Automated gating of flow cytometry data via robust model-based clustering.
The capability of flow cytometry to offer rapid quantification of multidimensional characteristics for millions of cells has made this technology indispensable for health research, medical diagnosis, and treatment. However, the lack of statistical and bioinformatics tools to parallel recent high-throughput technological advancements has hindered this technology from reaching its full potential. We propose a flexible statistical model-based clustering approach for identifying cell populations in flow cytometry data based on t-mixture models with a Box-Cox transformation. This approach generalizes the popular Gaussian mixture models to account for outliers and allow for nonelliptical clusters. We describe an Expectation-Maximization (EM) algorithm to simultaneously handle parameter estimation and transformation selection. Using two publicly available datasets, we demonstrate that our proposed methodology provides enough flexibility and robustness to mimic manual gating results performed by an expert researcher. In addition, we present results from a simulation study, which show that this new clustering framework gives better results in terms of robustness to model misspecification and estimation of the number of clusters, compared to the popular mixture models. The proposed clustering methodology is well adapted to automated analysis of flow cytometry data. It tends to give more reproducible results, and helps reduce the significant subjectivity and human time cost encountered in manual gating analysis.


Increased Gain in the Auditory Pathway, Alzheimer's Disease Continuum, and Air Pollution: Peripheral and Central Auditory System Dysfunction Evolves Across Pediatric and Adult Urbanites.
A major impediment in early diagnosis of Alzheimer's disease (AD) is the lack of robust non-invasive biomarkers of early brain dysfunction. Metropolitan Mexico City (MMC) children and young adults show hyperphosphorylated tau, amyloid-β, and α-synuclein within auditory and vestibular nuclei and marked dysmorphology in the ventral cochlear nucleus and superior olivary complex. Based on early involvement of auditory brainstem centers, we believe brainstem auditory evoked potentials can provide early AD biomarkers in MMC young residents. We measured brainstem auditory evoked potentials in MMC clinically healthy children (8.52±3.3 years) and adults (21.08±3.0 years, 42.48±8.5 years, and 71.2±6.4 years) compared to clean air controls (6.5±0.7 years) and used multivariate analysis adjusting for age, gender, and residency. MMC children had decreased latency to wave I, delays in waves III and V, and longer latencies for interwave intervals, consistent with delayed central conduction time of brainstem neural transmission. In sharp contrast, young adults have significantly shortened interwave intervals I-III and I-V. By the 5th decade, wave V and interval I-V were significantly shorter, while the elderly cohort had significant delay in mean latencies and interwave intervals. Compensatory plasticity, increased auditory gain, cochlear synaptopathy, neuroinflammation, and AD continuum likely play a role in the evolving distinct auditory pathology in megacity urbanites. Understanding auditory central and peripheral dysfunction in the AD continuum evolving and progressing in pediatric and young adult populations may shed light on the complex mechanisms of AD development and help identify strong noninvasive biomarkers. AD evolving from childhood in air pollution environments ought to be preventable.


From connectivity models to region labels: identifying foci of a neurological disorder.
We propose a novel approach to identify the foci of a neurological disorder based on anatomical and functional connectivity information. Specifically, we formulate a generative model that characterizes the network of abnormal functional connectivity emanating from the affected foci. This allows us to aggregate pairwise connectivity changes into a region-based representation of the disease. We employ the variational expectation-maximization algorithm to fit the model and subsequently identify both the afflicted regions and the differences in connectivity induced by the disorder. We demonstrate our method on a population study of schizophrenia.


High-osmolarity signalling in Saccharomyces cerevisiae is modulated in a carbon-source-dependent fashion.
High-osmolarity-induced expression of the small heat-shock gene HSP12 is regulated by the HOG (high-osmolarity glycerol) pathway and PKA (protein kinase A). To analyse the regulatory input of both signal transduction pathways, high-salt-induced HSP12 expression in different genetic backgrounds on glucose-, ethanol- and glycerol-based culture media was examined. Upon exposure to high-osmolarity stress, the kinetics of induction of HSP12 in cells growing on the non-fermentable carbon sources are strikingly different from those on glucose. Derepression of HSP12 gene expression under non-stress conditions was observed in cells growing on non-fermentable carbon sources. High-salt challenge resulted in a lower induction of the HSP12 mRNA levels in ethanol-grown cells as compared to glucose-grown cells, whereas in glycerol-grown cells hardly any high-salt induction of HSP12 mRNA levels could be detected. Analysis of signalling through the HOG pathway suggested that glycerol may influence the activity of this signalling route, possible via negative feedback. Furthermore, the cellular level of PKA activity was found to have a great impact on stress-responsive gene transcription. On the basis of the data obtained it was concluded that modulation of PKA activity plays a major role in the stress response. A glucose-dependent, PKA-regulated cellular component is postulated to affect high-osmolarity-induced HSP12 expression.


[Mosquito microbiota and its influence on disease vectorial transmission].
Mosquitoes (Diptera: Culicidae) are found worldwide. Around 100 among 3500 mosquito species are known to be vectors of parasites and viruses, responsible for infectious diseases including malaria and dengue. Mosquitoes host diverse microbial communities that influence disease transmission, either by direct interference or via affecting host immunity and physiology. These microbial communities are present within diverse tissues, including the digestive tract, and vary depending on the sex of the mosquito, its developmental stage, and ecological factors. This review summarizes the current knowledge about the mosquito microbiota, defined as a community of commensal, symbiotic or pathogenic microbes harboured by a host. We first describe the current knowledge on the diversity of the microbiota, that includes bacteria, fungi, parasites and viruses and on its modes of acquisition throughout the mosquito life cycle. We then focus on microbial interactions within the mosquito gut, which notably affect vector competence, and on host-microbe interactions affecting mosquito fitness. Finally, we discuss current or potential methods based on the use of microbes or microbial products to interfere with pathogen transmission or to reduce mosquito lifespan and reproduction.


Anthocyanin-rich plum juice reduces ambulatory blood pressure but not acute cognitive function in younger and older adults: a pilot crossover dose-timing study.
Consumption of anthocyanins from fruit sources may exert protection against hypertension and improve cognition. However, the effect of dose timing in studies is rarely considered. We hypothesized that timed-dose consumption of juice from an anthocyanin-rich Japanese plum variety (Queen Garnet plum, QGP) will have acute and dose-timing effects on cardiovascular responses, cognition, and urinary anthocyanin excretion profiles. Our study objective was to investigate the impact of plum juice on these health parameters. Twelve older (65+ years) and 12 younger (18-45 years) adults participated in an acute crossover study. Participants received, randomly, either 1 × 300 mL or 3 × 100 mL plum juice over 3 hours on 2 different occasions with a 2-week washout period. A battery of cognitive tasks was administered at 0 and 6 hours on each study day. Blood pressure (BP) and urinary anthocyanin/metabolite excretion profiles were measured over 24 hours. Area under the curve for BP was calculated (0-6 hours). A significant reduction in BP and cardiovascular responses was observed in both age groups which was more obvious in the older age group on the single dose for systolic BP, diastolic BP, mean arterial pressure, and heart rate (P values = .035, .028, .017, and .006, respectively). No significant difference was observed between dose-timing regimens for either age group. There was no observed effect on cognition. Native QGP anthocyanins, as well as methylated/glucuronidated metabolites, were detected in urine with no significant differences between age groups or dose timing. High-anthocyanin plum juice significantly reduced BP, but dose timing did not appear to be a significant factor in the potential acute BP-lowering effect of QGP juice.


T cell receptor genes in a series of class I major histocompatibility complex-restricted cytotoxic T lymphocyte clones specific for a Plasmodium berghei nonapeptide: implications for T cell allelic exclusion and antigen-specific repertoire.
We report here the first extensive study of a T cell repertoire for a class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocyte (CTL) response. We have found that the T cell receptors (TCRs) carried by 28 H-2Kd-restricted CTL clones specific for a single Plasmodium berghei circumsporozoite nonapeptide are highly diverse in terms of V alpha, J alpha, and J beta segments and aminoacid composition of the junctional regions. However, despite this extensive diversity, a high proportion of the TCRs contain the same V beta segment. These results are in contrast to most previously reported T cell responses towards class II MHC-peptide complexes, where the TCR repertoires appeared to be much more limited. In our study, the finding of a dominant V beta in the midst of otherwise highly diverse TCRs suggests the importance of the V beta segment in shaping the T cell repertoire specific for a given MHC-peptide complex. As an additional finding, we observed that nearly all clones have rearranged both TCR alpha loci. Moreover, as many as one-third of the CTL clones that we analyzed apparently display two productive alpha rearrangements. This argues against a regulated model of sequential recombination at the alpha locus and consequently raises the question of whether allelic exclusion of the TCR alpha chain is achieved at all.


A critical role of CD30 ligand/CD30 in controlling inflammatory bowel diseases in mice.
A CD30-ligand (CD30L) is a 40-kilodalton, type II membrane-associated glycoprotein belonging to the tumor necrosis factor family. Serum levels of soluble CD30 increased in inflammatory bowel diseases (IBD), suggesting that CD30L/CD30 signaling is involved in the pathogenesis of IBD. In this study, we investigated the role of CD30L in oxazolone (OXA)- and trinitrobenzene sulfonic acid (TNBS)-induced colitis in CD30L knockout (KO) mice. Colitis was induced by OXA or TNBS in CD30LKO mice with BALB/c or C57BL/6 background, respectively, and diverse clinical signs of the disease were evaluated. Cytokine production from lamina propria T cells of the colon was assessed by enzyme-linked immunosorbent assay. Anti-interleukin (IL)-4 monoclonal antibody (mAb) or agonistic anti-CD30 mAb was inoculated in mice with colitis induced by OXA or TNBS. CD30LKO mice were susceptible to OXA-induced colitis but resistant to TNBS-induced acute colitis. The levels of T helper cell 2 type cytokines such as IL-4 and IL-13 in the LP T cells were significantly higher, but the levels of interferon gamma were lower in OXA- or TNBS-treated CD30LKO mice than in wild-type mice. In vivo administration of agonistic anti-CD30 mAb ameliorated OXA-induced colitis but aggravated TNBS-induced colitis in CD30LKO mice. These results suggest that CD30L/CD30 signaling is involved in development of both OXA- and TNBS-induced colitis. Modulation of CD30L/CD30 signaling by mAb could be a novel biologic therapy for IBD.


The genesis of empathy in human development: a phenomenological reconstruction.
In phenomenology, theories of empathy are intimately connected with the question of how it is possible to have insight into the mind of the other person. In this article, the author wants to show why it is self-evident for us that the other person is having experiences. In order to do so, it is not enough to discuss the phenomenon of empathy with a starting point in the already constituted adult person; instead the article presents a genetic approach to human development. The author thus contrasts Edith Stein's discussion of Einfühlung (empathy), which takes its starting point in the experience of the grown-up, with Max Scheler's discussion of Einsfühlung (feeling of oneness), where the relation between mother and infant is taken as one example. Maurice Merleau-Ponty's discussion of the world of the infant is read as one way of developing Scheler's theory of intersubjectivity and of Einsfühlung. This genetic approach is developed further into a phenomenological analysis of the experience of the fetus and of birth. The author argues that the analysis of the fetus highlights the distinction between knowing that another person is having experiences, and knowing the specific content of the other person's experiences. The fetus does not experience different persons, but has a pre-subjective experience of life that includes what is later experienced as belonging to "another." Later in life, the experience of empathy, as an experience of a specific content, can be developed from this experience. In this way empathy and Einsfühlung can be understood as complementary rather than as competing phenomena.


Functional bracing for the treatment of fractures of the humeral diaphysis.
Nonoperatively treated fractures of the humeral diaphysis have a high rate of union with good functional results. However, there are clinical situations in which operative treatment is more appropriate, and, though interest in plate osteosynthesis has decreased, intramedullary nailing has gained popularity in recent years. We report the results of treating fractures of the humeral diaphysis with a prefabricated brace that permits full motion of all joints and progressive use of the injured extremity. Between 1978 and 1990, 922 patients who had a fracture of the humeral diaphysis were treated with a prefabricated brace that permitted motion of adjacent joints. The injured extremities were initially stabilized in an above-the-elbow cast or a coaptation splint for an average of nine days (range, zero to thirty-five days) prior to the application of the prefabricated brace. Orthopaedic residents, supervised by teaching staff, provided follow-up care in a special outpatient clinic. Radiographs were made at each follow-up visit until the fracture healed. We were able to follow 620 (67 percent) of the 922 patients. Four hundred and sixty-five (75 percent) of the fractures were closed, and 155 (25 percent) were open. Nine patients (6 percent) who had an open fracture and seven (less than 2 percent) who had a closed fracture had a nonunion after bracing. In 87 percent of the 565 patients for whom anteroposterior radiographs were available, the fracture healed in less than 16 degrees of varus angulation, and in 81 percent of the 546 for whom lateral radiographs were available, it healed in less than 16 degrees of anterior angulation. At the time of brace removal, 98 percent of the patients had limitation of shoulder motion of 25 degrees or less. We were unable to follow most of the patients long-term, as they did not return to the clinic once the fracture had united and use of the brace had been discontinued. Functional bracing for the treatment of fractures of the humeral diaphysis is associated with a high rate of union, particularly when used for closed fractures. The residual angular deformities are usually functionally and aesthetically acceptable. The present study illustrates the difficulties encountered in carrying out long-term follow-up of indigent patients treated in charity hospitals that are affiliated with teaching institutions. These difficulties are also becoming common with patients insured under managed-care organizations and are frequent in our peripatetic population.


Point-of-care screening for hepatitis B virus infection in pregnant women at an antenatal clinic: A South African experience.
Elimination of HIV and syphilis mother-to-child transmission (MTCT) has received much attention but little consideration has been given to the possibility of elimination of HBV MTCT. In sub-Saharan Africa, HBV vertical transmission continues to be reported and it remains an important public health problem. This study aimed to assess the feasibility of screening pregnant women for HBV using a point-of-care (POC) test and implementing interventions to prevent HBV MTCT. In this observational prospective cohort study, HIV-uninfected pregnant women who consented to testing were screened for HBV using a rapid POC test for HBsAg. Positive results were laboratory-confirmed and tested for HBV DNA and serological markers. Women with viral loads ≥ 20 000 IU/ml received tenofovir (TDF) treatment and all infants received birth-dose HBV vaccine. Two blood samples collected six months apart from HBV-exposed infants within their first year of life were tested for HBV DNA. Of 144 women who were approached, 134 consented to participating (93% acceptance rate of HBV POC test). Six women tested positive for HBsAg (4.5%; 95% CI 0.99%-8.01%), all confirmed by laboratory testing. Two mothers, M1 and M4, were treated with TDF during their third trimester of pregnancy. Six HBV-exposed infants received the HBV vaccine within 24 hours of birth, of whom two were lost to follow-up and four (including the two born to M1 and M4) had undetectable levels of HBV DNA when tested at the two time points. We found that HBV screening using POC testing fulfilled the criteria considered necessary for implementation. It has acceptable performance, is inexpensive, reliable, and was well accepted by the study participants. Screening pregnant women as part of the HBV MTCT prevention strategy is therefore feasible in a South African clinical setting.


MEK inhibition leads to PI3K/AKT activation by relieving a negative feedback on ERBB receptors.
The phosphoinositide 3-kinase (PI3K)/AKT and RAF/MEK/ERK signaling pathways are activated in a wide range of human cancers. In many cases, concomitant inhibition of both pathways is necessary to block proliferation and induce cell death and tumor shrinkage. Several feedback systems have been described in which inhibition of one intracellular pathway leads to activation of a parallel signaling pathway, thereby decreasing the effectiveness of single-agent targeted therapies. In this study, we describe a feedback mechanism in which MEK inhibition leads to activation of PI3K/AKT signaling in EGFR and HER2-driven cancers. We found that MEK inhibitor-induced activation of PI3K/AKT resulted from hyperactivation of ERBB3 as a result of the loss of an inhibitory threonine phosphorylation in the conserved juxtamembrane domains of EGFR and HER2. Mutation of this amino acid led to increased ERBB receptor activation and upregulation of the ERBB3/PI3K/AKT signaling pathway, which was no longer responsive to MEK inhibition. Taken together, these results elucidate an important, dominant feedback network regulating central oncogenic pathways in human cancer.


Do the morphological characteristics of arteriovenous malformations affect the results of radiosurgery?
The authors sought to determine which morphological features of arteriovenous malformations (AVMs) are statistically predictive of preradiosurgical hemorrhage, postradiosurgical hemorrhage, and neuroimaging-defined failure of radiosurgical treatment. In addition, correlation between computerized tomography (CT) scanning and angiography for the identification of AVM structures was investigated. Archived CT dosimetry and available angiographic and clinical data for 268 patients in whom AVMs were treated with linear accelerator radiosurgery were retrospectively reviewed. Many of the morphological features of AVMs, including location, volume, compact or diffuse nidus, neovascularity, ease of nidus identification, number of feeding arteries, location (deep or superficial) of feeding arteries, number of draining veins, deep or superficial venous drainage, venous stenoses, venous ectasias, and the presence of intranidal aneurysms, were analyzed. In addition, a number of patient and treatment factors, including patient age, presenting symptoms, radiation dose, repeated treatment, and radiological outcome, were subjected to multivariate analyses. Two hundred twenty-seven patients were treated with radiosurgery for the first time and 41 patients underwent repeated radiosurgery. Eighty-one patients presented with a history of AVM hemorrhage and 91 patients had AVMs in a periventricular location. Twenty-six patients (10%) experienced a hemorrhage following radiosurgery. Of the 268 patients, 81 (30%) experienced angiographically defined cures, and 37 (14%) experienced MR imaging-defined cures. Eighty-six patients (32%) experienced neuroimaging-defined treatment failure, and 64 underwent insufficiently long follow up. A larger AVM volume (odds ratio [OR] 0.349; p = 0.004) was associated with a decreased rate of pretreatment hemorrhage, whereas periventricular location (OR 6.358; p = 0.000) was associated with an increased rate of pretreatment hemorrhage. None of the analyzed factors was predictive of hemorrhage following radiosurgery. A higher radiosurgical dose was strongly correlated with neuroimaging-defined success (OR 3.743; p = 0.006), whereas a diffuse nidus structure (OR 0.246; p = 0.008) and associated neovascularity (OR 0.428; p = 0.048) were each associated with a lower neuroimaging-defined cure rate. A strong correlation between CT scanning and angiography was noted for both nidus structure (p = 0.000; Fisher exact test) and neovascularity (p = 0.002; Fisher exact test). Patients presenting with AVMs that are small or periventricular were at higher risk for experiencing hemorrhage. A higher radiosurgical dose correlated strongly with neuroimaging-defined success. Patients in whom the AVM had a diffuse structure or associated neovascularity were at higher risk for neuroimaging-defined failure of radiosurgery. A strong correlation between CT scanning and angiography in the assessment of AVM structure was demonstrated.


The ConCom Safety Management Scale: developing and testing a measurement instrument for control-based and commitment-based safety management approaches in hospitals.
Nursing management is considered important for patient safety. Prior research has predominantly focused on charismatic leadership styles, although it is questionable whether these best characterise the role of nurse managers. Managerial control is also relevant. Therefore, we aimed to develop and test a measurement instrument for control-based and commitment-based safety management of nurse managers in clinical hospital departments. A cross-sectional survey design was used to test the newly developed questionnaire in a sample of 2378 nurses working in clinical departments. The nurses were asked about their perceptions of the leadership behaviour and management practices of their direct supervisors. Psychometric properties were evaluated using confirmatory factor analysis and reliability estimates. The final 33-item questionnaire showed acceptable goodness-of-fit indices and internal consistency (Cronbach's α of the subscales range: 0.59-0.90). The factor structure revealed three subdimensions for control-based safety management: (1) stressing the importance of safety rules and regulations; (2) monitoring compliance; and (3) providing employees with feedback. Commitment-based management consisted of four subdimensions: (1) showing role modelling behaviour; (2) creating safety awareness; (3) showing safety commitment; and (4) encouraging participation. Construct validity of the scale was supported by high factor loadings and provided preliminary evidence that control-based and commitment-based safety management are two distinct yet related constructs. The findings were reconfirmed in a cross-validation procedure. The results provide initial support for the construct validity and reliability of our ConCom Safety Management Scale. Both management approaches were found to be relevant for managing patient safety in clinical hospital departments. The scale can be used to deepen our understanding of the influence of patient safety management on healthcare professionals' safety behaviour as well as patient safety outcomes.


Latest evidence on transcatheter aortic valve implantation vs. surgical aortic valve replacement for the treatment of aortic stenosis in high and intermediate-risk patients.
The goal of this review is to summarize the current evidence supporting the use of transcatheter aortic valve implantation (TAVI) in high and intermediate-risk patients. The focus is on the five randomized controlled trials comparing TAVI with surgical aortic valve replacement (SAVR) published to date, as well as two recent meta-analyses. TAVI has profoundly transformed the treatment of elderly patients presenting with symptomatic severe aortic stenosis. In experienced hands, the procedure has become well tolerated and the results more predictable. So far, two trials using two different devices [Placement of Aortic Transcatheter Valve (PARTNER) 1A and US CoreValve High Risk] have shown that TAVI is able to compete in terms of mortality with SAVR in high-risk patients. These findings have been extended to the intermediate-risk population in two recently published randomized controlled trials [PARTNER 2 and Nordic Aortic Valve Intervention (NOTION)]. The two meta-analyses suggested improved survival in both high and intermediate-risk patients during the first 2 years following the intervention. The survival benefit was only found in patients treated via the transfemoral access, and appeared more pronounced in women. Individual randomized trials enrolling high and intermediate-risk patients have established the noninferiority of TAVI in comparison with SAVR, whereas subsequent meta-analyses suggest superiority of transfemoral TAVI in terms of a sustained survival benefit 2 years after valve implantation irrespective of the surgical risk category. The benefit of TAVI appears more pronounced in women than in men.


Open-ended recursive calculation of single residues of response functions for perturbation-dependent basis sets.
We present theory, implementation, and applications of a recursive scheme for the calculation of single residues of response functions that can treat perturbations that affect the basis set. This scheme enables the calculation of nonlinear light absorption properties to arbitrary order for other perturbations than an electric field. We apply this scheme for the first treatment of two-photon circular dichroism (TPCD) using London orbitals at the Hartree-Fock level of theory. In general, TPCD calculations suffer from the problem of origin dependence, which has so far been solved by using the velocity gauge for the electric dipole operator. This work now enables comparison of results from London orbital and velocity gauge based TPCD calculations. We find that the results from the two approaches both exhibit strong basis set dependence but that they are very similar with respect to their basis set convergence.


Delivery and impact bypass in a karst aquifer with high phosphorus source and pathway potential.
Conduit and other karstic flows to aquifers, connecting agricultural soils and farming activities, are considered to be the main hydrological mechanisms that transfer phosphorus from the land surface to the groundwater body of a karstified aquifer. In this study, soil source and pathway components of the phosphorus (P) transfer continuum were defined at a high spatial resolution; field-by-field soil P status and mapping of all surface karst features was undertaken in a > 30 km(2) spring contributing zone. Additionally, P delivery and water discharge was monitored in the emergent spring at a sub-hourly basis for over 12 months. Despite moderate to intensive agriculture, varying soil P status with a high proportion of elevated soil P concentrations and a high karstic connectivity potential, background P concentrations in the emergent groundwater were low and indicative of being insufficient to increase the surface water P status of receiving surface waters. However, episodic P transfers via the conduit system increased the P concentrations in the spring during storm events (but not >0.035 mg total reactive P L(-1)) and this process is similar to other catchments where the predominant transfer is via episodic, surface flow pathways; but with high buffering potential over karst due to delayed and attenuated runoff. These data suggest that the current definitions of risk and vulnerability for P delivery to receiving surface waters should be re-evaluated as high source risk need not necessarily result in a water quality impact. Also, inclusion of conduit flows from sparse water quality data in these systems may over-emphasise their influence on the overall status of the groundwater body.


The pharmacokinetics of oral indoramin during pregnancy.
1. The pharmacokinetics of indoramin (Baratol; Wyeth) and its active metabolite 6-hydroxyindoramin administered to 13 women as a single 37.5 mg dose of indoramin were compared under pregnant and post-partum conditions. 2. No significant differences were observed between values determined under pregnant and post-partum conditions, for any pharmacokinetic parameter. Cmax and AUC values in individual subjects differed, in most cases, by no more than two-fold, i.e. the same order of magnitude as intra-subject variation seen in volunteers dosed repeatedly with indoramin. 3. Median values of Cmax, AUC(0.24) and t 1/2,z were of a similar order to values seen previously in normal volunteers after a single 37.5 mg dose. 4. It is concluded that in treating hypertension associated with pregnancy, this study does not provide evidence to depart from the usual clinical practice of titrating indoramin dosage with control of blood pressure as the end-point, keeping in mind the restriction of the dose-limiting side-effect of sedation.


Decentralization of Acid Fast Bacilli (AFB) External Quality Assurance Using Blind Rechecking for Sputum Smear Microscopy in Ethiopia.
Ethiopia achieved a rapid expansion of TB microscopic centers for acid fast bacilli (AFB). However, external quality assurance (EQA) services were, until recently, limited to few regional and sub-regional laboratories. In this paper, we describe the decentralization experience and the result of EQA using random blinded rechecking. The routine EQA quarterly report was compiled and analyzed. A positive result by the microscopic center while the EQA center reported negative result is categorized as false positive (FP). A negative result by the microscopic center while the EQA center reported positive is considered false negative (FN). The reading of EQA centers was considered a gold standard to compute the sensitivity, specificity, positive predictive (PPV) and negative predictive values (NPV) of the readings of microscopic centers. We decentralized sputum smear AFB EQA from 4 Regional Laboratories (RRLs) to 82 EQA centers and enrolled 956 health facilities in EQA schemes. Enrollment of HFs in EQA was gradual because it required training and mentoring laboratory professionals, institutionalizing internal QA measures, equipping all HFs to perform diagnosis, and establishing more EQA centers. From 2012 to 2014 (Phase I), the FP rate declined from 0.6% to 0.2% and FN fell from as high as 7.6% to 1.6% in supported health facilities (HFs). In HFs that joined in Phase II, FN rates ranged from 5.6 to 7.3%. The proportion of HFs without errors has increased from 77.9% to 90.5% in Phase I HFs and from 82.9% to 86.9% in Phase II HFs. Overall sensitivity and specificity were 95.0% and 99.7%, respectively. PPV and NPV were 93.3% and 99.7%, respectively. Decentralizing blinded rechecking of sputum smear microscopy is feasible in low-income settings. While a comprehensive laboratory improvement strategy enhanced the quality of microscopy, laboratory professionals' capacity in slide reading and smear quality requires continued support.


Journalists and Depressive Symptoms: A Systematic Literature Review.
Mental health research focusing on journalists has largely tended to give precedence to trauma exposure and subsequent symptoms of posttraumatic stress disorder. However, there are several occupational factors that may be associated with the development of depressive symptoms in journalists. This systematic literature review aims to provide a concise, comprehensive, and systematic review of the quantitative literature relating to journalists' experiences of depressive symptoms. The systematic literature review method adopted was based on that prescribed by Fink and contains three main elements: sampling the literature, screening the literature, and extracting data. Key terms were developed and used to source appropriate studies from several electronic databases, a hand search of reference lists was conducted, and authors were contacted to suggest examples of their own work not yet sampled. The sample included 13 quantitative studies reported in English language. Journalists most at risk of experiencing depressive symptoms had (1) greater exposure to work-related and personal trauma, (2) experienced threats to themselves or their family, and (3) reduced levels of family and peer support, social acknowledgment, and education. An area for further investigation is the prevalence and experiences of specific depressive disorders within the journalist population. There are a number of theoretical and methodological issues that can be addressed in future research.


A mushroom fruiting body-inducing substance inhibits activities of replicative DNA polymerases.
We found and isolated two natural products in the extract from a basidiomycete, Ganoderma lucidum, as eukaryotic DNA polymerase inhibitors. The compounds were identified as cerebrosides, (4E,8E)-N-D-2'-hydroxypalmitoyl- 1-O-beta-D-glucopyranosyl-9-methyl-4,8-sphingadienine and (4E,8E)-N-D-2'-hydroxystearoyl-1-O-beta-D-glucopyranos yl-9-methyl- 4,8-sphingadienine and were found to be identical to the mushroom fruiting body-inducing substances (FIS) reported. These cerebrosides selectively inhibited the activities of replicative DNA polymerases, especially the alpha-type, from phylogenetically broad eukaryotic species, whereas they hardly influenced the activities of DNA polymerase beta, prokaryotic DNA polymerases, terminal deoxynucleotidyl transferase, HIV reverse transcriptase, RNA polymerase, deoxyribonuclease I, and ATPase. The inhibition of another replicative polymerase, the delta-type, was moderate. The inhibitions of the replicative polymerases were dose-dependent, and the IC50 for animal or mushroom DNA polymerase alpha was achieved at approximately 12 micrograms/ml (16.2 microM) and for animal DNA polymerase delta at 57 micrograms/ml (77.2 microM). FIS is possibly a DNA polymerase inhibitor specific to the replicative enzyme group, and the fruiting body formation may be required for the suppression of the DNA replication or the vegetative growth of the mycelium.


Antifungal therapy of Aspergillus invasive otitis externa: efficacy of voriconazole and review.
Invasive otitis externa (IOE) due to Aspergillus is a rare, potentially life-threatening, invasive fungal infection affecting immunocompromised patients. The invasive process may lead to skull base osteomyelitis with progressive cranial nerve palsies and can result in irreversible hearing and neurological impairment. We report two cases of Aspergillus IOE treated with voriconazole alone and a literature review of antifungal therapy of Aspergillus IOE. Twenty-five patients, including the two described in the present report, were analyzed. Eighteen patients were treated with amphotericin B, and nine of them received itraconazole as an additional agent. Three patients received initial therapy with itraconazole, and one patient was treated with both voriconazole and caspofungin therapy. The two patients in the present report received voriconazole therapy alone with good clinical and biological tolerance despite prolonged treatment. The last patient did not receive antifungal therapy, as the diagnosis was made postmortem. Eighteen patients underwent an initial extensive surgical debridement. The majority of the patients had a favorable outcome, 17 patients experienced a complete recovery, and 6 showed a partial improvement. Both of the patients reported on here had favorable outcomes, and no aggressive surgical debridement was required. Although voriconazole has been shown to be effective for the treatment of invasive aspergillosis, its precise role in the management of Aspergillus IOE had not been documented. These observations demonstrate that voriconazole could be an effective and well-tolerated therapeutic option for the management of Aspergillus IOE.


Influence of N-linked oligosaccharide chains on the processing, cell surface expression and function of the measles virus fusion protein.
The fusion (F) glycoprotein of measles virus, a structural component of the virion envelope, contains four potential sites for attachment of N-linked oligosaccharides. Three are located in the F2 subunit of the protein and one in the signal peptide. Four mutants were constructed by oligonucleotide-directed mutagenesis, in each case changing one N-linked glycosylation site from Asn-X-Ser/Thr to Ser-X-Ser/Thr. The wild-type and altered forms of the F protein were expressed in BHK-21 and HeLa T4 cells by use of the recombinant vaccinia virus-encoding T7 polymerase system. Analysis of these proteins revealed that three (residues 29, 61 and 67) potential sites for addition of N-linked glycans in the F2 subunit are actually utilized. The functional glycosylation sites were systematically removed in all possible combinations from the F protein to form a panel of mutants from which the role of carbohydrates, singly or in various combinations, could be evaluated. One single-site mutant protein lacking the glycosylation site of Asn-67 was processed, transported to the cell surface and could induce cell fusion. However, the other two single-site mutant proteins with deletions of glycosylation sites Asn-29 or Asn-61 exhibited a defect in processing, were not transported to cell surface and thus induced no cell fusion. The absence of any two of the three or of all three glycosylation sites resulted in protein retention in the endoplasmic reticulum. Therefore, it appears that glycosylation of sites Asn-29 and Asn-61 has important roles in maintaining the native structure of the F protein.


Quantitative spinal cord MRI in MOG-antibody disease, neuromyelitis optica and multiple sclerosis.
Spinal cord involvement is a hallmark feature of multiple sclerosis, neuromyelitis optica with AQP4 antibodies and MOG-antibody disease. In this cross-sectional study we use quantitative spinal cord MRI to better understand these conditions, differentiate them and associate with relevant clinical outcomes. Eighty participants (20 in each disease group and 20 matched healthy volunteers) underwent spinal cord MRI (cervical cord: 3D T1, 3D T2, diffusion tensor imaging and magnetization transfer ratio; thoracic cord: 3D T2), together with disability, pain and fatigue scoring. All participants had documented spinal cord involvement and were at least 6 months post an acute event. MRI scans were analysed using publicly available software. Those with AQP4-antibody disease showed a significant reduction in cervical cord cross-sectional area (P = 0.038), thoracic cord cross-sectional area (P = 0.043), cervical cord grey matter (P = 0.011), magnetization transfer ratio (P ≤ 0.001), fractional anisotropy (P = 0.004) and increased mean diffusivity (P = 0.008). Those with multiple sclerosis showed significantly increased mean diffusivity (P = 0.001) and reduced fractional anisotropy (P = 0.013), grey matter volume (P = 0.002) and magnetization transfer ratio (P = 0.011). In AQP4-antibody disease the damage was localized to areas of the cord involved in the acute attack. In multiple sclerosis this relationship with lesions was absent. MOG-antibody disease did not show significant differences to healthy volunteers in any modality. However, when considering only areas involved at the time of the acute attack, a reduction in grey matter volume was found (P = 0.023). This suggests a predominant central grey matter component to MOG-antibody myelitis, which we hypothesize could be partially responsible for the significant residual sphincter dysfunction. Those with relapsing MOG-antibody disease showed a reduction in cord cross-sectional area compared to those with monophasic disease, even when relapses occurred elsewhere (P = 0.012). This suggests that relapsing MOG-antibody disease is a more severe phenotype. We then applied a principal component analysis, followed by an orthogonal partial least squares analysis. MOG-antibody disease was discriminated from both AQP4-antibody disease and multiple sclerosis with moderate predictive values. Finally, we assessed the clinical relevance of these metrics using a multiple regression model. Cervical cord cross-sectional area associated with disability scores (B = -0.07, P = 0.0440, R2 = 0.20) and cervical cord spinothalamic tract fractional anisotropy associated with pain scores (B = -19.57, P = 0.016, R2 = 0.55). No spinal cord metric captured fatigue. This work contributes to our understanding of myelitis in these conditions and highlights the clinical relevance of quantitative spinal cord MRI.


Neutral lipid alterations in human herpesvirus 8-infected HUVEC cells and their possible involvement in neo-angiogenesis.
Human Herpesvirus 8 (HHV8), the causative agent of Kaposi's sarcoma, induces an intense modification of lipid metabolism and enhances the angiogenic process in endothelial cells. In the present study, neutral lipid (NL) metabolism and angiogenesis were investigated in HHV8-infected HUVEC cells. The viral replication phases were verified by rtPCR and also by K8.1 and LANA immunostaining. Lipid droplets (Nile Red) were higher in all phases and NL staining (LipidTOX) combined with viral-antigen detection (immunofluorescence) demonstrated a NL content increase in infected cells. In particular, triglyceride synthesis increases in the lytic phase, whereas cholesteryl ester synthesis rises in the latent one. Moreover, the inhibition of cholesterol esterification reduces neo-tubule formation mainly in latently infected cells. We suggest that a reprogramming of cholesteryl ester metabolism is involved in regulating neo-angiogenesis in HHV8-infected cells and plays a likely role in the high metastatic potential of derived-tumours.


Spin the Abstracts of Systematic Reviews and Meta-Analyses Regarding the Treatment of Ménière's Disease.
To identify, quantify, and characterize the presence of spin-specific strategies leading to misrepresentation of study results-in the abstracts of systematic reviews and meta-analyses of Ménière's disease treatment. Using a cross-sectional design, we searched MEDLINE and Embase on May 28, 2020, for systematic reviews and meta-analyses focused on Ménière's disease treatment. Returned searches were screened, and data were extracted in a masked, duplicate fashion. Our sample included 36 systematic reviews and meta-analyses. Of the 36 included studies, 22 (61.1%) abstracts contained spin while 14 (38.9%) did not. The most common spin types were selective reporting of benefit (10/36, 27.8%) or harm (8/36, 22.2%). Other types of spin occurred when findings were extrapolated to the global improvement of the disease (5/36, 13.9%), beneficial effects were reported with high risk of bias in primary studies (3/36, 8.3%), and when beneficial effects were extrapolated to an entire class of interventions (1/36, 2.8%). No instances of other spin types occurred. Abstracts containing spin were substantively associated with studies of critically low methodological quality compared with studies with low and moderate quality. No studies had a methodological rating of high quality. No associations were observed between spin and intervention types, journal recommendation of adhering to Preferred Reporting Items for Systematic Reviews and Meta-Analyses, or funding. We found a negative correlation (r = -.31) between abstract word limit and presence of spin. Our study highlights that spin in the abstracts of systematic reviews of Ménière's disease is common, and it further enhances the discussion surrounding spin in abstracts of scientific research. Spin in an abstract does not discredit a study's findings; however, its occurrence should be eliminated.


A methodological framework for evaluating mobile health devices.
Mobile devices such as PDAs are increasingly used as a part of healthcare interventions. Although they can provide powerful communication and self-management tools for patients with chronic illness, relatively little is known about their usability. A methodological framework for evaluating mobile health devices is proposed. It adapts the well-known methodologies of cognitive task analysis and usability testing. The framework is illustrated in the context of an analysis of an advanced function glucose meter. The analyses documented a range of problems which highlights the need to carefully scrutinize the usability of mobile health devices.


The neuronal correlate of bidirectional synesthesia: a combined event-related potential and functional magnetic resonance imaging study.
The neuronal correlate of a rare explicit bidirectional synesthesia was investigated with numerical and physical size comparison tasks using both functional magnetic resonance imaging and event-related potentials. Interestingly, although participant I.S. exhibited similar congruity effects for both tasks at the behavioral level, subsequent analyses of the imaging data revealed that different brain areas were recruited for each task, and in different time windows. The results support: (1) the genuineness of bidirectional synesthesia at the neuronal level, (2) the possibility that discrepancy in the neuronal correlates of synesthesia between previous studies might be task-related, and (3) the possibility that synesthesia might not be a unitary phenomenon.


Diagnostic Reasoning of Resident Physicians in the Age of Clinical Pathways.
Development of skills in diagnostic reasoning is paramount to the transition from novice to expert clinicians. Efforts to standardize approaches to diagnosis and treatment using clinical pathways are increasingly common. The effects of implementing pathways into systems of care during diagnostic education and practice among pediatric residents are not well described. To characterize pediatric residents' perceptions of the tradeoffs between clinical pathway use and diagnostic reasoning. We conducted a qualitative study from May to December 2019. Senior pediatric residents from a high-volume general pediatric inpatient service at an academic hospital participated in semi-structured interviews. We utilized a basic interpretive qualitative approach informed by a dual process diagnostic reasoning framework. Nine residents recruited via email were interviewed. Residents reported using pathways when admitting patients and during teaching rounds. All residents described using pathways primarily as management tools for patients with a predetermined diagnosis, rather than as aids in formulating a diagnosis. As such, pathways primed residents to circumvent crucial steps of deliberate diagnostic reasoning. However, residents relied on bedside assessment to identify when patients are "not quite fitting the mold" of the current pathway diagnosis, facilitating recalibration of the diagnostic process. This study identifies important educational implications at the intersection of residents' cognitive diagnostic processes and use of clinical pathways. We highlight potential challenges clinical pathways pose for skill development in diagnostic reasoning by pediatric residents. We suggest opportunities for educators to leverage clinical pathways as a framework for development of these skills.


Temperature regulation of marine heterotrophic prokaryotes increases latitudinally as a breach between bottom-up and top-down controls.
Planktonic heterotrophic prokaryotes make up the largest living biomass and process most organic matter in the ocean. Determining when and where the biomass and activity of heterotrophic prokaryotes are controlled by resource availability (bottom-up), predation and viral lysis (top-down) or temperature will help in future carbon cycling predictions. We conducted an extensive survey across subtropical and tropical waters of the Atlantic, Indian and Pacific Oceans during the Malaspina 2010 Global Circumnavigation Expedition and assessed indices for these three types of controls at 109 stations (mostly from the surface to 4,000 m depth). Temperature control was approached by the apparent activation energy in eV (ranging from 0.46 to 3.41), bottom-up control by the slope of the log-log relationship between biomass and production rate (ranging from -0.12 to 1.09) and top-down control by an index that considers the relative abundances of heterotrophic nanoflagellates and viruses (ranging from 0.82 to 4.83). We conclude that temperature becomes dominant (i.e. activation energy >1.5 eV) within a narrow window of intermediate values of bottom-up (0.3-0.6) and top-down 0.8-1.2) controls. A pervasive latitudinal pattern of decreasing temperature regulation towards the Equator, regardless of the oceanic basin, suggests that the impact of global warming on marine microbes and their biogeochemical function will be more intense at higher latitudes. Our analysis predicts that 1°C ocean warming will result in increased biomass of heterotrophic prokaryoplankton only in waters with <26°C of mean annual surface temperature.


ABA signaling is necessary but not sufficient for RD29B transcriptional memory during successive dehydration stresses in Arabidopsis thaliana.
Plants subjected to a prior dehydration stress were seen to have altered transcriptional responses during a subsequent dehydration stress for up to 5 days after the initial stress. The abscisic acid (ABA) inducible RD29B gene of Arabidopsis thaliana was strongly induced after the first stress and displayed transcriptional memory with transcript levels nine-fold higher during the second dehydration stress. These increased transcript levels were due to an increased rate of transcription and are associated with an altered chromatin template during the recovery interval between the dehydration stresses. Here we use a combination of promoter deletion/substitutions, mutants in the trans-acting transcription factors and their upstream protein kinases, and treatments with exogenous ABA or dehydration stress to advance our understanding of the features required for transcriptional memory of RD29B. ABA Response Elements (ABREs) are sufficient to confer transcriptional memory on a minimal promoter, although there is a context effect from flanking sequences. Different mutations in Snf1 Related Protein Kinase 2 (SnRK2) genes positively and negatively affected the response, suggesting that this effect is important for transcriptional memory. Although exogenous ABA treatments could prime transcriptional memory, a second ABA treatment was not sufficient to activate transcriptional memory. Therefore, we concluded that transcriptional memory requires ABA and an ABA-independent factor that is induced or activated by a subsequent dehydration stress and directly or indirectly results in a more active RD29B chromatin template. These results advance our knowledge of the cis- and trans-acting factors that are required for transcriptional memory of RD29B.


Delayed egg-laying and shortened incubation duration of Arctic-breeding shorebirds coincide with climate cooling.
Biological impacts of climate change are exemplified by shifts in phenology. As the timing of breeding advances, the within-season relationships between timing of breeding and reproductive traits may change and cause long-term changes in the population mean value of reproductive traits. We investigated long-term changes in the timing of breeding and within-season patterns of clutch size, egg volume, incubation duration, and daily nest survival of three shorebird species between two decades. Based on previously known within-season patterns and assuming a warming trend, we hypothesized that the timing of clutch initiation would advance between decades and would be coupled with increases in mean clutch size, egg volume, and daily nest survival rate. We monitored 1,378 nests of western sandpipers, semipalmated sandpipers, and red-necked phalaropes at a subarctic site during 1993-1996 and 2010-2014. Sandpipers have biparental incubation, whereas phalaropes have uniparental incubation. We found an unexpected long-term cooling trend during the early part of the breeding season. Three species delayed clutch initiation by 5 days in the 2010s relative to the 1990s. Clutch size and daily nest survival showed strong within-season declines in sandpipers, but not in phalaropes. Egg volume showed strong within-season declines in one species of sandpiper, but increased in phalaropes. Despite the within-season patterns in traits and shifts in phenology, clutch size, egg volume, and daily nest survival were similar between decades. In contrast, incubation duration did not show within-season variation, but decreased by 2 days in sandpipers and increased by 2 days in phalaropes. Shorebirds demonstrated variable breeding phenology and incubation duration in relation to climate cooling, but little change in nonphenological components of traits. Our results indicate that the breeding phenology of shorebirds is closely associated with the temperature conditions on breeding ground, the effects of which can vary among reproductive traits and among sympatric species.


HuR stabilizes HTT mRNA via interacting with its exon 11 in a mutant HTT-dependent manner.
Huntington's Disease (HD) is a monogenetic neurodegenerative disorder mainly caused by the cytotoxicity of the mutant HTT protein (mHTT) encoded by the mutant HTT gene. Lowering HTT mRNA has been extensively studied as a potential therapeutic strategy, but how its level is regulated endogenously has been unclear. Here we report that the RNA-binding protein (RBP) HuR interacts with and stabilizes HTT mRNA in an mHTT-dependent manner. In HD cells but not wild-type cells, siRNA knockdown or CRISPR-induced heterozygous knockout of HuR decreased HTT mRNA stability. HuR interacted with HTT mRNA at a conserved site in exon 11 rather than the 3'-UTR region of the mRNA. Interestingly, this interaction was dependent on the presence of mHTT, likely via the activation of MAPK11, which enhanced cytosolic localization of the HuR protein. Thus, mHTT, MAPK11 and HuR may form a positive feedback loop that stabilizes HTT mRNA and enhances mHTT accumulation, which may contribute to HD progression. Our data reveal a novel regulatory mechanism of HTT mRNA via non-canonical binding of HuR.


Regulation of organelle movement in melanophores by protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2A (PP2A).
We used melanophores, cells specialized for regulated organelle transport, to study signaling pathways involved in the regulation of transport. We transfected immortalized Xenopus melanophores with plasmids encoding epitope-tagged inhibitors of protein phosphatases and protein kinases or control plasmids encoding inactive analogues of these inhibitors. Expression of a recombinant inhibitor of protein kinase A (PKA) results in spontaneous pigment aggregation. alpha-Melanocyte-stimulating hormone (MSH), a stimulus which increases intracellular cAMP, cannot disperse pigment in these cells. However, melanosomes in these cells can be partially dispersed by PMA, an activator of protein kinase C (PKC). When a recombinant inhibitor of PKC is expressed in melanophores, PMA-induced pigment dispersion is inhibited, but not dispersion induced by MSH. We conclude that PKA and PKC activate two different pathways for melanosome dispersion. When melanophores express the small t antigen of SV-40 virus, a specific inhibitor of protein phosphatase 2A (PP2A), aggregation is completely prevented. Conversely, overexpression of PP2A inhibits pigment dispersion by MSH. Inhibitors of protein phosphatase 1 and protein phosphatase 2B (PP2B) do not affect pigment movement. Therefore, melanosome aggregation is mediated by PP2A.


Assessing population health care need using a claims-based ACG morbidity measure: a validation analysis in the Province of Manitoba.
To assess the ability of an Adjusted Clinical Group (ACG)-based morbidity measure to assess the overall health service needs of populations. Data Sources/Study Setting. Three population-based secondary data sources: registration and health service utilization data from fiscal year 1995-1996; mortality data from vital statistics reports from 1996-1999; and Canadian census data. The study included all continuously enrolled residents in the universal health care plan in Manitoba. Using 60 small geographic areas as the units of analysis, we compared a population-based "ACG morbidity index," derived from individual ACG assignments in fiscal year 1995-1996, with the standardized mortality ratio (ages < 75 years) for 1996-1999. Key variables included a population-based socioeconomic status measure and age- and sex-standardized physician utilization ratios. The ACGs were assigned based on the complement of diagnoses assigned to persons on physician claims and hospital separation abstracts. The ACG index was created by weighting the ACGs using average health care expenditures. The ACG morbidity index had a strong positive linear relationship with the subsequent rate of premature death in the small areas of Manitoba. The ACG index was able to explain the majority of the relationships between mortality and both socioeconomic status and physician utilization. In Manitoba, ACGs are closely related to premature mortality, commonly accepted as the best single indicator for health service need in populations. Issues in applying ACGs in settings where needs adjustment is a primary objective are discussed.


[Recent approaches of quantification of interstitial fibrosis in renal transplantation].
Chronic allograft injury can be diagnosed early at a pre-clinical stage by its histopathological changes. Interstitial fibrosis (IF), one of its main histopathological features, is currently assessed by semi-quantitative analysis according to the Banff classification. Subjective interpretation by the pathologist is the main limiting factor of such a method. Different morphometric approaches have been used to quantify IF. Point counting methods have been published but their use is very tedious. Thus, semi-automatic computerized measurements of IF in biopsy specimens immunostained for collagen III have been proposed. Most authors used Sirius Red-stained tissue examined under polarized or non-polarized light. However, morphometric methods are time-consuming. Automatic color segmentation image analysis is a new, rapid, and robust method to quantify IF on renal biopsies stained by light green trichrome. It can be recommended as being reliable and reproducible for routine use. Methodology based on second harmonic generation microscopy allows specific quantitative imaging of interstitial fibrosis with high reproducibility and may bring complementary informations in the future. The prognostic value of quantitative image analysis might be increased by techniques addressing the dynamics of fibrosis matrix generation.


Mouse hepatitis virus is cleared from the central nervous systems of mice lacking perforin-mediated cytolysis.
Perforin-deficient [perforin (-/-)] mice were infected with two strains of JHM virus (JHMV) to analyze the role of perforin-mediated cytotoxicity in acute lethal and subacute central nervous system (CNS) infections. During both acute and subacute infections, the overall mortality of the perforin (-/-) mice was not different from that of the controls. Perforin (-/-) mice survived longer than the controls, consistent with reduced morbidity. Both strains of virus were cleared from the perforin (-/-) mice as in the controls; however, the rate of clearance was delayed in the perforin (-/-) mice, indicating that perforin-mediated cytolysis is involved in viral clearance. The absence of perforin-mediated cytolysis did not prevent encephalomyelitis or extensive demyelination. Cells undergoing apoptosis were detected in the CNS of both the perforin (-/-) and control groups, indicating that perforin is not essential for programmed cell death. Neutralizing antibodies were not detected in either group of mice until day 9 postinfection, when the majority of the virus had been cleared. These data further confirm the importance of cell-mediated cytotoxicity and suggest that additional components of the immune response contribute to the clearance of JHMV from the CNS.


Effect of particle size of bronchodilator aerosols on lung distribution and pulmonary function in patients with chronic asthma.
The particle size of bronchodilator aerosols may be important in determining the site of deposition in the lung and their therapeutic effect. The distribution of aerosols (labelled with technetium-99m diethylene triamine pentacetic acid) of two different particle sizes has been studied by gamma camera imaging. The particles had mass median aerodynamic diameters (geometric standard deviations) of 1.4 (1.4) and 5.5 (2.3) micron, and they were administered from a jet nebuliser to eight patients with chronic severe stable asthma. There was no significant difference in peripheral lung deposition with the two aerosols in any patient. The bronchodilator effect of the two aerosols was determined from cumulative dose-response studies. To avoid large doses that might mask possible differences in effect due to aerosol size, small, precisely determined incremental amounts of salbutamol (25-250 micrograms total lung dose) were used. The two doses were given via a nebuliser on separate occasions and the bronchodilator response was measured from FEV1, forced vital capacity, and peak expiratory flow 30 minutes after each dose. Bronchodilatation was similar with the two aerosols at each dose of salbutamol. There was therefore no difference in distribution within the lung or any difference in bronchodilator effect between an aerosol of small (1.4 micron) particle size and an aerosol of 5.5 microns in patients with severe but stable asthma.


BILIARY COMPLICATIONS AFTER LIVER TRANSPLANTATION.
Biliary reconstitution has been considered the Achilles's heel of liver transplantations due to its high rate of postoperative complications. To evaluate the risk factors for occurrence of biliary strictures and leakages, and the most efficient methods for their treatment. Of 310 patients who underwent liver transplantation between 2001 and 2015, 182 medical records were retrospectively analyzed. Evaluated factors included demographic profile, type of transplantation and biliary reconstitution, presence of vascular and biliary complications, their treatment and results. 153 (84.07%) deceased donor and 29 (15.93%) living donor transplantations were performed. Biliary complications occurred in 49 patients (26.92%): 28 strictures (15.38%), 14 leakages (7.7%) and seven leakages followed by strictures (3.85%). Hepatic artery thrombosis was present in 10 patients with biliary complications (20.4%; p=0,003). Percutaneous and endoscopic interventional procedures (including balloon dilation and stent insertion) were the treatment of choice for biliary complications. In case of radiological or endoscopic treatment failure, surgical intervention was performed (biliodigestive derivation or retransplantation (32.65%). Complications occurred in 25% of patients treated with endoscopic or percutaneous procedures and in 42.86% of patients reoperated. Success was achieved in 45% of patients who underwent endoscopic or percutaneous procedures and in 61.9% of those who underwent surgery. Biliary complications are frequent events after liver transplantation. They often require new interventions: endoscopic and percutaneous procedures at first and surgical treatment when needed. Hepatic artery thrombosis increases the number of biliary complications.


Site-specific GlcNAcylation of human erythrocyte proteins: potential biomarker(s) for diabetes.
O-linked N-acetylglucosamine (O-GlcNAc) is upregulated in diabetic tissues and plays a role in insulin resistance and glucose toxicity. Here, we investigated the extent of GlcNAcylation on human erythrocyte proteins and compared site-specific GlcNAcylation on erythrocyte proteins from diabetic and normal individuals. GlcNAcylated erythrocyte proteins or GlcNAcylated peptides were tagged and selectively enriched by a chemoenzymatic approach and identified by mass spectrometry. The enrichment approach was combined with solid-phase chemical derivatization and isotopic labeling to detect O-GlcNAc modification sites and to compare site-specific O-GlcNAc occupancy levels between normal and diabetic erythrocyte proteins. The enzymes that catalyze the cycling (addition and removal) of O-GlcNAc were detected in human erythrocytes. Twenty-five GlcNAcylated erythrocyte proteins were identified. Protein expression levels were compared between diabetic and normal erythrocytes. Thirty-five O-GlcNAc sites were reproducibly identified, and their site-specific O-GlcNAc occupancy ratios were calculated. GlcNAcylation is differentially regulated at individual sites on erythrocyte proteins in response to glycemic status. These data suggest not only that site-specific O-GlcNAc levels reflect the glycemic status of an individual but also that O-GlcNAc site occupancy on erythrocyte proteins may be eventually useful as a diagnostic tool for the early detection of diabetes.


Chromosome 2p shows significant linkage to antihypertensive response in the British Genetics of Hypertension Study.
There is a lack of consistently linked loci influencing blood pressure and hypertension status, and this may be because of genetic or phenotypic heterogeneity. We hypothesize that stratification of subjects by response to antihypertensive drug groups could be used to stringently define subsets that will have reduced genetic and etiologic heterogeneity, by partitioning contrasting mechanisms of hypertension and, thus, enhancing gene finding. We investigated the British Genetics of Hypertension Study population, which is composed of 2142 severely hypertensive white affected sibling pairs. Nonresponse to antihypertensive therapy was defined as an on-treatment blood pressure of >140/90 mm Hg or a difference between prediagnosis and on-treatment blood pressure of <20 mm Hg. Of the nonresponders, there were 89 sibling pairs (AB) who were both on antihypertensive therapy that inhibit the renin-angiotensin system (angiotensin-converting enzyme inhibitors, angiotensin II type-1 receptor blockers, or beta-blockers), and 76 sibling pairs (CD) who were both on drugs that do not (calcium channel blockers or diuretics). Nonparametric linkage analysis carried out using markers from a 10-cM genome scan and additional "grid tightening" markers showed significant linkage in the AB group on chromosome 2p (logarithm of odds=4.84 at 90.68 Kosambi cM) and suggestive linkage for the CD group on chromosome 10q (logarithm of odds=2.83 at 125.96 Kosambi cM). The AB linkage locus attained genomewide significance after simulation using 10,000 replicates (P=0.005). This locus may contain a gene for the salt-sensitive form of hypertension and/or a pharmacogenetic locus affecting drug response. We have demonstrated for the first time identification of a significant locus by partitioning different pathways of hypertension using drug response.


Factor V (Quebec): a bleeding diathesis associated with a qualitative platelet Factor V deficiency.
Studies were performed on a French-Canadian family afflicted with a bleeding disorder exhibiting an autosomal dominant inheritance pattern and a severe bleeding diathesis after trauma. Clinical laboratory coagulation tests were unimpressive; the only persistent abnormalities include mild thrombocytopenia and moderately reduced Factor V clotting activities. Some individuals had prolonged Stypven times when platelet-rich plasma was used, suggesting that their platelets could not support functional prothrombinase complex assembly. Detailed studies were performed by use of plasma and isolated, washed platelets from a sister and brother. Bioassay data indicate that both individuals had Factor V activities of approximately 40 and 36% of normal, respectively. A comparison of the Factor V radioimmunoassay and bioassay data on the brother's plasma indicated that the circulating amount of Factor V functional activity was low relative to Factor V antigen concentration (approximately 65-75%). In both individuals, the platelet Factor V functional activities were extremely low (2-4%) relative to antigen levels present as determined by radioimmunoassay. These discrepancies between Factor V activities and antigen concentration do not appear to be due to an unstable Factor V molecule or to the presence of a Factor V or Factor Va inhibitor or inactivator. Kinetics of prothrombin activation by use of purified clotting factors indicated that thrombin-activated platelets from both individuals supported prothrombinase complex assembly identical to controls in the presence of added purified Factor Va. Consequently, their bleeding diathesis appears to reflect their platelet, rather than their plasma, Factor V activity. These results suggest that platelet Factor V is an essential component in maintaining stable and prolonged hemostasis after trauma.


Induced differentiation in HT29, a human colon adenocarcinoma cell line.
The human colon adenocarcinoma cell line HT29 displays an undifferentiated phenotype under standard growth conditions. When these cells were cultured for 21 days and then treated with forskolin, most of the cells formed brush borders on their apical surfaces. Brush border formation was inhibited by cytochalasin D but not by colchicine. Colchicine, nocodazole and taxol were found to induce differentiation and apoptosis in HT29 cells. Differentiation was characterized by flattening of the cells, formation of brush borders on apical surfaces and tight junctions between adjacent cells. Apoptosis was characterized by detachment of round cells from the cell layer, condensation of nuclear DNA and annexin V binding to cell surfaces. Treatment with colchicine or forskolin induced the association of E-cadherin to the cytoskeleton fraction of subconfluent HT29 cells. This effect was less prominent in post confluent cells. Our data indicate that microtubule-interfering agents may serve as an important tool in the study of differentiation and apoptosis in intestinal carcinoma.


Fluorometric measurements of conformational changes in glutamate transporters.
Glutamate transporters remove glutamate from the synaptic cleft to maintain efficient synaptic communication between neurons and to prevent extracellular glutamate concentrations from reaching neurotoxic levels (1). It is thought that glutamate transporters mediate glutamate transport through a reaction cycle with conformational changes between the two major access states that alternatively expose glutamate-binding sites to the extracellular or to the intracellular solution. However, there is no direct real-time evidence for the conformational changes predicted to occur during the transport cycle. In the present study, we used voltage-clamp fluorometry to measure conformational changes in the neuronal excitatory amino acid transporter (EAAT) 3 glutamate transporter covalently labeled with a fluorescent reporter group. Alterations in glutamate and cotransported ion concentrations or in the membrane voltage induced changes in the fluorescence that allowed detection of conformational rearrangements occurring during forward and reverse transport. In addition to the transition between the two major access states, our results show that there are significant Na(+)-dependent conformational changes preceding glutamate binding. We furthermore show that Na(+) and H(+) are cotransported with glutamate in the forward part of the transport cycle. The data further suggest that an increase in proton concentrations slows the reverse transport of glutamate, which may play a neuro-protective role during ischemia.


Pharmacist-led interventions to reduce unplanned admissions for older people: a systematic review and meta-analysis of randomised controlled trials.
medication problems are thought to cause between 10 and 30% of all hospital admissions in older people. This systematic review aimed to evaluate the effectiveness of interventions led by hospital or community pharmacists in reducing unplanned hospital admissions for older people. eighteen databases were searched with a customised search strategy. Relevant websites and reference lists of included trials were checked. Randomised controlled trials were included that evaluated pharmacist-led interventions compared with usual care, with unplanned admissions or readmissions as an outcome. Two authors independently extracted data and assessed methodological quality. twenty-seven randomised controlled trials (RCTs) were identified; seven trials were excluded. The 20 included trials comprised 16 for older people and 4 for older people with heart failure. Interventions led by hospital pharmacists (seven trials) or community pharmacists (nine trials) did not reduce unplanned admissions in the older population (risk ratios 0.97 95% CI: 0.88, 1.07; 1.07 95% CI: 0.96, 1.20). Three trials in older people with heart failure showed that interventions delivered by a hospital pharmacist reduced the relative risk of admissions. However, these trials were heterogeneous in intensity and duration of follow-up. One trial had a high risk of bias. evidence from three randomised controlled trials suggests that interventions led by hospital pharmacists reduce unplanned hospital admissions in older patients with heart failure, although these trials were heterogeneous. Data from 16 trials do not support the concept that interventions led by hospital or community pharmacists for the general older population reduces unplanned admissions.


Factors predicting successful DNA recovery from archival cervical smear samples.
Polymerase chain reaction (PCR)-based DNA testing of archival cervical smear slides is a useful method of retrospectively establishing the presence of the human papillomavirus (HPV) in cervical cells. A cellular DNA recovery test is performed in parallel to HPV DNA testing to ensure that sufficient cells are present and purification of sample DNA has been successfully performed. Previous studies have not comprehensively assessed DNA recovery rates in slides older than 13 years. We undertook a study to determine the factors impacting DNA recovery in 436 UK slides dating from 11 to 33 years prior to testing. Overall, a low cellular DNA recovery success rate of 29% was obtained but a strong trend was observed with increasing recovery rates the older the slides (P < 0.001). Recovery rates increased from 22% in the most recent slides collected from 1988 to 1992, to 61% in the oldest slides, collected in 1970-72. It is likely that fixation compounds incorporating acetic acid, introduced in the UK through the 1980s, have compromised subsequent attempts at PCR amplification. These findings emphasize the importance of the original fixation method in the success of DNA recovery from archival smear samples.


The impact of heat exposure and repeated exercise on circulating stress hormones.
To determine if heat exposure alters the hormonal responses to moderate, repeated exercise, 11 healthy male subjects [age = 27.1 (3.0) years; maximal oxygen consumption, VO2max = 47.6 (6.2) ml x kg x min(-1); mean (SD)] were assigned to four different experimental conditions according to a randomized-block design. While in a thermoneutral (23 degrees C) or heated (40 degrees C, 30% relative humidity) climatic chamber, subjects performed either cycle ergometer exercise (two 30-min bouts at approximately 50% VO2max, separated by a 45-min recovery interval, CEx and HEx conditions), or remained seated for 3 h (CS and HS conditions). Blood samples were analyzed for various exercise stress hormones [epinephrine (E), norepinephrine (NE), dopamine, cortisol and human growth hormone (hGH)]. Passive heating did not alter the concentrations of any of these hormones significantly. During both environmental conditions, exercise induced significant (P < 0.001) elevations in plasma E, NE and hGH levels. At 23 degrees C during bout 1: E = 393 (199) pmol x l(-1) (CEx) vs 174 (85) pmol x l(-1) (CS), NE = 4593 (2640) pmol x l(-1) (CEx) vs 1548 (505) pmol x l(-1) (CS), and hGH = 274 (340) pmol x l(-1) (CEx)vs 64 (112) pmol x l(-1) (CS). At 40 degrees C, bout 1: E = 596 (346) pmol x l(-1) (HEx) vs 323 (181) pmol x l(-1) (HS), NE = 7789 (5129) pmol x l(-1) (HEx) vs 1527 (605) pmol x l(-1) (HS), and hGH = 453 (494) pmol x l(-1) (HEx) vs 172 (355) pmol x l(-1) (HS). However, concentrations of plasma cortisol were increased only in response to exercise in the heat [HEx = 364 (168) nmol x l(-1) vs HS = 295 (114) nmol x l(-1)]. Compared to exercise at room temperature, plasma levels of E, NE and cortisol were all higher during exercise in the heat (P < 0.001 in all cases). The repetition of exercise did not significantly alter the pattern of change in cortisol or hGH levels in either environmental condition. However, repetition of exercise in the heat increased circulatory and psychological stress, with significantly (P < 0.001) higher plasma concentrations of E and NE. These results indicate a differential response of the various stress hormones to heat exposure and repeated moderate exercise.


Eculizumab in Asian patients with anti-aquaporin-IgG-positive neuromyelitis optica spectrum disorder: A subgroup analysis from the randomized phase 3 PREVENT trial and its open-label extension.
Background Eculizumab, a terminal complement inhibitor, significantly reduced the risk of relapse compared with placebo in patients with anti-aquaporin-4 immunoglobulin G-positive (AQP4+) neuromyelitis optica spectrum disorder (NMOSD) in the PREVENT trial. We report efficacy and safety analyses in Asian patients in PREVENT and its open-label extension (OLE). Methods PREVENT was a double-blind, randomized, phase 3 trial. Patients with AQP4+ NMOSD were randomly assigned (2:1) to receive intravenous eculizumab (maintenance dose, 1200 mg/2 weeks) or placebo. Patients who completed PREVENT could receive eculizumab in an OLE. Analyses were performed in a prespecified subgroup of Asian patients. Results Of 143 patients enrolled, 52 (36.4%) were included in the Asian subgroup (eculizumab, n = 37; placebo, n = 15); 45 Asian patients received eculizumab in the OLE. Most Asian patients (86.5%) received concomitant immunosuppressive therapy. During PREVENT, one adjudicated relapse occurred in patients receiving eculizumab and six occurred in patients receiving placebo in the Asian subgroup (hazard ratio, 0.05; 95% confidence interval: 0.01-0.35; p = 0.0002). An estimated 95.2% of Asian patients remained relapse-free after 144 weeks of eculizumab treatment. Upper respiratory tract infections, headache, and nasopharyngitis were the most common adverse events with eculizumab in the Asian subgroup. Conclusion Eculizumab reduces the risk of relapse in Asian patients with AQP4+ NMOSD, with a benefit-risk profile similar to the overall PREVENT population. The benefits of eculizumab were maintained during long-term therapy. Clinical trial registration ClinicalTrials.gov identifiers: NCT01892345 (PREVENT); NCT02003144 (open-label extension).


Bone morphogenetic protein-2, -6, and -7 differently regulate osteogenic differentiation of human periodontal ligament stem cells.
The utility of adult stem cells for bone regeneration may be an attractive alternative in the treatment of extensive injury, congenital malformations, or diseases causing large bone defects. To create an environment that is supportive of bone formation, signals from molecules such as the bone morphogenetic proteins (BMPs) are required to engineer fully viable and functional bone. We therefore determined whether BMP-2, -6, and -7 differentially regulate the (1) proliferation, (2) mineralization, and (3) mRNA expression of bone/mineralized tissue associated genes of human periodontal ligament stem cells (hPDLSCs), which were obtained from periodontal ligament tissue of human impacted third molars. hPDLSCs from six participants were isolated and characterized using histochemical and immunohistochemical methods. A real-time cell analyzer was used to evaluate the effects of BMP-2, -6, and -7 on the proliferation of hPDLSCs. hPDLSCs were treated with Dulbecco's modified Eagle's medium containing different concentrations of BMP-2, -6, and -7 (10, 25, 50, 100 ng/mL) and monitored for 264 hours. After dose-response experiments, 50 and 100 ng/mL concentrations of BMPs were used to measure bone/mineralized tissue-associated gene expression. Type I collagen, bone sialoprotein, osteocalcin, osteopontin, and osteoblastic transcription factor Runx2 mRNA expression of hPDLSCs treated with BMP-2, -6, and -7, were evaluated using quantitative RT-PCR. Biomineralization of hPDLSCs was assessed using von Kossa staining. This study demonstrated that BMPs at various concentrations differently regulate the proliferation, mineralization, and mRNA expression of bone/mineralized tissue associated genes in hPDLSCs. BMPs regulate hPDLSC proliferation in a time and dose-dependent manner when compared to an untreated control group. BMPs induced bone/mineralized tissue-associated gene mRNA expression and biomineralization of hPDLSCs. The most pronounced induction occurred in the BMP-6 group in the biomineralization of the hPDLSCs. Our data suggest that BMP-2, -6, and -7 are potent regulators of hPDLSC gene expression and biomineralization. Employing BMPs with hPDLSCs isolated from periodontal ligament tissues provides a promising strategy for bone tissue engineering.


FGFR3 P250R mutation increases the risk of reoperation in apparent 'nonsyndromic' coronal craniosynostosis.
Many patients with a clinical diagnosis of "nonsyndromic" coronal craniosynostosis have been found to be heterozygous for the fibroblast growth factor receptor 3 (FGFR3) mutation Pro250Arg. The phenotype associated with this mutation is variable and lacks highly distinctive features, so it is difficult to diagnose on clinical examination alone. The authors present a retrospective study of 76 patients with isolated coronal synostosis who were operated on in a single dedicated craniofacial unit over 25 years. The authors investigated whether any single factor, including the presence of a FGFR3 Pro250Arg mutation, predisposed to an increased transcranial reoperation rate. Eight patients had repeat transcranial surgery for a functional indication. Heterozygosity for the FGFR3 Pro250Arg mutation, present in 29 patients in the cohort, was the only factor found to have a significant association (P=0.048) with the transcranial reoperation rate. Six patients (20.7%) with the mutation underwent reoperation on the basis of raised intracranial pressure, as compared with two patients (4.3%) without the mutation. This highlights the need for genetic analysis and long-term clinical follow-up in apparently "isolated" coronal synostosis.


A novel GPER antagonist protects against the formation of estrogen-induced cholesterol gallstones in female mice.
Many clinical studies and epidemiological investigations have clearly demonstrated that women are twice as likely to develop cholesterol gallstones as men, and oral contraceptives and other estrogen therapies dramatically increase that risk. Further, animal studies have revealed that estrogen promotes cholesterol gallstone formation through the estrogen receptor (ER) α, but not ERβ, pathway. More importantly, some genetic and pathophysiological studies have found that G protein-coupled estrogen receptor (GPER) 1 is a new gallstone gene, Lith18, on chromosome 5 in mice and produces additional lithogenic actions, working independently of ERα, to markedly increase cholelithogenesis in female mice. Based on computational modeling of GPER, a novel series of GPER-selective antagonists were designed, synthesized, and subsequently assessed for their therapeutic effects via calcium mobilization, cAMP, and ERα and ERβ fluorescence polarization binding assays. From this series of compounds, one new compound, 2-cyclohexyl-4-isopropyl-N-(4-methoxybenzyl)aniline (CIMBA), exhibits superior antagonism and selectivity exclusively for GPER. Furthermore, CIMBA reduces the formation of 17β-estradiol-induced gallstones in a dose-dependent manner in ovariectomized mice fed a lithogenic diet for 8 weeks. At 32 μg/day/kg CIMBA, no gallstones are found, even in ovariectomized ERα (-/-) mice treated with 6 μg/day 17β-estradiol and fed the lithogenic diet for 8 weeks. In conclusion, CIMBA treatment protects against the formation of estrogen-induced cholesterol gallstones by inhibiting the GPER signaling pathway in female mice. CIMBA may thus be a new agent for effectively treating cholesterol gallstone disease in women.


Surveillance of surgical site infection: more accurate definitions and intensive recording needed.
Surgical site infection (SSI) continues to be a burden on systems that deliver healthcare and on patients who suffer morbidity, and mortality, associated with this complication of medical intervention. Surveillance of SSI is often an integral part of organizational infection prevention and control activities, but unless post-discharge surveillance is carried out in a robust manner the data may be inaccurate and misleading. Coupled with a lack of robust application of definitions, variations in methods of case-finding and incomplete follow-up, the results may lead to a false sense of security or conversely cause unnecessary anxieties. Data from national surveillance schemes that purport to be suitable for benchmarking are often at odds with published rates from well-designed studies and the reasons for this should be examined. If benchmarking is truly desirable and if clinicians are to have confidence in the outputs, surveillance schemes should ensure that participating organizations adopt a consistent approach to definitions, case-finding methodologies following discharge, and to robust follow-up, to ensure that every opportunity is taken to maximize the return rate and enhance data validity.


Left ventricular systolic dysfunction, heart failure, and the risk of stroke and systemic embolism in patients with atrial fibrillation: insights from the ARISTOTLE trial.
We examined the risk of stroke or systemic embolism (SSE) conferred by heart failure (HF) and left ventricular systolic dysfunction (LVSD) in the Apixaban for Reduction in Stroke and Other Thromboembolic Events in Atrial Fibrillation Trial (ARISTOTLE), as well as the effect of apixaban versus warfarin. The risk of a number of outcomes, including the composite of SSE or death (to take account of competing risks) and composite of SSE, major bleeding, or death (net clinical benefit) were calculated in 3 patient groups: (1) no HF/no LVSD (n=8728), (2) HF/no LVSD (n=3207), and (3) LVSD with/without symptomatic HF (n=2736). The rate of both outcomes was highest in patients with LVSD (SSE or death 8.06; SSE, major bleeding, or death 10.46 per 100 patient-years), intermediate for HF but preserved LV systolic function (5.32; 7.24), and lowest in patients without HF or LVSD (1.54; 5.27); each comparison P<0.0001. Each outcome was less frequent in patients treated with apixaban: in all ARISTOTLE patients, the apixaban/warfarin hazard ratio for SSE or death was 0.89 (95% confidence interval, 0.81-0.98; P=0.02); for SSE, major bleed, or death it was 0.85 (0.78-0.92; P<0.001). There was no heterogeneity of treatment effect across the 3 groups. Patients with LVSD (with/without HF) had a higher risk of SSE or death (but similar rate of SSE) compared with patients with HF but preserved LV systolic function; both had a greater risk than patients without either HF or LVSD. Apixaban reduced the risk of both outcomes more than warfarin in all 3 patient groups. URL: http://www.clinicaltrials.gov. Unique identifier: NCT00412984.


The incidence of venous thromboembolic events in trauma patients after tranexamic acid administration: an EAST multicenter study.
To determine if there is a significant association between administration of tranexamic acid (TXA) in severely bleeding, injured patients, and venous thromboembolism (VTE), myocardial infarction (MI), or cerebrovascular accident (CVA). A multicenter, retrospective study was performed. Inclusion criteria were: age 18-80 years old and need for 5 units or more of blood in the first 24 h after injury. Exclusion criteria included: death within 24 h, pregnancy, administration of TXA more than 3 h following injury, and routine ultrasound surveillance for deep venous thrombosis. Incidence of VTE was the primary outcome. Secondary outcomes included MI, CVA, and death. A power analysis found that a total of 830 patients were needed to detect a true difference in VTE risk. 1333 patients (TXA = 887, No-TXA = 446 patients) from 17 centers were enrolled. There were no differences in age, shock index, Glasgow coma score, pelvis/extremity abbreviated injury score, or paralysis. Injury severity score was higher in the No-TXA group. Incidence of VTE, MI, or CVA was similar between the groups. The TXA group required significantly less transfusion (P < 0.001 for all products) and had a lower mortality [adjusted odds ratio 0.67 (95% confidence interval 0.45-0.98)]. Despite having a higher extremity/pelvis abbreviated injury score, results did not change when evaluating only patients with blunt injury. Use of TXA in bleeding, injured patients is not associated with VTE, MI, or CVA but is associated with a lower transfusion need and mortality.


Emerging roles for RNA polymerase II CTD in Arabidopsis.
Post-translational modifications of the carboxy-terminal domain of the largest subunit of RNA polymerase II (RNAPII CTD) provide recognition marks to coordinate recruitment of numerous nuclear factors controlling transcription, cotranscriptional RNA processing, chromatin remodeling, and RNA export. Compared with the progress in yeast and mammals, deciphering the regulatory roles of position-specific combinatorial CTD modifications, the so-called CTD code, is still at an early stage in plants. In this review, we discuss some of the recent advances in understanding of the molecular mechanisms controlling the deposition and recognition of RNAPII CTD marks in plants during the transcriptional cycle and highlight some intriguing differences between regulatory components characterized in yeast, mammals, and plants.


A randomized study of erythropoietin and granulocyte colony-stimulating factor (G-CSF) versus placebo and G-CSF for patients with Hodgkin's and non-Hodgkin's lymphoma undergoing autologous bone marrow transplantation.
Anemia is a universal finding in patients undergoing autologous bone marrow transplantation (BMT). Effective therapies to increase the number of autologous red blood cells could result in a lower morbidity and mortality associated with red blood cell transfusions. We examined whether the addition of erythropoietin (Epo) to intensive therapy supported by progenitor cell transplantation and granulocyte colony-stimulating factor (G-CSF) would result in a lower requirement for red blood cell transfusions. Thirty-five patients with lymphoma were randomized to receive Epo versus placebo. Epo (600 U/kg three times per week) or placebo was begun 3 weeks before administration of high-dose therapy. Epo was held during the week of the preparatory regimen, and restarted on the day after BMT. All patients also received G-CSF following BMT. No significant differences were noted between the two groups in terms of patient characteristics at pretreatment or post-BMT evaluation. There were no differences in the total number of red blood cell units transfused (median Epo: 8 v placebo: 6, P = .22) nor the number of platelet transfusions given (median Epo: 12 v placebo 5, P = .14). Engraftment of granulocytes (absolute neutrophil count > or = 500/microL) occurred in a median of 12 days (range, 9 to 33) for the patients receiving Epo and G-CSF, compared with a median of 10 days (range, 8 to 22) for those receiving placebo and G-CSF (P = .70). Likewise, there were no differences in the time to platelet count > or = 20,000/microL without further transfusions with a median of 22 days (range, 15 to 150+) for those receiving Epo and G-CSF compared with a median of 20 days (range, 11 to 54) for those patients receiving placebo and G-CSF (P = .28). The combination of G-CSF and Epo as administered in this study appears to be safe but does not result in an improvement in the total number of red blood cell transfusions or total number of single donor platelet units transfused.


The role of sleep quality on the relationship between posttraumatic stress symptoms and pain in women.
Pain frequently co-occurs with elevated posttraumatic stress symptoms (PTSS); women are at elevated risk for their co-occurrence. PTSS and pain are associated with poor sleep quality; yet, little research has examined how sleep impacts their co-occurrence. The current study examines the indirect role of sleep on the relationship between PTSS and pain. A community sample of 182 women completed psychometrically-sound questionnaires assessing PTSS, sleep quality, pain characteristics, depression and anxiety symptoms, and anxiety sensitivity. We examined how sleep quality impacted associations among PTSS and pain intensity and pain interference, while controlling for key psychological factors. Greater PTSS was associated with worse pain interference, and poor sleep quality had a significant indirect effect on this relationship. Sleep may represent a modifiable behavioral mechanism that contributes to the mutual maintenance of PTSS and pain in women. Future research is needed to further clarify the role of sleep quality in their co-occurrence.


Importance of the Ca2+-binding sites in the N-catalytic domain of a family I.3 lipase for activity and stability.
A family I.3 lipase from Pseudomonas sp. MIS38 (PML) contains three Ca(2+)-binding sites (Ca1-Ca3) in the N-catalytic domain. Of them, the Ca1 site is formed only in an open conformation. To analyze the role of these Ca(2+)-binding sites, three mutant proteins D157A-PML, D275A-PML and D337A-PML, which are designed to remove the Ca1, Ca2 and Ca3 sites, respectively, were constructed. Of them, the crystal structures of D157A-PML and D337A-PML in a closed conformation were determined. Both structures are nearly identical to that of the wild-type protein, except that the Ca3 site is missing in the D337A-PML structure. D157A-PML was as stable as the wild-type protein. Nevertheless, it exhibited little lipase and very weak esterase activities. D275A-PML was less stable than the wild-type protein by approximately 5 degrees C in T(1/2). It exhibited weak but significant lipase and esterase activities when compared with the wild-type protein. D337A-PML was also less stable than the wild-type protein by approximately 5 degrees C in T(1/2) but was fully active. These results suggest that the Ca1 site is required to make the active site fully open by anchoring lid 1. The Ca2 and Ca3 sites contribute to the stabilization of PML. The Ca2 site is also required to make PML fully active.


Blood vessels and the satellite cell niche.
The fate of stem cell is regulated by cues received from the surrounding area. Recently, the concept of "stem cell zone"--rather than a predefined niche--introduced the notion of dynamic and permanent interactions between stem cells and their microenvironment. In adult skeletal muscle, satellite cells are considered as the main stem cells responsible for muscle repair and maintenance. They are localized close to vessels regardless their state of activation and differentiation. Moreover, the number of satellite cells is positively correlated to the capillarization of the myofiber. Angiogenesis has been known for a long time to be essential for muscle repair. However, relationships between vessel cells and satellite/myogenic cells that govern myogenic cell expansion, myogenesis, and angiogenesis have been only recently investigated. In this chapter, we discuss the possible existence of a vascular amplifying/differentiating niche, in an attempt to reconciliate several recent observations showing that satellite/myogenic cells interact with various cell types during the time course of muscle regeneration. Indeed, endothelial cells (ECs) stimulate myogenic cell growth and, inversely, differentiating myogenic cells promote angiogenesis. However, stromal cells may also provide some proliferating or differentiating cues to satellite/myogenic cells in this vascular area. Although some molecular effectors have been identified, including growth factors and cytokines, molecular regulations that occur within this vascular amplifying/differentiating niche requires further investigation. At the end of muscle repair, maturation of newly formed vessels takes place. In this context, we discuss the potential quiescence niche of satellite cells and the specific role of periendothelial cells. Indeed, periendothelial cells promote the return to quiescence of a subset of satellite/myogenic cells and maintain their quiescence (through Angiopoietin-1/Tie-2 signaling). We ask to what extent the environment may control the fate choice of satellite/myogenic cells and we also question the "hypoxic niche" in skeletal muscle, such a quiescence niche having being observed in the bone marrow.