin production, respectively. TxeR and UviA define a new sub-group of the sigma(70) family of RNA polymerase initiation factors. In addition, the C. botulinum genome contains predicted two-component system genes, some of which are possibly involved in regulation of toxinogenesis.


Frequent overcrowding in U.S. emergency departments.
To describe the definition, extent, and factors associated with overcrowding in emergency departments (EDs) in the United States as perceived by ED directors. Surveys were mailed to a random sample of EDs in all 50 states. Questions included ED census, frequency, impact, and determination of overcrowding. Respondents were asked to rank perceived causes using a five-point Likert scale. Of 836 directors surveyed, 575 (69%) responded, and 525 (91%) reported overcrowding as a problem. Common definitions of overcrowding (>70%) included: patients in hallways, all ED beds occupied, full waiting rooms >6 hours/day, and acutely ill patients who wait >60 minutes to see a physician. Overcrowding situations were similar in academic EDs (94%) and private hospital EDs (91%). Emergency departments serving populations < or =250,000 had less severe overcrowding (87%) than EDs serving larger areas (96%). Overcrowding occurred most often several times per week (53%), but 39% of EDs reported daily overcrowding. On a 1-5 scale (+/-SD), causes of overcrowding included high patient acuity (4.3 +/- 0.9), hospital bed shortage (4.2 +/- 1.1), high ED patient volume (3.8 +/- 1.2), radiology and lab delays (3.3 +/- 1.2), and insufficient ED space (3.3 +/- 1.3). Thirty-three percent reported that a few patients had actual poor outcomes as a result of overcrowding. Episodic, but frequent, overcrowding is a significant problem in academic, county, and private hospital EDs in urban and rural settings. Its causes are complex and multifactorial.