Role of metabotropic glutamate receptors in ventrolateral medulla of hypertensive rats.
Evidence is accumulating for the role of metabotropic, as well as ionotropic, glutamate receptors in cardiovascular regulation. We sought to determine whether stimulation of metabotropic glutamate receptors in the rostral ventrolateral medulla would evoke enhanced cardiovascular responses in spontaneously hypertensive rats (SHR). Thus, we microinjected (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD], a selective agonist of metabotropic glutamate receptors, into the rostral ventrolateral medulla of urethane-anesthetized adult SHR and age-matched Wistar-Kyoto (WKY) rats. Microinjection of (1S,3R)-ACPD (1 nmol/50 nL) produced increases in mean arterial pressure and splanchnic sympathetic nerve activity in SHR (+41 +/- 6 mm Hg and +34 +/- 4%, respectively) that were significantly greater than those observed in WKY rats (+18 +/- 3 mm Hg and +22 +/- 3%, P < .005 and P < .05, respectively). The pressor responses evoked by microinjection of L-glutamate (2 nmol), N-methyl-D-aspartate (20 pmol), or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (5 pmol) were also significantly (P < .001) augmented in SHR (+55 +/- 3, +61 +/- 7, and +53 +/- 5 mm Hg, respectively, in SHR versus +31 +/- 1, +30 +/- 3, and +28 +/- 2 mm Hg in WKY rats). Results indicate that stimulation of metabotropic, as well as ionotropic, glutamate receptors in the rostral ventrolateral medulla evokes enhanced cardiovascular responses in SHR, which may contribute to hypertension in this model.


Influenza virus reassortment occurs with high frequency in the absence of segment mismatch.
Reassortment is fundamental to the evolution of influenza viruses and plays a key role in the generation of epidemiologically significant strains. Previous studies indicate that reassortment is restricted by segment mismatch, arising from functional incompatibilities among components of two viruses. Additional factors that dictate the efficiency of reassortment remain poorly characterized. Thus, it is unclear what conditions are favorable for reassortment and therefore under what circumstances novel influenza A viruses might arise in nature. Herein, we describe a system for studying reassortment in the absence of segment mismatch and exploit this system to determine the baseline efficiency of reassortment and the effects of infection dose and timing. Silent mutations were introduced into A/Panama/2007/99 virus such that high-resolution melt analysis could be used to differentiate all eight segments of the wild-type and the silently mutated variant virus. The use of phenotypically identical parent viruses ensured that all progeny were equally fit, allowing reassortment to be measured without selection bias. Using this system, we found that reassortment occurred efficiently (88.4%) following high multiplicity infection, suggesting the process is not appreciably limited by intracellular compartmentalization. That co-infection is the major determinant of reassortment efficiency in the absence of segment mismatch was confirmed with the observation that the proportion of viruses with reassortant genotypes increased exponentially with the proportion of cells co-infected. The number of reassortants shed from co-infected guinea pigs was likewise dependent on dose. With 10⁶ PFU inocula, 46%-86% of viruses isolated from guinea pigs were reassortants. The introduction of a delay between infections also had a strong impact on reassortment and allowed definition of time windows during which super-infection led to reassortment in culture and in vivo. Overall, our results indicate that reassortment between two like influenza viruses is efficient but also strongly dependent on dose and timing of the infections.


Identification and Characterization of Resistance to Phytophthora infestans in Leaves, Stems, Flowers, and Tubers of Potato Clones in the Pacific Northwest.
Resistance to Phytophthora infestans in leaves, stems, flowers, and tubers of eight cultivars grown commercially in the Columbia Basin and 29 advanced clones from the Tri-State Potato Variety Development Program was quantified. Foliage of all eight cultivars was susceptible in field and greenhouse tests. Six advanced clones had high levels of resistance in stems and leaves in the field at Mount Vernon, WA, but only two of these six clones had high levels of resistance in leaves and stems in greenhouse tests. Flowers of most plants with high to moderate levels of foliar resistance were susceptible to infection in both the field and greenhouse. Tubers of Umatilla Russet, Russet Legend, Gem Russet, and nine advanced clones, including A90586-11, had high levels of resistance in most laboratory tests. Level of foliar resistance against US-8 and US-11 clonal lineages of P. infestans was variable among clones, but tuber resistance by pathogen clonal lineage varied little among clones. Clones with high resistance expressed quantitative differences compared with susceptible cultivars and likely possess a high level of partial resistance.


Novel betapapillomavirus associated with hand and foot papillomas in a cynomolgus macaque.
Betapapillomavirus is a genus of papillomaviruses (PVs) commonly found in human skin and associated with both benign and malignant skin lesions. Only 2 previous beta-PVs have been fully characterized in nonhuman species. This report describes a novel beta-PV, named Macaca fascicularis PV type 2 (MfPV2), isolated from exophytic skin papillomas on the hands and feet of a 2-year-old male cynomolgus monkey (M. fascicularis). On histology the papillomas were composed of diffusely thickened epidermis with superficial foci of cytomegaly, cytoplasmic pallor, marginalized chromatin, and rare eosinophilic intranuclear inclusion bodies. Positive immunostaining for p16 and the proliferation marker Ki67 was present multifocally within affected epidermis, most prominently within basal-type cells. Complete sequence identity (100%) was noted between PV genomes fully sequenced from hand and foot lesions. The MfPV2 genome was 7632 base pairs in length and included putative open reading frames (ORFs) for E1, E2, E4, E6, E7, L1, and L2 genes, similar to other PVs. The closest relatives to MfPV2 based on the L1 ORF sequence were all beta-PVs. These included human PV (HPV) 9, HPV115, HPV76, HPV75, and MfPV1 (60-70% pairwise identity for all), the latter of which was also isolated from hand and foot papillomas in a cynomolgus macaque. Phylogenetic analysis placed MfPV2 in a new species group (beta-6), distinct from HPVs (beta-1 to beta-5) and MfPV1 (beta-1). These findings characterize a new nonhuman beta-PV and provide additional support for the idea that tissue tropism among ancestral primate PVs developed prior to divergence of certain Old World primate lineages.


Abundant GFP expression and LTP in hippocampal acute slices by in vivo injection of sindbis virus.
Virus-mediated gene transfer into neurons is a powerful tool for the analysis of neuronal structure and function. Recombinant sindbis virus has been previously used to study protein function in hippocampal neuron cultures as well as in hippocampal organotypic slice cultures. Nevertheless, some concern still exists about the physiological relevance of these cultured preparations. Acute hippocampal slices are a widely used preparation for the study of synaptic transmission, but currently recombinant gene delivery is usually achieved only through time-consuming transgenic techniques. In this study, we show that a subregion of the CA1 area in acute hippocampal slices can be specifically altered to express a gene of interest. A sindbis virus vector carrying an enhanced green fluorescent protein (EGFP) reporter was injected in vivo into the hippocampus of adult rats. After 18 h, rats were killed, and acute hippocampal slices, infected in the CA1 field, were analyzed morphologically and electrophysiologically. Infected slices showed healthy and stable electrophysiological responses as well as long-term potentiation. In addition, infected pyramidal cells were readily recognized in living slices by two-photon imaging. Specifically, the introduction of an EGFP-Actin fusion protein greatly enhanced the detection of fine processes and dendritic spines. We propose this technique as an efficient tool for studying gene function in adult hippocampal neurons.


Distribution and prognostic impact of microglia/macrophage subpopulations in gliomas.
While the central nervous system is considered an immunoprivileged site and brain tumors display immunosuppressive features, both innate and adaptive immune responses affect glioblastoma (GBM) growth and treatment resistance. However, the impact of the major immune cell population in gliomas, represented by glioma-associated microglia/macrophages (GAMs), on patients' clinical course is still unclear. Thus, we aimed at assessing the immunohistochemical expression of selected microglia and macrophage markers in 344 gliomas (including gliomas from WHO grade I-IV). Furthermore, we analyzed a cohort of 241 IDH1R132H-non-mutant GBM patients for association of GAM subtypes and patient overall survival. Phenotypical properties of GAMs, isolated from high-grade astrocytomas by CD11b-based magnetic cell sorting, were analyzed by immunocytochemistry, mRNA microarray, qRT-PCR and bioinformatic analyses. A higher amount of CD68-, CD163- and CD206-positive GAMs in the vital tumor core was associated with beneficial patient survival. The mRNA expression profile of GAMs displayed an upregulation of factors that are considered as pro-inflammatory M1 (eg, CCL2, CCL3L3, CCL4, PTGS2) and anti-inflammatory M2 polarization markers (eg, MRC1, LGMN, CD163, IL10, MSR1), the latter rather being associated with phagocytic functions in the GBM microenvironment. In summary, we present evidence that human GBMs contain mixed M1/M2-like polarized GAMs and that the levels of different GAM subpopulations in the tumor core are positively associated with overall survival of patients with IDH1R132H-non-mutant GBMs.


Key factors affecting people's unwillingness to be confined during the COVID-19 pandemic in Spain: a large-scale population study.
Population confinements have been one of the most widely adopted non-pharmaceutical interventions (NPIs) implemented by governments across the globe to help contain the spread of the SARS-CoV-2 virus. While confinement measures have been proven to be effective to reduce the number of infections, they entail significant economic and social costs. Thus, different policy makers and social groups have exhibited varying levels of acceptance of this type of measures. In this context, understanding the factors that determine the willingness of individuals to be confined during a pandemic is of paramount importance, particularly, to policy and decision-makers. In this paper, we study the factors that influence the unwillingness to be confined during the COVID-19 pandemic by the means of a large-scale, online population survey deployed in Spain. We perform two types of analyses (logistic regression and automatic pattern discovery) and consider socio-demographic, economic and psychological factors, together with the 14-day cumulative incidence per 100,000 inhabitants. Our analysis of 109,515 answers to the survey covers data spanning over a 5-month time period to shed light on the impact of the passage of time. We find evidence of pandemic fatigue as the percentage of those who report an unwillingness to be in confinement increases over time; we identify significant gender differences, with women being generally less likely than men to be able to sustain long-term confinement of at least 6 months; we uncover that the psychological impact was the most important factor to determine the willingness to be in confinement at the beginning of the pandemic, to be replaced by the economic impact as the most important variable towards the end of our period of study. Our results highlight the need to design gender and age specific public policies, to implement psychological and economic support programs and to address the evident pandemic fatigue as the success of potential future confinements will depend on the population's willingness to comply with them.


Aerosolized Pseudomonas elastase and lung fluid balance in anesthetized sheep.
The role of the lung epithelium in lung fluid balance was studied by ventilating anesthetized sheep with an aerosol of 20 mg of elastase from Pseudomonas aeruginosa (Ps. elastase) to increase lung epithelial permeability without affecting lung endothelial permeability or lung vascular pressures. Ps. elastase had no effect on the lung vascular pressures, the alveolar-arterial PO2 gradient (A-aPO2), the flow or protein concentration of the lung lymph, or the postmortem water volume of the lungs. The morphological alveolar flooding score in these sheep was 2.5 times the control level, but this was only marginally significant. Elevation of the left atrial pressure by 20 cmH2O alone increased the postmortem lung water volume but had no effect on A-aPO2, the alveolar flooding score, or the lung epithelial permeability assessed by the clearance of 99mTc-labeled human serum albumin. Addition of aerosolized Ps. elastase to these sheep had no effect on the total lung water volume, but it caused a redistribution of water into the air spaces, as evidenced by significant increases in the alveolar flooding score and A-aPO2 (P less than 0.01). Elevation of the left atrial pressure by 40 cmH2O without elastase caused the same response as elevation of the left atrial pressure by 20 cmH2O with elastase, except the higher pressure caused a greater increase in the total lung water volume. We conclude that alteration of the integrity of the lung epithelium with aerosolized Ps. elastase causes a redistribution of lung water into the alveoli without affecting the total lung water volume.(ABSTRACT TRUNCATED AT 250 WORDS)


Klinefelter's syndrome (47,XXY) is in excess among men with Sjögren's syndrome.
Primary Sjögren's syndrome (pSS) has a strong female bias. We evaluated an X chromosome dose effect by analyzing 47,XXY (Klinefelter's syndrome, 1 in 500 live male births) among subjects with pSS. 47,XXY was determined by examination of fluorescence intensity of single nucleotide polymorphisms from the X and Y chromosomes. Among 136 pSS men there were 4 with 47,XXY. This was significantly different from healthy controls (1 of 1254 had 47,XXY, p=0.0012 by Fisher's exact test) as well men with rheumatoid arthritis (0 of 363 with 47,XXY), but not different compared to men with systemic lupus erythematosus (SLE) (4 of 136 versus 8 of 306, Fisher's exact test p=NS). These results are consistent with the hypothesis that the number of X chromosomes is critical for the female bias of pSS, a property that may be shared with SLE but not RA.


Pleural Drainage Strategy After IPC Placement: A Focused Clinical Review.
The first indwelling pleural catheter (IPC) received Federal Drug Administration approval in 1997, nearly 40 years after John Chambers first described instillation of talc into the pleural space for palliation of malignant pleural effusions. Since then IPCs have revolutionized the management of malignant pleural effusions, providing an effective means of controlling dyspnea in the end stages of life without the pain and prolonged hospital stays long associated with chemical sclerosing agents. While palliation of symptoms is the primary purpose of IPCs, development of pleurodesis in the outpatient setting is often an important secondary goal. Historically, decisions regarding IPC drainage frequency have been largely arbitrary and only recently has the evidence started to emerge regarding the effects of specific drainage strategies on both symptom control and pleurodesis development. This focused clinical review explores the current literature regarding IPC drainage strategies as well as novel methods designed to improve the efficacy of IPCs in a thorough and systematic manner. In addition, this review provides an in-depth analysis and synthesis of the data focusing on key considerations needed to develop patient-centered and cost-effective strategies for the use of IPCs in malignancy.


Clinical veterinary students' perceptions of a 'Day one' skills guide.
Veterinary schools in the UK are required by the Royal College of Veterinary Surgeons to ensure that their students have attained essential 'Day one' skills (DOS) by graduation. To aid students' understanding of how they could satisfy this requirement, the Royal Veterinary College produced a DOS guide, which was distributed in the 2007 to 2008 academic year to students in the third and fourth years of the professional veterinary course. The same students were surveyed towards the end of the 2008 to 2009 session about the guide, when they were in the fourth and final years, to rate its usefulness and to indicate their perceived levels of competence, both currently and expected at graduation. Focus group discussions were subsequently conducted to explore the findings of the survey. Responses from 88 fourth-year (39 per cent) and 174 (87 per cent) final-year students revealed that while almost all respondents were aware of the DOS guide, their use of it was low. Final-year students rated themselves as being more competent than fourth-year students, but were less optimistic about their expected level of competence regarding several invasive procedures, which some students considered to be 'unattainable' by graduation, despite the apparent value of extramural studies and intramural rotations in providing opportunities for learning.


Collision metastasis of prostatic adenocarcinoma and urothelial carcinoma of the bladder.
The incidence of concomitant prostate adenocarcinoma found in patients with muscle-invasive bladder carcinoma is not uncommon, reaching up to 21%-28%. However, the presence of collision metastasis involving prostate cancer and bladder cancer within the same lymph node is exceedingly rare, with only 5 cases reported to date in the literature. We report a case of collision metastasis of prostate adenocarcinoma and urothelial carcinoma of the bladder in a 73-year-old man who underwent cystoprostatectomy with bilateral pelvic lymph node dissection for high-grade muscle-invasive urothelial carcinoma. Final pathology revealed a pT3aN2 high-grade urothelial carcinoma and pT3N1 Gleason 4 + 4 = 8 adenocarcinoma of the prostate with 12/40 pelvic lymph nodes positive for urothelial carcinoma. One node was positive for both urothelial carcinoma and prostate adenocarcinoma, confirmed by positive staining by p40 and prostate specific antigen(PSA), respectively. Immunohistochemistry is the sole method of confirming collision metastasis of two primary cancers. In this case, we describe immunohistochemical markers for urothelial carcinoma and prostate adenocarcinoma and their clinical implications. One month postoperatively, our patient began adjuvant leuprolide therapy and cycle 1 of gemcitabine and cisplatin chemotherapy, which he is tolerating well.


Picrotoxin- and bicuculline-sensitive inhibition of cardiac vagal reflexes.
Transmission in the cardiac vagal reflex pathway can be inhibited by stimulation of the hypothalamic defense region or somatic afferent nerves. A pharmacological analysis of inhibitory modulation of reflex vagal bradycardia was undertaken in the present study. Picrotoxin (0.5--1.5 mg/kg i.v.) or bicuculline (0.5--1.5 mg/kg i.v.) produced a dose-related blockade of inhibition of reflex vagal bradycardia elicited by stimulation of the lateral hypothalamus or branches of the brachial plexus in spinal (C1 or C8 transected) cats. In contrast, strychnine and pentylenetetrazol failed to change the heart rate responses produced by stimulation of the hypothalamus or brachial plexus afferents. Picrotoxin and bicuculline also blocked inhibition of reflex vagal bradycardia produced by stimulation of the inferior olive in decerebrate spinal cats. This observation supports the contention that these agents act in the brain stem to block inhibitory modulation of reflex vagal bradycardia. In addition, picrotoxin and bicuculline lowered basal heart rate in spinal cats but not in decerebrate spinal cats. This observation suggests that tonic suprabulbar inhibition of reflex vagal bradycardia also is sensitive to blockade by picrotoxin and bicuculline.


Predicting Imminent Progression to Clinically Significant Memory Decline Using Volumetric MRI and FDG PET.
Brain imaging measurements can provide evidence of possible preclinical Alzheimer's disease (AD). Their ability to predict individual imminent clinical conversion remains unclear. To investigate the ability of pre-specified volumetric magnetic resonance imaging (MRI) and fluorodeoxyglucose positron emission tomography (FDG-PET) measurements to predict which cognitively unimpaired older participants would subsequently progress to amnestic mild cognitive impairment (aMCI) within 2 years. From an apolipoprotein E4 (APOE4) enriched prospective cohort study, 18 participants subsequently progressed to the clinical diagnosis of aMCI or probable AD dementia within 1.8±0.8 years (progressors); 20 participants matched for sex, age, education, and APOE allele dose remained cognitively unimpaired for at least 4 years (nonprogressors). A complementary control group not matched for APOE allele dose included 35 nonprogressors. Groups were compared on baseline FDG-PET and MRI measures known to be preferentially affected in the preclinical and clinical stages of AD and by voxel-wise differences in regional gray matter volume and glucose metabolism. Receiver Operating Characteristic, binary logistic regression, and leave-one-out procedures were used to predict clinical outcome for the a priori measures. Compared to non-progressors and regardless of APOE-matching, progressors had significantly reduced baseline MRI and PET measurements in brain regions preferentially affected by AD and reduced hippocampal volume was the strongest predictor of an individual's imminent progression to clinically significant memory decline (79% sensitivity/78% specificity among APOE-matched cohorts). Regional MRI and FDG-PET measurements may be useful in predicting imminent progression to clinically significant memory decline.


Partial Loss of USP9X Function Leads to a Male Neurodevelopmental and Behavioral Disorder Converging on Transforming Growth Factor β Signaling.
The X-chromosome gene USP9X encodes a deubiquitylating enzyme that has been associated with neurodevelopmental disorders primarily in female subjects. USP9X escapes X inactivation, and in female subjects de novo heterozygous copy number loss or truncating mutations cause haploinsufficiency culminating in a recognizable syndrome with intellectual disability and signature brain and congenital abnormalities. In contrast, the involvement of USP9X in male neurodevelopmental disorders remains tentative. We used clinically recommended guidelines to collect and interrogate the pathogenicity of 44 USP9X variants associated with neurodevelopmental disorders in males. Functional studies in patient-derived cell lines and mice were used to determine mechanisms of pathology. Twelve missense variants showed strong evidence of pathogenicity. We define a characteristic phenotype of the central nervous system (white matter disturbances, thin corpus callosum, and widened ventricles); global delay with significant alteration of speech, language, and behavior; hypotonia; joint hypermobility; visual system defects; and other common congenital and dysmorphic features. Comparison of in silico and phenotypical features align additional variants of unknown significance with likely pathogenicity. In support of partial loss-of-function mechanisms, using patient-derived cell lines, we show loss of only specific USP9X substrates that regulate neurodevelopmental signaling pathways and a united defect in transforming growth factor β signaling. In addition, we find correlates of the male phenotype in Usp9x brain-specific knockout mice, and further resolve loss of hippocampal-dependent learning and memory. Our data demonstrate the involvement of USP9X variants in a distinctive neurodevelopmental and behavioral syndrome in male subjects and identify plausible mechanisms of pathogenesis centered on disrupted transforming growth factor β signaling and hippocampal function.


Apigenin Attenuates Experimental Autoimmune Myocarditis by Modulating Th1/Th2 Cytokine Balance in Mice.
This study aims to investigate the protective effect of apigenin on the development of experimental autoimmune myocarditis (EAM) and the underlying mechanisms. An EAM model was induced in BALB/c mice by the injection of porcine cardiac myosin. Apigenin was orally administered from day 1 to 21. The severity of myocarditis was assessed by determination of heart weight/body weight ratio (HW/BW) and histopathological evaluation. Echocardiography was conducted to evaluate the cardiac function and heart structure. Antigen-specific T cell proliferation responses to cardiac myosin were evaluated by the lymphocyte proliferation assay. ELISA was used to determine serum levels of type 1 helper (Th1) and Th2 cytokines. Apigenin treatment significantly decreased HW/BW. Histopathologic analysis showed that the infiltration of inflammatory cells was reduced significantly by apigenin treatment. Meanwhile, apigenin administration effectively ameliorated autoimmune myocarditis-induced cardiac hypertrophy and cardiac dysfunction as well as inhibited lymphocyte proliferation in mice immunized with myosin. Furthermore, Th1 cytokines tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), and interleukin-2 (IL-2) were significantly downregulated, while Th2 cytokines IL-4 and IL-10 were markedly upregulated. The results indicated that apigenin can alleviate EAM due to its immunomodulatory reactions in modification of helper T cell balance.


Lipolysis of different oils using crude enzyme isolate from the intestinal tract of rainbow trout, Oncorhynchus mykiss.
Crude enzyme isolate was prepared from the intestine of rainbow trout. Positional specificity of the crude enzyme isolate was determined from both 1(3)- and 2-MAG products after in vitro lipolysis of radioactive-labeled triolein. The ratio of 2-MAG/1(3)-MAG was 2:1, suggesting that the overall lipase specificity of the enzyme isolate from rainbow trout tended to be 1,3-specific; however, activity against the sn-2 position also was shown. In vitro lipolysis of four different unlabeled oils was performed with the crude enzyme isolate. The oils were: structured lipid [SL; containing the medium-chain FA (MCFA) 8:0 in the sn-1,3 positions and long-chain FA (LCFA) in the sn-2 position], DAG oil (mainly 1,3-DAG), fish oil (FO), and triolein (TO). MCFA were rapidly hydrolyzed from the SL oil. LCFA including n-3 PUFA were, however, preserved in the sn-2 position and therefore found in higher amounts in 2-MAG of SL compared with 2-MAG of FO, DAG, and TO. Lipolysis of the DAG oil produced higher amounts of MAG than the TAG oils, and 1(3)-MAG mainly was observed after lipolysis of the DAG oil. The positional specificity determined and the results from the hydrolysis of the different oils suggest that n-3 very long-chain PUFA from structured oils may be used better by aquacultured fish than that from fish oils.


Coverage effects on the palladium-catalyzed synthesis of vinyl acetate: comparison between theory and experiment.
The high adsorbate coverages that form on the surfaces of many heterogeneous catalysts under steady-state conditions can significantly lower the activation energies for reactions that involve the coupling of two adsorbed intermediates while increasing those which result in adsorbate bond-breaking reactions. The influence of the surface coverage on the kinetics of metal-catalyzed reactions is often ignored in theoretical and even in some ultrahigh vacuum experimental studies. Herein, first principle density functional theoretical calculations are combined with experimental surface titration studies carried out over well-defined Pd(111) surfaces to explicitly examine the influence of coverage on the acetoxylation of ethylene to form vinyl acetate over Pd. The activation energies calculated for elementary steps in the Samanos and Moiseev pathways for vinyl acetate synthesis carried out on acetate-saturated palladium surfaces reveal that the reaction proceeds via the Samanos mechanism which is consistent with experimental results carried out on acetate-saturated Pd(111) surfaces. The rate-limiting step involves a beta-hydride elimination from the adsorbed acetoxyethyl intermediate, which proceeds with an apparent calculated activation barrier of 53 kJ/mol which is in very good agreement with the experimental barrier of 55 +/- 4 kJ/mol determined from kinetic measurements.


Analysis of proteins synthesized by Salmonella typhimurium during growth within a host macrophage.
Salmonella typhimurium is a facultative intracellular pathogen, able both to invade and to survive within eukaryotic cells and to grow in various extracellular environments. To compare the bacterial responses to these disparate environments and to shed light on the nature of the intracellular environment, we have examined the pattern of protein synthesis by two-dimensional polyacrylamide gel electrophoresis. The levels of approximately 40 proteins were observed to increase during growth within macrophage-like U937 cells, while approximately 100 proteins exhibited levels that were repressed relative to those of an extracellular control culture. To aid in the interpretation of these results, the patterns of proteins made by S. typhimurium exposed to various environmental conditions in the laboratory were determined. The intracellular protein pattern was then compared with each of these benchmark protein patterns. This analysis revealed that, as expected, the intracellular environment appears to impose numerous stresses on the bacteria, but unexpectedly, the macrophage-induced response was not a simple sum of the individual stress responses displayed during extracellular growth.


Influence of light-curing protocols on polymerization shrinkage and shrinkage force of a dual-cured core build-up resin composite.
This study investigated the influence of time delay and duration of photo-activation on linear polymerization shrinkage, shrinkage force, and hardening of a dual-cured core build-up resin composite. The test material (Rebilda DC) was light-cured for 20 or 60 s either early (2 min) or late (7 min) after the start of mixing. Non-irradiated self-cured specimens served as controls. Linear shrinkage and shrinkage force were measured for 60 min using custom-made devices. Knoop hardness was determined at the end of the observation period. Self-cured controls, showing a linear shrinkage similar to that of specimens early light-cured for 20 s generated the lowest shrinkage force and hardness. A shorter light exposure time (20 s vs. 60 s) reduced linear shrinkage, shrinkage force, and hardness when early light-curing was performed, but did not affect the three properties in specimens light-cured late after the start of mixing. Late photo-activation increased linear shrinkage, irrespective of irradiation time, and resulted in a higher shrinkage force and hardness for short light exposure time. A moderate correlation was found between the two shrinkage properties studied (r(2) = 0.65). In conclusion, improvements in shrinkage behavior of the tested core build-up material were associated with inferior hardening, making it important to adapt curing protocols to the clinical situation.


Thirty-Day and 5-Year Readmissions following First Psychiatric Hospitalization: A System-Level Study of Ontario's Psychiatric Care.
Analyses of representative, system-level data to examine trends in short- and longer-term readmission rates for psychiatric illnesses are largely absent. The objective of this article is to examine key trends and variables with implications for inpatient care as indicated by 30-day readmission and outpatient care as reflected by readmission within 5 years. Using OMHRS data from 2005 to 2015, patients who had their first inpatient admission were followed for 5 years to examine their subsequent 30-day and overall admission rates stratified by discharge time and diagnosis. The study cohort consisted of 42,280 patients. The 30-day and 5-year readmission rates for the entire cohort were 7.2% and 35.1%, respectively. Using a time course analysis of readmission for discharges in different years, both 30-day readmission and 5-year readmission rates decreased in a linear manner from 2005 to 2010, primarily because of readmission patterns for patients diagnosed with mood disorders and schizophrenia/other psychotic disorders. It was also evident that both demographic considerations such as age and gender and variables reflective of social determinants such as education level and employment were predictive of rehospitalization risk. The trends of decreasing readmission rates may be reflective of improvements in the quality of hospital and community-based outpatient care. Such system-level indicators warrant tracking and may inform more effective tertiary prevention.


A model for predicting depression in victims of rape.
This article proposes a model for understanding the factors contributing to long-standing depression in women who have been raped. A path analysis of data obtained from 71 women who had been raped revealed that women with generalized beliefs that they could not control events in their lives were more likely to attribute responsibility for their rape to permanent intrapsychic factors and were more likely to be depressed. Women who perceived that they had higher levels of internal control tended to have higher levels of education, were more likely to be employed, and were less likely to be depressed more than one year after having been raped. Childhood sexual abuse was not associated with internal control or attributions of causality or depression in this analysis. Implications for the determination of prognosis and treatment recommendations in civil litigation assessments are discussed.


Ondansetron does not modify emergence agitation in children.
In this prospective, placebo-controlled study, we evaluated the effect of prophylactic ondansetron therapy on emergence agitation of children who underwent minor surgery below the umbilicus. Seventy children aged one to six years and American Society of Anesthesiologists physical status I were studied. Children were premedicated with midazolam rectally and were randomly assigned to receive either ondansetron (Group O) or placebo (Group P) in combination with caudal anaesthesia. Children in Group O received intravenous ondansetron (0.1 mg/kg for children weighing < 40 kg, 4 mg for children weighing > 40 kg) and Group P (n = 35) received normal saline 2 ml following anaesthesia induction with sevoflurane. Airway management was provided with LMA-Proseal without muscle relaxation and anaesthesia maintenance was provided with a 60:40 N2O:O2 mixture and sevoflurane. Emergence agitation was evaluated with a ten point scale and pain level was assessed every 10 minutes for the first 30 minutes after admission to the recovery room. There were no significant differences between the placebo and ondansetron groups with respect to demographic, anaesthetic and surgical details. Incidences of emergence agitation in ondansetron and placebo groups were similar (32.4% and 30.3% at 10 minutes respectively). Mean modified Children's Hospital of Eastern Ontario pain scale scores and mean ten-point scale scores and emergence agitation incidences decreased similarly after 10 minutes in both groups. Ready time for discharge was similar between the groups. Agitated patients had significantly increased ready time for discharge compared to non-agitated patients (P = 0.001). Prophylactic intravenous ondansetron administration does not reduce emergence agitation comparing to placebo after sevoflurane anaesthesia.


Patients' use of GPs and community pharmacists in minor illness: a cross-sectional questionnaire-based study.
Despite pharmacists having increased involvement in managing minor illness, many patients continue to attend their GP with problems that could be managed by community pharmacists. Our aim was to investigate the prevalence of visits to the GP that GPs felt could be managed by a pharmacist, and to explore patients' reasons for such visits. This cross-sectional questionnaire study was conducted at 13 general practices in West Sussex, UK. A questionnaire was given to all patients attending appointments with their GP in these practices over a 1-week period, asking what the presenting problem was and whether the advice of a pharmacist had been sought. If patients had not sought the advice of a pharmacist, they were asked why not. The GP was then asked to indicate whether, in their opinion, the patient's problem could have been managed by a community pharmacist. The response rate was 94% (3984), representing 87% of all patients consulting their doctor during the week of the study. GPs felt that only 7% (260) of these visits could have been managed by a community pharmacist. The proportion of 'unnecessary' visits was significantly higher (P < 0.001) amongst young adults, those presenting with new medical problems and those consulting about a child's health. Skin and musculoskeletal problems were the most common causes of 'unnecessary' visits to the GP. The majority of patients making 'unnecessary' visits (59%) disagreed with the GP and felt that the pharmacist would not have been appropriate for their problem. GPs and patients were, on the whole, in agreement over which conditions were appropriate for GP attention. There is, however, a need for education to increase awareness of the roles of pharmacists, aimed particularly at young adults and at those with children.


Understanding the Elevated Prevalence of Intimate Partner Violence in Interracial Relationships.
Research from the United States and Canada suggest that interracial relationships tend to have an elevated prevalence of intimate partner violence (IPV). Among seven extant studies, only one empirically examined speculations in the literature for this relationship. Based on analyses of data from Statistics Canada's 2009 General Social Survey (GSS), Brownridge was not able to fully account for the elevated odds of IPV in interracial relationships. The current study used data on 16,706 Canadians (731 in interracial relationships and 15,975 in noninterracial relationships) from the 2014 iteration of Statistics Canada's GSS to determine whether the risk of IPV in interracial relationships had changed since 2009 and to explore risk factors that may account for the elevated odds of IPV in interracial relationships. Results showed that individuals in interracial relationships faced elevated odds of IPV victimization relative to monoracial relationships in the 5-year reporting period prior to the study (odds ratio [OR] = 2.37; 95% confidence interval [CI] = [1.40, 4.02]; p < .001). This was similar to what Brownridge found in the 2009 GSS data, indicating that the elevated risk of IPV in interracial relationships had remained stable in Canada over a 10-year reporting period. Risk factors from three levels of an ecological model were explored, and logistic multiple regression analyses showed that characteristics of the partner (young age, drug use, and jealous behavior) fully accounted for the significantly elevated odds of IPV victimization in interracial unions. Although the leading speculation for this relationship implicates stressors that are unique to these relationships, the current study suggested that this phenomenon is mostly due to characteristics of individuals with whom those in interracial relationships are more likely to be coupled.


In situ push-pull method to determine ground water denitrification in riparian zones.
To quantify ground water denitrification in discrete locations of riparian aquifers, we modified and evaluated an in situ method based on conservative tracers and 15N-enriched nitrate. Ground water was "pushed" (i.e., injected) into a mini-piezometer and then "pulled" (i.e., extracted) from the same mini-piezometer after an incubation period. This push-pull method was applied in replicate mini-piezometers at two Rhode Island riparian sites, one fresh water and one brackish water. Conservative tracer pretests were conducted to determine incubation periods, ranging from 5 to 120 h, to optimize recovery of introduced plumes. For nitrate push-pull tests, we used two conservative tracers, sulfur hexafluoride and bromide, to provide insight into plume recovery. The two conservative tracers behaved similarly. The dosing solutions were amended with 15N-enriched nitrate that enabled us to quantify the mass of denitrification gases generated during the incubation period. The in situ push-pull method detected substantial denitrification rates at a site where we had previously observed high denitrification rates. At our brackish site, we found high rates of ground water denitrification in marsh locations and minimal denitrification in soils fringing the marsh. The push-pull method can provide useful insights into spatial and temporal patterns of denitrification in riparian zones. The method is robust and results are not seriously affected by dilution or degassing from ground water to soil air. In conjunction with measurements of ground water flow-paths, this method holds promise for evaluating the influence of site and management factors on the ground water nitrate removal capacity of riparian zones.


The CONSTANCES Cohort Biobank: An Open Tool for Research in Epidemiology and Prevention of Diseases.
"General-purpose cohorts" in epidemiology and public health are designed to cover a broad scope of determinants and outcomes, in order to answer several research questions, including those not defined at study inception. In this context, the general objective of the CONSTANCES project is to set up a large population-based cohort that will contribute to the development of epidemiological research by hosting ancillary projects on a wide range of scientific domains, and to provide public health information. CONSTANCES was designed as a randomly selected sample of French adults aged 18-69 years at study inception; 202,045 subjects were included over an 8-year period. At inclusion, the selected participants are invited to attend one of the 24 participating Health Prevention Centers (HPCs) for a comprehensive health examination. The follow-up includes a yearly self-administered questionnaire, and a periodic visit to an HPC. Procedures have been developed to use the national healthcare databases to allow identification and validation of diseases over the follow-up. The biological collection (serum, lithium heparinized plasma, EDTA plasma, urine and buffy coat) began gradually in June 2018. At the end of the inclusions, specimens from 83,000 donors will have been collected. Specimens are collected according to a standardized protocol, identical in all recruitment centers. All operations relating to bio-banking have been entrusted by Inserm to the Integrated Biobank of Luxembourg (IBBL). A quality management system has been put in place. Particular attention has been paid to the traceability of all operations. The nature of the biological samples stored has been deliberately limited due to the economic and organizational constraints of the inclusion centers. Some research works may require specific collection conditions, and can be developed on request for a limited number of subjects and in specially trained centers. The biological specimens that are collected will allow for a large spectrum of biomarkers studies and genetic and epigenetic markers through candidate or agnostic approaches. By linking the extensive data on personal, lifestyle, environmental, occupational and social factors with the biomarker data, the CONSTANCES cohort offers the opportunity to study the interplays between these factors using an integrative approach and state-of-the-art methods.


Comparing 3T and 1.5T MRI for mapping hippocampal atrophy in the Alzheimer's Disease Neuroimaging Initiative.
Prior MR imaging studies, primarily at 1.5T, established hippocampal atrophy as a biomarker for Alzheimer disease. 3T MR imaging offers a higher contrast and signal-to-noise ratio, yet distortions and intensity uniformity are harder to control. We applied our automated hippocampal segmentation technique to 1.5T and 3T MR imaging data, to determine whether hippocampal atrophy detection was enhanced at 3T. We analyzed baseline MR imaging data from 166 subjects from the Alzheimer's Disease Neuroimaging Initiative-1 (37 with Alzheimer disease, 76 with mild cognitive impairment, and 53 healthy controls) scanned at 1.5T and 3T. Using multiple linear regression, we analyzed the effect of clinical diagnosis on hippocampal radial distance, while adjusting for sex. 3D statistical maps were adjusted for multiple comparisons by using permutation-based statistics at a threshold of P < .01. Bilaterally significant radial distance differences in the areas corresponding to the cornu ammonis 1, cornu ammonis 2, and subiculum were detected for Alzheimer disease versus healthy controls and mild cognitive impairment versus healthy controls at 1.5T and more profoundly at 3T. Comparison of Alzheimer disease with mild cognitive impairment did not reveal significant differences at either field strength. Subjects who converted from mild cognitive impairment to Alzheimer disease within 3 years of the baseline scan versus nonconverters showed significant differences in the area corresponding to cornu ammonis 1 of the right hippocampus at 3T but not at 1.5T. While hippocampal atrophy patterns in diagnostic comparisons were similar at 1.5T and 3T, 3T showed a superior signal-to-noise ratio and detected atrophy with greater effect size compared with 1.5T.


Assessment of spiritual suffering in the cancer context: A systematic literature review.
An important goal of cancer medicine is relief of patients' suffering. In view of the clinical challenges of identifying suffering patients, we sought to identify valid instruments for assessing the spiritual suffering of people diagnosed with cancer. A systematic review of the literature was conducted in the Medline, Embase, the Cochrane Library, and PsycINFO databases seeking assessment instruments that measure either suffering or one of its synonyms or symptoms. The psychometric properties of the identified measures were compared. A total of 90 articles were identified that supplied information about 58 measures. The constructs examined were: suffering, hopelessness/demoralization, hope, meaning, spiritual well-being, quality of life where a spiritual/existential dimension was included, distress in the palliative care setting and pain, distress or struggle of a spiritual nature. The Pictorial Representation of Illness and Self Measure (PRISM) (patient completed) was the most promising measure identified for measuring the burden of suffering caused by illness due to its ease of use and the inclusion of a subjective component. Although the appropriateness of any measure for the assessment of spiritual suffering in cancer patients will depend on the context in which it is intended to be utilized, the PRISM is promising for measuring the burden of suffering due to illness.


Methods to assess the contribution of diseases to disability using cross-sectional studies: comparison of different versions of the attributable fraction and the attribution method.
This study aims to illustrate the differences between approaches proposed for apportioning disability to different diseases in a multicausal situation, i.e. the unadjusted attributable fraction (AF), the adjusted AF, the average AF and the attribution method (AM). This information is useful to better interpret results obtained from cross-sectional data and help policy makers decide on public health strategies. Data for 29 931 individuals, representative of the French household population, who participated in the 2008-09 cross-sectional Disability-Health Survey, were included. Disability was defined as any limitation reported with the Global Activity Limitation Indicator. Unadjusted AFs were calculated using Levin's formula. Adjusted AFs were estimated for each disease by calculating predicted probabilities of disability for each individual in the dataset, under the assumption that the individual is unexposed to this specific disease (logistic model). Average AFs are based on the same methodology, but have the additional advantage that the average AFs for different diseases sum to the total AF associated with eliminating all diseases. AM accounts for competing risks and partitions total disability prevalence into additive contributions of different diseases and background disability (additive model). All methods obtained similar results with respect to the estimates of the disease contribution to disability prevalences and to ranking of the diseases, except unadjusted AFs, as the method ignores multimorbidity. Confounders other than diseases, such as age and gender, should be accurately taken into account. Conceptual differences, strengths and limitations of the different approaches were discussed.


Data mining for proteins characteristic of clades.
A synapomorphy is a phylogenetic character that provides evidence of shared descent. Ideally a synapomorphy is ubiquitous within the clade of related organisms and nonexistent outside the clade, implying that it arose after divergence from other extant species and before the last common ancestor of the clade. With the recent proliferation of genetic sequence data, molecular synapomorphies have assumed great importance, yet there is no convenient means to search for them over entire genomes. We have developed a new program called Conserv, which can rapidly assemble orthologous sequences and rank them by various metrics, such as degree of conservation or divergence from out-group orthologs. We have used Conserv to conduct a largescale search for molecular synapomorphies for bacterial clades. The search discovered sequences unique to clades, such as Actinobacteria, Firmicutes and gamma-Proteobacteria, and shed light on several open questions, such as whether Symbiobacterium thermophilum belongs with Actinobacteria or Firmicutes. We conclude that Conserv can quickly marshall evidence relevant to evolutionary questions that would be much harder to assemble with other tools.


Tissue-Factor Targeted Peptide Amphiphile Nanofibers as an Injectable Therapy To Control Hemorrhage.
Noncompressible torso hemorrhage is a leading cause of mortality in civilian and battlefield trauma. We sought to develop an i.v.-injectable, tissue factor (TF)-targeted nanotherapy to stop hemorrhage. Tissue factor was chosen as a target because it is only exposed to the intravascular space upon vessel disruption. Peptide amphiphile (PA) monomers that self-assemble into nanofibers were chosen as the delivery vehicle. Three TF-binding sequences were identified (EGR, RLM, and RTL), covalently incorporated into the PA backbone, and shown to self-assemble into nanofibers by cryo-transmission electron microscopy. Both the RLM and RTL peptides bound recombinant TF in vitro. All three TF-targeted nanofibers bound to the site of punch biopsy-induced liver hemorrhage in vivo, but only RTL nanofibers reduced blood loss versus sham (53% reduction, p < 0.05). Increasing the targeting ligand density of RTL nanofibers yielded qualitatively better binding to the site of injury and greater reductions in blood loss in vivo (p < 0.05). In fact, 100% RTL nanofiber reduced overall blood loss by 60% versus sham (p < 0.05). Evaluation of the biocompatibility of the RTL nanofiber revealed that it did not induce RBC hemolysis, did not induce neutrophil or macrophage inflammation at the site of liver injury, and 70% remained intact in plasma after 30 min. In summary, these studies demonstrate successful binding of peptides to TF in vitro and successful homing of a TF-targeted PA nanofiber to the site of hemorrhage with an associated decrease in blood loss in vivo. Thus, this therapeutic may potentially treat noncompressible hemorrhage.


A method for evaluating image quality of monochrome and color displays based on luminance by use of a commercially available color digital camera.
The aim of this study is to propose a method for the quantitative evaluation of image quality of both monochrome and color liquid-crystal displays (LCDs) using a commercially available color digital camera. The intensities of the unprocessed red (R), green (G), and blue (B) signals of a camera vary depending on the spectral sensitivity of the image sensor used in the camera. For consistent evaluation of image quality for both monochrome and color LCDs, the unprocessed RGB signals of the camera were converted into gray scale signals that corresponded to the luminance of the LCD. Gray scale signals for the monochrome LCD were evaluated by using only the green channel signals of the camera. For the color LCD, the RGB signals of the camera were converted into gray scale signals by employing weighting factors (WFs) for each RGB channel. A line image displayed on the color LCD was simulated on the monochrome LCD by using a software application for subpixel driving in order to verify the WF-based conversion method. Furthermore, the results obtained by different types of commercially available color cameras and a photometric camera were compared to examine the consistency of the authors' method. Finally, image quality for both the monochrome and color LCDs was assessed by measuring modulation transfer functions (MTFs) and Wiener spectra (WS). The authors' results demonstrated that the proposed method for calibrating the spectral sensitivity of the camera resulted in a consistent and reliable evaluation of the luminance of monochrome and color LCDs. The MTFs and WS showed different characteristics for the two LCD types owing to difference in the subpixel structure. The MTF in the vertical direction of the color LCD was superior to that of the monochrome LCD, although the WS in the vertical direction of the color LCD was inferior to that of the monochrome LCD as a result of luminance fluctuations in RGB subpixels. The authors' method based on the use of a commercially available color camera is useful to evaluate and understand the display performances of both monochrome and color LCDs in radiology departments.


Reliability of a two-dimensional footprint measurement approach.
Although footprint evidence can be taken from the scene of a crime, the science underpinning such measurement in forensic science has not been fully explored. A literature search revealed various measuring approaches, all of which demonstrated either little or no measurement rigour in terms of reliability. The aim of this study was to apply a robust measurement approach for testing the reliability of two-dimensional footprint impressions. Three dynamic and three static footprints were taken from the right foot of thirty female and thirty one male volunteers using the 'Inkless Shoeprint Kit'. The images were digitised. Lengths, widths and angles were measured using a selection of currently employed methods. An investigation of the reliability of the chosen measuring method suggested high intra-rater agreement: for example, the length measurement suggested an intraclass correlation coefficient (ICC) 0.99, 95% Confidence Interval (CI) -0.28 to 0.01, standard error of measurement (SEM) 0.07, Limits of Agreement (LOA) -0.91 to 0.65. Inter-rater reliability between three operators was also high: SEM ranged from 0.05 mm to 0.07 mm, ICC 0.99. Our study has established a reliable two-dimensional measuring technique that could be used for footprint comparison in further research.


Extraction and inhibition of enzymatic activity of botulinum neurotoxins /B1, /B2, /B3, /B4, and /B5 by a panel of monoclonal anti-BoNT/B antibodies.
Botulism is caused by botulinum neurotoxins (BoNTs), extremely toxic proteins which can induce respiratory failure leading to long-term intensive care or death. Treatment for botulism includes administration of antitoxins, which must be administered early in the course of the intoxication; therefore, rapid determination of human exposure to BoNT is an important public health goal. In previous work, our laboratory reported on Endopep-MS, a mass spectrometry-based activity method for detecting and differentiating BoNT/A, /B, /E, and /F in clinical samples. We also demonstrated that antibody-capture is effective for purification and concentration of BoNTs from complex matrices such as clinical samples. However, some antibodies inhibit or neutralize the enzymatic activity of BoNT, so the choice of antibody for toxin extraction is critical. In this work, we evaluated 24 anti-BoNT/B monoclonal antibodies (mAbs) for their ability to inhibit the in vitro activity of BoNT/B1, /B2, /B3, /B4, and /B5 and to extract those toxins. Among the mAbs, there were significant differences in ability to extract BoNT/B subtypes and inhibitory effect on BoNT catalytic activity. Some of the mAbs tested enhanced the in vitro light chain activity of BoNT/B, suggesting that BoNT/B may undergo conformational change upon binding some mAbs. In addition to determining in vitro inhibition abilities of a panel of mAbs against BoNT/B1-/B5, this work has determined B12.2 and 2B18.2 to be the best mAbs for sample preparation before Endopep-MS. These mAb characterizations also have the potential to assist with mechanistic studies of BoNT/B protection and treatment, which is important for studying alternative therapeutics for botulism.


Thromboinflammatory mechanisms in sickle cell disease - challenging the hemostatic balance.
Sickle cell disease (SCD) is an inherited hemoglobinopathy that is caused by the presence of abnormal hemoglobin S (HbS) in red blood cells, leading to alterations in red cell properties and shape, as the result of HbS dexoygenation and subsequent polymerization. SCD pathophysiology is characterized by chronic inflammatory processes, triggered by hemolytic and vaso-occlusive events, which lead to the varied complications, organ damage and elevated mortality seen in individuals with the disease. In association with activation of the endothelium and leukocytes, hemostatic alterations and thrombotic events are well-documented in SCD. Here we discuss the role for inflammatory pathways in modulating coagulation and inducing platelet activation in SCD, due to tissue factor activation, adhesion molecule expression, inflammatory mediator production and the induction of innate immune responses, amongst other mechanisms. Thromboinflammatory pathways may play a significant role in some of the major complications of SCD, such as stroke, venous thromboembolism and possibly acute chest syndrome, besides exacerbating the chronic inflammation and cellular interactions that trigger vaso-occlusion, ischemia-reperfusion processes, and eventually organ damage.


Trajectories of psychiatric disorders in a cohort of children with cerebral palsy across four years.
Risk of psychiatric disorders has been reported for children and adolescents with cerebral palsy (CP) at different ages, however little is known regarding the long-term trajectories of these disorders. The aim of this study was to assess the trajectories of psychiatric disorders in children with CP, and to explore their association to risk factors. We assessed a cohort of children with CP at age seven and again at age eleven with a child psychiatric diagnostic instrument, and parents were informants. We assessed type of CP, Gross Motor Function Classification System (GMFCS) levels, and co-occurring medical conditions in a medical examination, through the medical records, and in an interview with the parents at the onset of the study. We found a significant increase in the prevalence of emotional disorders from seven to eleven years of age (p 0.01), whereas the prevalence of behavioral disorders was stable. Half of the cohort met criteria for a psychiatric disorder at both assessment points. Type of CP, spastic bilateral or unilateral, dyskinetic or ataxic, and co-occurring medical conditions were non-significant predictors of psychiatric disorders. Subthreshold psychiatric disorders at age seven were predictive of psychiatric disorders at age eleven. We found a persistently elevated prevalence of psychiatric disorders in children with CP. Prevalence of behavioral disorders was stable, whereas we found a significant four-fold increase in emotional disorders. Sub-threshold psychiatric disorders predicted later psychiatric disorders. Increased focus on early mental health symptoms as well as more knowledge regarding emotional disorders in children with CP seems warranted.


Two-Stage Isothermal Enzymatic Amplification for Concurrent Multiplex Molecular Detection.
The wide array of pathogens responsible for infectious diseases makes it difficult to identify causative pathogens with single-plex tests. Although multiplex PCR detects multiple targets, it is restricted to centralized laboratories, which delays test results or makes multiplexing unavailable, depriving healthcare providers of critical, real-time information. To address the need for point-of-care (POC) highly multiplexed tests, we propose the 2-stage, nested-like, rapid (<40 min) isothermal amplification assay, dubbed rapid amplification (RAMP). RAMP's first-stage uses outer loop-mediated isothermal amplification (LAMP) primers to amplify all targets with recombinase polymerase amplification (RPA). First-stage amplicons are aliquoted to second stage reactors, each specialized for a specific target, to undergo LAMP. The assay is implemented in a microfluidic chip. LAMP amplicons are detected in situ with colorimetric dye or with a fluorescent dye and a smartphone. In experiments on a benchtop and in a microfluidic format, RAMP demonstrated high level of multiplexing (≥16); high sensitivity (i.e., 1 plaque-forming unit of Zika virus) and specificity (no false positives or negatives); speed (<40 min); ease of use; and ability to cope with minimally processed samples. RAMP is a hybrid, 2-stage, rapid, and highly sensitive and specific assay with extensive multiplexing capabilities, combining the advantages of RPA and LAMP, while circumventing their respective shortcomings. RAMP can be used in the lab, but one of its distinct advantages is amenability to simple implementation in a microfluidic format for use at the POC, providing healthcare personnel with an inexpensive, highly sensitive tool to detect multiple pathogens in a single sample, on site.


Human cystic echinococcosis in the West Bank of Palestine: surgical incidence and seroepidemiological study.
The surgical incidence of cystic echinococcosis (CE) due to Echinococcus granulosus was investigated in hospitals of the West Bank, Palestinian Authority between January 1990 and December 1997. Serum samples from school-children in Yata town, which showed the highest surgical incidence, were tested for anti-hydatid antibodies. A total of 390 surgically confirmed cases were recorded throughout the 8-year period, with an overall mean annual surgical incidence (MASI) of 3.1 per 100,000. A high MASI of 4.9, 5.0 and 5.1 per 100,000 was found in Hebron, Jericho and Bethlehem Governorates, respectively. Yata town, Hebron governorate, showed the highest MASI, at 16.8 per 100,000. The highest incidence was found in age groups 11-20 and 21-30 years, at 27.4% and 21.5% of the total number of cases. While there was no significant gender difference in the number of cases in the age groups of 20 years or less, the male to female case ratio was 1:3.2-4.1 in the older age groups. The liver was the most common site of hydatid cysts in 69.9% of cases. Lung cysts were predominant in younger age groups (20 years or less). The seropositivity for CE in the school-children of Yata was 2.4% and 2.1% using enzyme-linked immunosorbent assay and the indirect haemagglutination test, respectively. CE is a significant endemic disease throughout the West Bank. The disease is acquired early in life and is more prevalent among females than males. Behaviour and life-style favour the spread of the disease.


Cell assay using a two-photon-excited europium chelate.
We report application of two-photon excitation of europium chelates to immunolabeling of epidermal growth factor receptor (EGFR) cell surface proteins on A431 cancer cells. The europium chelates are excited with two photons of infrared light and emit in the visible. Europium chelates are conjugated to antibodies for EGFR. A431 (human epidermoid carcinoma) cells are labeled with this conjugate and imaged using a multiphoton microscope. To minimize signal loss due to the relatively long-lived Eu(3+) emission, the multiphoton microscope is used with scanning laser two-photon excitation and non-scanning detection with a CCD. The chelate labels show very little photobleaching (less than 1% during continuous illumination in the microscope for 20 minutes) and low levels of autofluorescence (less than 1% of the signal from labeled cells). The detection limit of the europium label in the cell assay is better than 100 zeptomoles.


Aberrantly elevated microRNA-34a in obesity attenuates hepatic responses to FGF19 by targeting a membrane coreceptor β-Klotho.
MicroRNA-34a (miR-34a) is the most highly elevated hepatic miR in obese mice and is also substantially elevated in patients who have steatosis, but its role in obesity and metabolic dysfunction remains unclear. After a meal, FGF19 is secreted from the ileum; binds to a hepatic membrane receptor complex, FGF19 receptor 4 and coreceptor β-Klotho (βKL); and mediates postprandial responses under physiological conditions, but hepatic responses to FGF19 signaling were shown to be impaired in patients with steatosis. Here, we show an unexpected functional link between aberrantly elevated miR-34a and impaired βKL/FGF19 signaling in obesity. In vitro studies show that miR-34a down-regulates βKL by binding to the 3' UTR of βKL mRNA. Adenoviral-mediated overexpression of miR-34a in mice decreased hepatic βKL levels, impaired FGF19-activated ERK and glycogen synthase kinase signaling, and altered expression of FGF19 metabolic target genes. Consistent with these results, βKL levels were decreased and hepatic responses to FGF19 were severely impaired in dietary obese mice that have elevated miR-34a. Remarkably, in vivo antisense inhibition of miR-34a in obese mice partially restored βKL levels and improved FGF19 target gene expression and metabolic outcomes, including decreased liver fat. Further, anti-miR-34a treatment in primary hepatocytes of obese mice restored FGF19-activated ERK and glycogen synthase kinase signaling in a βKL-dependent manner. These results indicate that aberrantly elevated miR-34a in obesity attenuates hepatic FGF19 signaling by directly targeting βKL. The miR-34a/βKL/FGF19 axis may present unique therapeutic targets for FGF19-related human diseases, including metabolic disorders and cancer.


Lipoprotein-associated phospholipase A2 (Lp-PLA2) and future risk of type 2 diabetes: results from the Cardiovascular Health Study.
Lipoprotein-associated phospholipase A2 (Lp-PLA(2)) has been consistently associated with cardiovascular disease (CVD) risk factors and predictive of CVD outcomes; furthermore, it is consistently higher among type 2 diabetics than nondiabetics. However, the relationships of circulating Lp-PLA(2) mass and activity with incident type 2 diabetes mellitus have not been examined. Therefore, the purpose of this study was to determine the association of Lp-PLA(2) mass and activity with type 2 diabetes among older adults. We conducted analyses of Lp-PLA(2) and prevalent and incident diabetes among 5474 men and women from the Cardiovascular Health Study (1989-2007). Lp-PLA(2) mass and activity were measured in baseline plasma. Diabetes status was ascertained annually with medication inventories and repeated blood glucose measurements. Generalized linear and Cox proportional hazards models were used to adjust for confounding factors including body mass index and inflammation. At baseline, the top two quintiles of Lp-PLA(2) activity were significantly associated with prevalent type 2 diabetes with a multivariable relative risk = 1.35 [95% confidence interval (CI) = 1.11-1.63] for quintile 4, and relative risk = 1.33 (95% CI = 1.07-1.66) for quintile 5. Among participants free of diabetes at baseline, we found a significant positive association with both the homeostatic model assessment for insulin resistance and β-cell function per SD increase in Lp-PLA(2) activity (P values for both <0.01). In prospective analyses, the risk of incident type 2 diabetes was significantly higher among those in the highest quintile of Lp-PLA(2) activity [multivariable hazard ratio = 1.45 (95% CI = 1.01-2.07)] compared with the lowest quintile. Lp-PLA(2) mass was not significantly associated with incident type 2 diabetes. Lp-PLA(2) activity is positively associated with insulin resistance and predicts incident type 2 diabetes among older adults independent of multiple factors associated with diabetes pathogenesis.


Global characterization of T cells in non-small-cell lung cancer by single-cell sequencing.
Cancer immunotherapies have shown sustained clinical responses in treating non-small-cell lung cancer1-3, but efficacy varies and depends in part on the amount and properties of tumor infiltrating lymphocytes4-6. To depict the baseline landscape of the composition, lineage and functional states of tumor infiltrating lymphocytes, here we performed deep single-cell RNA sequencing for 12,346 T cells from 14 treatment-naïve non-small-cell lung cancer patients. Combined expression and T cell antigen receptor based lineage tracking revealed a significant proportion of inter-tissue effector T cells with a highly migratory nature. As well as tumor-infiltrating CD8+ T cells undergoing exhaustion, we observed two clusters of cells exhibiting states preceding exhaustion, and a high ratio of "pre-exhausted" to exhausted T cells was associated with better prognosis of lung adenocarcinoma. Additionally, we observed further heterogeneity within the tumor regulatory T cells (Tregs), characterized by the bimodal distribution of TNFRSF9, an activation marker for antigen-specific Tregs. The gene signature of those activated tumor Tregs, which included IL1R2, correlated with poor prognosis in lung adenocarcinoma. Our study provides a new approach for patient stratification and will help further understand the functional states and dynamics of T cells in lung cancer.


Promotion of corneal epithelial wound healing in vitro and in vivo by annexin A5.
To investigate the effect of annexin A5, a calcium-dependent phospholipid-binding protein, on corneal epithelial wound healing. The effect of annexin A5 on migration of rabbit corneal epithelial (RCE) cells in vitro was examined in scrape-wounded cell monolayers. The effect of annexin A5 on the release of urokinase-type plasminogen activator (uPA) from cultured RCE cells was determined by zymography, fluorogenic assay of PA activity, and enzyme-linked immunosorbent assay. The proliferation of RCE cells was assessed by measurement of [3H]thymidine incorporation. The effect of annexin A5 on corneal wound closure in rabbits was investigated after removal of the corneal epithelium, either by exposure to iodine vapor or surgically. Eye drops containing annexin A5 were instilled into one eye and vehicle into the other. The area of the epithelial defect was measured at various times after wounding, and the healing rate was calculated by linear regression analysis. Annexin A5 significantly promoted the migration of RCE cells in a wounded monolayer. However, annexin A5 had no effect on RCE cell proliferation. Annexin A5 also increased the release of uPA both from wounded RCE cell monolayers and from nonwounded semiconfluent RCE cells. In both models of corneal wound closure, the healing rate was significantly increased by instillation of eye drops containing annexin A5 compared with that apparent in the eyes that received vehicle. Annexin A5 promoted corneal epithelial wound healing both in vitro and in vivo. Upregulation of uPA release from corneal epithelial cells may contribute to this effect of annexin A5.


The CtsR regulator of Listeria monocytogenes contains a variant glycine repeat region that affects piezotolerance, stress resistance, motility and virulence.
A spontaneous high hydrostatic pressure (HHP)-tolerant mutant of Listeria monocytogenes ScottA, named AK01, was isolated previously. This mutant was immotile and showed increased resistance to heat, acid and H2O2 compared with the wild type (wt) (Karatzas, K.A.G. and Bennik, M.H.J. 2002 Appl Environ Microbiol 68: 3183-3189). In this study, we conclusively linked the increased HHP and stress tolerance of strain AK01 to a single codon deletion in ctsR (class three stress gene repressor) in a region encoding a highly conserved glycine repeat. CtsR negatively regulates the expression of the clp genes, including clpP, clpE and the clpC operon (encompassing ctsR itself), which belong to the class III heat shock genes. Allelic replacement of the ctsR gene in the wt background with the mutant ctsR gene, designated ctsRDeltaGly, rendered mutants with phenotypes and protein expression profiles identical to those of strain AK01. The expression levels of CtsR, ClpC and ClpP proteins were significantly higher in ctsRDeltaGly mutants than in the wt strain, indicative of the CtsRDeltaGly protein being inactive. Further evidence that the CtsRDeltaGly protein lacks its repressor function came from the finding that the Clp proteins in the mutant were not further induced upon heat shock, and that HHP tolerance of a ctsR deletion strain was as high as that of a ctsRDeltaGly mutant. The high HHP tolerance possibly results from the increased expression of the clp genes in the absence of (active) CtsR repressor. Importantly, the strains expressing CtsRDeltaGly show significantly attenuated virulence compared with the wt strain; however, no indication of disregulation of PrfA in the mutant strains was found. Our data highlight an important regulatory role of the glycine-rich region of CtsR in stress resistance and virulence.


Effects of aging on arithmetic problem-solving: an event-related brain potential study.
Younger and older participants were asked to indicate if 240 complex two-digit addition problems were smaller than 100 or not. Half of the problems were small-split problems (i.e., the proposed sums were 2% or 5% away from 100; e.g., 53 + 49) and half were large-split problems (i.e., proposed sums were 10% or 15% away from 100; 46 + 39). Behavioral and event-related potential (ERP) data revealed that (a) both groups showed a split effect on both reaction times and percent errors, (b) split effects were smaller for older than for younger adults in ERPs, and (c) the hemispheric asymmetry (left hemisphere advantage) reported for younger adults was reduced in older adults (age-related hemispheric asymmetry reduction). These results suggest that older adults tend to use only one strategy to solve all problems, whereas younger adults flexibly and adaptively use different strategies for small and large-split problems. Implications of these findings for our understanding of age-related similarities and differences in arithmetic problem-solving are discussed.


Serum free testosterone and metabolic syndrome in Japanese men.
To examine the association between late-onset hypogonadism (LOH) and metabolic syndrome (Mets) or insulin resistance in the Japanese adult male population, we evaluated anthropometric parameters, indices of glucose and lipid metabolism, and hormones related to sexual function in 274 men (mean age: 46.0 +/- 11 years) who underwent general health checks. Seventy subjects (25.5%) were diagnosed as having Mets, while the frequency of LOH was 8.0%. Glycated hemoglobin was normal in the majority of participants (94.9%). The serum free testosterone (FT) level was significantly lower in the Mets (+) group than in the Mets (-) group (11.7 +/- 4.0 vs. 14.7 +/- 4.6 pg/mL, p<0.0001). FT decreased significantly along with an increase in the number of Mets components. Likewise, the number of Mets components showed a significant difference among the eugonadal, borderline, and hypogonadal groups (2.2 +/- 1.4, 1.5 +/- 1.4, and 0.9 +/- 1.1, respectively). After adjustment for age, body mass index (BMI), and waist circumference (WC), FT was still significantly correlated with Mets (standard partial regression coefficient = - 0.0971; 95 % confidence interval = - 0.1936 approximately - 0.0006; p = 0.048). A compensatory increase of gonadotropins was not seen in the hypogonadal group. Among Japanese men who were mainly without diabetes, FT was associated with Mets independently of age, BMI, and WC. Mets and insulin resistance may decrease serum testosterone via induction of hypogonadotrophic hypogonadism, and the reduction of testosterone may in turn cause further obesity and insulin resistance, consequently initiating a vicious cycle.


Controlled biocide release from hierarchically-structured biogenic silica: surface chemistry to tune release rate and responsiveness.
Biocides are essential for crop protection, packaging and several other biosystem applications. Therein, properties such as tailored and controlled release are paramount in the development of sustainable biocide delivery systems. We explore the self-similar nano-organized architecture of biogenic silica particles to achieve high biocide payload. The high surface area accessibility of the carrier allowed us to develop an efficient, low energy loading strategy, reaching significant dynamic loadings of up to 100 mg·g-1. The release rate and responsiveness were tuned by manipulating the interfaces, using either the native hydroxyl surfaces of the carrier or systems modified with amines or carboxylic acids in high density. We thoroughly evaluated the impact of the carrier-biocide interactions on the release rate as a function of pH, ionic strength and temperature. The amine and carboxyl functionalization strategy led to three-fold decrease in the release rate, while higher responsiveness against important agro-industrial variables. Key to our discoveries, nanostructuring thymol in the biogenic silica endowed systems with controlled, responsive release promoting remarkable, high and localized biocidal activity. The interfacial factors affecting related delivery were elucidated for an increased and localized biocidal activity, bringing a new light for the development of controlled release systems from porous materials.


Reticuloendotheliosis virus and avian leukosis virus subgroup J synergistically increase the accumulation of exosomal miRNAs.
Co-infection with avian leukosis virus subgroup J and reticuloendotheliosis virus induces synergistic pathogenic effects and increases mortality. However, the role of exosomal miRNAs in the molecular mechanism of the synergistic infection of the two viruses remains unknown. In this study, exosomal RNAs from CEF cells infected with ALV-J, REV or both at the optimal synergistic infection time were analysed by Illumina RNA deep sequencing. A total of 54 (23 upregulated and 31 downregulated) and 16 (7 upregulated and 9 downregulated) miRNAs were identified by comparing co-infection with two viruses, single-infected ALV-J and REV, respectively. Moreover, five key miRNAs, including miR-184-3p, miR-146a-3p, miR-146a-5p, miR-3538 and miR-155, were validated in both exosomes and CEF cells by qRT-PCR. GO annotation and KEGG pathway analysis of the miRNA target genes showed that the five differentially expressed miRNAs participated in virus-vector interaction, oxidative phosphorylation, energy metabolism and cell growth. We demonstrated that REV and ALV-J synergistically increased the accumulation of exosomal miRNAs, which sheds light on the synergistic molecular mechanism of ALV-J and REV.


Noninvasive assessment of wall-shear rate and vascular elasticity using combined ARFI/SWEI/spectral Doppler imaging system.
The progression of atherosclerotic disease is a complex process believed to be a function of the localized mechanical properties and hemodynamic loading associated with the arterial wall. It is hypothesized that measurements of cardiovascular stiffness and wall-shear rate (WSR) may provide important information regarding vascular remodeling, endothelial function and the growth of soft lipid-filled plaques that could help a clinician better predict the occurrence of clinical events such as stroke. Two novel ARFI based imaging techniques, combined on-axis/off-axis ARFI/Spectral Doppler Imaging (SAD-SWEI) and Gated 2D ARFI/Spectral Doppler Imaging (SAD-Gated), were developed to form co-registered depictions of B-mode echogenicity, ARFI displacements, ARF-excited transverse wave velocity estimates and estimates ofwall-shear rate throughout the cardiac cycle. Implemented on a commercial ultrasound scanner, the developed techniques were evaluated in tissue-mimicking and steady-state flow phantoms and compared with conventional techniques, other published study results and theoretical values. Initial in vivo feasibility of the method is demonstrated with results obtained from scanning the carotid arteries of five healthy volunteers. Cyclic variations over the cardiac cycle were observed in on-axis displacements, off-axis transverse-wave velocities and wall-shear rates.


Excitation Intensity Driven PL Shifts of SiGe Islands on Patterned and Planar Si(001) Substrates: Evidence for Ge-rich Dots in Islands.
For randomly nucleated SiGe/Si(001) islands, a significantly stronger blue-shift of the PL spectra as a function of the excitation intensity is observed when compared to islands grown on patterned substrates side by side within the same run in a solid source molecular beam epitaxy chamber. We ascribe this different PL behavior to the much larger inhomogeneity of the Ge distribution in islands on planar substrates when compared to islands grown on pit-patterned ones, as observed previously. 3D band-structure calculations show that Ge-rich inclusions of approximately 5 nm diameter at the apex of the islands can account for the observed differences in the PL spectra. The existence of such inclusions can be regarded as a quantum dot in an island and is in agreement with recent nano-tomography experiments.


Microsomal prostaglandin E synthase-1 protects against Fas-induced liver injury.
Microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme for the synthesis of prostaglandin E2 (PGE2), a proproliferative and antiapoptotic lipid molecule important for tissue regeneration and injury repair. In this study, we developed transgenic (Tg) mice with targeted expression of mPGES-1 in the liver to assess Fas-induced hepatocyte apoptosis and acute liver injury. Compared with wild-type (WT) mice, the mPGES-1 Tg mice showed less liver hemorrhage, lower serum alanine transaminase (ALT) and aspartate transaminase (AST) levels, less hepatic necrosis/apoptosis, and lower level of caspase cascade activation after intraperitoneal injection of the anti-Fas antibody Jo2. Western blotting analysis revealed increased expression and activation of the serine/threonine kinase Akt and associated antiapoptotic molecules in the liver tissues of Jo2-treated mPGES-1 Tg mice. Pretreatment with the mPGES-1 inhibitor (MF63) or the Akt inhibitor (Akt inhibitor V) restored the susceptibility of the mPGES-1 Tg mice to Fas-induced liver injury. Our findings provide novel evidence that mPGES-1 prevents Fas-induced liver injury through activation of Akt and related signaling and suggest that induction of mPGES-1 or treatment with PGE2 may represent important therapeutic strategy for the prevention and treatment of Fas-associated liver injuries.


Risks of deep vein thrombosis, pulmonary embolism, and bleeding after covid-19: nationwide self-controlled cases series and matched cohort study.
To quantify the risk of deep vein thrombosis, pulmonary embolism, and bleeding after covid-19. Self-controlled case series and matched cohort study. National registries in Sweden. 1 057 174 people who tested positive for SARS-CoV-2 between 1 February 2020 and 25 May 2021 in Sweden, matched on age, sex, and county of residence to 4 076 342 control participants. Self-controlled case series and conditional Poisson regression were used to determine the incidence rate ratio and risk ratio with corresponding 95% confidence intervals for a first deep vein thrombosis, pulmonary embolism, or bleeding event. In the self-controlled case series, the incidence rate ratios for first time outcomes after covid-19 were determined using set time intervals and the spline model. The risk ratios for first time and all events were determined during days 1-30 after covid-19 or index date using the matched cohort study, and adjusting for potential confounders (comorbidities, cancer, surgery, long term anticoagulation treatment, previous venous thromboembolism, or previous bleeding event). Compared with the control period, incidence rate ratios were significantly increased 70 days after covid-19 for deep vein thrombosis, 110 days for pulmonary embolism, and 60 days for bleeding. In particular, incidence rate ratios for a first pulmonary embolism were 36.17 (95% confidence interval 31.55 to 41.47) during the first week after covid-19 and 46.40 (40.61 to 53.02) during the second week. Incidence rate ratios during days 1-30 after covid-19 were 5.90 (5.12 to 6.80) for deep vein thrombosis, 31.59 (27.99 to 35.63) for pulmonary embolism, and 2.48 (2.30 to 2.68) for bleeding. Similarly, the risk ratios during days 1-30 after covid-19 were 4.98 (4.96 to 5.01) for deep vein thrombosis, 33.05 (32.8 to 33.3) for pulmonary embolism, and 1.88 (1.71 to 2.07) for bleeding, after adjusting for the effect of potential confounders. The rate ratios were highest in patients with critical covid-19 and highest during the first pandemic wave in Sweden compared with the second and third waves. In the same period, the absolute risk among patients with covid-19 was 0.039% (401 events) for deep vein thrombosis, 0.17% (1761 events) for pulmonary embolism, and 0.101% (1002 events) for bleeding. The findings of this study suggest that covid-19 is a risk factor for deep vein thrombosis, pulmonary embolism, and bleeding. These results could impact recommendations on diagnostic and prophylactic strategies against venous thromboembolism after covid-19.


Histopathological effects of Thiodan on the freshwater snail, Galba truncatula (gastropoda, pulmonata).
Histopathological alterations in three tissues, namely, digestive gland, foot and mantle of the freshwater snail Galba truncatula induced by Thiodan 35 EC, a commercial preparation of endosulfan, were studied in the laboratory. The snails were exposed to five sublethal concentrations of Thiodan, two of them higher concentrations at 0.33% and 0.65% in glass jars for short-term exposure (96 h) and three of them lower concentrations at 0.08%, 0.16% and 0.27% for long-term exposure (10, 20 and 30 days). Thiodan caused significant histopathological changes in the tissues of the snail, irrespective of the concentration of the pesticide and its exposure period. The histopathological changes included accumulation of amoebocytes in the hemolymphatic spaces between the tubules of the digestive gland, exudation in the lumen of tubules, expansion of hemolymphatic spaces between the tubules and increase of vacuolation and necrotic changes in the digestive cells. Disruption of columnar muscle fibers and alteration of epithelial layers were detected in the foot and mantle of the treated specimens. The number and size of mucous and protein cells in the foot and mantle also changed.


An expansive human regulatory lexicon encoded in transcription factor footprints.
Regulatory factor binding to genomic DNA protects the underlying sequence from cleavage by DNase I, leaving nucleotide-resolution footprints. Using genomic DNase I footprinting across 41 diverse cell and tissue types, we detected 45 million transcription factor occupancy events within regulatory regions, representing differential binding to 8.4 million distinct short sequence elements. Here we show that this small genomic sequence compartment, roughly twice the size of the exome, encodes an expansive repertoire of conserved recognition sequences for DNA-binding proteins that nearly doubles the size of the human cis-regulatory lexicon. We find that genetic variants affecting allelic chromatin states are concentrated in footprints, and that these elements are preferentially sheltered from DNA methylation. High-resolution DNase I cleavage patterns mirror nucleotide-level evolutionary conservation and track the crystallographic topography of protein-DNA interfaces, indicating that transcription factor structure has been evolutionarily imprinted on the human genome sequence. We identify a stereotyped 50-base-pair footprint that precisely defines the site of transcript origination within thousands of human promoters. Finally, we describe a large collection of novel regulatory factor recognition motifs that are highly conserved in both sequence and function, and exhibit cell-selective occupancy patterns that closely parallel major regulators of development, differentiation and pluripotency.


Artemisinin combination therapies for treatment of uncomplicated malaria in Uganda.
To compare the efficacy and safety of artemisinin combination therapies for the treatment of uncomplicated falciparum malaria in Uganda. Randomized single-blind controlled trial. Tororo, Uganda, an area of high-level malaria transmission. Children aged one to ten years with confirmed uncomplicated P. falciparum malaria. Amodiaquine + artesunate or artemether-lumefantrine. Risks of recurrent symptomatic malaria and recurrent parasitemia at 28 days, unadjusted and adjusted by genotyping to distinguish recrudescences and new infections. Of 408 participants enrolled, 403 with unadjusted efficacy outcomes were included in the per-protocol analysis. Both treatment regimens were highly efficacious; no recrudescences occurred in patients treated with amodiaquine + artesunate, and only two occurred in those treated with artemether-lumefantrine. However, recurrent malaria due to new infections was common. The unadjusted risk of recurrent symptomatic malaria was significantly lower for participants treated with artemether-lumefantrine than for those treated with amodiaquine + artesunate (27% versus 42%, risk difference 15%, 95% CI 5.9%-24.2%). Similar results were seen for the risk of recurrent parasitemia (51% artemether-lumefantrine versus 66% amodiaquine + artesunate, risk difference 16%, 95% CI 6.2%-25.2%). Amodiaquine + artesunate and artemether-lumefantrine were both well-tolerated. Serious adverse events were uncommon with both regimens. Amodiaquine + artesunate and artemether-lumefantrine were both highly efficacious for treatment of uncomplicated malaria. However, in this holoendemic area, despite the excellent performance of both regimens in terms of efficacy, many patients experienced recurrent parasitemia due to new infections. Artemether-lumefantrine was superior to amodiaquine + artesunate for prevention of new infections. To maximize the benefit of artemisinin combination therapy in Africa, treatment should be integrated with strategies to prevent malaria transmission. The impact of frequent repeated therapy on the efficacy, safety, and cost-effectiveness of new artemisinin regimens should be further investigated.


Efficacy of soyfoods and soybean isoflavone supplements for alleviating menopausal symptoms is positively related to initial hot flush frequency.
Soy has received attention as an alternative to conventional hormone replacement therapy (HRT) largely because it is a unique dietary source of isoflavones. Isoflavones are diphenolic compounds that have both hormonal and nonhormonal properties and are considered to be selective estrogen receptor modulators. The estrogen-like effects of isoflavones in combination with the low reported frequency of hot flushes in Japan has prompted investigation of the effect of soy on menopausal symptoms. The purpose of this review is to evaluate the efficacy of soyfoods and isoflavone supplements for the alleviation of hot flushes. Nineteen trials (13 using a parallel design) involving more than 1,700 women were identified. Six trials were excluded from analysis: two that involved breast cancer patients, two that reported data on severity but not hot flush frequency, one that was not blinded, and one that did not include a control group. Based on a simple regression analysis of the remaining data set (13 trials), there was a statistically significant relationship (P =.01) between initial hot flush frequency and treatment efficacy. Initial hot flush frequency explained about 46% of the treatment effects, and hot flush frequency decreased by about 5% (above placebo or control effects) for every additional initial hot flush per day in women whose initial hot flush frequency was five or more per day. Although conclusions based on this analysis should be considered tentative, the available data justify the recommendation that patients with frequent hot flushes consider trying soyfoods or isoflavone supplements for the alleviation of their symptoms.


Surfactant therapy of pulmonary conditions excluding those with primary surfactant deficiency and bronchoscopy as delivery method: an overview of Russian patents and publications.
Preparations of pulmonary surfactant are used for the treatment of respiratory distress syndrome in a newborn. Their applicability as a method of routine for lung diseases beyond the neonatal period is questionable. Some publications from the former Soviet Union (SU) have reported on successful surfactant therapy of ARDS in children and adults as well as for inhalation injuries, pneumonia, and tuberculosis. Bronchoscopy was used and recommended as a method of surfactant delivery for ARDS, some types of pneumonia and tuberculosis. Manufacturing processes of surfactant preparations from bovine lung and amniotic fluid, described by Russian patents, and bronchoscopy as a delivery mode are discussed here. A concluding point is that some reports from the former SU about administration of exogenous surfactant in pulmonary conditions, excluding those with primary surfactant deficiency, are only partly confirmed by the international literature.


Fixed-dose combination therapy for type 2 diabetes: sitagliptin plus pioglitazone.
Type 2 diabetes is typically associated with insulin resistance and dysfunction of insulin-secreting pancreatic beta-cells. Addressing these defects often requires therapy with a combination of differently acting antidiabetic agents. A potential novel combination in development brings together the dipeptidyl peptidase-4 (DPP-4) inhibitor sitagliptin with the thiazolidinedione pioglitazone into a fixed-dose single-tablet combination. The former component acts mainly to increase prandial insulin secretion; the latter improves insulin sensitivity. To date, clinical trials conducted in type 2 diabetes patients have used combinations of sitagliptin (100 mg/day) and pioglitazone (30 - 45 mg/day) as separate tablets. These trials have shown that the combinations offer additive efficacy in reducing blood glucose when given as initial antidiabetic therapy and as add-on therapy when pioglitazone alone fails to maintain glycemic control. Initial therapy with a combination of sitagliptin (100 mg/day) and pioglitazone (30 mg/day) reduced HbA1c by > 2% starting from a baseline > 9%. Adding sitagliptin (50 - 100 mg/day) to patients inadequately controlled on pioglitazone reduced HbA1c by 0.7 - 1.4% from a baseline of 8 - 8.5%. The combination did not increase the risk of hypoglycemia and produced similar or slightly more weight gain than pioglitazone alone when introduced as initial antidiabetic therapy. The combination of sitagliptin and pioglitazone was well tolerated in these trials, and would appear to be suited to a fixed-dose single-tablet combination for once-daily administration.


A microflow superfusion system for use with excised membrane patches.
A new method for rapidly changing the solution superfusing excised membrane patches is described. The main advantage of this method over previous rapid exchange methods is that it is possible to use very small quantities of solution (minimal volume about 100 microliters). The method consists of inserting the pipette tip, after obtaining an excised patch, into a small tube which can be independently perfused. Air bubbles are used to separate different solutions and to produce rapid "step" changes of solution. To test this method we examined the rate of change of: the pipette resistance produced in response to changing to a solution of lower conductivity, the amplitude of single K-channel currents in response to changing external K. The results suggest that the solution exchange is essentially a step function and is complete within 10-20 ms. The recording is briefly interrupted during the time taken for the passage of the air bubble past the pipette tip (less than 250 ms). In general, the passage of the air bubble did not affect the seal resistance. This method will be particularly useful for studies of the interactions of single ionic channels with chemicals which can only be obtained in small quantities.