Structural Elucidation of Intact Rough-Type Lipopolysaccharides using Field Asymmetric Ion Mobility Spectrometry and Kendrick Mass Defect Plots.
Lipopolysaccharide (LPS) is a hallmark virulence factor of Gram-negative bacteria. It is a complex, structurally heterogeneous mixture due to variations in number, type, and position of its simplest units: fatty acids and monosaccharides. Thus, LPS structural characterization by traditional mass spectrometry (MS) methods is challenging. Here, we describe the benefits of field asymmetric ion mobility spectrometry (FAIMS) for analysis of intact R-type lipopolysaccharide complex mixture (lipooligosaccharide; LOS). Structural characterization was performed using Escherichia coli J5 (Rc mutant) LOS, a TLR4 agonist widely used in glycoconjugate vaccine research. FAIMS gas phase fractionation improved the (S/N) ratio and number of detected LOS species. Additionally, FAIMS allowed the separation of overlapping isobars facilitating their tandem MS characterization and unequivocal structural assignments. In addition to FAIMS gas phase fractionation benefits, extra sorting of the structurally related LOS molecules was further accomplished using Kendrick mass defect (KMD) plots. Notably, a custom KMD base unit of [NaH] created a highly organized KMD plot that allowed identification of interesting and novel structural differences across the different LOS ion families; i.e., ions with different acylation degrees, oligosaccharides composition, and chemical modifications. Defining the composition of a single LOS ion by tandem MS along with the organized KMD plot structural network was sufficient to deduce the composition of 179 LOS species out of 321 species present in the mixture. The combination of FAIMS and KMD plots allowed in-depth characterization of the complex LOS mixture and uncovered a wealth of novel information about its structural variations.


Exosomes and their implications in central nervous system tumor biology.
Exosomes are 20-100 nm cellular derived vesicles that upon discovery, were thought to be a form of cellular recycling of intracellular contents. More recently, these vesicles are under investigation for their purported significant roles in intercellular communication in both healthy and diseased states. Herein, we focus on the secretion of exosomes associated with glioblastoma, as most exosome studies on brain tumors have been performed in this tumor type. However, we included exosomes secreted from other forms of brain tumors for comparison as available. Exosomes contain intracellular content that can be transferred to other cells in the tumor or to cells of the immune system and endothelial cells. These recipient cells may subsequently take on oncogenic properties, including therapeutic resistance, cancer progression, and angiogenesis. Genetic components (DNA, RNA and miRNA) of the cell of origin may be included in the secreted exosomes. The presence of genetic material in the exosomes could serve as a biomarker for mutations in tumors, potentially leading to novel treatment strategies. In the last decade, exosomes have been identified as having a major impact on multiple aspects of medicine and tumor biology, and appear to be primed for a critical position in cancer diagnosis, prognosis, and treatment.


Comparative Study on Insall-Salvati Index by Radiological and Clinical Methods in a Tertiary Care Centre of North Bengal, India.
Patellar height is an important parameter of patellar stability. Its importance is evident from various attempts to measure it, by different scientists from time to time and many indices have been derived among which Insall-Salvati index (IS index) is the most simple, accurate and easy to measure. In this study a new clinical method has been tried to measure patellar height by applying the same principle of IS index. To compare clinical method of measurement of IS index with the one derived radiologically and to establish the higher cut off value of the clinical method. In the present study 186 knee joints (93 subjects) were subjected to measurement of patellar height by both clinical and radiological methods. The IS index was measured bilaterally from lateral view of X-ray plates and it was measured clinically in every knee joint. Both the values were compared using students t-test and higher cut off values were set for normal knee joint for the clinical method. Among 93 study participants majority were females (54.83%), age of the patients varied from 10-68 years with mean age of 28 years and Standard deviation (SD) of 16.4. It was found that, there was no statistically significant difference between the mean values obtained by clinical method of measurement, compared to conventional one (IS Index) for both the genders and age groups on both the sides. A cut off value of 0.98 cm by clinical method gives sensitivity of 80% and specificity of 36% with area under the ROC curve 0.596. The subjects with patellar height of less than cut off value by clinical method can avoid radiological investigation as there was no statistically significant difference of IS index between radiological and clinical methods.


Identification of protein arginine methyltransferase 2 as a coactivator for estrogen receptor alpha.
In an attempt to isolate cofactors capable of influencing estrogen receptor alpha (ERalpha) transcriptional activity, we used yeast two-hybrid screening and identified protein arginine methyltransferase 2 (PRMT2) as a new ERalpha-binding protein. PRMT2 interacted directly with three ERalpha regions including AF-1, DNA binding domain, and hormone binding domain in a ligand-independent fashion. The ERalpha-interacting region on PRMT2 has been mapped to a region encompassing amino acids 133-275. PRMT2 also binds to ERbeta, PR, TRbeta, RARalpha, PPARgamma, and RXRalpha in a ligand-independent manner. PRMT2 enhanced both ERalpha AF-1 and AF-2 transcriptional activity, and the potential methyltransferase activity of PRMT2 appeared pivotal for its coactivator function. In addition, PRMT2 enhanced PR, PPARgamma, and RARalpha-mediated transactivation. Although PRMT2 was found to interact with two other coactivators, the steroid receptor coactivator-1 (SRC-1) and the peroxisome proliferator-activated receptor-interacting protein (PRIP), no synergistic enhancement of ERalpha transcriptional activity was observed when PRMT2 was coexpressed with either PRIP or SRC-1. In this respect PRMT2 differs from coactivators PRMT1 and CARM1 (coactivator-associated arginine methyltransferase). These results suggest that PRMT2 is a novel ERalpha coactivator.


Behavioral and neurochemical analysis of ongoing bone cancer pain in rats.
Cancer-induced bone pain is described as dull, aching ongoing pain. Ongoing bone cancer pain was characterized after intratibial injection of breast cancer cells in rats. Cancer produced time-dependent bone remodeling and tactile hypersensitivity but no spontaneous flinching. Conditioned place preference (CPP) and enhanced dopamine (DA) release in the nucleus accumbens (NAc) shell was observed after peripheral nerve block (PNB) selectively in tumor-bearing rats revealing nociceptive-driven ongoing pain. Oral diclofenac reversed tumor-induced tactile hypersensitivity but did not block PNB-induced CPP or NAc DA release. Tumor-induced tactile hypersensitivity, and PNB-induced CPP and NAc DA release, was blocked by prior subcutaneous implantation of a morphine pellet. In sham rats, morphine produced a modest but sustained increase in NAc DA release. In contrast, morphine produced a transient 5-fold higher NAc DA release in tumor bearing rats compared with sham morphine rats. The possibility that this increased NAc DA release reflected the reward of pain relief was tested by irreversible blockade of rostral anterior cingulate cortex (rACC) μ-opioid receptors (MORs). The rACC MOR blockade prevented the morphine-induced transient increased NAc DA release in tumor bearing rats but did not affect morphine-induced effects in sham-operated animals. Consistent with clinical experience, ongoing cancer pain was controlled by morphine but not by a dose of diclofenac that reversed evoked hypersensitivity. Additionally, the intrinsic reward of morphine can be dissociated from the reward of relief of cancer pain by blockade of rACC MOR. This approach allows mechanistic and therapeutic assessment of ongoing cancer pain with likely translation relevance.


Expression-based GWAS identifies variants, gene interactions and key regulators affecting intramuscular fatty acid content and composition in porcine meat.
The aim of this work is to better understand the genetic mechanisms determining two complex traits affecting porcine meat quality: intramuscular fat (IMF) content and its fatty acid (FA) composition. With this purpose, expression Genome-Wide Association Study (eGWAS) of 45 lipid-related genes associated with meat quality traits in swine muscle (Longissimus dorsi) of 114 Iberian × Landrace backcross animals was performed. The eGWAS identified 241 SNPs associated with 11 genes: ACSM5, CROT, FABP3, FOS, HIF1AN, IGF2, MGLL, NCOA1, PIK3R1, PLA2G12A and PPARA. Three expression Quantitative Trait Loci (eQTLs) for IGF2, ACSM5 and MGLL were identified, showing cis-acting effects, whereas 16 eQTLs had trans regulatory effects. A polymorphism in the ACSM5 promoter region associated with its expression was identified. In addition, strong candidate genes regulating ACSM5, FOS, PPARA, PIK3R1, PLA2G12A and HIF1AN gene expression were also seen. Notably, the analysis highlighted the NR3C1 transcription factor as a strong candidate gene involved in the regulation of the 45 genes analysed. Finally, the IGF2, MGLL, MC2R, ARHGAP6, and NR3C1 genes were identified as potential regulators co-localizing within QTLs for fatness and growth traits in the IBMAP population. The results obtained increase our knowledge in the functional regulatory mechanisms involved in these complex traits.


Lifestyle and nickel allergy in a Swedish adolescent population: effects of piercing, tattooing and orthodontic appliances.
The aim of this study was to estimate the prevalence of life-style practices in adolescents and their association with nickel allergy. Upper secondary school pupils (n = 4,376; 15-23 years) were patch-tested for nickel aller-gy, follow-ing completion of a questionnaire (answered by 6,095). Almost 86% girls and 21% of boys reported piercing. More girls (6%) than boys (3%) had a tattoo. Twenty-six percent of the girls and 18% of the boys were regular smokers. Vegetarian/vegan diets were reported by 20% of girls and by 6% of boys. Piercing, female gender, and vocational programme increased the risk of nickel allergy, whereas orthodontic appliance treat-ment prior to piercing reduced the risk of nickel allergy. Pupils in vocational programmes had the highest prevalence of nickel allergy. Lifestyle behaviours are interconnected and cluster in subgroups of adolescents. Female sex, piercing and choice of educational programme are prominent lifestyle markers. A trend shift is observed, where more girls than boys report tattooing.


HLA-DQ β1 alleles associated with Epstein-Barr virus (EBV) infectivity and EBV gp42 binding to cells.
Epstein-Barr virus (EBV) infects B cells and ~95% of adults are infected. EBV glycoprotein gp42 is essential for entry of virus into B cells. EBV gp42 binds to the β1 chain of HLA-DQ, -DR, and -DP on B cells, and uses these molecules for infection. To investigate if certain HLA-DQ alleles are associated with EBV seronegativity, we recruited ~3,300 healthy adult blood donors, identified 106 EBV-seronegative individuals, and randomly selected a control group of EBV-seropositive donors from the donor pool. A larger than expected proportion of EBV-seronegative subjects were HLA-DQ β1 *04/*05 and *06/*06, and to a lesser extent, *02/*03, compared with the control group, while a larger than expected portion of EBV-seropositive persons were HLA-DQ β1 *02/*02. We examined the ability of EBV gp42 to bind to different HLA-DQ molecules using human and mouse cells stably expressing these alleles. EBV gp42 bound less effectively to cells expressing HLA-DQ β1 *04/*05, *06/*06, or *03/*03 than to cells expressing HLA-DQ β1 *02/*02. These data are consistent with our observations of increased EBV seronegativity with DQ β1 *04/*05 or *06/*06 alleles. These findings emphasize the importance of a single genetic locus (HLA-DQ β1) to influence infectivity with EBV.


Part IV-sorption of hydrophobic organic contaminants.
Behavior of hydrophobic organic contaminants (HOCs) in the environment has attracted research interest for more than three decades. It has been clearly concluded that humic substances (HSs), which are the main content of soil/sediment organic matter (SOM) and dissolved organic matter (DOM), controls the sorption of HOCs in soils/sediments. In order to predict the movement of HOCs in the environment, many studies have been conducted to relate HOCs sorption characteristics with HS chemical properties. However, no consensus has been reached on precisely what HS chemical properties regulate HOC sorption, indicating that other HS properties (besides chemical properties) may also control HOC-HS interactions. Increasing amounts of research reveal that SOM physical properties can affect the accessibility of HOCs to SOM sorption sites and thus are of great importance for altering HOC-SOM interactions. Therefore, different from the past reviews on HOCs sorption in soils/sediments, this current one emphasizes physical conformation of HSs for both solid and dissolved forms. SOM chemical properties such as aromatic content, aliphatic content, polarity, and molecular weight have all been reported to affect HOC sorption. No general model has been proposed to predict SOM sorption characteristics from any individual chemical properties. Physical conformations of both solid SOM and DOM are of great importance for altering HOC-SOM interactions. The terms of glassy and rubbery domains have been used to describe physical conformations of solid SOM, and efficiency of chemical oxidation and glassy-rubbery transition temperature are indirect methods to describe SOM rigidity. Various techniques and parameters have been employed to study DOM conformation, such as microscopic images, pyrene-probing hydrophobic region, surface tension, and zeta potential. However, DOM nonideal sorption properties are not properly regarded. HOC-DOM interactions are investigated using solubility enhancement, gas-phase partitioning, fluorescence quenching, and dialysis equilibration methods. The limitations of all the methods are discussed in this review. Relatively, a dialysis equilibration experiment is a better design to study the true HOC-DOM interactions. Physical conformation of SOM are of the same importance as, if not more important than, SOM chemical properties for HOC sorption. Although increasing amounts of research focus on SOM physical conformation regarding HOC sorption, proper mathematical description of its physical conformation and the relationship between SOM physical conformation and its sorption properties are still unclear. Quantitative characterization of SOM conformation regarding its sorption properties with HOCs is a topic worth of further research. The HOC-DOM interactions could not be adequately addressed because of the inappropriate research approach; thus, a reevaluation of HOC-DOM interactions is also required.


Putative Biomarkers of Environmental Enteric Disease Fail to Correlate in a Cross-Sectional Study in Two Study Sites in Sub-Saharan Africa.
Environmental enteric dysfunction (EED) is an elusive, inflammatory syndrome of the small intestine thought to be associated with enterocyte loss and gut leakiness and lead to stunted child growth. To date, the gold standard for diagnosis is small intestine biopsy followed by histology. Several putative biomarkers for EED have been proposed and are widely used in the field. Here, we assessed in a cross-sectional study of children aged 2-5 years for a large set of biomarkers including markers of protein exudation (duodenal and fecal alpha-1-antitrypsin (AAT)), inflammation (duodenal and fecal calprotectin, duodenal, fecal and blood immunoglobulins, blood cytokines, C-reactive protein (CRP)), gut permeability (endocab, lactulose-mannitol ratio), enterocyte mass (citrulline) and general nutritional status (branched-chain amino acids (BCAA), insulin-like growth factor) in a group of 804 children in two Sub-Saharan countries. We correlated these markers with each other and with anemia in stunted and non-stunted children. AAT and calprotectin, CRP and citrulline and citrulline and BCAA correlated with each other. Furthermore, BCAA, citrulline, ferritin, fecal calprotectin and CRP levels were correlated with hemoglobin levels. Our results show that while several of the biomarkers are associated with anemia, there is little correlation between the different biomarkers. Better biomarkers and a better definition of EED are thus urgently needed.


NF-kappa B-inducing kinase is a common mediator of IL-17-, TNF-alpha-, and IL-1 beta-induced chemokine promoter activation in intestinal epithelial cells.
IL-17 expression is restricted to activated T cells, whereas the IL-17R is expressed in a variety of cell types including intestinal epithelial cells. However, the functional responses of intestinal epithelial cells to stimulation with IL-17 are unknown. Moreover, the signal transduction pathways activated by the IL-17R have not been characterized. IL-17 induced NF-kappa B protein-DNA complexes consisting of p65/p50 heterodimers in the rat intestinal epithelial cell line IEC-6. The induction of NF-kappa B correlated with the induction of CXC and CC chemokine mRNA expression in IEC-6 cells. IL-17 acted in a synergistic fashion with IL-1 beta to induce the NF-kappa B site-dependent CINC promoter. Induction of the CINC promoter by IL-17 in IEC-6 cells was TNF receptor-associated factor-6 (TRAF6), but not TRAF2, dependent. Furthermore, IL-17 induction of the CINC promoter could be inhibited by kinase-negative mutants of NF-kappa B-inducing kinase and I kappa B kinase-alpha. In addition to activation of the NF-kappa B, IL-17 regulated the activities of extracellular regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases in IEC-6 cells. Whereas the IL-17-mediated activation of extracellular regulated kinase mitogen-activated protein kinases was mediated through ras, c-Jun N-terminal kinase activation was dependent on functional TRAF6. These data suggest that NF-kappa B-inducing kinase serves as the common mediator in the NF-kappa B signaling cascades triggered by IL-17, TNF-alpha, and IL-1 beta in intestinal epithelial cells.


Post-mortem imaging as an alternative to autopsy in the diagnosis of adult deaths: a validation study.
Public objection to autopsy has led to a search for minimally invasive alternatives. Imaging has potential, but its accuracy is unknown. We aimed to identify the accuracy of post-mortem CT and MRI compared with full autopsy in a large series of adult deaths. This study was undertaken at two UK centres in Manchester and Oxford between April, 2006, and November, 2008. We used whole-body CT and MRI followed by full autopsy to investigate a series of adult deaths that were reported to the coroner. CT and MRI scans were reported independently, each by two radiologists who were masked to the autopsy findings. All four radiologists then produced a consensus report based on both techniques, recorded their confidence in cause of death, and identified whether autopsy was needed. We assessed 182 unselected cases. The major discrepancy rate between cause of death identified by radiology and autopsy was 32% (95% CI 26-40) for CT, 43% (36-50) for MRI, and 30% (24-37) for the consensus radiology report; 10% (3-17) lower for CT than for MRI. Radiologists indicated that autopsy was not needed in 62 (34%; 95% CI 28-41) of 182 cases for CT reports, 76 (42%; 35-49) of 182 cases for MRI reports, and 88 (48%; 41-56) of 182 cases for consensus reports. Of these cases, the major discrepancy rate compared with autopsy was 16% (95% CI 9-27), 21% (13-32), and 16% (10-25), respectively, which is significantly lower (p<0·0001) than for cases with no definite cause of death. The most common imaging errors in identification of cause of death were ischaemic heart disease (n=27), pulmonary embolism (11), pneumonia (13), and intra-abdominal lesions (16). We found that, compared with traditional autopsy, CT was a more accurate imaging technique than MRI for providing a cause of death. The error rate when radiologists provided a confident cause of death was similar to that for clinical death certificates, and could therefore be acceptable for medicolegal purposes. However, common causes of sudden death are frequently missed on CT and MRI, and, unless these weaknesses are addressed, systematic errors in mortality statistics would result if imaging were to replace conventional autopsy. Policy Research Programme, Department of Health, UK.


Characterization of mechanisms positioning costimulatory complexes in immune synapses.
Small immunoglobulin superfamily (sIGSF) adhesion complexes form a corolla of microdomains around an integrin ring and secretory core during immunological synapse (IS) formation. The corolla recruits and retains major costimulatory/checkpoint complexes, such as CD28, making forces that govern corolla formation of particular interest. Here, we investigated the mechanisms underlying molecular reorganization of CD2, an adhesion and costimulatory molecule of the sIGSF family during IS formation. Computer simulations showed passive distal exclusion of CD2 complexes under weak interactions with the ramified F-actin transport network. Attractive forces between CD2 and CD28 complexes relocate CD28 from the IS center to the corolla. Size-based sorting interactions with large glycocalyx components, such as CD45, or short-range CD2 self-attraction successfully explain the corolla 'petals.' This establishes a general simulation framework for complex pattern formation observed in cell-bilayer and cell-cell interfaces, and the suggestion of new therapeutic targets, where boosting or impairing characteristic pattern formation can be pivotal.


The effect of a hospital nurse staffing mandate on patient health outcomes: evidence from California's minimum staffing regulation.
We evaluate the impact of California Assembly Bill 394, which mandated maximum levels of patients per nurse in the hospital setting. When the law was passed, some hospitals already met the requirements, while others did not. Thus changes in staffing ratios from the pre- to post-mandate periods are driven in part by the legislation. We find persuasive evidence that AB394 had the intended effect of decreasing patient/nurse ratios in hospitals that previously did not meet mandated standards. However, these improvements in staffing ratios do not appear to be associated with relative improvements in measured patient safety in affected hospitals.


Effects of green tea supplementation on inflammation markers, antioxidant status and blood pressure in NaCl-induced hypertensive rat model.
Background: Recent studies indicate the important role of chronic inflammation and oxidative stress in the pathogenesis of hypertension. Green tea, due to the high content of catechins, shows high antioxidant activity. Objective: To determine the effect of supplementation with green tea extract on the blood pressure, on the concentration of selected parameters of inflammation and antioxidant status in the model of high-sodium-diet induced hypertension. Design: The study lasted 42 days. The experimental population consisted of 30 rats. The rats were divided into three groups. The rats in the control group were fed a standard diet with 35 g of NaCl per kg of diet, in the second group hypertensive rats were fed a standard diet with NaCl (35 g/kg diet) and with an extract of green tea (2 g/kg diet). The third group consisted of hypertensive rats fed a standard diet with NaCl (35 g/kg diet), and 4 g of green tea extract/kg diet. Results: Supplementation with green tea had no effect on body mass of rats on a high-sodium diet. At the end of the experiment systolic blood pressures in SH2 and SH4 groups were significantly lower than in the control group SK. The SH4 group was characterized by a significantly lower diastolic blood pressure value and concentration of TNF-α in comparison to the SK group. The rats from both SH2 and SH4 groups were characterized by higher total antioxidant status values compared to the control group. Discussion: The mechanism of the beneficial effects of green tea on blood pressure is not clear, but it is believed that it is related to its omnidirectional properties. Conclusions: Supplementation of green tea has a beneficial effect on blood pressure, markers of inflammation and antioxidant status in an experimental model of hypertension.


DNA polymerase III requirement for repair of DNA damage caused by methyl methanesulfonate and hydrogen peroxide.
The pcbA1 mutation allows DNA replication dependent on DNA polymerase I at the restrictive temperature in polC(Ts) strains. Cells which carry pcbA1, a functional DNA polymerase I, and a temperature-sensitive DNA polymerase III gene were used to study the role of DNA polymerase III in DNA repair. At the restrictive temperature for DNA polymerase III, these strains were more sensitive to the alkylating agent methyl methanesulfonate (MMS) and hydrogen peroxide than normal cells. The same strains showed no increase in sensitivity to bleomycin, UV light, or psoralen at the restrictive temperature. The sensitivity of these strains to MMS and hydrogen peroxide was not due to the pcbAl allele, and normal sensitivity was restored by the introduction of a chromosomal or cloned DNA polymerase III gene, verifying that the sensitivity was due to loss of DNA polymerase III alpha-subunit activity. A functional DNA polymerase III is required for the reformation of high-molecular-weight DNA after treatment of cells with MMS or hydrogen peroxide, as demonstrated by alkaline sucrose sedimentation results. Thus, it appears that a functional DNA polymerase III is required for the optimal repair of DNA damage by MMS or hydrogen peroxide.


A clinical score (RAPID) to identify those at risk for poor outcome at presentation in patients with pleural infection.
Pleural infection is associated with a high morbidity and mortality. Development of a validated clinical risk score at presentation to identify those at high risk of dying would enable patient triage and may help formulate early management strategies. A clinical risk score was derived based on data from patients entering the multicenter UK pleural infection trial (first Multicenter Intrapleural Sepsis Trial [MIST1], n=411). From 22 baseline clinical characteristics, model selection was undertaken to find variables predictive of poor clinical outcome. Outcomes were mortality at 3 months (primary), need for surgical intervention at 3 months, and time from randomization to discharge. The derived scoring system RAPID (renal, age, purulence, infection source, and dietary factors) was validated using patients enrolled in the subsequent MIST2 trial (n=191). Age, urea, albumin, hospital-acquired infection, and nonpurulence predicted poor outcome. Patients were stratified into low-risk (0-2), medium-risk (3-4), and high-risk (5-7) groups. Using the low-risk group as a reference, a RAPID score of 3 to 4 and >4 was associated with an OR of 24.4 (95% CI, 3.1-186.7; P=.002) and 192.4 (95% CI, 25.0-1480.4; P<.001), respectively, for death at 3 months. In the validation cohort (MIST2), a medium-risk RAPID score was nonsignificantly associated with mortality (OR, 3.2; 95% CI, 0.8-13.2; P=.11), and a high-risk score was associated with increased mortality (OR, 14.1; 95% CI, 3.5-56.8; P<.001). Hospitalization duration was associated with increasing RAPID score (score 0-2: median duration=7, interquartile range 6-13; score>5: median duration=15, interquartile range 9-28, P=.08). The RAPID score may permit risk stratification of patients with pleural infection at presentation.


Pain-related anxiety mediates the relationship between depressive symptoms and pain interference in veterans with hepatitis C.
Depression and chronic pain are common in persons chronically infected with the hepatitis C virus (HCV), although little is known about the rate of co-occurrence or mechanisms by which they are associated. We evaluated whether pain-related anxiety mediates the relationship between depressive symptoms and pain-related physical functioning in patients with HCV. Patients with HCV (n=175) completed self-report measures assessing demographic characteristics, pain-related function and mental health. Path analyses examined direct effects of cognitive-affective and somatic symptoms of depression on pain interference and indirect effects of these relationships via four subscales of the Pain Anxiety Symptoms Scale-20. Cognitive-affective and somatic symptoms of depression were positively and significantly related to pain interference. Pain-related anxiety mediated the relationship between both cognitive-affective and somatic symptoms of depression, and this mediation was predominantly accounted for by the escape-avoidance component of pain-related anxiety. Findings indicate a potential mediating role of pain-related anxiety, particularly escape-avoidance anxiety, on the relationship between depression and pain interference in patients with HCV. These findings suggest that escape-avoidance anxiety may be a particularly germane target for treatment in patients with HCV and chronic pain, particularly when depression, with characteristic features of withdrawal and inhibition, is a comorbid condition.


Role of fibroblasts in the regulation of proinflammatory interleukin IL-1, IL-6 and IL-8 levels induced by keratinocyte-derived IL-1.
In the epidermis, the keratinocytes are the first cells to be encountered by external stimuli and they are able to promote the inflammatory response by increased production and release of various cytokines. In their turn, these cytokines may directly affect the production of proinflammatory cytokines in human dermal fibroblasts. In addition, in both epithelial and mesenchymal cells cytokine production may be modulated by their mutual interaction, and thereby regulate the inflammatory response. The present study aimed to examine the role of fibroblasts in the regulation of proinflammatory IL-1, IL-6 and IL-8 levels induced by keratinocyte-derived IL-1. The data show that in fibroblasts exposed to conditioned media derived from cultures of normal human keratinocytes or squamous carcinoma cells (SCC-4), both the IL-8 and IL-6 mRNA expression as well as protein production were elevated. In addition, it was shown that these effects were induced by IL-1 alpha. The IL-1 alpha-induced increase in IL-8 and IL-6 production, both on the protein level as well as on the mRNA level, were concentration dependent and occurred almost simultaneously. While the induction of IL-6 and IL-8 occurred simultaneously, the IL-6 mRNA remained elevated for longer. In contrast to increased IL-6 and IL-8 production the IL-1 alpha levels markedly decreased upon culturing of fibroblasts in keratinocyte-derived conditioned medium. From internalization experiments it could be concluded that binding of IL-1 to IL-1 receptors, and its subsequent internalization and intracellular degradation is the most likely mechanism involved in the reduction of IL-1 levels by fibroblasts. Comparing the rate of IL-1 reduction in the presence of various cell types indicated that the rate of IL-1 reduction is directly related to the number of IL-1 receptors found on these cell types. In conclusion, these results indicate that the release of IL-1 alpha by activated keratinocytes may act as an inducer of IL-8 and IL-6 production in neighbouring fibroblasts. This may be an important pathway for the amplification of the inflammatory response. The amounts of both cytokines produced by fibroblasts were at least two to three orders of magnitude higher than those produced by keratinocytes, suggesting an important role of fibroblasts in the general inflammatory response. Furthermore, fibroblasts might be involved in turning off this inflammatory response by reducing IL-1 levels, most likely via IL-1 receptor-mediated uptake.


Hypoglycemia and Risk Factors for Death in 13 Years of Pediatric Admissions in Mozambique.
Hypoglycemia is a life-threatening complication of several diseases in childhood. We describe the prevalence and incidence of hypoglycemia among admitted Mozambican children, establishing its associated risk factors. We retrospectively reviewed clinical data of 13 years collected through an ongoing systematic morbidity surveillance in Manhiça District Hospital in rural Mozambique. Logistic regression was used to identify risk factors for hypoglycemia and death. Minimum community-based incidence rates (MCBIRs) for hypoglycemia were calculated using data from the demographic surveillance system. Of 49,089 children < 15 years hospitalized in Manhiça District Hospital, 45,573 (92.8%) had a glycemia assessment on admission. A total of 1,478 children (3.2%) presented hypoglycemia (< 3 mmol/L), of which about two-thirds (972) were with levels < 2.5 mmol/L. Independent risk factors for hypoglycemia on admission and death among hypoglycemic children included prostration, unconsciousness, edema, malnutrition, and bacteremia. Hypoglycemic children were significantly more likely to die (odds ratio [OR] = 7.11; P < 0.001), with an associated case fatality rate (CFR) of 19.3% (245/1,267). Overall MCBIR of hypoglycemia was 1.57 episodes/1,000 child years at risk (CYAR), significantly decreasing throughout the study period. Newborns showed the highest incidences (9.47 episodes/1,000 CYAR, P < 0.001). Hypoglycemia remains a hazardous condition for African children. Symptoms and signs associated to hypoglycemia should trigger the verification of glycemia and the implementation of life-saving corrective measures.


Workshop 8 (synthesis): challenges of expanding ecological sanitation into urban areas.
After seven papers discussing specific examples of ecological sanitation projects, the workshop considered the background, motivation and feasibility of this technology. It was agreed that ecological sanitation could help tackle water scarcity, health protection and, by recycling water and nutrients into agriculture, food scarcity. Dense cities are probably unsuitable for its implementation, but rural, town and peri-urban areas are promising. Key issues that remains are: the microbiological and chemical risks associated with the technology; the need for major information, education and support programmes; effective linking to reuse schemes; tailoring systems to meet local physical, socio-economic and cultural circumstances.


Prevention of opportunistic infections in HIV infection by pentoxiphylline.
Levels of tumour necrosis factor (TNF) are increased in patients with HIV infection leading to increased apoptosis and reduced CD4 cell life. Pentoxiphylline is a TNF inhibitor with properties that might make it useful for the treatment of HIV infection. These include improved cell mediated immunity and inhibition of viral replication. We carried out this study to determine the therapeutic utility of pentoxiphylline in improving constitutional manifestations, preventing opportunistic infections and sustaining CD4 counts among asymptomatic HIV infected individuals (i.e., those with no opportunistic infection). Individuals with HIV infection who were over 18 yr of age and free of opportunistic infections were recruited in the study and followed up 4 weekly. CD4 counts were measured using a flowcytometer using anti-human CD4 intervals. Pentoxiphylline was prescribed in a dose of 400 mg thrice daily. Thirty three (18 males) patients with HIV infection were studied. During their follow up (mean 12.5 +/- 5.6 months) one patient each developed cryptococcal meningitis and fibrocavitary tuberculosis. Weight increased from 51.3 +/- 7.4 kg at baseline to 55.3 +/- 7.4 kg (P<0.05). Malaise, fatigue and appetite improved in all those with these complaints, except the two with opportunistic infections. Mean CD4 counts were 184 +/- 36.4/microl at baseline and increased to 210 +/- 28.6/microl3 at four weeks (P<0.05). The patients had stable CD4 counts over the follow up period since then, i.e., within 25 per cent of the previous levels. Pentoxiphylline therapy in HIV infected individuals, who were free of opportunistic infections, improved their body weight, minimized opportunistic infections, increased and sustained CD4 counts. Given the low cost of the drug it could be recommended for the use in individuals who are at a high risk of developing opportunistic infections.


Co-relationship between sexual dysfunction and high-risk sexual behavior in patients receiving buprenorphine and naltrexone maintenance therapy for opioid dependence.
People suffering from substance dependence suffer from various sexual dysfunctions and are at risk for indulging in various high-risk sexual behaviors and thus are vulnerable to acquire various infections such as HIV/AIDS and other sexually transmitted infections. The aim of the study was to evaluate the correlation between sexual dysfunction and high-risk sexual behavior in opioid-dependent men receiving buprenorphine and naltrexone maintenance therapy. Semi-structured questionnaire, brief male sexual functioning inventory and HIV-risk taking behavior scale was administered to a sample of 60 sexually active men, receiving buprenorphine (n = 30) and naltrexone (n = 30) maintenance therapy for opioid dependence. The main outcomes are correlation between severity of sexual dysfunction and HIV-risk taking behavior. The study results showed 83% of the men on buprenorphine and 90% on naltrexone reported at least one of the sexual dysfunction symptoms. There was a negative correlation between sexual dysfunction and HIV-risk taking behavior that suggest severe the dysfunction, higher the risk taking behavior. Significant correlation was present with overall sexual dysfunction and HIV-risk taking behavior (P = 0.028 and in naltrexone receiving group premature ejaculation versus HIV-risk taking behavior however, (P = 0.022, P < 0.05) there were no significant differences among both the groups except above findings. Conclusion was treatment is associated with sexual dysfunctions and HIV-risk taking behavior, which has clinical implication. Future research should explore this further using biochemical analyses.


Interaction of heptanol and pressure on sodium and chloride transport by toad skin.
We examined the interaction of heptanol and hydrostatic pressure on Na+ and Cl- transport in isolated toad skin. In the presence of Cl-, heptanol decreased short-circuit current (Isc) and total transepithelial resistance (Rt). However, in the absence of Cl- in the mucosal bath, heptanol increased Rt, although it retained the same inhibitory effect on Isc. When transepithelial active Na+ transport was blocked by amiloride, heptanol had no effect on Isc whether or not Cl- was present, whereas it decreased the shunt resistance (Rs) only in the presence of Cl- in the mucosal bath. Moreover, this effect of heptanol on Rs was significantly smaller in the presence of diphenylamine-2-carboxylate (DPC), a known Cl- channel blocker. Pressure also decreased Isc through inhibition of active Na+ transport, but it increased Rs. When heptanol and pressure were applied together, their inhibitory effects on Isc were additive, but their effects on Rs were antagonistic. Furthermore, when a transepithelial Cl- current was produced by reducing the Cl- concentration of the serosal bath, heptanol stimulated this current, which was reversibly inhibited by pressure or DPC addition to the mucosal bath. When the heptanol-stimulated Cl- current was first inhibited by pressure, subsequent DPC addition had less or no effect. These results suggest that one site of an antagonistic interaction of heptanol and pressure in toad skin is an apical membrane Cl- conductance.


Similar Squamous Cell Carcinoma Epithelium microRNA Expression in Never Smokers and Ever Smokers.
The incidence of oral tumors in patients who never used mutagenic agents such as tobacco is increasing. In an effort to better understand these tumors we studied microRNA (miRNA) expression in tumor epithelium of never tobacco users, tumor epithelium of ever tobacco users, and nonpathological control oral epithelium. A comparison of levels among 372 miRNAs in 12 never tobacco users with oral squamous cell carcinoma (OSCC) versus 10 healthy controls was made using the reverse transcription quantitative polymerase chain reaction. A similar analysis was done with 8 ever tobacco users with OSCC. These comparisons revealed miR-10b-5p, miR-196a-5p, and miR-31-5p as enriched in the tumor epithelium in OSCC of both never and ever tobacco users. Examination of The Cancer Genome Atlas (TCGA) project miRNA data on 305 OSCCs and 30 controls revealed 100% of those miRNAs enriched in never smoker OSCCs in this patient group were also enriched in ever smoker OSCCs. Nonsupervised clustering of TCGA OSCCs was suggestive of two or four subgroups of tumors based on miRNA levels with limited evidence for differences in tobacco exposure among the groups. Results from both patient groups together stress the importance of miR196a-5p in OSCC malignancy in both never and ever smokers, and emphasize the overall similarity of miRNA expression in OSCCs in these two risk groups. It implies that there may be great similarity in etiology of OSCC in never and ever smokers and that classifying OSCC based on tobacco exposure may not be helpful in the clinic.


Alzheimer's disease beta-secretase BACE1 is not a neuron-specific enzyme.
The brains of Alzheimer's disease (AD) patients are morphologically characterized by neurofibrillar abnormalities and by parenchymal and cerebrovascular deposits of beta-amyloid peptides. The generation of beta-amyloid peptides by proteolytical processing of the amyloid precursor protein (APP) requires the enzymatic activity of the beta-site APP cleaving enzyme 1 (BACE1). The expression of this enzyme has been localized to the brain, in particular to neurons, indicating that neurons are the major source of beta-amyloid peptides in brain. Astrocytes, on the contrary, are known to be important for beta-amyloid clearance and degradation, for providing trophic support to neurons, and for forming a protective barrier between beta-amyloid deposits and neurons. However, under certain conditions related to chronic stress, the role of astrocytes may not be beneficial. Here we present evidence demonstrating that astrocytes are an alternative source of BACE1 and therefore may contribute to beta-amyloid plaque formation. While resting astroyctes in brain do not express BACE1 at detectable levels, cultured astrocytes display BACE1 promoter activity and express BACE1 mRNA and enzymatically active BACE1 protein. Additionally, in animal models of chronic gliosis and in brains of AD patients, there is BACE1 expression in reactive astrocytes. This would suggest that the mechanism for astrocyte activation plays a role in the development of AD and that therapeutic strategies that target astrocyte activation in brain may be beneficial for the treatment of AD. Also, there are differences in responses to chronic versus acute stress, suggesting that one consequence of chronic stress is an incremental shift to different phenotypic cellular states.


Duplex-Specific Nuclease-Amplified Detection of MicroRNA Using Compact Quantum Dot-DNA Conjugates.
Advances in nanotechnology have provided new opportunities for the design of next-generation nucleic acid biosensors and diagnostics. Indeed, combining advances in functional nanoparticles, DNA nanotechnology, and nuclease-enzyme-based amplification can give rise to new assays with advantageous properties. In this work, we developed a microRNA (miRNA) assay using bright fluorescent quantum dots (QDs), simple DNA probes, and the enzyme duplex-specific nuclease. We employed an isothermal target-recycling mechanism, where a single miRNA target triggers the cleavage of many DNA signal probes. The incorporation of DNA-functionalized QDs enabled a quantitative fluorescent readout, mediated by Förster resonance energy transfer (FRET)-based interaction with the DNA signal probes. Our approach splits the reaction in two, performing the enzyme-mediated amplification and QD-based detection steps separately such that each reaction could be optimized for performance of the active components. Target recycling gave ca. 3 orders of magnitude amplification, yielding highly sensitive detection with a limit of 42 fM (or 1.2 amol) of miR-148, with excellent selectivity versus mismatched sequences and other miRNAs. Furthermore, we used an alternative target (miR-21) and FRET pair for direct and absolute quantification of miR-21 in RNA extracts from human cancer and normal cell lines.


Structure and regulation of the human Nek2 centrosomal kinase.
The dimeric Ser/Thr kinase Nek2 regulates centrosome cohesion and separation through phosphorylation of structural components of the centrosome, and aberrant regulation of Nek2 activity can lead to aneuploid defects characteristic of cancer cells. Mutational analysis of autophosphorylation sites within the kinase domain identified by mass spectrometry shows a complex pattern of positive and negative regulatory effects on kinase activity that are correlated with effects on centrosomal splitting efficiency in vivo. The 2.2-A resolution x-ray structure of the Nek2 kinase domain in complex with a pyrrole-indolinone inhibitor reveals an inhibitory helical motif within the activation loop. This helix presents a steric barrier to formation of the active enzyme and generates a surface that may be exploitable in the design of specific inhibitors that selectively target the inactive state. Comparison of this "auto-inhibitory" conformation with similar arrangements in cyclin-dependent kinase 2 and epidermal growth factor receptor kinase suggests a role for dimerization-dependent allosteric regulation that combines with autophosphorylation and protein phosphatase 1c phosphatase activity to generate the precise spatial and temporal control required for Nek2 function in centrosomal maturation.


The role and effectiveness of computer-assisted learning in physical therapy education: a systematic review.
Physical therapy educators seek teaching methodologies to efficiently educate students. The purpose of this article was to perform a systematic review of the literature pertaining to the use and effectiveness of computer-assisted learning (CAL) in physical therapy education. Criteria were established for article selection. Articles related to use included those reporting at least a 30% survey response return and a sample of at least 50. Articles regarding effectiveness included only randomized control studies related to use in physical therapy and physical therapy assistant education. Six articles related to the use and 17 articles related to the effectiveness were identified. Early published investigations were focused in the area of anatomy. The survey studies related to use indicated that educators highly valued CAL, but they reported initial use to be low due to cost. Expanding software development has resulted in higher contemporary use. CAL can effectively convey content material compared to traditional methods of instruction. CAL is largely underresearched in the field of physical therapy compared to other health professions. Recommendations for future research include larger studies, broader representation of the practice field, and development of interactive programming.


Low-grade clear cell renal cell carcinoma mimicking hemangioma of the kidney: a series of 4 cases.
Clear cell renal cell carcinoma (CCRCC) has a rich, sinusoid-like vascularity frequently used as a diagnostic criterion. CCRCC with predominantly vascular architecture has not been described. To describe 4 unusual CCRCC cases, primarily presenting with hemangioma-like morphologic pattern. Clinicopathologic and selected immunohistochemical analysis of 4 cases of CCRCC mimicking hemangioma. Cases were seen in 1 woman and 3 men (average age, 48.8 years; range, 40-66 years). Grossly, tumors were red-brown (3 of 4) with scant bright-yellow foci in 1. The average tumor size was 4 cm (range, 2.5-5.5 cm). Microscopically, all were composed of varying proportions of a rich, arborizing, sinusoid-like vasculature with focal hobnail appearance of endothelial cells. Entrapment of renal tubules between blood vessels was seen at the periphery of the tumors. This morphology was reminiscent of anastomosing hemangioma. Isolated tumor cells resembling lymphocytes with clear halos were sparsely interspersed between vessels. Cytokeratin immunostain confirmed the diagnosis of CCRCC. Extensive sampling and immunohistochemical workup of what is deemed to be a benign vascular neoplasm of the kidney is needed to rule out the presence of individual carcinoma cells or small viable carcinoma cell clusters.


Preoperative diagnosis of vagal and sympathetic cervical schwannomas based on radiographic findings.
OBJECTIVE Vagus nerve and sympathetic chain cervical schwannomas (VNCSs and SCCSs) are benign nerve sheath tumors that arise in the head and neck. Despite similar presentations that make accurate preoperative diagnosis more difficult, the potential for morbidity following resection is significantly higher for patients with VNCS. Therefore, the authors analyzed a retrospective case series and performed a comparative analysis of the literature to establish diagnostic criteria to facilitate more accurate preoperative diagnoses. METHODS The authors conducted a blinded review of imaging studies from retrospectively collected, operatively confirmed cases of VNCS and SCCS. They also performed a systematic review of published series that reported patient-specific preoperative imaging findings in VNCS or SCCS. RESULTS Nine patients with VNCS and 11 with SCCS were identified. In the study cohort, splaying of the internal carotid artery (ICA) and internal jugular vein (IJV) did not significantly predict the nerve of origin (p = 0.06); however, medial and lateral ICA displacement were significantly associated with VNCS and SCCS, respectively (p = 0.01 and p = 0.003, respectively). Multivariate analysis demonstrated that ICA and IJV splaying with medial ICA displacement carried an 86% probability of VNCS (p = 0.001), while the absence of splaying with lateral ICA displacement carried a 91% probability of SCCS (p = 0.006). The presence of vocal cord symptoms or peripheral enhancement significantly augmented the predictive probability of VNCS, as did Horner's syndrome or homogeneous enhancement for SCCS. A review of the literature produced 25 publications that incorporated a total of 106 patients, including the present series. Splaying of the ICA and IJV was significantly, but not uniquely, associated with VNCS (p < 0.0001); multivariate analysis demonstrated that ICA and IJV splaying with medial ICA displacement carries a 75% probability of VNCS (p < 0.0001), while the absence of such splaying with lateral ICA displacement carries an 87% probability of SCCS (p = 0.0003). CONCLUSIONS ICA and IJV splaying frequently predicts VNCS; however, this finding is also commonly observed in SCCS and, among the 9 cases in the present study, was observed more often than previously reported. When congruent with splaying, medial or lateral ICA displacement significantly enhances the reliability of preoperative predictions, empowering more accurate prognostication.


Ultrasmall demultiplexer by use of one-dimensional photonic crystal.
Silica-based one-dimensional photonic crystal (1D-PC) is fabricated by use of a high-spatial-frequency grating with input and output surfaces tilted with respect to its periodic direction. An incident beam is coupled with the first photonic band in the second Brillouin zone of the 1D-PC. The output beam angle changes 3 degrees with a wavelength change of 1%. A prototype of an ultrasmall demultiplexer is demonstrated by use of a silica slab waveguide with 1D-PC.


Automated Quantification of Nonperfusion Areas in 3 Vascular Plexuses With Optical Coherence Tomography Angiography in Eyes of Patients With Diabetes.
Diabetic retinopathy (DR) is a leading cause of vision loss that is managed primarily through qualitative clinical examination of the retina. Optical coherence tomography angiography (OCTA) may offer an objective and quantitative method of evaluating DR. To quantify capillary nonperfusion in 3 vascular plexuses in the macula of eyes patients with diabetes of various retinopathy severity using projection-resolved OCTA (PR-OCTA). Cross-sectional study at a tertiary academic center comprising 1 eye each from healthy control individuals and patients with diabetes at different severity stages of retinopathy. Data were acquired and analyzed between January 2015 and December 2017. Foveal avascular zone area, extrafoveal avascular area (EAA), and the sensitivity of detecting levels of retinopathy. The study included 39 control individuals (20 women [51%]; mean [SD] age, 43.41 [19.37] years); 16 patients with diabetes without retinopathy (8 women [50%]; mean [SD] age, 56.50 [12.43] years); 23 patients with mild to moderate nonproliferative DR (18 women [78%] ; mean [SD] age, 62.48 [10.55] years); and 32 patients with severe nonproliferative DR or proliferative DR (12 women [38%]; mean age, 53.41 [14.05] years). Mean (SD) foveal avascular zone area was 0.203 (0.103) mm2 for control individuals, 0.192 (0.084) mm2 for patients with diabetes without retinopathy, 0.243 [0.079] mm2 for mild to moderate nonproliferative DR, and 0.359 (0.275) mm2 for severe nonproliferative DR or proliferative DR. Mean (SD) EAA in whole inner retinal slab in these groups, respectively, were 0.020 (0.031) mm2, 0.034 (0.047) mm2, 0.038 (0.040) mm2, and 0.237 (0.235) mm2. The mean (SD) sum of EAA from 3 segmented plexuses in each of the respective groups were 0.103 (0.169) mm2, 0.213 (0.242) mm2, 0.451 (0.243) mm2, and 1.325 (1.140) mm2. With specificity fixed at 95%, using EAA in inner retinal slab, the sensitivity of detecting patients with diabetes from healthy control individuals was 28% (95% CI, 18%-40%), 31% for patients with DR (95% CI, 19%-45%), and 47% for patients with severe DR (95% CI, 29%-65%) from whole inner retinal EAA. With the sum of EAA from 3 individual plexuses, the sensitivities were 69% (95% CI, 57%-80%), 82% (95% CI, 70%-91%), and 97% (95% CI, 85%-100%), respectively. Avascular areas were not associated with signal strength index. The commercial vessel density from the 2-plexus scheme distinguished the groups with lower sensitivity and were dependent on SSI. Automatically quantified avascular areas from a 3-layer segmentation scheme using PR-OCTA distinguished levels of retinopathy with a greater sensitivity than avascular areas from unsegmented inner retinal slab or measurements from a commercially available vessel density in 2-layer scheme. Additional studies are needed to investigate the applicability of nonperfusion parameters in clinical settings.


Invaginated Pancreaticojejunostomy via the Space Behind the Root of Superior Mesenteric Vessels.
This study was to explore a safe and effective procedure to prevent pancreatic fistula (PF) after pancreaticoduodenectomy (PD). Forty-three modified PD with pancreaticojejunostomy by direct invagination of the pancreas to the jejunum that was brought up via the space behind the root of superior mesenteric vessel were performed between January 2003 and June 2006, and were compared to the fifty-six conventional PD (Child' method). There was no pancreatic fistula after PD in the modified group. Two cases developed biliary fistula that were successfully treated with complete drainage for 2 to 3 weeks; 2 cases abdominal infection managed with anti-infection and completely drainage; 4 cases stress ulcer cured with Losec and coagulant. Three cases in the Child group developed PF of different severities, with amylase level > 9000 U/L in the abdominal drainage fluid. Two of the PF were treated with Stilamin, parenteral nutrition, fasting and completely drainage and cured after 21 to 32 days. The other 82-year-old patient died. Other complications had no significant difference between the two groups (P > 0.05). The modified PD can effectively prevent PF and is a safe and effective procedure for periampullary neoplasm. Further studies of its clinical use are warranted.


AND-1, a natural chimeric DNA-binding protein, combines an HMG-box with regulatory WD-repeats.
Using a specific monoclonal antibody (mAb AND-1/23-5-14) we have identified, cDNA-cloned and characterized a novel DNA-binding protein of the clawed toad, Xenopus laevis, that is accumulated in the nucleoplasm of oocytes and various other cells. This protein comprises 1,127 amino acids, with a total molecular mass of 125 kDa and a pI of 5.27. It is encoded by a mRNA of approximately 4 kb and contains, in addition to clusters of acidic amino acids, two hallmark motifs: the amino-terminal part harbours seven consecutive 'WD-repeats', which are sequence motifs of about 40 amino acids that are characteristic of a large group of regulatory proteins involved in diverse cellular functions, while the carboxy terminal portion possesses a 63-amino-acid-long 'HMG-box', which is typical of a family of DNA-binding proteins involved in regulation of chromatin assembly, transcription and replication. The DNA-binding capability of the protein was demonstrated by DNA affinity chromatography and electrophoretic mobility shift assays using four-way junction DNA. Protein AND-1 (acidic nucleoplasmic DNA-binding protein) appears as an oligomer, probably a homodimer, and has been localized throughout the entire interchromatinic space of the interphase nucleoplasm, whereas during mitosis it is transiently dispersed over the cytoplasm. We also identified a closely related, perhaps orthologous protein in mammals. The unique features of protein AND-1, which is a 'natural chimera' combining properties of the WD-repeat and the HMG-box families of proteins, are discussed in relation to its possible nuclear functions.


Modulation of Coordinated Activity across Cortical Layers by Plasticity of Inhibitory Synapses.
In the neocortex, synaptic inhibition shapes all forms of spontaneous and sensory evoked activity. Importantly, inhibitory transmission is highly plastic, but the functional role of inhibitory synaptic plasticity is unknown. In the mouse barrel cortex, activation of layer (L) 2/3 pyramidal neurons (PNs) elicits strong feedforward inhibition (FFI) onto L5 PNs. We find that FFI involving parvalbumin (PV)-expressing cells is strongly potentiated by postsynaptic PN burst firing. FFI plasticity modifies the PN excitation-to-inhibition (E/I) ratio, strongly modulates PN gain, and alters information transfer across cortical layers. Moreover, our LTPi-inducing protocol modifies firing of L5 PNs and alters the temporal association of PN spikes to γ-oscillations both in vitro and in vivo. All of these effects are captured by unbalancing the E/I ratio in a feedforward inhibition circuit model. Altogether, our results indicate that activity-dependent modulation of perisomatic inhibitory strength effectively influences the participation of single principal cortical neurons to cognition-relevant network activity.


Progressive feline pressure-overload: noninvasive assessment correlates with abnormalities in single cells.
Serial echocardiography and Doppler were used to monitor the progression of pressure-overload produced by banding the ascending aortas of young cats. The peak Doppler gradient across the band increased (as the animals grew in size) from 42 +/- 4.2 mmHg at 1 wk to 78 +/- 4.5 mmHg at 2-3 mos. Echocardiographic measurements of septal wall thickness increased significantly at 1 wk. Global ventricular function was unaltered in banded cats versus shams at each time point. However, in the subgroup of animals with an aortic-constricted area of < 0.025 cm2 at 1 wk, fractional shortening decreased by 40% at 2-3 mos. Contractile abnormalities were present in isolated myocytes from hypertrophied hearts. Mechanical function was more profoundly depressed in cells from hearts with echocardiographic evidence of ventricular decompensation. Echocardiographic and Doppler studies assessed cardiac size and function and identified indexes predictive of global and cellular myocardial dysfunction. The use of noninvasive techniques as a predictor of failure makes the feline model of progressive left ventricular pressure-overload useful for studies of cellular and molecular factors regulating not only the development of cardiac hypertrophy but also the transition from compensated hypertrophy to myocardial failure.


The prognostic impact of RAP2A expression in patients with early and locoregionally advanced nasopharyngeal carcinoma in an endemic area.
By data mining from published transcriptomic databases, we identified RAP2A as a significantly upregulated gene in nasopharyngeal carcinoma (NPC) tissues. RAP2A, a member of the RAS oncogene family, is involved in the process of GTP binding and GTPase activity. The aim of this study was to evaluate the expression of RAP2A and its prognostic impact in patients with early and locoregionally advanced NPC. RAP2A immunohistochemistry was performed for 124 NPC patients who were receiving standard treatment and had no initial distal metastasis. We also performed Western blotting to evaluate the endogenous protein expression of RAP2A in NPC cells and non-neoplastic mucosal cells. The result of RAP2A expression was further correlated with clinicopathological variables, disease-specific survival (DSS), distant metastasis-free survival (DMeFS), and local recurrence-free survival (LRFS). High expression of RAP2A was significantly associated with advanced primary tumor status (P = 0.024) and advanced TNM stage (P = 0.006). In univariate analysis, high expression of RAP2A served as a significant prognostic factor for inferior DSS (P < 0.0001), DMeFS (P < 0.0001), and LRFS (P < 0.0001). In multivariate analysis, RAP2A overexpression still independently predicted worse DSS (hazard ratio [HR] = 2.976, P < 0.001), DMeFS (HR = 4.233, P < 0.001), and LRFS (HR = 4.156, P < 0.001). Moreover, Both HONE1 and TW01 NPC cells, but not non-neoplastic DOK cells demonstrated significantly increased RAP2A expression. Overexpression of RAP2A is associated with advanced disease status and may therefore be an important prognosticator for poor outcomes in NPC, as well as a potential therapeutic target to aid in developing effective treatment modalities.


Peer review and gender bias: A study on 145 scholarly journals.
Scholarly journals are often blamed for a gender gap in publication rates, but it is unclear whether peer review and editorial processes contribute to it. This article examines gender bias in peer review with data for 145 journals in various fields of research, including about 1.7 million authors and 740,000 referees. We reconstructed three possible sources of bias, i.e., the editorial selection of referees, referee recommendations, and editorial decisions, and examined all their possible relationships. Results showed that manuscripts written by women as solo authors or coauthored by women were treated even more favorably by referees and editors. Although there were some differences between fields of research, our findings suggest that peer review and editorial processes do not penalize manuscripts by women. However, increasing gender diversity in editorial teams and referee pools could help journals inform potential authors about their attention to these factors and so stimulate participation by women.


Laparoscopic incisional and ventral hernia repair.
It has been more than a decade, since the introduction of laparoscopic management of ventral and incisional hernia. The purpose of this article was to systematically review the literature, analyze the results of Laparoscopic repair of ventral and incisional hernia and to ascertain its role. Pubmed was used for identifying the original articles. Both incisional and ventral hernia repair were included. Out of 145 articles extracted from Pubmed, 34 original studies were considered for review. More than three thousand patients were included in the review. Variables analyzed in the review were inpatient stay, defect size, mesh size, hematoma, seroma, wound infection, bowel perforation, obstruction, ileus, recurrence and pain. Qualitative analysis of the variables was carried out. Seromas (5.45%) and post operative pain (2.75%) are the two common complications associated with this procedure. Recurrence rate was found to be 3.67%. Overall complication rate was 19.24%, with two deaths reported. The results suggest laparoscopic repair of ventral and incisional hernia as an effective procedure. Faster recovery and shorter in patient stay - makes it a feasible alternative to open repair.


Model for measurement of water layer thickness under lipid bilayers by surface plasmon resonance.
A multilayered-substrate model is proposed for surface plasmon resonance (SPR)-based measurement of the thickness of a water layer sandwiched between a lipid bilayer and an underlying support. To calculate sensitivity, a 473 nm-wavelength excitation source and a silver layer are used for the SPR sensor. It is theoretically shown that the multilayered substrate design achieves sufficient sensitivity for such measurements and that sensitivity is enhanced with a SiO(2) layer of appropriate thickness and a buffer solution of high refractive index.


Use of Submicron Vaterite Particles Serves as an Effective Delivery Vehicle to the Respiratory Portion of the Lung.
Nano- and microencapsulation has proven to be a useful technique for the construction of drug delivery vehicles for use in vascular medicine. However, the possibility of using these techniques within the lung as an inhalation delivery mechanism has not been previously considered. A critical element of particle delivery to the lung is the degree of penetrance that can be achieved with respect to the airway tree. In this study we examined the effectiveness of near infrared (NIR) dye (Cy7) labeled calcium carbonate (vaterite) particles of 3.15, 1.35, and 0.65 μm diameter in reaching the respiratory portion of the lung. First of all, it was shown that, interaction vaterite particles and the components of the pulmonary surfactant occurs a very strong retardation of the recrystallization and dissolution of the particles, which can subsequently be used to create systems with a prolonging release of bioactive substances after the particles penetrate the distal sections of the lungs. Submicro- and microparticles, coated with Cy7 labeled albumin as a model compound, were delivered to mouse lungs via tracheostomy with subsequent imaging performed 24, 48, and 72 h after delivery by in vivo fluorescence. 20 min post administration particles of all three sizes were visible in the lung, with the deepest penetrance observed with 0.65 μm particles. In vivo biodistribution was confirmed by fluorescence tomography imaging of excised organs post 72 h. Laser scanning confocal microscopy shows 0.65 μm particles reaching the alveolar space. The delivery of fluorophore to the blood was assessed using Cy7 labeled 0.65 μm particles. Cy7 labeled 0.65 μm particles efficiently delivered fluorescent material to the blood with a peak 3 h after particle administration. The pharmacokinetics of NIR fluorescence dye will be shown. These studies establish that by using 0.65 μm particles loaded with Cy7 we can efficiently access the respiratory portion of the lung, which represents a potentially efficient delivery mechanism for both the lung and the vasculature.


Complete mitochondrial genome sequence of black mustard (Brassica nigra; BB) and comparison with Brassica oleracea (CC) and Brassica carinata (BBCC).
Crop species of Brassica (Brassicaceae) consist of three monogenomic species and three amphidiploid species resulting from interspecific hybridizations among them. Until now, mitochondrial genome sequences were available for only five of these species. We sequenced the mitochondrial genome of the sixth species, Brassica nigra (nuclear genome constitution BB), and compared it with those of Brassica oleracea (CC) and Brassica carinata (BBCC). The genome was assembled into a 232 145 bp circular sequence that is slightly larger than that of B. oleracea (219 952 bp). The genome of B. nigra contained 33 protein-coding genes, 3 rRNA genes, and 17 tRNA genes. The cox2-2 gene present in B. oleracea was absent in B. nigra. Although the nucleotide sequences of 52 genes were identical between B. nigra and B. carinata, the second exon of rps3 showed differences including an insertion/deletion (indel) and nucleotide substitutions. A PCR test to detect the indel revealed intraspecific variation in rps3, and in one line of B. nigra it amplified a DNA fragment of the size expected for B. carinata. In addition, the B. carinata lines tested here produced DNA fragments of the size expected for B. nigra. The results indicate that at least two mitotypes of B. nigra were present in the maternal parents of B. carinata.


Ambulance use in Pakistan: an analysis of surveillance data from emergency departments in Pakistan.
The utilization of ambulances in low- and middle-income countries is limited. The aim of this study was to ascertain frequency of ambulance use and characteristics of patients brought into emergency departments (EDs) through ambulance and non-ambulance modes of transportation. The Pakistan National Emergency Departments Surveillance (Pak-NEDS) was a pilot active surveillance conducted in seven major tertiary-care EDs in six main cities of Pakistan between November 2010 and March 2011. Univariate and multivariate logistic regression was performed to investigate the factors associated with ambulance use. Out of 274,436 patients enrolled in Pak-NEDS, the mode of arrival to the ED was documented for 94. 9% (n = 260,378) patients, of which 4.1% (n = 10,546) came to EDs via ambulances. The mean age of patients in the ambulance group was significantly higher compared to the mean age of the non-ambulance group (38 ± 18.4 years versus 32.8 ± 14.9 years, p-value < 0.001). The most common presenting complaint in the ambulance group was head injury (12%) while among non-ambulance users it was fever (12%). Patients of all age groups were less likely to use an ambulance compared to those >45 years of age (p-value < 0.001) adjusted for gender, cities, hospital type, presenting complaint group and disposition. The adjusted odds ratio of utilizing ambulances for those with injuries was 3.5 times higher than those with non-injury complaints (p-value < 0.001). Patients brought to the ED by ambulance were 7.2 times more likely to die in the ED than non-ambulance patients after adjustment for other variables in the model. Utilization of ambulances is very low in Pakistan. Ambulance use was found to be more among the elderly and those presenting with injuries. Patients presenting via ambulances were more likely to die in the ED.


Structural insights into the substrate specificity and transglycosylation activity of a fungal glycoside hydrolase family 5 β-mannosidase.
β-Mannosidases are exo-acting glycoside hydrolases (GHs) that catalyse the removal of the nonreducing end β-D-mannose from manno-oligosaccharides or mannoside-substituted molecules. They play important roles in fundamental biological processes and also have potential applications in various industries. In this study, the first fungal GH family 5 β-mannosidase (RmMan5B) from Rhizomucor miehei was functionally and structurally characterized. RmMan5B exhibited a much higher activity against manno-oligosaccharides than against p-nitrophenyl β-D-mannopyranoside (pNPM) and had a transglycosylation activity which transferred mannosyl residues to sugars such as fructose. To investigate its substrate specificity and transglycosylation activity, crystal structures of RmMan5B and of its inactive E202A mutant in complex with mannobiose, mannotriose and mannosyl-fructose were determined at resolutions of 1.3, 2.6, 2.0 and 2.4 Å, respectively. In addition, the crystal structure of R. miehei β-mannanase (RmMan5A) was determined at a resolution of 2.3 Å. Both RmMan5A and RmMan5B adopt the (β/α)8-barrel architecture, which is globally similar to the other members of GH family 5. However, RmMan5B shows several differences in the loop around the active site. The extended loop between strand β8 and helix α8 (residues 354-392) forms a `double' steric barrier to `block' the substrate-binding cleft at the end of the -1 subsite. Trp119, Asn260 and Glu380 in the β-mannosidase, which are involved in hydrogen-bond contacts with the -1 mannose, might be essential for exo catalytic activity. Moreover, the structure of RmMan5B in complex with mannosyl-fructose has provided evidence for the interactions between the β-mannosidase and D-fructofuranose. Overall, the present study not only helps in understanding the catalytic mechanism of GH family 5 β-mannosidases, but also provides a basis for further enzymatic engineering of β-mannosidases and β-mannanases.


A case of bronchiectasis needing lung isolation for cerebello pontine angle tumor excision: Anesthetic challenges.
The main goals of neuroanesthesia are the maintenance of adequate cerebral perfusion pressure, avoidance of hypercarbia, hypoxemia, and to provide better brain relaxation. Providing anesthesia for a patient with bronchiectasis needing lung isolation for craniotomy can be challenging. A 56-year-old male patient, case of right lung bronchiectasis with a right cerebello pontine angle tumor underwent excision in the left lateral position. Since he had severe bronchiectasis of the right lung, we had isolated the right lung using right-sided double lumen tube to avoid spillage. Intraoperative split lung test was performed to assess the right lung contribution on carbon dioxide (CO2) elimination and found that there was a significant contribution from the right lung. Hence, both lungs were ventilated to control CO2. The importance of lung isolation to prevent spillage and avoidance of one lung ventilation to control the arterial CO2 are highlighted in this case report. By providing a balanced anesthetic keeping both, the neurosurgical and thoracic concerns are important for better postoperative outcome.


3D gelatin-chitosan hybrid hydrogels combined with human platelet lysate highly support human mesenchymal stem cell proliferation and osteogenic differentiation.
Bone marrow and adipose tissue human mesenchymal stem cells were seeded in highly performing 3D gelatin-chitosan hybrid hydrogels of varying chitosan content in the presence of human platelet lysate and evaluated for their proliferation and osteogenic differentiation. Both bone marrow and adipose tissue human mesenchymal stem cells in gelatin-chitosan hybrid hydrogel 1 (chitosan content 8.1%) or gelatin-chitosan hybrid hydrogel 2 (chitosan 14.9%) showed high levels of viability (80%-90%), and their proliferation and osteogenic differentiation was significantly higher with human platelet lysate compared to fetal bovine serum, particularly in gelatin-chitosan hybrid hydrogel 1. Mineralization was detected early, after 21 days of culture, when human platelet lysate was used in the presence of osteogenic stimuli. Proteomic characterization of human platelet lysate highlighted 59 proteins mainly involved in functions related to cell adhesion, cellular repairing mechanisms, and regulation of cell differentiation. In conclusion, the combination of our gelatin-chitosan hybrid hydrogels with hPL represents a promising strategy for bone regenerative medicine using human mesenchymal stem cells.


An intelligent rollator for mobility impaired persons, especially stroke patients.
An intelligent rollator (IRO) was developed that aims at obstacle detection and guidance to avoid collisions and accidental falls. The IRO is a retrofit four-wheeled rollator with an embedded computer, two solenoid brakes, rotation sensors on the wheels and IR-distance sensors. The value reported by each distance sensor was compared in the computer to a nominal distance. Deviations indicated a present obstacle and caused activation of one of the brakes in order to influence the direction of motion to avoid the obstacle. The IRO was tested by seven healthy subjects with simulated restricted and blurred sight and five stroke subjects on a standardised indoor track with obstacles. All tested subjects walked faster with intelligence deactivated. Three out of five stroke patients experienced more detected obstacles with intelligence activated. This suggests enhanced safety during walking with IRO. Further studies are required to explore the full value of the IRO.


Genetic structure in natural populations of barley/cereal yellow dwarf virus isolates from Alaska.
The genetic structure of natural populations of Alaskan barley yellow dwarf virus (BYDV)-PAV, BYDV-PAS, and cereal yellow dwarf virus (CYDV)-RPV from barley (Hordeum vulgare L.) and oats (Avena sativa L.) in Alaska were analyzed between 2002 and 2004. PCR products spanning the viral coat protein gene of 187 isolates were cloned and sequenced. The majority (78%) were similar to BYDV-PAS, 19% were similar to CYDV-RPV, and only about 3% resembled BYDV-PAV. The CYDV-RPV isolates clustered in three groups: 44, 17, and 39% resembled RPS-like CP from Mexico, resembled RPV-like CP from New York, or formed a unique clade that was RPV/RPS recombinant CP, respectively. The patterns of genetic variation of PAS and RPV varied little over time or with respect to host plant. The difference in spatial and temporal population genetic structures of the PAS and RPV isolates suggests that these two viruses are influenced by different agroecological factors. Sequence of PCR products spanning the carboxyl terminus of the polymerase gene, the intergenic region, and most of the coat protein gene of RPV revealed two probable ancestral recombination events for some isolates.


Advances in real-time flood forecasting.
This paper discusses the modelling of rainfall-flow (rainfall-run-off) and flow-routeing processes in river systems within the context of real-time flood forecasting. It is argued that deterministic, reductionist (or 'bottom-up') models are inappropriate for real-time forecasting because of the inherent uncertainty that characterizes river-catchment dynamics and the problems of model over-parametrization. The advantages of alternative, efficiently parametrized data-based mechanistic models, identified and estimated using statistical methods, are discussed. It is shown that such models are in an ideal form for incorporation in a real-time, adaptive forecasting system based on recursive state-space estimation (an adaptive version of the stochastic Kalman filter algorithm). An illustrative example, based on the analysis of a limited set of hourly rainfall-flow data from the River Hodder in northwest England, demonstrates the utility of this methodology in difficult circumstances and illustrates the advantages of incorporating real-time state and parameter adaption.


Surface antigen profiles of leukocytes and melanoma cells in lymph node metastases are associated with survival in AJCC stage III melanoma patients.
There is an urgent need to identify more accurate prognostic biomarkers in melanoma patients, particularly in those with metastatic disease. This study aimed to identify melanoma and leukocyte surface antigens predictive of survival in a prospective series of AJCC stage IIIb/c melanoma patients (n = 29). Live cell suspensions were prepared from melanoma metastases within lymph nodes (LN). The suspensions were immuno-magnetically separated into CD45(+) (leukocyte) and CD45(-) (non-hematopoietic, enriched melanoma cell) fractions. Surface antigens on CD45(-) and CD45(+) cell populations were profiled using DotScan™ microarrays (Medsaic Pty. Ltd.) and showed differential abundance levels for 52 and 78 antigens respectively. Associations of the surface profiles with clinicopathologic and outcome data (median follow-up 35.4 months post LN resection) were sought using univariate (log-rank test) and multivariate (Wald's test; modelled with patient's age, gender and AJCC staging at LN recurrence) survival models. CD9 (p = 0.036), CD39 (p = 0.004) and CD55 (p = 0.005) on CD45(+) leukocytes were independently associated with distant metastasis-free survival using multivariate analysis. Leukocytes with high CD39 levels were also significantly associated with increased overall survival (OS) in multivariate analysis (p = 0.016). LNs containing leukocytes expressing CD11b (p = 0.025), CD49d (p = 0.043) and CD79b (p = 0.044) were associated with reduced OS on univariate analysis. For enriched melanoma cells (CD45(-) cell populations), 11 surface antigens were significantly correlated with the disease-free interval (DFI) between diagnosis of culprit primary melanoma and LN metastasis resection. Nine antigens on CD45(+) leukocytes also correlated with DFI. Following validation in independent datasets, surface markers identified here should enable more accurate determination of prognosis in stage III melanoma patients and provide better risk stratification of patients entering clinical trials.


ActiGait implantable drop foot stimulator in multiple sclerosis: a new indication.
OBJECTIVE Direct stimulation of the peroneal nerve by the ActiGait implantable drop foot stimulator is a potent therapy that was described previously for stroke-related drop foot. The authors report here successful long-term application of the ActiGait implantable drop foot stimulator in patients with multiple sclerosis (MS). METHODS Six patients with MS and 2 years of persisting central leg paresis received an implantable ActiGait drop foot stimulator after successful surface test stimulation. Ten weeks and 1 year after surgery, their gait speed, endurance, and safety were evaluated. Patient satisfaction was assessed with a questionnaire. RESULTS In the 20-m gait test, stimulation with the ActiGait stimulator significantly reduced the time needed, on average, by approximately 23.6% 10 weeks after surgery, and the time improved further by 36.3% after 1 year. The median distance covered by patients with the stimulator after 6 minutes of walking increased significantly from 217 m to 321 m and remained stable for 1 year; the distance covered by patients after surface stimulation was 264 m. Patients with an implanted ActiGait stimulator noticed pronounced improvement in their mobility, social participation, and quality of life. CONCLUSIONS The ActiGait implantable drop foot stimulator improved gait speed, endurance, and quality of life in all patients over a period of 1 year. It may serve as a new therapeutic option for patients with MS-related drop foot.


Theory-of-mind in individuals with Alström syndrome is related to executive functions, and verbal ability.
This study focuses on cognitive prerequisites for the development of theory-of-mind (ToM), the ability to impute mental states to self and others in young adults with Alström syndrome (AS). AS is a rare and quite recently described recessively inherited ciliopathic disorder which causes progressive sensorineural hearing loss and juvenile blindness, as well as many other organ dysfunctions. Two cognitive abilities were considered; Phonological working memory (WM) and executive functions (EF), both of importance in speech development. Ten individuals (18-37 years) diagnosed with AS, and 20 individuals with no known impairment matched for age, gender, and educational level participated. Sensory functions were measured. Information about motor functions and communicative skills was obtained from responses to a questionnaire. ToM was assessed using Happés strange stories, verbal ability by a vocabulary test, phonological WM by means of an auditory presented non-word serial recall task and EF by tests of updating and inhibition. The AS group performed at a significantly lower level than the control group in both the ToM task and the EF tasks. A significant correlation was observed between recall of non-words and EF in the AS group. Updating, but not inhibition, correlated significantly with verbal ability, whereas both updating and inhibition were significantly related to the ability to initiate and sustain communication. Poorer performance in the ToM and EF tasks were related to language perseverance and motor mannerisms. The AS group displayed a delayed ToM as well as reduced phonological WM, EF, and verbal ability. A significant association between ToM and EF, suggests a compensatory role of EF. This association may reflect the importance of EF to perceive and process input from the social environment when the social interaction is challenged by dual sensory loss. We argue that limitations in EF capacity in individuals with AS, to some extent, may be related to early blindness and progressive hearing loss, but maybe also to gene specific abnormalities.


Magnetic brain stimulation can improve clinical outcome in incomplete spinal cord injured patients.
Preliminary longitudinal clinical trial. To test the efficacy of repetitive transcranial magnetic stimulation (rTMS) in modulating corticospinal inhibition and improving recovery in stable incomplete spinal cord injury (iSCI). National Spinal Injuries Centre, Stoke Mandeville Hospital, Bucks, UK and Division of Neuroscience, Imperial College Faculty of Medicine, Charing Cross Hospital, London, UK. Four stable iSCI patients were treated with rTMS over the occipital cortex (sham treatment) and then over the motor cortex (real treatment). Patients were assessed using electrophysiological, clinical and functional measures before treatment, during sham treatment, during the therapeutic treatment and during a 3-week follow-up period. Cortical inhibition was reduced during the treatment week. Perceptual threshold to electrical stimulation of the skin, ASIA clinical measures of motor and sensory function and time to complete a peg-board improved and remained improved into the follow-up period. In this preliminary trial, rTMS has been shown to alter cortical inhibition in iSCI and improve the clinical and functional outcome. This work was supported by the International Spinal Research Trust.


BREEDING AND GENETICS SYMPOSIUM: Breeding for resilience to heat stress effects in dairy ruminants. A comprehensive review.
Selection for heat tolerant (HT) animals in dairy production has been so far linked to estimation of declines in production using milk recording and meteorological information on the day of control using reaction norms. Results from these models show that there is a reasonable amount of genetic variability in the individual response to high heat loads, which makes feasible selection of HT animals at low costs. However, the antagonistic relationship between level of production and response to heat stress (HS) implies that selection for HT animals under this approach must be done with caution so that productivity is not damaged. Decomposition of the genetic variability in principal components (PC) can provide selection criteria independent of milk production level although biological interpretation of PC is difficult. Moreover, given that response to heat stress for each animal is estimated with very sparse information collected under different physiological and management circumstances, biased (normally underestimation) and lack of accuracy may be expected. Alternative phenotypic characterization of HT can come from the use of physiological traits, which have also shown moderate heritability. However, costs of a large scale implementation based on physiological characteristics has precluded its use. Another alternative is the use of biomarkers that define heat tolerance. A review of biomarkers of HS from more recent studies is provided. Of particular interest are milk biomarkers, which together with infrared spectra prediction equations can provide useful tools for genetic selection. In the 'omics' era, genomics, transcriptomics, proteomics and metabolomics have been already used to detect genes affecting HT. A review of findings in these areas is also provided. Except for the slick hair gene, there are no other genes for which variants have been clearly associated with HT. However, integration of omics information could help in pointing at knots of the HS control network and, in the end, to a panel of markers to be used in the selection of HT animals. Overall, HT is a complex phenomenon that requires integration of fine phenotypes and omics information to provide accurate tools for selection without damaging productivity. Technological developments to make on-farm implementation feasible and with greater insight into the key biomarkers and genes involved in HT are needed.


MicroRNA-224-5p regulates adipocyte apoptosis induced by TNFα via controlling NF-κB activation.
Tumor necrosis factor (TNF) α can induce cell apoptosis and activate nuclear transcription (NF)-κB in different cell types. Activated NF-κB further promotes or suppresses cellular apoptosis in different cases. The present study explored the effect of activated NF-κB on adipocyte apoptosis induced by TNFα and which microRNAs (miRNAs) were involved in the process. Our findings demonstrated that treatment of differentiated 3T3-L1 adipocytes with TNFα (20 ng/mL) rapidly activated NF-κB and induced moderate apoptosis. Pyrrolidinedithiocarbamic acid (PDTC, 60 µM), a specific NF-κB inhibitor, abated NF-κB activation that rendered the adipocytes vulnerable to TNFα-induced apoptosis. Dozens of miRNAs exhibited significant expression changes following TNFα treatment and the addition of PDTC. In which, miRNA-224-5p (miR-224) was up-regulated by TNFα exposure but down-regulated by PDTC addition. Furthermore, over-expression of miR-224 promoted NF-κB activation and prevented the adipocyte apoptosis induced by TNFα, while miR-224 deficiency showed the opposite effects. The TRAF-associated NF-κB activator (TANK) gene was identified as a direct target of miR-224 by computational and luciferase reporter assays. Additionally, silencing the TANK gene by the small interfering RNA similarly promoted NF-κB activation and attenuated the cellular apoptosis. In conclusion, these findings demonstrate that miR-224 plays an essential role in adipocyte apoptosis caused by TNFα through control of NF-κB activation via targeting the TANK gene.


Serial prostate specific antigen, free-to-total prostate specific antigen ratio and complexed prostate specific antigen for the diagnosis of prostate cancer.
The free-to-total prostate specific antigen (PSA) ratio and complexed PSA have been introduced as adjuncts to total PSA for prostate cancer screening. Little data exist on the use of these tests for serial PSA screening. We compared serial total PSA, the free-to-total PSA ratio and calculated complexed PSA in men diagnosed with prostate cancer and matched controls in a population based study. We identified 90 men diagnosed with prostate cancer between 1988 and 1996 with at least 3 serial serum samples obtained at 2-year intervals who were participants in the beta-Carotene and Retinol Efficacy Trial for the prevention of lung cancer. Samples were available up to 10 years before diagnosis. A total of 90 age matched men from the same cohort without prostate carcinoma were identified as controls. Free and total PSA was measured by the Abbott AxSYM system. Baseline demographics of cases and controls were similar. At baseline and diagnosis the men with prostate cancer had higher total and complexed PSA, and a lower free-to-total PSA ratio than controls. Mean followup was 5.2 years in cases and 5.5 in controls. The yearly change in PSA parameters in cases versus controls was 20.7% versus 3.5% for total, -3.4% versus 0.2% for free-to-total and 21.5% versus 3.4% for complexed PSA (p <0.0001). At diagnosis PSA alone was estimated to perform with more than 90% specificity in our model. In this population based study total PSA was superior to the free-to-total PSA ratio for predicting the development of prostate cancer. While serial changes in free-to-total PSA ratios with time were statistically significantly different in men diagnosed with prostate cancer and controls, the magnitude of these serial changes were slight enough to render them clinically insignificant.


A tubulin alpha 8 mouse knockout model indicates a likely role in spermatogenesis but not in brain development.
Tubulin alpha 8 (Tuba8) is the most divergent member of the highly conserved alpha tubulin family, and uniquely lacks two key post-translational modification sites. It is abundantly expressed in testis and muscle, with lower levels in the brain. We previously identified homozygous hypomorphic TUBA8 mutations in human subjects with a polymicrogyria (PMG) syndrome, suggesting its involvement in development of the cerebral cortex. We have now generated and characterized a Tuba8 knockout mouse model. Homozygous mice were confirmed to lack Tuba8 protein in the testis, but did not display PMG and appeared to be neurologically normal. In response to this finding, we re-analyzed the human PMG subjects using whole exome sequencing. This resulted in identification of an additional homozygous loss-of-function mutation in SNAP29, suggesting that SNAP29 deficiency, rather than TUBA8 deficiency, may underlie most or all of the neurodevelopmental anomalies in these subjects. Nonetheless, in the mouse brain, Tuba8 specifically localised to the cerebellar Purkinje cells, suggesting that the human mutations may affect or modify motor control. In the testis, Tuba8 localisation was cell-type specific. It was restricted to spermiogenesis with a strong acrosomal localization that was gradually replaced by cytoplasmic distribution and was absent from spermatozoa. Although the knockout mice were fertile, the localisation pattern indicated that Tuba8 may have a role in spermatid development during spermatogenesis, rather than as a component of the mature microtubule-rich flagellum itself.


Semiparametric Estimation with Data Missing Not at Random Using an Instrumental Variable.
Missing data occur frequently in empirical studies in health and social sciences, often compromising our ability to make accurate inferences. An outcome is said to be missing not at random (MNAR) if, conditional on the observed variables, the missing data mechanism still depends on the unobserved outcome. In such settings, identification is generally not possible without imposing additional assumptions. Identification is sometimes possible, however, if an instrumental variable (IV) is observed for all subjects which satisfies the exclusion restriction that the IV affects the missingness process without directly influencing the outcome. In this paper, we provide necessary and sufficient conditions for nonparametric identification of the full data distribution under MNAR with the aid of an IV. In addition, we give sufficient identification conditions that are more straightforward to verify in practice. For inference, we focus on estimation of a population outcome mean, for which we develop a suite of semiparametric estimators that extend methods previously developed for data missing at random. Specifically, we propose inverse probability weighted estimation, outcome regression-based estimation and doubly robust estimation of the mean of an outcome subject to MNAR. For illustration, the methods are used to account for selection bias induced by HIV testing refusal in the evaluation of HIV seroprevalence in Mochudi, Botswana, using interviewer characteristics such as gender, age and years of experience as IVs.


Suppression of in-vitro lymphoproliferative responses in acute malaria patients can be partially reversed by indomethacin.
In-vitro lymphoproliferative responses to malaria antigens are suppressed in patients with acute Plasmodium falciparum infection. Studies with other parasitic diseases have suggested that monocyte/macrophage-derived prostaglandins may be responsible for immunosuppression. Since acute malaria infection is characteristically associated with fever it is likely that prostaglandin E production will also be enhanced in these patients. In this study, indomethacin, a cyclooxygenase inhibitor which blocks the synthesis of prostaglandins, was added to the culture medium during assays of lymphoproliferative responses to malaria antigens and other soluble proteins. Responses to several antigens were enhanced in the presence of indomethacin, indicating that prostaglandins may have a generalized immunosuppressive role in malaria-infected individuals. However, responses to malaria antigens were particularly enhanced by indomethacin, suggesting that malaria-specific T-cells are especially sensitive to the effects of prostaglandin, possibly due to prior activation in vivo by circulating malaria antigens.


Parathyroid hormone concentration in geriatric cats with various degrees of renal function.
To determine whether cats in the nonazotemic stages of chronic kidney disease have increased plasma parathyroid hormone (PTH) concentrations as a compensatory physiologic mechanism to maintain plasma phosphate concentration within the reference interval. Prospective longitudinal study. 118 client-owned geriatric cats with various degrees of renal function. For each cat, a blood sample was obtained for plasma biochemical analysis and determination of plasma PTH concentration, and a urine sample was obtained for determination of urine specific gravity at study entry (baseline) and after 12 months. For a subset of 30 cats, plasma calcitriol concentration was determined at baseline. Cats were categorized into 1 of 3 groups on the basis of kidney function at the end of 12 months. At baseline and after 12 months, plasma concentrations of variables associated with calcium homeostasis were compared between the 3 groups and also within groups over time. Multivariable linear regression was used to identify variables associated with plasma PTH concentration. Plasma PTH concentration was significantly increased in cats that developed azotemia, compared with PTH concentration in cats that remained nonazotemic, and PTH concentration increased before changes in plasma calcium and phosphate concentrations were detected. A moderate positive association between plasma calcitriol and PTH concentrations was identified. Plasma PTH concentration was associated with age and plasma urea, creatinine, and total calcium concentrations in the final multivariable model. Results suggested that renal secondary hyperparathyroidism can develop prior to azotemia in cats, even in the absence of hyperphosphatemia and hypocalcemia.