Reinforcement effect of histamine on the differentiation of murine myeloblasts and promyelocytes: externalization of granulocyte colony-stimulating factor receptors induced by histamine.
Histamine and recombinant granulocyte colony-stimulating factor (rG-CSF) stimulated the differentiation of murine myeloblasts and promyelocytes to mature neutrophils. In connection with this, myeloperoxidase activity of these progenitor cells was decreased by either histamine or rG-CSF treatment. After pretreatment with histamine at 1 microM, both differentiation and the decrease in myeloperoxidase activity of myeloblasts and promyelocytes induced by rG-CSF were significantly augmented. Binding assays using 125I-labeled rG-CSF showed that the number of rG-CSF binding sites on the surface of neutrophil progenitor cells increased after histamine treatment. The histamine-induced increase in rG-CSF binding appeared to be definitely through H2 receptors. Furthermore, the increase in rG-CSF binding sites due to histamine treatment seemed to take place in association with the externalization of G-CSF receptors, because 1) the binding increase was observed in the presence of cycloheximide, 2) no concomitant increase in [3H]leucine uptake was elicited, and 3) colchicine and cytochalasin D effectively prevented the increase in rG-CSF binding due to histamine. In neutrophil progenitors, cAMP contents increased very rapidly and significantly after either histamine or rG-CSF treatment. Moreover, dibutyryl-cAMP increased rG-CSF binding to neutrophil progenitor cells in a dose-dependent fashion. However, when progenitor cells were pretreated with protein kinase A inhibitors, the histamine-induced increase in rG-CSF binding was remarkably decreased. This result seems to indicate that the stimulatory effects of histamine on rG-CSF binding to progenitor cells are intimately related to the cAMP-protein kinase A system in neutrophil progenitors. Moreover, c-myc mRNA expression in neutrophil progenitors was markedly reduced by either histamine or rG-CSF treatment. It was concluded that rG-CSF-induced differentiation of murine neutrophil progenitors was augmented by histamine pretreatment mainly due to an increase in rG-CSF receptors on these cells and this increase might be related to the externalization of rG-CSF receptors.


Purine enzymes in rheumatoid arthritis: possible association with response to azathioprine. A pilot study.
To study the possible association of purine enzyme activities with response to azathioprine (AZA) treatment in rheumatoid arthritis (RA) and their correlation with parameters of disease activity. Lymphocyte activities of hypoxanthine-guanine phosphoribosyl-transferase (HGPRT), adenine phosphoribosyltransferase (APRT), purine nucleoside phosphorylase (PNP) and 5'-nucleotidase (5NT), and erythrocyte activities of thiopurine methyltransferase (TPMT) were measured in 14 healthy controls and 36 patients with RA. Eight patients had not previously been treated with AZA. Response to AZA therapy in 28 patients, determined in a prospective trial, was considered good in nine (group 1), insufficient in seven (group 2). In 12 patients AZA was withdrawn because of adverse reactions (group 3). Disease activity parameters were obtained simultaneously with purine enzyme measurements. Purine enzyme levels in the different groups were compared. Levels of 5NT activity were significantly lower in patients with RA than in healthy controls. PNP activity was higher in patients with RA not using prednisone compared with those who did and healthy controls. No clear correlation between purine enzyme levels and disease activity parameters was found. 5NT activities were significantly higher in group one than in group three (p = 0.012; alpha = 0.017), and almost significantly higher than in group two (p = 0.03; alpha = 0.017). The results indicate that purine enzyme activities in patients with RA differ from healthy controls, are associated with the outcome of AZA treatment and seem not to be associated with disease activity. Our findings may offer a clue to predict the response to AZA therapy in RA.


Community health workers for universal health-care coverage: from fragmentation to synergy.
To achieve universal health coverage, health systems will have to reach into every community, including the poorest and hardest to access. Since Alma-Ata, inconsistent support of community health workers (CHWs) and failure to integrate them into the health system have impeded full realization of their potential contribution in the context of primary health care. Scaling up and maintaining CHW programmes is fraught with a host of challenges: poor planning; multiple competing actors with little coordination; fragmented, disease-specific training; donor-driven management and funding; tenuous linkage with the health system; poor coordination, supervision and support, and under-recognition of CHWs' contribution. The current drive towards universal health coverage (UHC) presents an opportunity to enhance people's access to health services and their trust, demand and use of such services through CHWs. For their potential to be fully realized, however, CHWs will need to be better integrated into national health-care systems in terms of employment, supervision, support and career development. Partners at the global, national and district levels will have to harmonize and synchronize their engagement in CHW support while maintaining enough flexibility for programmes to innovate and respond to local needs. Strong leadership from the public sector will be needed to facilitate alignment with national policy frameworks and country-led coordination and to achieve synergies and accountability, universal coverage and sustainability. In moving towards UHC, much can be gained by investing in building CHWs' skills and supporting them as valued members of the health team. Stand-alone investments in CHWs are no shortcut to progress.


The role of extended venous thromboembolism prophylaxis for major urological cancer operations.
Venous thromboembolism (VTE), consisting of both pulmonary embolism (PE) and deep vein thromboses (DVT), remains a well-recognised complication of major urological cancer surgery. Several international guidelines recommend extended thromboprophylaxis (ETP) with LMWH, whereby the period of delivery is extended to the post-discharge period, where the majority of VTE occurs. In this literature review we investigate whether ETP should be indicated for all patients undergoing major urological cancer surgery, as well procedure specific data that may influence a clinician's decision. We performed a search of six databases (PubMed, Cochrane, EMBASE, Cumulative Index to Nursing and Allied Health Literature (CINAHL), PsycINFO, and British Nursing Index (BNI)) from inception to June 2019, for studies looking at adult patients who received VTE prophylaxis after surgery for a major urological malignancy. Eighteen studies were analysed. VTE risk is highest in open and robotic Radical Cystectomy (RC) (2.6-11.6%) and ETP demonstrates a significant reduction in risk of VTE, but not a significant difference in Pulmonary Embolism (PE) or mortality. Risk of VTE in open Radical Prostatectomy (RP) (0.8-15.7%) is comparable to RC, but robotic RP (0.2-0.9%), open partial/radical nephrectomy (1.0-4.4%) and robotic partial/radical nephrectomy (0.7-3.9%) were lower risk. It has not been shown that ETP reduces VTE risk specifically for RP or nephrectomy. The decision to use ETP is a fine balance between variables such as VTE incidence, bleeding risk and perioperative morbidity/mortality. This balance should be assessed for each specific procedure type. While ETP still remains of net benefit for open RP as well as open and robotic RC, the balance is closer for minimally invasive RP as well as radical and partial nephrectomy. Due to a lack of procedure specific evidence for the use of ETP, adherence with national guidelines remains poor. Therefore, we advocate further studies directly comparing ETP vs standard prophylaxis, for specific procedure types, in order to allow clinicians to make a more informed decision in future.


First-principles computation of electron transfer and reaction rate at a perovskite cathode for hydrogen production.
The focus of this research is on the electron transfer and its reaction rate at the perovskite cathode of a photoelectrochemical cell for hydrogen production. By employing the density functional theory (DFT), the electron density, projected density of states (PDOS), electron distribution and electron transfer path between [Fe-Fe] hydrogenase and the perovskite cathode can be obtained. Simulation results show that the perovskite cathode is better than traditional cathodes for hydrogen production. Before transmission to the [Fe-Fe] hydrogenase, electron clouds mainly aggregate at the periphery of amine molecules. Simulations also show that the key to hydrogen production at the perovskite structure lies in the organic molecules. Electrons are transferred to the hydrocarbon structural chain before reaching the Fe atoms. The Rice, Ramsperger, Kassel and Marcus (RRKM) theory was used to predict the reaction rates at different temperatures. It was found that the reaction rates are in good agreement with the experimental results. This research provides more physical insight into the electron transfer mechanism during the hydrogen production process.


Sexual function of women surviving cervical cancer.
The objective of this study was to evaluate the sexual function of survivors of cervical cancer (CC) in comparison to the control group of women without a history of cancer. This was an observational, analytical, case-control study. In the cancer group, women subjected to CC treatment of at least 3 months in the past were included (n = 37). For each survivor, one random selection from a base population control group was made (n = 37) of a woman without a history of cancer and with similar socioeconomic and demographic characteristics. The sexual function was evaluated through the female sex function index (FSFI) instrument. Data collection occurred through the application of questions in a face-to-face interview. In the cancer group, 64.9 % related vaginal stenosis or shortening; 59.5 % were not sexually active and of those which had sexual relations, 80 % showed dysfunction. The total FSFI score varied between 9.60 and 35.10 in the cancer group and 23.90 and 36.00 in the control group. The means of the cancer group were statistically inferior (P < 0.05) to the control group in all the FSFI domains and in the total score. The mean total score was 21.72 in the cancer group, classified as sexual dysfunction when considering a score of 26 as the cutoff point. CC treatment was found to have a negative impact on the sexual function of women. Further, sexual function should be monitored routinely by interdisciplinary teams to provide comprehensive care with the objective of an improved quality of life post-cancer.


Spray freeze drying: Emerging applications in drug delivery.
Spray freeze drying is relatively a recent drying technique involving heterogeneous set of steps which includes droplet formation, freezing, and sublimation. It has proven benefits over other drying methods in terms of producing products with improved structural integrity, superior quality, and better shelf stability. With such merits, spray freeze drying has found numerous applications in the field of drug delivery. Spray freeze drying yields particles of sizes and densities that show higher stability in the lungs, nasal mucosa, intestine, and skin, as compared to other drying technologies. These particles also possess the vital trait of sustained release and specificity through various delivery routes as compared to conventional drying techniques. This drives the market for commercialization of spray freeze dried drugs. The focus of this paper is on manufacturing approaches of spray freeze dried powders, with emphasis on its application in drug delivery systems. An overview of other applications of spray freeze drying is also presented.


The relation of alcoholic myopathy to cardiomyopathy.
To evaluate the relation between skeletal muscle disease (myopathy) and degenerative changes in cardiac muscle (cardiomyopathy) in patients with chronic alcoholism. A cross-sectional study. University medical center. Group A included 24 patients with chronic alcoholism who had dilated cardiomyopathy; group B, 24 patients with chronic alcoholism who had normal cardiac function; group C, 12 patients with dilated cardiomyopathy because of coronary heart disease; group D, 12 patients with idiopathic dilated cardiomyopathy; group E, 24 normal participants; and group F, 5 young men who died suddenly in traffic accidents. Clinical assessment of muscle strength, echocardiography, radionuclide cardiac angiography (groups A to E), muscle biopsy (groups A, B, E), endomyocardial biopsy of the left ventricle (group A), and examination of postmortem specimens of the left ventricle (group F). Alcoholic patients with cardiomyopathy had less muscle strength than did alcoholic patients with normal cardiac function, patients with idiopathic dilated cardiomyopathy, and patients with coronary heart disease (all P < 0.01). Among alcoholic patients with cardiomyopathy, 20 of 24 (83%) had histologic findings of skeletal myopathy compared with 1 of 24 (4%) alcoholic patients with normal cardiac function (P < 0.001). Interstitial fibrosis occurred in all cardiac biopsy specimens, hypertrophy of the myocytes occurred in 95%, and myocytolysis occurred in 83%. Those patients with more severe cellular hypertrophy and interstitial fibrosis of the myocardium had a greater decrease in deltoid muscle strength and had worse histologic myopathy. Diseases of skeletal and cardiac muscle in patients with chronic alcoholism are clinically and histologically related. The presence of muscle weakness in an alcoholic person suggests the likelihood of an accompanying cardiomyopathic abnormality.


Clinical and morphologic factors associated with suture anchor refixation of labral tears in the hip.
The acetabular labrum is critical to hip function. Surgical options for treatment of a damaged labrum include removal, debridement, and refixation using suture anchors. The purpose of this study is to determine if certain patient demographic and osseous morphological factors result in increased labral damage requiring refixation. Data was collected prospectively from a consecutive series of 334 procedures performed from August 2010 to June 2011 for femoroacetabular impingement. Demographic data, including age, sex, and race, was collected from patient charts. Three-dimensional (3D) CT scans were reviewed to retrieve alpha angles, acetabular version, femoral version, and lateral center edge angle on the symptomatic hip. In 238 (71.3%) of the procedures, the labrum required refixation using suture anchors with a mean of 2.74 anchors being used. Of males, 78.8% required suture anchors and 62.3% of females required suture anchors. Among procedures requiring suture anchors, significantly more suture anchors were used in males (2.92) than females (2.47). Regression analysis showed a positive association between alpha angle, acetabular retroversion at 1 and 2 o'clock, and the number of suture anchors used. The mean alpha angle in the cohort that required suture anchors (63.1°) was significantly greater than the cohort that did not (59.4°). This study found femoral deformities to contribute more to labral damage than acetabular deformities and highlighted the importance of preoperative 3D CT scans. This study provides demographic and morphologic factors to review preoperatively to evaluate if extensive labral damage is present and if suture anchor refixation will be required.


CMR Parametric Mapping as a Tool for Myocardial Tissue Characterization.
Cardiovascular magnetic resonance (CMR) is the current gold standard for imaging cardiac anatomy, function, and advanced myocardial tissue characterization. After cine, late gadolinium enhancement (LGE), and perfusion imaging, parametric mapping is widely regarded as the 4th era of myocardial CMR development. In contrast to conventional CMR tissue characterization techniques, which rely on relative variations in image intensities to highlight abnormal tissues, parametric mapping provides direct visualization of tissue MR properties such as T1, T2 and T2* in absolute denominations (e.g. in milliseconds). Presentation as pixel-wise parametric maps adds spatial information for a more complete assessment of the myocardium. Advantages of parametric mapping include direct, quantitative comparisons inter- and within-individuals, as well as detection of diffuse disease not evident on conventional CMR imaging, without the need for contrast agents. CMR parametric mapping methods have matured over the past decade into clinical tools, demonstrating not only clinical utility but added value in a wide range of cardiac diseases. They are particularly useful for the evaluation of acute myocardial injury, suspected infiltration and heart failure of unclear etiology. This review discusses the background of parametric mapping, particularly T1-, T2- and ECV-mapping, general magnetic resonance physics principles, clinical applications (including imaging protocols, image analysis and reporting guidelines), current challenges and future directions. CMR parametric mapping is increasingly available on routine clinical scanners, and promises to deliver advanced myocardial tissue characterization beyond conventional CMR techniques, ultimately helping clinicians to benefit patients in their clinical management.


Increased salivary calcium levels as an indicator of digoxin intoxication.
One hundred ten individuals were divided into patients with digoxin intoxication; patients treated with digoxin; patients treated with digoxin and diuretics; patients treated with diuretics; and control subjects. Measurement of salivary potassium and calcium levels showed that 81% of the patients with digoxin intoxication had noticeable elevation of the salivary calcium level. In 22%, elevation of the salivary calcium level preceded clinical manifestations of intoxication. The high calcium level in the saliva was not accompanied by changes in serum or urinary calcium levels. The elevation of salivary calcium levels can be used not only as an additional indicator of digoxin intoxication but also for detecting impending intoxication in patients treated with this drug.


Formate Supplementation Enhances Antitumor CD8+ T-cell Fitness and Efficacy of PD-1 Blockade.
The tumor microenvironment (TME) restricts antitumor CD8+ T-cell function and immunotherapy responses. Cancer cells compromise the metabolic fitness of CD8+ T cells within the TME, but the mechanisms are largely unknown. Here we demonstrate that one-carbon (1C) metabolism is enhanced in T cells in an antigen-specific manner. Therapeutic supplementation of 1C metabolism using formate enhances CD8+ T-cell fitness and antitumor efficacy of PD-1 blockade in B16-OVA tumors. Formate supplementation drives transcriptional alterations in CD8+ T-cell metabolism and increases gene signatures for cellular proliferation and activation. Combined formate and anti-PD-1 therapy increases tumor-infiltrating CD8+ T cells, which are essential for enhanced tumor control. Our data demonstrate that formate provides metabolic support to CD8+ T cells reinvigorated by anti-PD-1 to overcome a metabolic vulnerability in 1C metabolism in the TME to further improve T-cell function. This study identifies that deficiencies in 1C metabolism limit the efficacy of PD-1 blockade in B16-OVA tumors. Supplementing 1C metabolism with formate during anti-PD-1 therapy enhances CD8+ T-cell fitness in the TME and CD8+ T-cell-mediated tumor clearance. These findings demonstrate that formate supplementation can enhance exhausted CD8+ T-cell function. See related commentary by Lin et al., p. 2507. This article is featured in Selected Articles from This Issue, p. 2489.


Analysis of conifer FLOWERING LOCUS T/TERMINAL FLOWER1-like genes provides evidence for dramatic biochemical evolution in the angiosperm FT lineage.
In flowering plants, homologs of the Arabidopsis phosphatidylethanolamine-binding protein (PEBP) FLOWERING LOCUS T (FT) are key components in controlling flowering time. We show here that, although FT homologs are found in all angiosperms with completed genome sequences, there is no evidence to date that FT-like genes exist in other groups of plants. Through phylogeny reconstructions and heterologous expression, we examined the biochemical function of the Picea (spruces) and Pinus (pines) PEBP families - two gymnosperm taxa phylogenetically distant from the angiosperms. We have defined a lineage of gymnosperm PEBP genes, termed the FT/TERMINAL FLOWER1 (TFL1)-like genes, that share sequence characteristics with both the angiosperm FT- and TFL1-like clades. When expressed in Arabidopsis, FT/TFL1-like genes repressed flowering, indicating that the proteins are biochemically more similar to the angiosperm TFL1-like proteins than to the FT-like proteins. This suggests that the regulation of the vegetative-to-reproductive switch might differ in gymnosperms compared with angiosperms. Molecular evolution studies suggest that plasticity at exon 4 contributes to the divergence of FT-like function in floral promotion. In addition, the presence of FT-like genes in basal angiosperms indicates that the FT-like function emerged at an early stage during the evolution of flowering plants as a means to regulate flowering time.


Model Organisms in G Protein-Coupled Receptor Research.
The study of G protein-coupled receptors (GPCRs) has benefited greatly from experimental approaches that interrogate their functions in controlled, artificial environments. Working in vitro, GPCR receptorologists discovered the basic biologic mechanisms by which GPCRs operate, including their eponymous capacity to couple to G proteins; their molecular makeup, including the famed serpentine transmembrane unit; and ultimately, their three-dimensional structure. Although the insights gained from working outside the native environments of GPCRs have allowed for the collection of low-noise data, such approaches cannot directly address a receptor's native (in vivo) functions. An in vivo approach can complement the rigor of in vitro approaches: as studied in model organisms, it imposes physiologic constraints on receptor action and thus allows investigators to deduce the most salient features of receptor function. Here, we briefly discuss specific examples in which model organisms have successfully contributed to the elucidation of signals controlled through GPCRs and other surface receptor systems. We list recent examples that have served either in the initial discovery of GPCR signaling concepts or in their fuller definition. Furthermore, we selectively highlight experimental advantages, shortcomings, and tools of each model organism.


Enhancing Clinical Content and Race/Ethnicity Data in Statewide Hospital Administrative Databases: Obstacles Encountered, Strategies Adopted, and Lessons Learned.
Eight grant teams used Agency for Healthcare Research and Quality infrastructure development research grants to enhance the clinical content of and improve race/ethnicity identifiers in statewide all-payer hospital administrative databases. Grantees faced common challenges, including recruiting data partners and ensuring their continued effective participation, acquiring and validating the accuracy and utility of new data elements, and linking data from multiple sources to create internally consistent enhanced administrative databases. Successful strategies to overcome these challenges included aggressively engaging with providers of critical sources of data, emphasizing potential benefits to participants, revising requirements to lessen burdens associated with participation, maintaining continuous communication with participants, being flexible when responding to participants' difficulties in meeting program requirements, and paying scrupulous attention to preparing data specifications and creating and implementing protocols for data auditing, validation, cleaning, editing, and linking. In addition to common challenges, grantees also had to contend with unique challenges from local environmental factors that shaped the strategies they adopted. The creation of enhanced administrative databases to support comparative effectiveness research is difficult, particularly in the face of numerous challenges with recruiting data partners such as competing demands on information technology resources. Excellent communication, flexibility, and attention to detail are essential ingredients in accomplishing this task. Additional research is needed to develop strategies for maintaining these databases when initial funding is exhausted.


Estrogen alters spine number and morphology in prefrontal cortex of aged female rhesus monkeys.
Long-term cyclic treatment with 17beta-estradiol reverses age-related impairment in ovariectomized rhesus monkeys on a test of cognitive function mediated by the prefrontal cortex (PFC). Here, we examined potential neurobiological substrates of this effect using intracellular loading and morphometric analyses to test the possibility that the cognitive benefits of hormone treatment are associated with structural plasticity in layer III pyramidal cells in PFC area 46. 17beta-Estradiol did not affect several parameters such as total dendritic length and branching. In contrast, 17beta-estradiol administration increased apical and basal dendritic spine density, and induced a shift toward smaller spines, a response linked to increased spine motility, NMDA receptor-mediated activity, and learning. These results document that, although the aged primate PFC is vulnerable in the absence of factors such as circulating estrogens, it remains responsive to long-term cyclic 17beta-estradiol treatment, and that increased dendritic spine density and altered spine morphology may contribute to the cognitive benefits of such treatment.


Effects of Plyometric Training and Beta-Alanine Supplementation on Maximal-Intensity Exercise and Endurance in Female Soccer Players.
Plyometric training and beta-alanine supplementation are common among soccer players, although its combined use had never been tested. Therefore, a randomized, double-blind, placebo-controlled trial was conducted to compare the effects of a plyometric training program, with or without beta-alanine supplementation, on maximal-intensity and endurance performance in female soccer players during an in-season training period. Athletes (23.7 ± 2.4 years) were assigned to either a plyometric training group receiving a placebo (PLACEBO, n = 8), a plyometric training group receiving beta-alanine supplementation (BA, n = 8), or a control group receiving placebo without following a plyometric training program (CONTROL, n = 9). Athletes were evaluated for single and repeated jumps and sprints, endurance, and change-of-direction speed performance before and after the intervention. Both plyometric training groups improved in explosive jumping (ES = 0.27 to 1.0), sprinting (ES = 0.31 to 0.78), repeated sprinting (ES = 0.39 to 0.91), 60 s repeated jumping (ES = 0.32 to 0.45), endurance (ES = 0.35 to 0.37), and change-of-direction speed performance (ES = 0.36 to 0.58), whereas no significant changes were observed for the CONTROL group. Nevertheless, compared to the CONTROL group, only the BA group showed greater improvements in endurance, repeated sprinting and repeated jumping performances. It was concluded that beta-alanine supplementation during plyometric training may add further adaptive changes related to endurance, repeated sprinting and jumping ability.


Using genomics data to reconstruct transmission trees during disease outbreaks.
Genetic sequence data from pathogens present a novel means to investigate the spread of infectious disease between infected hosts or infected premises, complementing traditional contact-tracing approaches, and much recent work has gone into developing methods for this purpose. The objective is to recover the epidemic transmission tree, which identifies who infected whom. This paper reviews the various approaches that have been taken. The first step is to define a measure of difference between sequences, which must be done while taking into account such factors as recombination and convergent evolution. Three broad categories of method exist, of increasing complexity: those that assume no withinhost genetic diversity or mutation, those that assume no within-host diversity but allow mutation, and those that allow both. Until recently, the assumption was usually made that every host in the epidemic could be identified, but this is now being relaxed, and some methods are intended for sparsely sampled data, concentrating on the identification of pairs of sequences that are likely to be the result of direct transmission rather than inferring the complete transmission tree. Many of the procedures described here are available to researchers as free software.


Release of magnetic nanoparticles from cell-encapsulating biodegradable nanobiomaterials.
The future of tissue engineering requires development of intelligent biomaterials using nanoparticles. Magnetic nanoparticles (MNPs) have several applications in biology and medicine; one example is Food and Drug Administration (FDA)-approved contrast agents in magnetic resonance imaging. Recently, MNPs have been encapsulated within cell-encapsulating hydrogels to create novel nanobiomaterials (i.e., M-gels), which can be manipulated and assembled in magnetic fields. The M-gels can be used as building blocks for bottom-up tissue engineering to create 3D tissue constructs. For tissue engineering applications of M-gels, it is essential to study the release of encapsulated MNPs from the hydrogel polymer network and the effect of MNPs on hydrogel properties, including mechanical characteristics, porosity, swelling behavior, and cellular response (e.g., viability, growth). Therefore, we evaluated the release of MNPs from photocrosslinkable gelatin methacrylate hydrogels as the polymer network undergoes biodegradation using inductively coupled plasma atomic emission spectroscopy. MNP release correlated linearly with hydrogel biodegradation rate with correlation factors (Pearson product moment correlation coefficient) of 0.96 ± 0.03 and 0.99 ± 0.01 for MNP concentrations of 1% and 5%, respectively. We also evaluated the effect of MNPs on hydrogel mechanical properties, porosity, and swelling behavior, as well as cell viability and growth in MNP-encapsulating hydrogels. Fibroblasts encapsulated with MNPs in hydrogels remained viable (>80% at t = 144 h) and formed microtissue constructs in culture (t = 144 h). These results indicated that MNP-encapsulating hydrogels show promise as intelligent nanobiomaterials, with great potential to impact broad areas of bioengineering, including tissue engineering, regenerative medicine, and pharmaceutical applications.


Inhibition of tumor cell proliferation in human uterine leiomyomas by vitamin D via Wnt/β-catenin pathway.
To assess the effect of vitamin D (VitD) on human uterine leiomyomas through Wnt/β-catenin pathway inhibition, apoptosis induction, and cell growth arrest. A prospective study comparing leiomyoma vs. myometrium tissues. Paired design study comparing human uterine leiomyoma primary (HULP) cells treated with or without VitD. University hospital. Human uterine leiomyoma and myometrium were collected from women (aged 35-52 years) without hormonal treatment. Samples were collected from women undergoing surgery due to symptomatic uterine leiomyoma pathology. Uterine leiomyoma and myometrium tissues were analyzed by western blot (WB) to determine proliferation, Wnt/β-catenin, and apoptosis pathways. HULP cells were used to study VitD effect in cell proliferation (WB), cell cycle (flow cytometry), Wnt/β-catenin and apoptosis genes (polymerase chain reaction arrays), Wnt-related proteins (protein array), and apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling [TUNEL] assay). Human leiomyoma tissues compared with matched myometrium showed higher proliferation (fold change = 8.16; P=.0006) and altered Wnt/β-catenin pathway (fold change = 5.5; P<.0001), whereas no differences in apoptosis were observed. VitD induced cell growth arrest and decreased proliferation in HULP cells (fold change = 0.74; P=.007). Moreover, VitD decreased Wnt-pathway expression in HULP cells at gene (activity score = -0.775; P<.001) and protein levels. However, VitD did not induce apoptosis expression. Increased proliferation and Wnt/β-catenin pathway deregulation play a role in the development and growth of leiomyomas, whereas apoptosis appears not to contribute. VitD exerts an antiproliferative action on HULP cells through cell growth arrest and Wnt/β-catenin pathway inhibition, but not through apoptosis regulation, suggesting VitD as an effective therapy to stabilize leiomyoma size and prevent its growth.


Novel long-chain neurotoxins from Bungarus candidus distinguish the two binding sites in muscle-type nicotinic acetylcholine receptors.
αδ-Bungarotoxins, a novel group of long-chain α-neurotoxins, manifest different affinity to two agonist/competitive antagonist binding sites of muscle-type nicotinic acetylcholine receptors (nAChRs), being more active at the interface of α-δ subunits. Three isoforms (αδ-BgTx-1-3) were identified in Malayan Krait (Bungarus candidus) from Thailand by genomic DNA analysis; two of them (αδ-BgTx-1 and 2) were isolated from its venom. The toxins comprise 73 amino acid residues and 5 disulfide bridges, being homologous to α-bungarotoxin (α-BgTx), a classical blocker of muscle-type and neuronal α7, α8, and α9α10 nAChRs. The toxicity of αδ-BgTx-1 (LD50 = 0.17-0.28 µg/g mouse, i.p. injection) is essentially as high as that of α-BgTx. In the chick biventer cervicis nerve-muscle preparation, αδ-BgTx-1 completely abolished acetylcholine response, but in contrast with the block by α-BgTx, acetylcholine response was fully reversible by washing. αδ-BgTxs, similar to α-BgTx, bind with high affinity to α7 and muscle-type nAChRs. However, the major difference of αδ-BgTxs from α-BgTx and other naturally occurring α-neurotoxins is that αδ-BgTxs discriminate the two binding sites in the Torpedo californica and mouse muscle nAChRs showing up to two orders of magnitude higher affinity for the α-δ site as compared with α-ε or α-γ binding site interfaces. Molecular modeling and analysis of the literature provided possible explanations for these differences in binding mode; one of the probable reasons being the lower content of positively charged residues in αδ-BgTxs. Thus, αδ-BgTxs are new tools for studies on nAChRs.


Protein expression changes in ovarian cancer during the transition from benign to malignant.
Epithelial ovarian carcinoma has in general a poor prognosis since the vast majority of tumors are genomically unstable and clinically highly aggressive. This results in rapid progression of malignancy potential while still asymptomatic and thus in late diagnosis. It is therefore of critical importance to develop methods to diagnose epithelial ovarian carcinoma at its earliest developmental stage, that is, to differentiate between benign tissue and its early malignant transformed counterparts. Here we present a shotgun quantitative proteomic screen of benign and malignant epithelial ovarian tumors using iTRAQ technology with LC-MALDI-TOF/TOF and LC-ESI-QTOF MS/MS. Pathway analysis of the shotgun data pointed to the PI3K/Akt signaling pathway as a significant discriminatory pathway. Selected candidate proteins from the shotgun screen were further confirmed in 51 individual tissue samples of normal, benign, borderline or malignant origin using LC-MRM analysis. The MRM profile demonstrated significant differences between the four groups separating the normal tissue samples from all tumor groups as well as perfectly separating the benign and malignant tumors with a ROC-area of 1. This work demonstrates the utility of using a shotgun approach to filter out a signature of a few proteins only that discriminates between the different sample groups.


Definition of the Beijing/W lineage of Mycobacterium tuberculosis on the basis of genetic markers.
Mycobacterium tuberculosis Beijing genotype strains are highly prevalent in Asian countries and in the territory of the former Soviet Union. They are increasingly reported in other areas of the world and are frequently associated with tuberculosis outbreaks and drug resistance. Beijing genotype strains, including W strains, have been characterized by their highly similar multicopy IS6110 restriction fragment length polymorphism (RFLP) patterns, deletion of spacers 1 to 34 in the direct repeat region (Beijing spoligotype), and insertion of IS6110 in the genomic dnaA-dnaN locus. In this study the suitability and comparability of these three genetic markers to identify members of the Beijing lineage were evaluated. In a well-characterized collection of 1,020 M. tuberculosis isolates representative of the IS6110 RFLP genotypes found in The Netherlands, strains of two clades had spoligotypes characteristic of the Beijing lineage. A set of 19 Beijing reference RFLP patterns was selected to retrieve all Beijing strains from the Dutch database. These reference patterns gave a sensitivity of 98.1% and a specificity of 99.7% for identifying Beijing strains (defined by spoligotyping) in an international database of 1,084 strains. The usefulness of the reference patterns was also assessed with large DNA fingerprint databases in two other European countries and for identification strains from the W lineage found in the United States. A standardized definition for the identification of M. tuberculosis strains belonging to the Beijing/W lineage, as described in this work, will facilitate further studies on the spread and characterization of this widespread genotype family of M. tuberculosis strains.


Constitutive activation of mitogen-activated protein (MAP) kinases in human renal cell carcinoma.
Mitogen-activated protein kinases (MAPKs) play a pivotal role in the mitogenic signal transduction pathway and are essential components of the MAPK cascade, which includes MEK (also known as MAP kinase kinase), Raf-1, and Ras. In this study, we examined whether constitutive activation of the MAPK cascade was associated with the carcinogenesis of human renal cell carcinomas in a series of 25 tumors and in corresponding normal kidneys. Constitutive activation of MAPKs in tumor tissue, as determined by the appearance of phosphorylated forms, was found in 12 cases (48%), and this activation was confirmed by a direct in vitro kinase assay of immunoprecipitate using myelin basic protein as the substrate. The phosphorylation of MEK and of Raf-1, as monitored by a mobility shift in SDS-PAGE, which is reportedly associated with the activation of these kinases, occurred in 9 of 18 cases (50%) and in 6 of 11 cases (55%) respectively. The activation of MAPKs was correlated with MEK activation (P = 0.0045) and with Raf-1 activation (P = 0.067). Furthermore, overexpression of MEK was found in 13 of 25 cases (52%) by Western blot analysis, and this overexpression was associated significantly with MAPK activation (P = 0.034). No mutations were noted in H-,K-, or N-ras genes by PCR direct sequencing in any of the 25 tumor samples. Of the patients studied, 8 of 18 (44%) stage pT2 patients and four of six (67%) stage pT3 patients showed MAPK activation. The single stage pT1 patient did not evidence MAPK activation. Furthermore, one of seven (14%) grade 1 patients, 9 of 13 (69%) grade 2 patients, and two of five (40%) grade 3 patients showed MAPK activation (grade 1 versus grades 2 and 3, P = 0.046). Our results suggest that constitutive activation of MAPKs may be associated with the carcinogenesis of human RCCs.


Four N-(E)-cinnamoyl (cinnamamide) derivatives of aminoalkanols with promising anticonvulsant and analgesic activity.
Epilepsy and neuropathic pain are frequent neurological disorders with pathomechanism based on abnormal neuronal discharges. Secondary tissue impairment observed after traumatic brain injury is also connected with neuronal dysfunction. Those three neurological disorders are ineffectively treated with currently available pharmacotherapy options so great effort is made in searching for new effective drugs. Four N-(E)-cinnamoyl (cinnamamide) derivatives of aminoalkanols: S-(2E)-N-(1-hydroxypropan-2-yl)-3-(2-methylphenyl)prop-2-enamide (1), R,S-(2E)-3-(4-chlorophenyl)-N-(1-hydroxybutan-2-yl)prop-2-enamide (2), R,S-(2E)-3-(4-chlorophenyl)-N-(2-hydroxypropyl)prop-2-enamide (3), (2E)-3-(4-chlorophenyl)-N-(4-hydroxycyclohexyl)prop-2-enamide (4) were evaluated in vivo and in vitro for anticonvulsant, neuroprotective and/or analgesic activity. In intravenous metrazol seizure threshold test compounds 1-3 did not show pro-convulsive effect but proved anticonvulsant potential. In corneal kindled mice model the tested compounds showed beneficial anticonvulsant properties with ED50 of 36.8 mg/kg for 1, 25.7 mg/kg for 2, and 51.1 mg/kg for 3. Compound 2 tested in vitro in spontaneously bursting rat hippocampal slice model significantly reduced burst rate. Compounds 1 and 2 did not decrease lesion volume in acute model of traumatic brain injury. In formalin test of hyperalgesia in mice, compound 1 was active in the acute phase of the test, while compound 4 caused reduction of the time of licking of the affected paw by approx. 88% during the acute phase and 100% during the inflammatory phase. In rat sciatic ligation model of neuropathic pain, compound 1 significantly increased the paw withdrawal threshold starting from one hour after oral administration and the activity continued up to six hours. Reported here four N-(E)-cinnamoyl derivatives of aminoalkanols possess promising activity as anticonvulsant and/or analgesic agents.


Clarify Sit-to-Stand Muscle Synergy and Tension Changes in Subacute Stroke Rehabilitation by Musculoskeletal Modeling.
Post-stroke patients exhibit distinct muscle activation electromyography (EMG) features in sit-to-stand (STS) due to motor deficiency. Muscle activation amplitude, related to muscle tension and muscle synergy activation levels, is one of the defining EMG features that reflects post-stroke motor functioning and motor impairment. Although some qualitative findings are available, it is not clear if and how muscle activation amplitude-related biomechanical attributes may quantitatively reflect during subacute stroke rehabilitation. To better enable a longitudinal investigation into a patient's muscle activation changes during rehabilitation or an inter-subject comparison, EMG normalization is usually applied. However, current normalization methods using maximum voluntary contraction (MVC) or within-task peak/mean EMG may not be feasible when MVC cannot be obtained from stroke survivors due to motor paralysis and the subject of comparison is EMG amplitude. Here, focusing on the paretic side, we first propose a novel, joint torque-based normalization method that incorporates musculoskeletal modeling, forward dynamics simulation, and mathematical optimization. Next, upon method validation, we apply it to quantify changes in muscle tension and muscle synergy activation levels in STS motor control units for patients in subacute stroke rehabilitation. The novel method was validated against MVC-normalized EMG data from eight healthy participants, and it retained muscle activation amplitude differences for inter- and intra-subject comparisons. The proposed joint torque-based method was also compared with the common static optimization based on squared muscle activation and showed higher simulation accuracy overall. Serial STS measurements were conducted with four post-stroke patients during their subacute rehabilitation stay (137 ± 22 days) in the hospital. Quantitative results of patients suggest that maximum muscle tension and activation level of muscle synergy temporal patterns may reflect the effectiveness of subacute stroke rehabilitation. A quality comparison between muscle synergies computed with the conventional within-task peak/mean EMG normalization and our proposed method showed that the conventional was prone to activation amplitude overestimation and underestimation. The contributed method and findings help recapitulate and understand the post-stroke motor recovery process, which may facilitate developing more effective rehabilitation strategies for future stroke survivors.


Opsin-like immunoreaction in the retinae and pineal organs of four mammalian species.
Opsin-like immunoreactivity was observed in the retinae and pineal organs of the mouse, rat and guinea pig, and the pineal organ of the cat. In the retina the immunoreaction was restricted to photoreceptor cells, which displayed immunostaining in their perikarya and outer and inner segments. Distinct pinealocytes endowed with characteristic processes were labelled in the pineal organs of the mouse and cat. However, in the cat the number of immunoreactive pinealocytes was very limited. In the pineal organs of the rat and guinea pig immunoreaction was very weak and diffuse. No immunoreaction was observed when the antibody was preabsorbed with purified bovine (rhod)opsin. These findings are in accord with the results of previous studies indicating molecular similarities between retinal photoreceptors and pinealocytes in mammals.


Prediction of tinnitus masking benefit within a case series using a spiking neural network model.
Masking has been widely used as a tinnitus therapy, with large individual differences in its effectiveness. The basis of this variation is unknown. We examined individual tinnitus and psychological responses to three masking types, energetic masking (bilateral broadband static or rain noise [BBN]), informational masking (BBN with a notch at tinnitus pitch and 3-dimensional cues) and a masker combining both effects (BBN with spatial cues). Eleven participants with chronic tinnitus were followed for 12 months, each person used each masking approach for 3 months with a 1 month washout-baseline. The Tinnitus Functional Index (TFI), Tinnitus Rating Scales, Positive and Negative Affect Scale and Depression Anxiety Stress Scales, were measured every month of treatment. Electroencephalography (EEG) and psychoacoustic assessment was undertaken at baseline and following 3 months of each masking sound. The computational modeling of EEG data was based on the framework of brain-inspired Spiking Neural Network (SNN) architecture called NeuCube, designed for this study for mapping, learning, visualizing and classifying of brain activity patterns. EEG was related to clinically significant change in the TFI using the SNN model. The SNN framework was able to predict sound therapy responders (93% accuracy) from non-responders (100% accuracy) using baseline EEG recordings. The combination of energetic and informational masking was an effective treatment sound in more individuals than the other sounds used. Although the findings are promising, they are preliminary and require confirmation in independent and larger samples.


Angiotensin converting enzyme activity and evolution of pulmonary vascular disease in rats with monocrotaline pulmonary hypertension.
We have investigated the role of angiotensin converting enzyme (ACE) in the development of pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular disease in rats given a single subcutaneous injection of the pyrrolizidine alkaloid monocrotaline. Thirty-six young female Wistar rats were divided into a test group of 27 animals and a control group of nine animals. Each test rat was given a single subcutaneous injection of monocrotaline (60 mg/kg body weight). On the first, third, fifth, seventh, tenth, twelfth, fourteenth, seventeenth, and twenty-second days after the injection of monocrotaline the mean right ventricular systolic blood pressure was measured in one control and three test rats. The animals were then killed and we measured the specific activity of ACE in serum and lung homogenate. We also evaluated muscularisation of pulmonary arterioles, medial hypertrophy of muscular pulmonary arteries, and right ventricular hypertrophy. The sequence of changes was as follows: muscularisation of pulmonary arterioles and medial hypertrophy of muscular pulmonary arteries were apparent seven days after administration of monocrotaline; pulmonary hypertension and reduced lung ACE activity occurred after 10 days; right ventricular hypertrophy was detected after 12 days. Serum ACE activity was unchanged. It is concluded that the reduction in lung ACE activity is a result rather than a cause of the pulmonary hypertension. This reduction in lung ACE activity may be a protective mechanism designed to limit the elevation of the pulmonary arterial pressure.


Migrant population and perinatal health.
The number of foreign migrants tends to grow. The situation of people living away from their own country is complex and has an impact on the public health system. The objective of this article was to analyze the perinatal risk of migrant mothers and their newborn infants in comparison to native Argentine mothers and their newborn infants. Cohort study that included 2000 mothers and their newborn infants (1000 migrants and 1000 natives) assisted at a public maternity hospital. Native mothers had a higher rate of low birth weight newborn infants (9.9% versus 5.3%; p <0.01), primiparity (42% versus 37%; p= 0.012), and adolescent pregnancy (6% versus 1.3%; p <0.001). Migrant women had a higher rate of poorly controlled pregnancies (21.7% versus 13.4%; p <0.01), positive serology for Chagas disease (3.2% versus 0.4%; p <0.01), and newborn infants requiring phototherapy (20.5% versus 14.3%; p =0.04). In our study, Argentine mothers had a higher rate of low birth weight newborn infants, primiparity and adolescent pregnancies, while immigrant women had higher rate of positive serology for Chagas disease, poor antenatal controls and more newborn infants requiring phototherapy.


Cobalt oxide-carbon nanocatalysts with highly enhanced catalytic performance for the green synthesis of nitrogen heterocycles through the Friedländer condensation.
A novel series of eco-sustainable catalysts developed by supporting CoO nanoparticles on different carbon supports, highly efficient in the synthesis of quinolines and naphthyridines, through the Friedländer condensation, are reported for the first time. Textural properties, dispersion and location of the Co-phase are influenced by the nature of the carbon support, Co-precursor salt and metal loading, having a significant impact on the catalytic performance. Thus, the presence of the mesopores and macropores in carbon aerogels together with the homogeneous distribution of the active phase favours the formation of product 3a as a function of the metal loading. However, an increase in the metal content when using CNTs indicates the formation of CoO aggregates and an optimal concentration of 3 wt% CoO was observed, providing the highest conversion values. The carbon-based catalysts herein reported can be considered to be a sustainable alternative having advantages such as easy preparation, superior stability and notably enhanced catalytic performance, operating at lower temperature and under solvent-free conditions.


Long-term continuous corticosterone treatment decreases VEGF receptor-2 expression in frontal cortex.
Stress and increased glucocorticoid levels are associated with many neuropsychiatric disorders including schizophrenia and depression. Recently, the role of vascular endothelial factor receptor-2 (VEGFR2/Flk1) signaling has been implicated in stress-mediated neuroplasticity. However, the mechanism of regulation of VEGF/Flk1 signaling under long-term continuous glucocorticoid exposure has not been elucidated. We examined the possible effects of long-term continuous glucocorticoid exposure on VEGF/Flk1 signaling in cultured cortical neurons in vitro, mouse frontal cortex in vivo, and in post mortem human prefrontal cortex of both control and schizophrenia subjects. We found that long-term continuous exposure to corticosterone (CORT, a natural glucocorticoid) reduced Flk1 protein levels both in vitro and in vivo. CORT treatment resulted in alterations in signaling molecules downstream to Flk1 such as PTEN, Akt and mTOR. We demonstrated that CORT-induced changes in Flk1 levels are mediated through glucocorticoid receptor (GR) and calcium. A significant reduction in Flk1-GR interaction was observed following CORT exposure. Interestingly, VEGF levels were increased in cortex, but decreased in serum following CORT treatment. Moreover, significant reductions in Flk1 and GR protein levels were found in postmortem prefrontal cortex samples from schizophrenia subjects. The alterations in VEGF/Flk1 signaling following long-term continuous CORT exposure represents a molecular mechanism of the neurobiological effects of chronic stress.


Mycobacterium kansasii pulmonary infection: a prospective study of the results of nine months of treatment with rifampicin and ethambutol. Research Committee, British Thoracic Society.
Pulmonary disease caused by Mycobacterium kansasii is reported in approximately 50 new patients in Britain annually. Rifampicin and ethambutol are effective in vitro but the optimal duration of treatment, and whether isoniazid should also be given, are uncertain. The British Thoracic Society has conducted a prospective, multicentre study of the treatment of this condition with rifampicin and ethambutol given for nine months. One hundred and seventy three patients with two or more positive cultures and radiological evidence of disease were recruited via the Mycobacterium Reference Unit (PHLS) in Cardiff from 113 physicians in England, Scotland, and Wales. Rifampicin and ethambutol were given for nine months, other antituberculosis drugs being discontinued once the culture was identified as M kansasii. Patients were reviewed, sputum cultured, and chest radiographs performed before, during, and at regular intervals for 51 months after chemotherapy. The mean (SD) age was 55.5 (11.7) years, 73% were men, and 50% had other lung problems. Cavitation was seen in 88%, bilateral shadowing in 48%, and three or more lung zones were affected in 46%. All cultures were sensitive to rifampicin and ethambutol but resistant to isoniazid and pyrazinamide. One patient who took chemotherapy irregularly still had positive cultures at seven and eight months. Fifteen patients developed positive cultures after the end of chemotherapy; factors which might account for the relapse were identified in eight. Reinfection rather than relapse was suspected in three of the 15. Radiographic improvement stabilised within three years in 80%. M kansasii pulmonary infection responds well to nine months of treatment with rifampicin and ethambutol but patients who contract this disease have a high mortality rate from other causes. Isoniazid does not appear to be a necessary part of the regimen.


MICAL-L1-related and unrelated mechanisms underlying elongated tubular endosomal network (ETEN) in human dendritic cells.
The endosomal pathway constitutes a highly dynamic intracellular transport system, which is composed of vesicular and tubular compartments. Endosomal tubules enable geometry-based discrimination between membrane and luminal content. Extended tubular endosomes were suggested to deliver a steady stream of membrane proteins to one location more reliable and effective than vesicular endosomes. Recently, we demonstrated that human dendritic cells (DCs) form a large elongated tubular endosomal network, e.g. ETEN, upon distinct triggers. LPS-stimulation triggered late endosomal tubulation. Additional clustering of class I MHC and ICAM-1 by a cognate interaction between antigen-laden DC and antigen-specific CD8(+) T-cells induces formation of transferrin-positive tubules emanating from the endosomal recycling compartment (ERC). We here discuss cell-biological mechanisms that are involved in membrane bending and possibly underlie initiation, elongation, and stabilization of ETEN in human DCs. Using a knock-down approach we demonstrate that MICAL-L1 is necessary for ETEN remodeling originating from ERC in human DCs.


Flour for Home Baking: A Cross-Sectional Analysis of Supermarket Products Emphasising the Whole Grain Opportunity.
Flour, typically derived from wheat, rye, corn and rice is a pantry staple, providing structure to bread and baked goods. This study aimed to provide a cross-sectional analysis of flour for home baking, highlighting the nutrition composition of whole grain flour and identifying novel categories. An audit was undertaken in February 2020, in four major supermarkets in metropolitan Sydney (Aldi, Coles, IGA and Woolworths). Ingredient lists, Nutrition Information Panel, claims, and country of origin were collected. The median and range were calculated for energy, protein, fat, saturated fat, carbohydrate, sugars, dietary fibre and sodium. Overall, 130 products were collected, including 26 plain flour, 12 self-raising, 17 plain wholemeal, 4 wholemeal self-raising, 20 bread-making mixes (4 were whole grain), 20 other refined grain (including corn and rice flour), 17 gluten-free, 3 legume, 4 fruit/vegetable, 4 coconut and 3 other non-grain (e.g., hemp seed, cricket flour) products. Plain wheat flour dominated the category, while whole grain (wholemeal) made up 19% of products, yet they contained significantly more dietary fibre (p < 0.001) and protein (p < 0.001). Self-raising flours were significantly higher in sodium (p < 0.001) and gluten-free products were lower in protein and dietary fibre, making legume, buckwheat and quinoa flour a better choice. Sustainability principles in fruit and vegetable production and novel insect products have driven new product development. There is a clear opportunity for further on-pack promotion of whole grain and dietary fibre within the category via food product labelling.


Dual-wavelength diode-seeded Ti:sapphire laser for differential absorption lidar applications.
A dual-wavelength, diode-seeded, flash-lamp-pumped Ti:sapphire laser system has been developed. Injection seeding allowed the attainment of high output energies (130 mJ), narrow linewidth (250 MHz), and high reliability. Furthermore, an original method is presented for dual-wavelength operation, using diode current sweeping. These specifications are ideal for differential absorption lidar applications, and the example of NO(2) detection is presented. Extension to other pollutants is discussed.


Imatinib Alters Agonists-mediated Cytoskeletal Biomechanics in Lung Endothelium.
The endothelium serves as a size-selective barrier and tightly controls the fluid exchange from the circulation to the surrounding tissues. In this study, a multiplexed microscopy characterization is developed to study the spatio-temporal effects of Abl kinases on endothelial cytoskeletal structure using AFM, SEM, and immunofluorescence. Sphingosine 1-phosphate (S1P) produces significant endothelial barrier enhancement by means of peripheral actin rearrangement. However, Abl kinase inhibition by imatinib reduces rapid redistribution of the important cytoskeletal proteins to the periphery and their association with the cortical actin ring. Herein, it moderates the thickness of the cortical actin ring, and diminishes the increase in elastic modulus at the periphery and cytoplasm. These findings demonstrate that imatinib attenuates multiple cytoskeletal changes associated with S1P-mediated endothelial barrier enhancement and suggest a novel role for Abl kinases in mediating these S1P effects. These observations bridge the gap between molecule dynamics, structure complexity and function connectivity across varied length-scales to improve our understanding on human pulmonary endothelial barrier regulation. Moreover, our study suggests a framework for understanding form-function relationships in other biomechanical subsystems, wherein complex hierarchical organization programmed from the molecular scale to the cellular and tissue levels has an intimate relationship to the overall physiological function.


First-spike latency information in single neurons increases when referenced to population onset.
It is well known that many stimulus parameters, such as sound location in the auditory system or contrast in the visual system, can modulate the timing of the first spike in sensory neurons. Could first-spike latency be a candidate neural code? Most studies measuring first-spike latency information assume that the brain has an independent reference for stimulus onset from which to extract latency. This assumption creates an obvious confound that casts doubt on the feasibility of first-spike latency codes. If latency is measured relative to an internal reference of stimulus onset calculated from the responses of the neural population, the information conveyed by the latency of single neurons might decrease because of correlated changes in latency across the population. Here we assess the effects of a realistic model of stimulus onset detection on the first-spike latency information conveyed by single neurons in the auditory system. Contrary to expectation, we find that on average, the information contained in single neurons does not decrease; in fact, the majority of neurons show a slight increase in the information conveyed by latency referenced to a population onset. Our results show that first-spike latency codes are a feasible mechanism for information transfer even when biologically plausible estimates of stimulus onset are taken into account.


Becoming a web-based learner: registered nurses' experiences.
The purpose of the study was to describe Registered Nurses' experiences when taking a web-based course from either the workplace or home, and the impact of their learning on clinical practice. Little is known about the web-based learners' experience, particularly when courses are accessed from the nursing practice setting. Even less is known about whether nurses transfer their web-based learning to clinical practice. A qualitative design employing focus group interviews was used. Participants included hospital and community nurses from three Canadian provinces and one territory. Data were collected at three points over a 6-month period and analysed using a thematic analysis process. These findings emanate from a larger study using survey method and focus group interviews. The focus group interviews captured the hurdles nurses faced during the first weeks when they struggled with technology, re-framed their views of teaching and adjusted to web-based learning from home and work. These first stressful weeks were followed by a period during which nurses developed relationships with the teacher and peers that enabled them to focus on learning and prevented attrition. Most nurses reported the web course was convenient and that they would be interested and comfortable using technology for learning and work purposes in the future. Six weeks after the course was completed, nurses articulated a number of ways the course had improved their practice. Initial weeks in a web-based course can be very challenging for novice Internet users, however, most nurses who completed the course reported a positive learning experience. Nurses, employers and educators should evaluate computer skills, computer access and the learning environment when preparing for web-based learning.


Estimating the full public health value of vaccination.
There is an enhanced focus on considering the full public health value (FPHV) of vaccination when setting priorities, making regulatory decisions and establishing implementation policy for public health activities. Historically, a therapeutic paradigm has been applied to the evaluation of prophylactic vaccines and focuses on an individual benefit-risk assessment in prospective and individually-randomized phase III trials to assess safety and efficacy against etiologically-confirmed clinical outcomes. By contrast, a public health paradigm considers the population impact and encompasses measures of community benefits against a range of outcomes. For example, measurement of the FPHV of vaccination may incorporate health inequity, social and political disruption, disruption of household integrity, school absenteeism and work loss, health care utilization, long-term/on-going disability, the development of antibiotic resistance, and a range of non-etiologically and etiologically defined clinical outcomes. Following an initial conference at the Fondation Mérieux in mid-2015, a second conference (December 2016) was held to further describe the efficacy of using the FPHV of vaccination on a variety of prophylactic vaccines. The wider scope of vaccine benefits, improvement in risk assessment, and the need for partnership and coalition building across interventions has also been discussed during the 2014 and 2016 Global Vaccine and Immunization Research Forums and the 2016 Geneva Health Forum, as well as in numerous publications including a special issue of Health Affairs in February 2016. The December 2016 expert panel concluded that while progress has been made, additional efforts will be necessary to have a more fully formulated assessment of the FPHV of vaccines included into the evidence-base for the value proposition and analysis of unmet medical need to prioritize vaccine development, vaccine licensure, implementation policies and financing decisions. The desired outcomes of these efforts to establish an alternative framework for vaccine evaluation are a more robust vaccine pipeline, improved appreciation of vaccine value and hence of its relative affordability, and greater public access and acceptance of vaccines.


The development of sleep/wake disruption and cataplexy as hypocretin/orexin neurons degenerate in male vs. female Orexin/tTA; TetO-DTA Mice.
Narcolepsy Type 1 (NT1), a sleep disorder with similar prevalence in both sexes, is thought to be due to loss of the hypocretin/orexin (Hcrt) neurons. Several transgenic strains have been created to model this disorder and are increasingly being used for preclinical drug development and basic science studies, yet most studies have solely used male mice. We compared the development of narcoleptic symptomatology in male vs. female orexin-tTA; TetO-DTA mice, a model in which Hcrt neuron degeneration can be initiated by removal of doxycycline (DOX) from the diet. EEG, EMG, subcutaneous temperature, gross motor activity, and video recordings were conducted for 24-h at baseline and 1, 2, 4, and 6 weeks after DOX removal. Female DTA mice exhibited cataplexy, the pathognomonic symptom of NT1, by Week 1 in the DOX(-) condition but cataplexy was not consistently present in males until Week 2. By Week 2, both sexes showed an impaired ability to sustain long wake bouts during the active period, the murine equivalent of excessive daytime sleepiness in NT1. Subcutaneous temperature appeared to be regulated at lower levels in both sexes as the Hcrt neurons degenerated. During degeneration, both sexes also exhibited the "Delta State", characterized by sudden cessation of activity, high delta activity in the EEG, maintenance of muscle tone and posture, and the absence of phasic EMG activity. Since the phenotypes of the two sexes were indistinguishable by Week 6, we conclude that both sexes can be safely combined in future studies to reduce cost and animal use.


Electrochemically Gated Long-Distance Charge Transport in Photosystem I.
The transport of electrons along photosynthetic and respiratory chains involves a series of enzymatic reactions that are coupled through redox mediators, including proteins and small molecules. The use of native and synthetic redox probes is key to understanding charge transport mechanisms and to the design of bioelectronic sensors and solar energy conversion devices. However, redox probes have limited tunability to exchange charge at the desired electrochemical potentials (energy levels) and at different protein sites. Herein, we take advantage of electrochemical scanning tunneling microscopy (ECSTM) to control the Fermi level and nanometric position of the ECSTM probe in order to study electron transport in individual photosystem I (PSI) complexes. Current-distance measurements at different potentiostatic conditions indicate that PSI supports long-distance transport that is electrochemically gated near the redox potential of P700, with current extending farther under hole injection conditions.


Design of a broadband UV-visible alpha-barium borate polarizer.
Until recently, the construction of polarizers for operation below approximately 260 nm were limited to materials such as magnesium fluoride and crystalline quartz. These materials have a much smaller birefringence than calcite, but unlike calcite they have good transmission below 200 nm. These materials are, however, not well suited for Glan-Taylor-type polarizer designs, as they do not produce a large angular separation of the polarized components. A new material, a-barium borate, has recently become available, which transmits to just below 200 nm and has a birefringence that approaches that of calcite. We analyze the performance of various polarizer designs that use this material. Results are presented that compare theory with experimental investigation of a manufactured device.


An improved scoring method for protein residue conservation and multiple sequence alignment.
One of the most fundamental operation in biological sequence analysis is multiple sequence alignment (MSA). Optimally aligning multiple sequences is an intractable problem; however, it is a critical tool for biologists to identify the relationships between species and also possibly predict the structure and functionality of biological sequences. The most fundamental step of assembling MSA results is identifying the best location to place the sequence residues. And the accuracy of the sequence assembly depends heavily on the reliability of a scoring function used. With an appropriate scoring function, an MSA program can boost its accuracy of multiple sequence alignment up to 25%. In this study, we present a new, fast, and biologically reliable scoring method, hierarchical expected matching probability (HEP), to use in protein multiple sequence alignment. The new scoring method eliminates the burden of gap cost selection process. And it has consistently proven to be more biologically reliable than all other tested scoring methods through all tests on four different theoretical and experimental benchmarks, Valdar's theoretical conservation benchmark, RT-OSM, BAliBASE3.0, and PREFAB4.0. An implementation of our new scoring method into progressive multiple sequence alignment, resembling the alignment algorithm in PIMA, ClustalW, and T-COFFEE, has shown an accuracy improvement up to 7% on BAliBASE3.0 and up to 5% on PREFAB4.0 benchmarks.


Expanding the genetic code of Escherichia coli with phosphoserine.
O-Phosphoserine (Sep), the most abundant phosphoamino acid in the eukaryotic phosphoproteome, is not encoded in the genetic code, but synthesized posttranslationally. Here, we present an engineered system for specific cotranslational Sep incorporation (directed by UAG) into any desired position in a protein by an Escherichia coli strain that harbors a Sep-accepting transfer RNA (tRNA(Sep)), its cognate Sep-tRNA synthetase (SepRS), and an engineered EF-Tu (EF-Sep). Expanding the genetic code rested on reengineering EF-Tu to relax its quality-control function and permit Sep-tRNA(Sep) binding. To test our system, we synthesized the activated form of human mitogen-activated ERK activating kinase 1 (MEK1) with either one or two Sep residues cotranslationally inserted in their canonical positions (Sep(218), Sep(222)). This system has general utility in protein engineering, molecular biology, and disease research.


Multi-level analysis of alcohol-related injury and drinking pattern: emergency department data from 19 countries.
Aim While drinking in the event is an important factor in injury occurrence, the pattern of usual drinking may also be important in risk of injury. Explored here is the relationship of an alcohol-related injury with an individual usual drinking pattern. Design Alcohol-related injury is examined using hierarchical linear models, taking into account individual usual volume of consumption over the past 12 months, as well as aggregate-level detrimental drinking pattern (DDP) and alcohol policy measures. Setting Data analyzed are from emergency departments (EDs) in 19 countries, comprising three collaborative studies on alcohol and injury, all of which used a similar methodology. Participants The sample comprised 14 132 injured drinkers across 46 emergency room (ER) studies. Measurements Alcohol-related injury was measured, separately, by any self-reported drinking prior to injury, a blood alcohol concentration (BAC) ≥ 0.08 and self-reported causal attribution of injury to drinking. Findings While individual usual volume strongly predicted an alcohol-related injury for all three measures, usual drinking pattern also predicted an alcohol-related injury (controlling for volume), with episodic heavy and frequent heavy drinking both more predictive of alcohol-related injury than other drinking patterns. When individual usual volume and drinking pattern were controlled, DDP was no longer a significant predictor of alcohol-related injury. Alcohol policy measures were predictive of both BAC and causal attribution (the stronger the policy the lower the rates of alcohol-related injury). Conclusions Volume of alcohol typically consumed and occurrence of heavy drinking episodes are associated independently with incidence of alcohol-related injury. The stronger the anti-alcohol policies in a country, the lower the rates of alcohol-related injury.


A Novel Method Combining Vitreous Aspiration and Intravitreal AAV2/8 Injection Results in Retina-Wide Transduction in Adult Mice.
Gene therapies to treat eye disorders have been extensively studied in the past 20 years. Frequently, adeno-associated viruses were applied to the subretinal or intravitreal space of the eye to transduce retinal cells with nucleotide sequences of therapeutic potential. In this study we describe a novel intravitreal injection procedure that leads to a reproducible adeno-associated virus (AAV)2/8-mediated transduction of more than 70% of the retina. Prior to a single intravitreal injection of a enhanced green fluorescent protien (GFP)-expressing viral suspension, we performed an aspiration of vitreous tissue from wild-type C57Bl/6J mice. One and one-half microliters of AAV2/8 suspension was injected. Funduscopy, optical coherence tomography (OCT), laser scanning microscopy of retinal flat mounts, cryosections of eye cups, and ERG recordings verified the efficacy and safety of the method. The combination of vitreous aspiration and intravitreal injection resulted in an almost complete transduction of the retina in approximately 60% of the eyes and showed transduced cells in all retinal layers. Photoreceptors and RPE cells were predominantly transduced. Eyes presented with well-preserved retinal morphology. Electroretinographic recordings suggested that the new combination of techniques did not cause significant alterations of the retinal physiology. We show a novel application technique of AAV2/8 to the vitreous of mice that leads to widespread transduction of the retina. The results of this study have implications for virus-based gene therapies and basic science; for example, they might provide an approach to apply gene replacement strategies or clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in vivo. It may further help to develop similar techniques for larger animal models or humans.


Catalytic hydrogenation by triruthenium clusters: a mechanistic study with parahydrogen-induced polarization.
The reactivity of the cluster family [Ru(3)(CO)(12-x)(L)(x)] (in which L=PMe(3), PMe(2)Ph, PPh(3) and PCy(3), x=1-3) towards hydrogen is described. When x=2, three isomers of [Ru(3)(H)(mu-H)(CO)(9)(L)(2)] are formed, which differ in the arrangement of their equatorial phosphines. Kinetic studies reveal the presence of intra- and inter-isomer exchange processes with activation parameters and solvent effects indicating the involvement of ruthenium-ruthenium bond heterolysis and CO loss, respectively. When x=3, reaction with H(2) proceeds to form identical products to those found with x=2, while when x=1 a single isomer of [Ru(3)(H)(mu-H)(CO)(10)(L)] is formed. Species [Ru(3)(H)(mu-H)(CO)(9)(L)(2)] have been shown to play a kinetically significant role in the hydrogenation of an alkyne substrate through initial CO loss, with rates of H(2) transfer being explicitly determined for each isomer. A less significant secondary reaction involving loss of L yields a detectable product that contains both a pendant vinyl unit and a bridging hydride ligand. Competing pathways that involve fragmentation to form [Ru(H)(2)(CO)(2)(L)(alkyne)] are also observed and shown to be favoured by nonpolar solvents. Kinetic data reveal that catalysis based on [Ru(3)(CO)(10)(PPh(3))(2)] is the most efficient although [Ru(3)(H)(mu-H)(CO)(9)(PMe(3))(2)] corresponds to the most active of the detected intermediates.


Attenuation of cocaine self-administration by chronic oral phendimetrazine in rhesus monkeys.
Chronic treatment with the monoamine releaser d-amphetamine has been consistently shown to decrease cocaine self-administration in laboratory studies and clinical trials. However, the abuse potential of d-amphetamine is an obstacle to widespread clinical use. Approaches are needed that exploit the efficacy of the agonist approach but avoid the abuse potential associated with dopamine releasers. The present study assessed the effectiveness of chronic oral administration of phendimetrazine (PDM), a pro-drug for the monoamine releaser phenmetrazine (PM), to decrease cocaine self-administration in four rhesus monkeys. Each day, monkeys pressed a lever to receive food pellets under a 50-response fixed-ratio (FR) schedule of reinforcement and self-administered cocaine (0.003-0.56 mg/kg per injection, i.v.) under a progressive-ratio (PR) schedule in the evening. After completing a cocaine self-administration dose-response curve, sessions were suspended and PDM was administered (1.0-9.0 mg/kg, p.o., b.i.d.). Cocaine self-administration was assessed using the PR schedule once every 7 days while food-maintained responding was studied daily. When a persistent decrease in self-administration was observed, the cocaine dose-effect curve was re-determined. Daily PDM treatment decreased cocaine self-administration by 30-90% across monkeys for at least 4 weeks. In two monkeys, effects were completely selective for cocaine. Tolerance developed to initial decreases in food-maintained responding in the third monkey and in the fourth subject, fluctuations were observed that were lower in magnitude than effects on cocaine self-administration. Cocaine dose-effect curves were shifted down and/or rightward in three monkeys. These data provide further support for the use of agonist medications for cocaine abuse, and indicate that the promising effects of d-amphetamine extend to a more clinically viable pharmacotherapy.


Surfaces Decorated with Enantiomorphically Pure Polymer Nanohelices via Hierarchical Chirality Transfer across Multiple Length Scales.
Mesoscale chiral materials are prepared by lithographic methods, assembly of chiral building blocks, and through syntheses in the presence of polarized light. Typically, these processes result in micrometer-sized structures, require complex top-down manipulation, or rely on tedious asymmetric separation. Chemical vapor deposition (CVD) polymerization of chiral precursors into supported films of liquid crystals (LCs) are discovered to result in superhierarchical arrangements of enantiomorphically pure nanofibers. Depending on the molecular chirality of the 1-hydroxyethyl [2.2]paracyclophane precursor, extended arrays of enantiomorphic nanohelices are formed from achiral nematic templates. Arrays of chiral nanohelices extend over hundreds of micrometers and consistently display enantiomorphic micropatterns. The pitch of individual nanohelices depends on the enantiomeric excess and the purity of the chiral precursor, consistent with the theoretical model of a doubly twisted LC director configuration. During CVD of chiral precursors into cholesteric LC films, aspects of molecular and mesoscale asymmetry combine constructively to form regularly twisted nanohelices. Enantiomorphic surfaces permit the tailoring of a wide range of functional properties, such as the asymmetric induction of weak chiral systems.


Adiabatic invariance in GRIN channel waveguides and its use in 3-dB cross couplers.
We have extended the local normal mode approach of Burns and Milton to derive the condition for adiabatic invariance in graded-index channel waveguides. The results show that in spite of the index-grading and 2-D confinement, the inequality describing the adiabatic condition is basically the same as that for step-index slab waveguides. To verify the predictions of the theory, we have fabricated, using ion exchange in glass, a cross coupler with two asymmetric input channel waveguides and two symmetric output guides. The experimental results show equal output power (3.0 +/- 0.1 dB) from the two symmetric guides when either of the two unequal guides is excited, demonstrating excellent agreement with the theory.


Patients with pain disorder show gray-matter loss in pain-processing structures: a voxel-based morphometric study.
To investigate whether the functional changes in pain disorder might be reflected by structural brain changes. Pain disorder assessed with the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM-IV) criteria is characterized by persistent and distressing chronic pain at one or more body sites which cannot be fully explained by a physiological process or somatic disorder. Psychological factors are thought to play a major role. Recent neuroimaging studies evidenced altered pain processing in patients suffering from this disorder. Fourteen right-handed women fulfilling the DSM-IV criteria for pain disorder and 25 healthy age-matched women were investigated with magnetic resonance imaging. In the voxel-based morphometry analysis, we compared both groups for changes of gray-matter density. We included age and Beck Depression Inventory scores as nuisance variables to minimize possible confounding effects of age or depressive comorbidity. In the patient group, we found significant gray-matter decreases in the prefrontal, cingulate, and insular cortex. These regions are known to be critically involved in the modulation of subjective pain experiences. In the context of similar results in patients with other functional pain syndromes, such as fibromyalgia and chronic back pain, we suggest that structural changes in fronto-limbic brain circuits represent not only an objective marker of these pain syndromes but also constitute a critical pathophysiological element. These findings represent a further proof of the important role of central changes in pain disorder.


O(1D) kinetic study of key ozone depleting substances and greenhouse gases.
A key stratospheric loss process for ozone depleting substances (ODSs) and greenhouse gases (GHGs) is reaction with the O((1)D) atom. In this study, rate coefficients, k, for the O((1)D) atom reaction were measured for the following key halocarbons: chlorofluorocarbons (CFCs) CFCl3 (CFC-11), CF2Cl2 (CFC-12), CFCl2CF2Cl (CFC-113), CF2ClCF2Cl (CFC-114), CF3CF2Cl (CFC-115); hydrochlorofluorocarbons (HCFCs) CHF2Cl (HCFC-22), CH3CClF2 (HCFC-142b); and hydrofluorocarbons (HFCs) CHF3 (HFC-23), CHF2CF3 (HFC-125), CH3CF3 (HFC-143a), and CF3CHFCF3 (HFC-227ea). Total rate coefficients, kT, corresponding to the loss of the O((1)D) atom, were measured over the temperature range 217-373 K using a competitive reactive technique. kT values for the CFC and HCFC reactions were >1 × 10(-10) cm(3) molecule(-1) s(-1), except for CFC-115, and the rate coefficients for the HFCs were in the range (0.095-0.72) × 10(-10) cm(3) molecule(-1) s(-1). Rate coefficients for the CFC-12, CFC-114, CFC-115, HFC-23, HFC-125, HFC-143a, and HFC-227ea reactions were observed to have a weak negative temperature dependence, E/R ≈ -25 K. Reactive rate coefficients, kR, corresponding to the loss of the halocarbon, were measured for CFC-11, CFC-115, HCFC-22, HCFC-142b, HFC-23, HFC-125, HFC-143a, and HFC-227ea using a relative rate technique. The reactive branching ratio obtained was dependent on the composition of the halocarbon and the trend in O((1)D) reactivity with the extent of hydrogen and chlorine substitution is discussed. The present results are critically compared with previously reported kinetic data and the discrepancies are discussed. 2D atmospheric model calculations were used to evaluate the local and global annually averaged atmospheric lifetimes of the halocarbons and the contribution of O((1)D) chemistry to their atmospheric loss. The O((1)D) reaction was found to be a major global loss process for CFC-114 and CFC-115 and a secondary global loss process for the other molecules included in this study.


Heroin-induced reinstatement is specific to compulsive heroin use and dissociable from heroin reward and sensitization.
Increased drug availability can precipitate a rapid transition to compulsive drug use in both vulnerable humans and laboratory animals. Recent studies have shown that despite equivalent levels of psychomotor sensitization, only rats with prolonged, but not limited, access to cocaine self-administration respond to the priming effects of cocaine on drug seeking, as measured in a within-session reinstatement model of drug craving. In this model, drug seeking is first extinguished and then reinstated by non-contingent presentations of the drug alone in the absence of response-contingent stimuli. Here, we assessed the generality of this observation in rats with daily short (1 h, ShA) vs long access (6 h, LgA) to i.v. heroin self-administration. As expected, heroin intake by LgA rats (n=24) increased over time to become excessive compared to heroin intake by ShA rats (n=24). After escalation, LgA rats tended to be less sensitive to heroin-induced locomotion (7.5-30 microg, i.v.) than ShA rats. In contrast, only LgA rats, not ShA rats, responded to the priming effects of heroin, as measured by the ability of heroin alone (7.5-30 microg, i.v.) to reinstate extinguished drug-seeking behavior. Finally, during the course of heroin intake escalation, a large proportion of LgA rats developed self-injury (mostly targeting the nails and digit tips of the forepaws), a negative consequence not seen in ShA rats. This study reproduces and extends previous research on compulsive cocaine use by showing that heroin-induced reinstatement is also specific to compulsive drug use and dissociable from heroin-induced reward and psychomotor sensitization.


Characteristics and Outcomes of Atrial Tachycardia Originating from the Sinus Venosus during Catheter Ablation of Atrial Fibrillation.
The sinus venosus (SV) is not a well known source of atrial tachycardia (AT), but it can harbor AT during catheter ablation of atrial fibrillation (AF). A total of 1223 patients who underwent catheter ablation for AF were reviewed. Electrophysiological and electrocardiographic characteristics and outcomes after catheter ablation of AT originating from the SV were investigated. Ten patients (0.82%) demonstrated AT from the SV (7 males, 53.9±16.0 years, 6 persistent) during ablation of AF. The mean cycle length was 281±73 ms. After pulmonary vein isolation and left atrial ablation, AF converted to AT from the SV during right atrial ablation in 2 patients, by rapid atrial pacing after AF termination in 7 patients, and during isoproterenol infusion in 1 patient. Positive P-waves in inferior leads were shown in most patients (90%). The activation sequence of AT was from proximal to distal in the superior vena cava and high to low in the right atrium, which was similar to that of AT from crista terminalis. Fragmented double potentials were recorded during sinus, and a second discrete potential preceded the onset of P wave by 80±37 ms during AT. Using 4.4±2.7 radiofrequency focal applications, ATs were terminated and became no longer inducible in all. After ablation procedure, two patients showed transient right phrenic nerve palsy. After 19.9±14.8 months, all but 1 patient were free of atrial tachyarrhythmia without complications. The AT which develops during AF ablation is rarely originated from SV, and its electrophysiologic characteristics may be helpful in guiding effective focal ablation.


Distinct functions of polysaccharide deacetylases in cell shape, neutral polysaccharide synthesis and virulence of Bacillus anthracis.
Peptidoglycan deacetylases (PGNG-dacs) belong to the Carbohydrate Esterase Family 4 (CE4) and have been described as required for bacterial evasion to lysozyme and innate immune responses. Interestingly, there is an unusual occurrence of 10 putative polysaccharide deacetylases, including five PGNG-dacs, in the Bacillus sp. genomes, especially B. cereus and B. anthracis. To elucidate the physiological role of these multiple deacetylases, we employed genetic analysis and protein localization studies of five putative PGNG-dacs from B. anthracis as well as biochemical analysis of their corresponding homologues from B. cereus. Our data confirm that three enzymes are PGNG-dacs. While BA1977, associated with lateral peptidoglycan synthesis, is a bona fide peptidoglycan deacetylase involved in resistance to host lysozyme and required for full virulence, BA1961 and BA3679 participate in the biogenesis of the peptidoglycan during both elongation and cell division. Furthermore, two enzymes are important for neutral polysaccharide attachment to PG and consequently anchoring of S-layer proteins (BA5436) and for polysaccharide modification (BA2944). Our results provide novel and fundamental insights into the function of polysaccharide deacetylases in a major bioterrorism agent.


Triple trans-splicing adeno-associated virus vectors capable of transferring the coding sequence for full-length dystrophin protein into dystrophic mice.
Recombinant adeno-associated virus (rAAV) vectors have been shown to permit very efficient widespread transgene expression in skeletal muscle after systemic delivery, making these increasingly attractive as vectors for Duchenne muscular dystrophy (DMD) gene therapy. DMD is a severe muscle-wasting disorder caused by DMD gene mutations leading to complete loss of dystrophin protein. One of the major issues associated with delivery of the DMD gene, as a therapeutic approach for DMD, is its large open reading frame (ORF; 11.1 kb). A series of truncated microdystrophin cDNAs (delivered via a single AAV) and minidystrophin cDNAs (delivered via dual-AAV trans-spliced/overlapping reconstitution) have thus been extensively tested in DMD animal models. However, critical rod and hinge domains of dystrophin required for interaction with components of the dystrophin-associated protein complex, such as neuronal nitric oxide synthase, syntrophin, and dystrobrevin, are missing; these dystrophin domains may still need to be incorporated to increase dystrophin functionality and stabilize membrane rigidity. Full-length DMD gene delivery using AAV vectors remains elusive because of the limited single-AAV packaging capacity (4.7 kb). Here we developed a novel method for the delivery of the full-length DMD coding sequence to skeletal muscles in dystrophic mdx mice using a triple-AAV trans-splicing vector system. We report for the first time that three independent AAV vectors carrying "in tandem" sequential exonic parts of the human DMD coding sequence enable the expression of the full-length protein as a result of trans-splicing events cojoining three vectors via their inverted terminal repeat sequences. This method of triple-AAV-mediated trans-splicing could be applicable to the delivery of any large therapeutic gene (≥11 kb ORF) into postmitotic tissues (muscles or neurons) for the treatment of various inherited metabolic and genetic diseases.


Association of immunity and tolerance to host H-2 determinants in irradiated F1 hybrid mice reconstituted with bone marrow cells from one parental strain.
Semiallogenetic radiation chimeras were prepared by injecting heavily irradiated F1 hybrid mice with bone marrow cells from one parental strain; the bone marrow cells were treated with anti-theta serum and complement to remove T cells and injected in large numbers (2 times 10-7 cells). The mice survived in excellent health until sacrifice 6 mo later. Thoracic duct cannulation at this stage showed that the mice possessed normal numbers of recirculating lymphocytes. Close to 100% of thoracic duct lymphocytes and lymph node cells were shown to be of donor strain origin. The capacity of lymphocytes from the chimeras to respond to host-type determinants was tested in mixed leukocyte culture and in an assay for cell-mediated lympholysis (CML). Mixed leukocyte reactions (MLR) were measured both in vitro and in vivo; tumor cells and phytohemmaglutinin-stimulated blast cells were used as target cells for measuring CML. While responding normally to third party determinants, cells from the chimeras gave a definite, though reduced MLR when exposed to host-type determinants. However, this proliferative response to host-type determinants, unlike that to third party determinants, was not associated with differentiation into cytotoxic lymphocytes. No evidence could be found that unresponsiveness in this situation was due to blocking serum factors or suppressor T cells. It is argued that the results support the concept that lymphocytes responsive in mixed leukocyte culture have a different specificity to those exerting cell-mediated lympholysis.


Diagnosis Setting and Colorectal Cancer Outcomes: The Impact of Cancer Diagnosis in the Emergency Department.
The rate of diagnosis of colorectal cancer (CRC) in the emergency department (ED), its characteristics, and its effect on outcomes have been poorly described. Chart review was conducted to identify presenting clinical setting leading to diagnosis, symptoms, and history of colonoscopy for patients diagnosed with CRC at a single institution from 2012-2014. Patients diagnosed with CRC as a result of an ED visit (EDDx) were compared with those diagnosed after presentation to other settings (non-EDDx). Of 638 patients meeting inclusion criteria, 271 (42.4%) were EDDx patients. These patients were more likely to be older than 80 y (29.89% versus 19.35%), have Medicare (59.78% versus 42.78%) or Medicaid (23.62% versus 12.81%) insurance, have stage IV cancer (45.02% versus 18.26%), and were symptomatic at the time of presentation (94.83% versus 64.03%). EDDx patients were less likely to ever have had a colonoscopy (21.77% versus 41.69%). In a model adjusted for patient demographics, cancer stage, presence of symptoms, and history of prior colonoscopy, EDDx was associated with increased mortality (hazard ratio, 1.89; 95% confidence interval, 1.3-2.8). On stratifying survival by stage, it was found that for all stages, EDDx was associated with decreased survival. More than 40% of patients with CRC received their diagnosis through the ED. EDDx was associated with a nearly twofold mortality risk increase. EDDx should be considered a marker of poor outcomes for CRC and may be related to unaccounted patient-level or systems-level factors. Efforts should be made to identify modifiable risks of cancer diagnosis in the ED to improve cancer outcomes.


Retinopathy of prematurity: applicability of international and national screening guidelines in the north of Iran.
To determine the applicability of current international and national retinopathy of prematurity (ROP) screening guidelines and to identify a suitable community-based screening criterion. A retrospective study on premature neonates (≤37 weeks gestation) referred to a tertiary eye hospital ROP clinic in the north of Iran was conducted over a 10-year period. Neonates were classified as no ROP, with ROP and type 1 ROP. Data consisting of birth weight (BW), gestational age (GA) and chief risk factors were evaluated. Various screening criteria and currently established screening guidelines were applied and compared for applicability using a receiver operating characteristic curve. A total of 716 neonates with a mean GA of 31.4 ± 2.8 weeks and BW of 1629 ± 502 grams were screened. The incidence of ROP was 22.9% and type 1 ROP requiring treatment was 0.28%. When applying the national Ministry of Health Guidelines, all neonates with type 1 ROP requiring treatment were identified; These criteria had a specificity of 7% for the diagnosis of type 1 ROP, and a large number of neonates (n=645) who are not at risk for type 1 ROP will be redundantly screened. Guidelines of the American Academy of Pediatrics and the UK would miss 4.5% of patients requiring ROP treatment. According to our data a threshold of GA≤32 weeks and/or BW ≤1600 grams demonstrated a sensitivity of 95.7% and specificity of 33.6% for the diagnosis of any ROP and a sensitivity of 100% and specificity of 26.8% for type 1 ROP requiring treatment. The ideal ROP screening guideline is one that is very sensitive and identifies patients requiring treatment without delay. To minimize redundant screening while maintaining optimum ROP requiring treatment diagnosis, we proposed a new local evidence-based screening guideline.