Does the "hygiene hypothesis" provide an explanation for the high prevalence of multiple sclerosis in Sardinia?
The "hygiene hypothesis" describes a hypothetical scenario in which the balance between TH1 (defending host against bacterial and viral infections) and TH2 (defending against parasitic infections) immune responses is pivotal and in which the consequence of reducing the infectious stressors during infancy is increased autoimmunity (TH1-mediated) and allergy (TH2-mediated). Many epidemiological observations confirm that allergic and autoimmune diseases are significantly increased in the "developed" countries and negatively associated with childhood infections. However, it has been recently revealed that immune elements associated with allergy are extensively involved also in the pathogenesis of autoimmune demyelination and that TH2- and TH1-mediated infections ameliorate the course of the disease confirming that the allergic root is also responsible for the escalation of autoimmune disorders, and both have a common immunological denominator. In the Italian island of Sardinia, MS and type-I diabetes frequencies have sharply increased in the last decades compared to other populations living in the same Mediterranean area. Initial observation led us to believe that environmental changes favoured the MS risk rise, thus sustaining the hygiene hypothesis. However, data on MS prevalence distribution in this territory suggest that other mechanisms than environment have also to be taken into great account. Our recent epidemiological studies reveal significant differences in the MS prevalence between rural and urban areas within the same province of Sassari but, contrarily to what expected from the hygiene hypothesis, MS prevalence is significantly higher in rural, genetically "archaic", areas where the westernalization process has been less pronounced. On this basis we believe that, beside hygiene-related factors, genetics could represent a more relevant determinant of Sardinian high susceptibility to MS.


[Hormone-dependent process of the maturation of the major structural proteins of the mouse mammary tumor virus].
The following findings were obtained by the radio-immunoprecipitation method with antisera to gp52 and p27. When the cells were cultivated in a hormone-free medium, they contained precursors of proteins of gene gag (Pr 73gag) and gene evn (gPr 70env). No mature structural MTV proteins were found. The addition of insulin to the growth medium had no effect on maturation of protein precursors. When dexamethazone was added to the medium, gPr 70env "maturated" to gp52 of the main envelope glycoprotein of virion coat whereas Pr 73gag was not cleaved into final products. When the cells were cultivated in the presence of insulin and dexamethazone, there was a marked stimulation of Pr 73gag (although its maturation did not occur), g Pr 70env, and, especially, gp 52. Extracellular particles were found to contain p27 which was precipitated by homologous monospecific antiserum, that is, in the system of clone F2 cells processing of Pr 73gag occurred mainly in extracellular particles. Thus, expression of all three known genes (gag, pol, and env) occurred in the cells of cloned F2 culture.


Targeting NADPH oxidase with a novel dual Nox1/Nox4 inhibitor attenuates renal pathology in type 1 diabetes.
Reactive oxygen species (ROS) generated by Nox NADPH oxidases may play a critical role in the pathogenesis of diabetic nephropathy (DN). The efficacy of the Nox1/Nox4 inhibitor GKT137831 on the manifestations of DN was studied in OVE26 mice, a model of type 1 diabetes. Starting at 4-5 mo of age, OVE26 mice were treated with GKT137831 at 10 or 40 mg/kg, once-a-day for 4 wk. At both doses, GKT137831 inhibited NADPH oxidase activity, superoxide generation, and hydrogen peroxide production in the renal cortex from diabetic mice without affecting Nox1 or Nox4 protein expression. The increased expression of fibronectin and type IV collagen was reduced in the renal cortex, including glomeruli, of diabetic mice treated with GKT137831. GKT137831 significantly reduced glomerular hypertrophy, mesangial matrix expansion, urinary albumin excretion, and podocyte loss in OVE26 mice. GKT137831 also attenuated macrophage infiltration in glomeruli and tubulointerstitium. Collectively, our data indicate that pharmacological inhibition of Nox1/4 affords broad renoprotection in mice with preexisting diabetes and established kidney disease. This study validates the relevance of targeting Nox4 and identifies GKT137831 as a promising compound for the treatment of DN in type 1 diabetes.


Biogenesis of the trypanosome endo-exocytotic organelle is cytoskeleton mediated.
Trypanosoma brucei is a protozoan parasite that is used as a model organism to study such biological phenomena as gene expression, protein trafficking, and cytoskeletal biogenesis. In T. brucei, endocytosis and exocytosis occur exclusively through a sequestered organelle called the flagellar pocket (FP), an invagination of the pellicular membrane. The pocket is the sole site for specific receptors thus maintaining them inaccessible to components of the innate immune system of the mammalian host. The FP is also responsible for the sorting of protective parasite glycoproteins targeted to, or recycling from, the pellicular membrane, and for the removal of host antibodies from the cell surface. Here, we describe the first characterisation of a flagellar pocket cytoskeletal protein, BILBO1. BILBO1 functions to form a cytoskeleton framework upon which the FP is made and which is also required and essential for FP biogenesis and cell survival. Remarkably, RNA interference (RNAi)-mediated ablation of BILBO1 in insect procyclic-form parasites prevents FP biogenesis and induces vesicle accumulation, Golgi swelling, the aberrant repositioning of the new flagellum, and cell death. Cultured bloodstream-form parasites are also nonviable when subjected to BILBO1 RNAi. These results provide the first molecular evidence for cytoskeletally mediated FP biogenesis.


Simulation modelling of population dynamics of mosquito vectors for rift valley Fever virus in a disease epidemic setting.
Rift Valley Fever (RVF) is weather dependent arboviral infection of livestock and humans. Population dynamics of mosquito vectors is associated with disease epidemics. In our study, we use daily temperature and rainfall as model inputs to simulate dynamics of mosquito vectors population in relation to disease epidemics. Time-varying distributed delays (TVDD) and multi-way functional response equations were implemented to simulate mosquito vectors and hosts developmental stages and to establish interactions between stages and phases of mosquito vectors in relation to vertebrate hosts for infection introduction in compartmental phases. An open-source modelling platforms, Universal Simulator and Qt integrated development environment were used to develop models in C++ programming language. Developed models include source codes for mosquito fecundity, host fecundity, water level, mosquito infection, host infection, interactions, and egg time. Extensible Markup Language (XML) files were used as recipes to integrate source codes in Qt creator with Universal Simulator plug-in. We observed that Floodwater Aedines and Culicine population continued to fluctuate with temperature and water level over simulation period while controlled by availability of host for blood feeding. Infection in the system was introduced by floodwater Aedines. Culicines pick infection from infected host once to amplify disease epidemic. Simulated mosquito population show sudden unusual increase between December 1997 and January 1998 a similar period when RVF outbreak occurred in Ngorongoro district. Findings presented here provide new opportunities for weather-driven RVF epidemic simulation modelling. This is an ideal approach for understanding disease transmission dynamics towards epidemics prediction, prevention and control. This approach can be used as an alternative source for generation of calibrated RVF epidemics data in different settings.


Mechanistic basis for the activation of plant membrane receptor kinases by SERK-family coreceptors.
Plant-unique membrane receptor kinases with leucine-rich repeat ectodomains (LRR-RKs) can sense small molecule, peptide, and protein ligands. Many LRR-RKs require SERK-family coreceptor kinases for high-affinity ligand binding and receptor activation. How one coreceptor can contribute to the specific binding of distinct ligands and activation of different LRR-RKs is poorly understood. Here we quantitatively analyze the contribution of SERK3 to ligand binding and activation of the brassinosteroid receptor BRI1 and the peptide hormone receptor HAESA. We show that while the isolated receptors sense their respective ligands with drastically different binding affinities, the SERK3 ectodomain binds the ligand-associated receptors with very similar binding kinetics. We identify residues in the SERK3 N-terminal capping domain, which allow for selective steroid and peptide hormone recognition. In contrast, residues in the SERK3 LRR core form a second, constitutive receptor-coreceptor interface. Genetic analyses of protein chimera between BRI1 and SERK3 define that signaling-competent complexes are formed by receptor-coreceptor heteromerization in planta. A functional BRI1-HAESA chimera suggests that the receptor activation mechanism is conserved among different LRR-RKs, and that their signaling specificity is encoded in the kinase domain of the receptor. Our work pinpoints the relative contributions of receptor, ligand, and coreceptor to the formation and activation of SERK-dependent LRR-RK signaling complexes regulating plant growth and development.


G-protein-coupled receptor kinase-5 mediates inflammation but does not regulate cellular infiltration or bacterial load in a polymicrobial sepsis model in mice.
NFκB-dependent signaling is an important modulator of inflammation in several diseases including sepsis. G-protein-coupled receptor kinase-5 (GRK5) is an evolutionarily conserved regulator of the NFκB pathway. We hypothesized that GRK5 via NFκB regulation plays an important role in the pathogenesis of sepsis. To test this we utilized a clinically relevant polymicrobial sepsis model in mice that were deficient in GRK5. We subjected wild-type (WT) and GRK5 knockout (KO) mice to cecal ligation and puncture (CLP)-induced polymicrobial sepsis and assessed the various events in sepsis pathogenesis. CLP induced a significant inflammatory response in the WT and this was markedly attenuated in the KO mice. To determine the signaling mechanisms and the role of NFκB activation in sepsis-induced inflammation, we assessed the levels of IκBα phosphorylation and expression of NFκB-dependent genes in the liver in the two genotypes. Both IκBα phosphorylation and gene expression were significantly inhibited in the GRK5 KO compared to the WT mice. Interestingly, however, GRK5 did not modulate either immune cell infiltration (to the primary site of infection) or local/systemic bacterial load subsequent to sepsis induction. In contrast GRK5 deficiency significantly inhibited sepsis-induced plasma corticosterone levels and the consequent thymocyte apoptosis in vivo. Associated with these outcomes, CLP-induced mortality was significantly prevented in the GRK5 KO mice in the presence of antibiotics. Together, our studies demonstrate that GRK5 is an important regulator of inflammation and thymic apoptosis in polymicrobial sepsis and implicate GRK5 as a potential molecular target in sepsis.


A novel mutation of the human luteinizing hormone receptor in 46XY and 46XX sisters.
We report a novel homozygous mutation of the LH receptor (LHR) gene in three siblings: two 46XY and one 46XX. The 46XY siblings presented with female external genitalia, primary amenorrhea, and lack of breast development. Hormonal evaluation revealed a markedly elevated LH level with a low testosterone level, which failed to increase after human CG stimulation. Enzymatic deficiencies of testosterone biosynthesis were eliminated as possible etiologies. Histologic analysis of the inguinal gonads in a 46XY sibling revealed no Leydig cells; Sertoli cells, spermatogonia, and primary spermatocytes were seen. The 46XX sibling had female external genitalia, normal breast development, and primary amenorrhea. Hormonal analyses showed markedly elevated LH levels and low plasma 17 beta-estradiol levels. Genetic analysis of the LHR revealed a homozygous missense mutation at exon 11 of the LHR gene. Guanine was replaced by adenine (GAA-->AAA), resulting in a substitution of lysine for glutamic acid (glu) at amino acid position 354 of the receptor. This mutation is located in the extracellular domain adjacent to the first transmembrane helix of the LHR. Glutamic acid at position 354 of the LHR has been highly conserved throughout evolution. Functional analysis of the LHR mutation, using an in vitro mutagenesis-transfection assay, demonstrated complete loss of function, indicated by the lack of cAMP production after human CG stimulation in transfected human embryonic kidney 293 cells. Screening of family members demonstrated heterozygosity for the mutation, indicating autosomal recessive inheritance. Delineation of the specific genetic defect in this family confirms recent reports that a single mutation in the LHR gene causes male pseudohermaphroditism in 46XY subjects and primary amenorrhea in 46XX subjects. More importantly, it also defines a new region of the LHR molecule that is critical for biologic activity.


Nuclear expression of Rac1 in cervical premalignant lesions and cervical cancer cells.
Abnormal expression of Rho-GTPases has been reported in several human cancers. However, the expression of these proteins in cervical cancer has been poorly investigated. In this study we analyzed the expression of the GTPases Rac1, RhoA, Cdc42, and the Rho-GEFs, Tiam1 and beta-Pix, in cervical pre-malignant lesions and cervical cancer cell lines. Protein expression was analyzed by immunochemistry on 102 cervical paraffin-embedded biopsies: 20 without Squamous Intraepithelial Lesions (SIL), 51 Low- grade SIL, and 31 High-grade SIL; and in cervical cancer cell lines C33A and SiHa, and non-tumorigenic HaCat cells. Nuclear localization of Rac1 in HaCat, C33A and SiHa cells was assessed by cellular fractionation and Western blotting, in the presence or not of a chemical Rac1 inhibitor (NSC23766). Immunoreacivity for Rac1, RhoA, Tiam1 and beta-Pix was stronger in L-SIL and H-SIL, compared to samples without SIL, and it was significantly associated with the histological diagnosis. Nuclear expression of Rac1 was observed in 52.9% L-SIL and 48.4% H-SIL, but not in samples without SIL. Rac1 was found in the nucleus of C33A and SiHa cells but not in HaCat cells. Chemical inhibition of Rac1 resulted in reduced cell proliferation in HaCat, C33A and SiHa cells. Rac1 is expressed in the nucleus of epithelial cells in SILs and cervical cancer cell lines, and chemical inhibition of Rac1 reduces cellular proliferation. Further studies are needed to better understand the role of Rho-GTPases in cervical cancer progression.


CD38 binding to human myeloid cells is mediated by mouse and human CD31.
Soluble forms of membrane receptors are emerging candidates as physiological regulators of leukocyte trafficking. In the present study, we found that the soluble form of the CD38 antigen (sCD38) bears a binding domain of low affinity for a cellular receptor on U937 cells. Cross-linking and peptide-mapping studies confirmed the physical association and the identification of the U937 receptor as a 130 kDa protein. The binding of sCD38 to the receptor was differentially inhibited by several monoclonal antibodies against the CD31 cell-adhesion molecule. Thus the interaction was analysed through direct association of soluble and membrane CD38 with soluble recombinant murine CD31 with three N-terminal and with all six extracellular Ig domains. Cross-linking experiments on U937 intact cells, and ligand blot assays of the immunoprecipitated CD38 molecule, indicated that (i) the recognized epitope is determined by the tertiary structure of the molecule, and that (ii) the binding domain involved resides in the ectocellular portion of the CD31 molecule, more precisely in the first three N-terminal domains. A comparative functional activity between murine and human CD31 was also explored. The data presented suggest that (i) human CD31 bears a highly functional similarity with its murine counterpart, as it is a receptor in myeloid cells with more than one ligand (the alphavbeta3 integrin and the CD38 molecule), and that (ii) the activity of sCD38 as decoy molecule for CD31 may play an important role in cell-cell interactions in physiological and pathological conditions.


Suicide-Specific Cognitions, Attentional Fixation, and Worst-Point Suicidal Ideation.
The fluid vulnerability theory of suicide posits that each person has a baseline risk for suicide, which is comprised of both stable and dynamic factors. The current study investigated the unique involvement of suicide-specific cognitions and attentional fixation on recent suicidal ideation (SI) and SI at its worst. Data were analyzed from a sample of N = 126 undergraduate students with a history of SI. Path analyses were used to analyze the relationship between suicide-specific cognitions, attentional fixation, and SI (current and worst point). Results revealed that suicide-specific cognitions were directly related to both recent SI and worst-point SI. Suicide-specific cognitions had a significant, indirect effect with worst-point SI through attentional fixation, but this effect was not significant when using recent SI. These data support the need for interventions to target cognitive contents and contexts (e.g., fixation) to reduce escalation of SI. Future work would benefit from replicating and extending results in studies that include prospective designs and the assessment of suicidal behaviors.


Taurine as a Natural Antioxidant: From Direct Antioxidant Effects to Protective Action in Various Toxicological Models.
Natural antioxidants have received tremendous attention over the last 3 decades. At the same time, the attitude to free radicals is slowly changing, and their signalling role in adaptation to stress has recently received a lot of attention. Among many different antioxidants in the body, taurine (Tau), a sulphur-containing non-proteinogenic β-amino acid, is shown to have a special place as an important natural modulator of the antioxidant defence networks. Indeed, Tau is synthesised in most mammals and birds, and the Tau requirement is met by both synthesis and food/feed supply. From the analysis of recent data, it could be concluded that the direct antioxidant effect of Tau due to scavenging free radicals is limited and could be expected only in a few mammalian/avian tissues (e.g., heart and eye) with comparatively high (>15-20 mM) Tau concentrations. The stabilising effects of Tau on mitochondria, a prime site of free radical formation, are characterised and deserve more attention. Tau deficiency has been shown to compromise the electron transport chain in mitochondria and significantly increase free radical production. It seems likely that by maintaining the optimal Tau status of mitochondria, it is possible to control free radical production. Tau's antioxidant protective action is of great importance in various stress conditions in human life, and is related to commercial animal and poultry production. In various in vitro and in vivo toxicological models, Tau showed AO protective effects. The membrane-stabilizing effects, inhibiting effects on ROS-producing enzymes, as well as the indirect AO effects of Tau via redox balance maintenance associated with the modulation of various transcription factors (e.g., Nrf2 and NF-κB) and vitagenes could also contribute to its protective action in stress conditions, and thus deserve more attention.


Content analysis and thematic analysis: Implications for conducting a qualitative descriptive study.
Qualitative content analysis and thematic analysis are two commonly used approaches in data analysis of nursing research, but boundaries between the two have not been clearly specified. In other words, they are being used interchangeably and it seems difficult for the researcher to choose between them. In this respect, this paper describes and discusses the boundaries between qualitative content analysis and thematic analysis and presents implications to improve the consistency between the purpose of related studies and the method of data analyses. This is a discussion paper, comprising an analytical overview and discussion of the definitions, aims, philosophical background, data gathering, and analysis of content analysis and thematic analysis, and addressing their methodological subtleties. It is concluded that in spite of many similarities between the approaches, including cutting across data and searching for patterns and themes, their main difference lies in the opportunity for quantification of data. It means that measuring the frequency of different categories and themes is possible in content analysis with caution as a proxy for significance.


Anti-allergic role of cholinergic neuronal pathway via α7 nicotinic ACh receptors on mucosal mast cells in a murine food allergy model.
The prevalence of food allergy (FA) has increased in developed countries over the past few decades. However, no effective drug therapies are currently available. Therefore, we investigated cholinergic anti-inflammatory pathway as a regulatory system to ameliorate disrupted mucosal immune homeostasis in the gut based on the pathophysiological elucidation of mucosal mast cells (MMCs) in a murine FA model. BALB/c mice sensitized with ovalbumin received repeated oral ovalbumin for the development of FA. FA mice developed severe allergic diarrhea and exhibited enhanced type 2 helper T (Th2) cell immune responses in both systemic immunity and mucosal immunity, along with MMCs hyperplasia in the colon. MMCs were localized primarily in the strategic position of the mucosal epithelium. Furthermore, the allergic symptoms did not develop in p85α disrupted phosphoinositide-3 kinase-deficient mice that lacked mast cells in the gut. Vagal stimulation by 2-deoxy-D-glucose and drug treatment with nicotinic ACh receptor (nAChR) agonists (nicotine and α7 nAChR agonist GTS-21) alleviated the allergic symptoms in the FA mice. Nicotine treatment suppressed MMCs hyperplasia, enhanced MPO and upregulated mRNA expression of Th1 and Th2 cytokines in the FA mice colon. MMCs, which are negatively regulated by α7 nAChRs, were often located in close proximity to cholinergic CGRP-immunoreactive nerve fibers in the FA mice colon. The present results reveal that the cholinergic neuroimmune interaction via α7 nAChRs on MMCs is largely involved in maintaining intestinal immune homeostasis and can be a target for a new therapy against mucosal immune diseases with homeostatic disturbances such as FA.


Childhood emotional and behavior problems and their associations with cesarean delivery.
To determine the prevalence of childhood emotional and behavioral problems and examine their associations with cesarean delivery. Our sample consisted of 8,900 preschoolers from 35 kindergartens in four cities in East China. Parents completed the Strengths and Difficulties Questionnaire (SDQ) and provided other information. Children's emotional and behavioral problems were assessed using five subscales of the SDQ. Mode of delivery was classified as vaginal or cesarean section (CS); in sub-analyses, we divided CS into elective or emergency delivery. Logistic regression was used to examine associations. A total of 1,209 (13.6%) children had a total SDQ score within abnormal range; 25.5% had peer problems within abnormal range, 9.0% had abnormal emotional symptoms, 13.9% had abnormal conduct problems, 18.9% had abnormal hyperactivity problems, and 16.2% were rated abnormal in pro-social behavior. Overall, 67.3% of the children who participated were delivered by CS. In fully adjusted analysis, CS was significantly associated with abnormal total SDQ score (OR = 1.27; 95%CI 1.10-1.46; p < 0.05) and pro-social behavior (OR = 1.27; 95%CI 1.12-1.45; p < 0.0001). No significant association was found between CS and risk of having conduct problems (OR 1.13; 95%CI 0.98-1.29), peer problems (OR 1.11; 95%CI 0.99-1.24), hyperactivity (OR 1.02; 95%CI 0.91-1.15), or emotional problems (OR 1.06; 95%CI 0.90-1.24). In this sample, CS was associated with risk of behavioral problems, but not with emotional problems. Further research is needed to better understand these associations.


Association between whole-grain consumption and carotid atherosclerosis: the Tianjin chronic low-grade systemic inflammation and health (TCLSIH) cohort study.
Background: Whole-grain contains a range of beneficial nutrients, which are thought to play a role in the prevention of chronic diseases. However, the association between whole-grain consumption and the risk of developing carotid atherosclerosis (CA) has not been sufficiently elucidated. We, therefore, conducted this study to investigate the relationship between whole-grain consumption and CA in the general adult population. Methods: This prospective cohort study included a total of 2166 participants (19.2-84.6 years, 55.0% men) without a history of cardiovascular disease, cancer, and CA at baseline. A validated food frequency questionnaire was used to assess whole-grain consumption. Measurements of CA include carotid intima-media thickness (IMT) and carotid plaque. IMT thickening is defined as: IMT ≥ 1.0 mm or a carotid bifurcation IMT ≥ 1.2 mm. Carotid plaque is defined as: distinct area protruding ≥1.5 mm into the vascular lumen of the carotid artery. Cox proportional hazards regression models were used to examine the association of whole-grain consumption with incident CA. Results: A total of 538 (341 men) first incident cases of CA occurred during 5585 person-years of follow-up (median follow-up: 4.2 years). After adjusting for demographic characteristics, lifestyle factors, dietary intake, individual and family history of disease, the multivariable HRs (95% CIs) for incident CA were 1.00 (reference) for <1 time per week, 1.10 (0.85, 1.43) for 1 time per week, 0.95 (0.75, 1.20) for 2-6 times per week, and 1.12 (0.80, 1.56) for ≥1 times per day, respectively (P for trend = 0.99). Similar results were observed in stratified analyses by the main covariates and sensitivity analyses. Conclusion: Our data indicate that whole-grain consumption had no significant association with the risk of CA in an adult Chinese population. In our study population, there is a low consumption of whole-grain, which may limit our ability to see an association. Further cohort studies or randomized controlled trials are needed to confirm our results.


Residual Cholesteatoma After Endoscope-guided Surgery in Children.
Endoscopes can facilitate surgery within tympanomastoid recesses that are not visible with the operating microscope. This study investigates whether use of endoscopes to guide dissection of cholesteatoma leads to lower rates of residual cholesteatoma than using the endoscope only for inspection after microscope-guided dissection. Comparative cohort study. Tertiary pediatric center. Two hundred thirty-five patients with acquired or congenital cholesteatoma in children <18 years having intact canal wall surgery and follow-up >12 months. Comparison of group (A) microscope surgery followed by endoscopic inspection, with group (B) endoscope-guided dissection. Residual cholesteatoma rates, controlling for site of initial cholesteatoma, detection by second-stage surgery, and length of follow-up. Analysis of all patients showed endoscopic dissection was associated with less residua in the middle ear (risk difference = 0.12; p = 0.026, Kaplan-Meier log rank analysis; number needed to treat = 9) but not at other sites. When restricting analysis to ears that were evaluated with second look surgery, no significant reduction in residual disease was found after endoscopic dissection at any site (e.g., retrotympanic residua: 12% Group A versus 7% Group B (NS, Fisher exact test). Endoscopic dissection allowed more permeatal surgery. No complications were attributable to endoscope use. Wound complications occurred in 4% of open cases. Endoscopes enhance surgical access to tympanomastoid recesses. In conjunction with the availability of the operating microscope, angled instruments, and KTP laser, endoscope-guided dissection provides a small incremental benefit for prevention of residual cholesteatoma, and facilitates a minimally invasive approach.


Antifungal susceptibility and phenotypic virulence markers of Candida species isolated from Nepal.
Candida species are part of the commensal microflora in many anatomical sites of the human body; however, breach in the integrity of the body part and impaired immunity of the host can lead to invasive candidiasis. A number of virulence determinants could contribute towards its pathogenicity. Thus we attempted to evaluate the in vitro expression of different virulence factors among clinical isolates of Candida species and assayed their susceptibility patterns against a range of antifungal agents. Of the total of 71 isolates we obtained, 48 (67.6%) were Candida albicans, 11 (15.49%) Candida tropicalis, 09 (12.67%) Candida glabrata and 03 (4.22%) were Candida krusei. Proteinase, phospholipase and esterase production could be revealed amongst 43 (60.56%), 44 (61.97%) and 49 (69.01%) isolates respectively. None of the isolates showed DNAase activity. Fifty-five (77.39%) isolates were biofilm producers, and 53 (74.6%) exhibited high cell surface hydrophobicity.


[Epidemiolgy of high blood pressure in France].
In France, more than 10 million people receive medications to treat hypotension. This simple figure underscores the dimension of hypertension for the country as a public health problem. Hypertension deserves careful attention not only for itself, but also because of the cardiac and vascular diseases which it may induce. Thus, renewed primary and secondary prevention strategies are needed. These must be based upon a better knowledge of the relationship between blood pressure level and cardiovascular diseases, upon new thresholds used to define hypertension, and upon an integrated approach to assess and reduce the overall cardiovascular risk in individuals. Eventually, low-cost population-based preventive strategies, still marginally used in our country, must receive a greater commitment from Public Health Authorities.


Safety and in-hospital outcomes of bivalirudin use in dialysis patients undergoing percutaneous coronary intervention.
Chronic dialysis-dependent patients undergoing percutaneous coronary intervention (PCI) are at a greater risk of bleeding and ischemic events. Bivalirudin has been associated with fewer bleeding complications than unfractionated heparin (UFH) in patients undergoing PCI in various clinical settings. These studies, however, have systematically excluded patients dependent on chronic dialysis. We sought to assess the safety, bleeding rates, and in-hospital outcomes of bivalirudin use compared to UFH use alone in patients requiring dialysis and undergoing PCI. A retrospective analysis of 396 dialysis-dependent patients undergoing PCI from January 2000 to March 2009 was performed. Patients treated with a dose-adjusted bivalirudin regimen (n = 267) were compared to those treated with UFH alone (n = 129). The primary end point of major bleeding (hematocrit decrease > or = 15%, gastrointestinal or intracerebral bleeding) and the composite end point of in-hospital death, nonfatal Q-wave myocardial infarction, and urgent target vessel revascularization were compared between groups. The baseline characteristics were similar between the 2 groups, except for the proportion of men and nonsmokers and body mass index, which were greater in patients treated with bivalirudin. The rate of major bleeding was similar between the bivalirudin and UFH groups (3.4% vs 3.1%, respectively, p = 0.9). The rate of the composite end point (death, Q-wave myocardial infarction, urgent target vessel revascularization) was not significantly different between the 2 groups (1.8% for bivalirudin vs 0.8% for UFH group, p = 0.7). After adjustment, bivalirudin use was not associated with major bleeding (odds ratio 1.23, 95% confidence interval 0.37 to 4.13, p = 0.7). In conclusion, a dose-adjusted bivalirudin anticoagulation regimen for patients requiring chronic dialysis undergoing PCI seems to be as safe and as effective as UFH use alone. These results do not suggest the superiority of bivalirudin over UFH.


Alcohol consumption and physical symptoms in a Mexican American population.
The negative health consequences of alcohol consumption are well established. The present study examines whether alcohol consumption is also associated with a series of self-reported physical symptoms. Analyses were performed on data from a three-generation study of Mexican Americans. Using four measures of alcohol consumption we found that alcohol consumption was related to physical symptoms only in the younger generation. While drinking levels in the older generation did not have sufficient variance to enable the assessment of their effects on symptoms, drinking levels in the middle generation were comparable to those in the younger generation. We hypothesized that the lack of association in the middle generation may reflect increased tolerance due to longer exposure to alcohol, to possible reduction of alcohol consumption by some middle-aged persons because of health problems, and to due possible removal from observation of certain middle-aged men whose heavy drinking led to health problems. Although alcohol consumption in the younger generation was not strongly associated with either psychosomatic or infectious symptoms, it was strongly related to upper respiratory symptoms among younger males. Among younger women, alcohol consumption was moderately related to physical symptoms with no symptom constellation having a particularly strong association with alcohol consumption.


Acute treatment of migraine with zolmitriptan 5 mg orally disintegrating tablet.
To determine the speed of onset of headache response and duration of response with zolmitriptan 5 mg orally disintegrating tablet (ODT) in the acute treatment of migraine. Rapid pain relief is important for migraine sufferers. The early efficacy (30 minutes) of the 5 mg dose of the zolmitriptan ODT formulation was evaluated. In this double-blind, parallel-group trial, 670 patients were randomised to receive either zolmitriptan 5 mg ODT (n=329) or matching placebo (n=341) to treat two migraine headaches of moderate or severe intensity. Zolmitriptan 5 mg ODT was significantly more effective than placebo in achieving a headache response (reduction in migraine headache intensity from moderate or severe to mild or no pain) at 30 minutes (16.5% vs 12.5%; p=0.048; primary endpoint), 1 hour (p <0.0001), and 2 hours (p <0.0001). Significantly more patients achieved a sustained headache response for 24 hours with zolmitriptan 5 mg ODT than with placebo (42.5% vs 16.4%; p <0.0001). Zolmitriptan 5 mg ODT also produced a higher pain-free rate than placebo at all timepoints (0.5, 1 and 2 hours post-dose), with the differences becoming significant at 1 hour. Adverse events were reported by 37.7% of the patients treated with zolmitriptan 5 mg ODT and 18.1% of the patients treated with placebo. Those that occurred most often for the patients treated with zolmitriptan 5 mg ODT were tightness (7.0%), dizziness (6.7%), somnolence (6.7%) and paraesthesias (6.1%). A significant headache response as early as 30 minutes post-dose and good maintenance of this headache response at 24 hours makes zolmitriptan 5 mg ODT an excellent acute treatment for migraine. Together with the relatively low adverse event rate, these data demonstrate that zolmitriptan 5 mg ODT is a valuable therapy for migraine sufferers.


Genomic epidemiology of SARS-CoV-2 in large university hospital cohort: the UnCoVER-Brazil project.
This work aimed to study the role of different SARS-CoV-2 lineages in the epidemiology of multiple waves of the COVID-19 pandemic in Ribeirão Preto (São Paulo state), with comparison within Brazil and globally. Viral genomic sequencing was combined with clinical and sociodemographic information of 2,379 subjects at a large Brazilian hospital. On the whole 2,395 complete SARS-CoV-2 genomes were obtained from April 2020 to January 2022. We report variants of concern (VOC) and interest (VOI) dynamics and the role of Brazilian lineages. We identified three World Health Organization VOCs (Gamma, Delta, Omicron) and one VOI (Zeta), which caused distinct waves in this cohort. We also identified 47 distinct Pango lineages. Consistent with the high prevalence of Gamma in Brazil, Pango lineage P.1 dominated infections in this cohort for half of 2021. Each wave of infection largely consisted of a single variant group, with each new group quickly and completely rising to dominance. Despite increasing vaccination in Brazil starting in 2021, this pattern was observed throughout the study and is consistent with the hypothesis that herd immunity tends to be SARS-CoV-2 variant-specific and does not broadly protect against COVID-19.


Prevalence and impact of pain at the greater trochanter after open surgery for the treatment of femoro-acetabular impingement.
Femoro-acetabular impingement can cause pain and degenerative changes of the hip joint. Traditionally, surgical dislocation of the hip joint has been performed for correction of pathologic abnormalities in the proximal part of the femur and the acetabulum. Failures of surgical treatment are often related to postoperative pain in the groin or in the area of the greater trochanter, associated with this surgical approach. The aim of our study was to determine the prevalence and functional impact of pain at the greater trochanter after surgical dislocation of the hip. Fifty consecutive patients in whom femoro-acetabular impingement had been treated with surgical dislocation of the hip at our institution were seen for clinical and radiographic follow-up at one year. The Merle d'Aubigné score was calculated preoperatively and at the time of follow-up. Pain in the groin or at the greater trochanter during activity and at rest was recorded. The mean Merle d'Aubigné score significantly improved from 14.4 points preoperatively to 17 points postoperatively. Preoperatively, eight patients (16%) had pain over the greater trochanter. At one year after surgery, twenty-three (46%) had such pain; these patients were primarily female (seventeen of the twenty-three). The mean Merle d'Aubigné score was 17.4 points for the patients without groin pain and 16.1 points for those with groin pain. The presence or absence of pain at the greater trochanter had no significant influence on the outcome, but groin pain was associated with inferior results. Tenderness or pain over the greater trochanter is frequent after osteotomy of the greater trochanter and has a distinct female predominance. However, it has no significant negative influence on outcome. This is in contrast to groin pain, which is associated with inferior results.


Childhood disability and socio-economic circumstances in low and middle income countries: systematic review.
The majority of children with disability live in low and middle income (LAMI) countries. Although a number of important reviews of childhood disability in LAMI countries have been published, these have not, to our knowledge, addressed the association between childhood disability and the home socio-economic circumstances (SEC). The objective of this study is to establish the current state of knowledge on the SECs of children with disability and their households in LAMI countries through a systematic review and quality assessment of existing research. Electronic databases (MEDLINE; EMBASE; PUBMED; Web of Knowledge; PsycInfo; ASSIA; Virtual Health Library; POPLINE; Google scholar) were searched using terms specific to childhood disability and SECs in LAMI countries. Publications from organisations including the World Bank, UNICEF, International Monetary Fund were searched for. Primary studies and reviews from 1990 onwards were included. Studies were assessed for inclusion, categorisation and quality by 2 researchers. 24 primary studies and 13 reviews were identified. Evidence from the available literature on the association between childhood disability and SECs was inconsistent and inconclusive. Potential mechanisms by which poverty and low household SEC may be both a cause and consequence of disability are outlined in the reviews and the qualitative studies. The association of poor SECs with learning disability and behaviour problems was the most consistent finding and these studies had low/medium risk of bias. Where overall disability was the outcome of interest, findings were divergent and many studies had a high/medium risk of bias. Qualitative studies were methodologically weak. This review indicates that, despite socially and biologically plausible mechanisms underlying the association of low household SEC with childhood disability in LAMI countries, the empirical evidence from quantitative studies is inconsistent and contradictory. There is evidence for a bidirectional association of low household SEC and disability and longitudinal data is needed to clarify the nature of this association.


Mechanisms underlying diminished novelty-seeking behavior in patients with probable Alzheimer's disease.
To better understand apathy and disengagement in patients with Alzheimer's disease (AD), the authors investigated possible behavioral mechanisms underlying diminished novelty-seeking activity in patients with probable AD. Apathy and disengagement have been shown to be the most common behavioral changes associated with AD. Patients and age-matched normal controls had their eye movements recorded while pairs of line drawings pitting an incongruous figure against a congruous figure were shown on a screen for 12 seconds. Characteristics of a subset of AD patients who were indifferent to novel visual stimuli as measured by exploratory eye movements were compared to those of a subset of AD patients who were attracted to novel stimuli to a degree similar to that of normal controls. The indifferent patients were judged by informants, who completed a personality questionnaire, to exhibit a greater degree of apathy. The two AD groups did not differ in overall dementia severity or performance on a Saccade-to-Target Task that required shifts of attention and gaze. In a separate task, the indifferent patients were able to accurately identify the more novel stimuli in 97.5% of trials. Normal control subjects exhibited a strong bias toward processing novel stimuli, directing a higher proportion of their first fixations and dwell time to the incongruous stimuli whether the analysis was run for 3, 6, or 12 seconds of viewing. Indifferent patients did not direct their initial fixation toward novel stimuli and distributed their looking time evenly between incongruous and congruous stimuli throughout all measured intervals. The results suggest that the indifference to novelty observed in some patients with probable AD cannot simply be attributed to global cognitive decline, more elementary attentional deficits, more rapid habituation of response to novel stimuli, or an inability to discriminate upon demand between stimuli of varying degrees of novelty. It is more likely that their behavior reflects a disruption, by AD pathology, of neural systems that modulate behavioral engagement and maintain attentional bias toward novel events in the environment.


Effect of the herbicide glyphosate on enzymatic activity in pregnant rats and their fetuses.
To prevent health risk from environmental chemicals, particularly for progeny, we have studied the effects of the herbicide glyphosate on several enzymes of pregnant rats. Glyphosate is an organophosphorated nonselective agrochemical widely used in many countries including Argentina and acts after the sprout in a systemic way. We have studied three cytosolic enzymes: isocitrate dehydrogenase-NADP dependent, glucose-6-phosphate dehydrogenase, and malic dehydrogenase in liver, heart, and brain of pregnant Wistar rats. The treatment was administered during the 21 days of pregnancy, with 1 week as an acclimation period. The results suggest that maternal exposure to agrochemicals during pregnancy induces a variety of functional abnormalities in the specific activity of the enzymes in the studied organs of the pregnant rats and their fetuses.


IgG4-related disease -Mechanistic insights from both clinical and immunologic understanding of this condition.
IgG4-related disease (IgG4-RD) is a chronic inflammatory disease characterized by tumescent lesions with characteristic storiform fibrosis, obliterative phlebitis and a marked lymphoplasmacytic infiltrate that includes a large number of IgG4 positive plasma cells. It's widely accepted that rituximab-mediated B cell depletion therapy is effective for this disease. Important mechanistic insights correlated with the pathogenesis of IgG4-RD have been gradually disclosed from studies of patients treated by B cell depletion. 1) IgG4-RD patients have the large clonal expansion of activated plasmablasts and CD4+CTLs, so this disease might be antigen-driven. 2) CD4+CTLs are the dominant population in affected tissues, on the other hands direct examination of TH1 and TH2 cells in tissues reveal that these subsets are sparse. 3) CD4+CTLs into affected lesions secret cytotoxic, inflammatory, and pro-fibrotic cytokines, indicating reactivation by antigen in tissue sites. 4) The decline in CD4+CTLs number by B cell depletion is associated with clinical remission of IgG4-RD patients. 5) CD4+CXCR5+TFH cells that express IL-4 are located outside germinal centers and specialized TFH cells that expanded dramatically in conditions with polarized class switching to IgG4. These results suggested that the disease pathogenesis might be based on orchestrating of activated plasmablasts, CD4+CTLs, and TFH cells.


Presubicular and parasubicular cortical neurons of the rat: functional separation of deep and superficial neurons in vitro.
1. The presubiculum and parasubiculum are retrohippocampal structures bordered by the subiculum and medial entorhinal cortex. Deep layer (IV-VI) neurons from this region exhibit stable synaptically triggered burst behaviour which distinguishes them from superficial layer (I-III) cells. This functional separation was examined with intracellular and field potential recordings from horizontal slices of rat brain. Neurobiotin labelling and rapid Golgi techniques were used to obtain anatomical evidence of axonal trajectories. 2. Extracellular stimulation of the subiculum, deep medial entorhinal cortex or superficial pre- or parasubiculum caused, in deep layer cells only, a short latency burst discharge which could be followed by one or more after-discharges. Bursts appeared after repetitive stimulation and were stable for the life of the slice. Each event was supported by giant excitatory postsynaptic potentials (EPSPs). Events were similar whether they were evoked in horizontal slices or slices cut perpendicular to the horizontal plane. 3. Bath application of the NMDA receptor antagonist 3-[2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP; 5 microM) elevated the threshold for evoking the giant EPSP. Higher concentrations (10-15 microM) reduced the amplitude and duration of the giant EPSP. Bath application of the AMPA receptor antagonist beta-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 5 microM) eliminated the evoked EPSP. 4. In intact slices, superficial layer neurons of pre- and parasubiculum could exhibit EPSPs coincident with bursts recorded in the deep layers. However, in isolated subsections of horizontal slices or in 'vertical slices', both of which contained only pre- and/or parasubiculum, evoked or picrotoxin-induced bursts occurred only in deep layer cells. Superficial layer cells in these subsections showed no response to deep layer events. 5. Antidromic population spikes confirmed projections from superficial cell layers of pre- and parasubiculum down to their deep cell layers. Reciprocal antidromic responses were absent. 6. Axons of superficial layer stellate and pyramidal cells had horizontal collaterals and at least one ascending and one descending collateral. Branches of the descending collaterals were given off in layer V and some axons were found to reach the angular bundle. Axons of deep layer stellate and pyramidal cells also had horizontal collaterals and descending collaterals which could be traced to the angular bundle. One ascending axon collateral was found among the thirty-one deep layer cells examined morphologically. 7. We conclude that the deep layer cells of the presubiculum and parasubiculum are richly interconnected with excitatory synapses. These interconnections can generate giant excitatory synaptic potentials that support the bursting behaviour exhibited by these neurons. Any of the excitatory inputs to deep layer cells can trigger the population bursts and specific inputs from entorhinal cortex produce the after-discharges. Further, connections between superficial and deep layer cells appear to be almost exclusively in the direction of superficial to deep. The absence of significant ascending input can account for the functional separation of superficial and deep layer neurons of presubiculum and parasubiculum.


Mapping loss of heterozygosity at chromosome 13q: loss at 13q12-q13 is associated with breast tumour progression and poor prognosis.
Several chromosome regions exhibit loss of heterozygosity (LOH) in human breast carcinoma and are thought to harbour tumour suppressor genes (TSG). At chromosome 13q, two TSGs have been identified, RB1 at 13q14 and BRCA2 at 13q12-q13. In this study, 139 sporadic breast tumours were analysed with 18 polymorphic microsatellite markers for detailed mapping of LOH at chromosome 13q and evaluation of an association with known progression factors. LOH with at least one marker was observed in 71 (51%) of the tumours analysed. The deletion mapping indicated three LOH target regions, 13q12-q13, 13q14 and 13q31-q34. LOH at chromosome 13q12-q13 was associated with low progesterone receptor content, a high S phase fraction and aneuploidy. Multivariate analysis adjusting for lymph node involvement and S phase fraction showed that patients with tumours exhibiting LOH at 13q12-q13 have a 3-4-fold increased risk of recurrence and death compared with other patients. Our results suggest there are at least three separate LOH target regions at chromosome 13q and inactivation of one or more genes at chromosome 13q12-q13 results in poor prognosis for breast cancer patients.


FEV1 as a Standalone Spirometric Predictor and the Attributable Fraction for Death in Older Persons.
Commonly used thresholds for staging FEV1 have not been evaluated as standalone spirometric predictors of death in older persons. Specifically, the proportion of deaths attributed to a reduced FEV1, when staged by commonly used thresholds in L, percent of predicted (% pred), and Z scores, has not been previously reported. In 4,232 white persons ≥ 65 y old, sampled from the Cardiovascular Health Study, FEV1 was stratified as stage 1 (FEV1 ≥ 2.00 L, ≥80% pred, and Z score ≥-1.64), stage 2 (FEV1 1.50-1.99 L, 50-79%pred, and Z score -2.55 to -1.63), and stage 3 (FEV1 < 1.50 L, < 50% pred, and Z score < -2.55). Notably, a Z score threshold of -1.64 defines normal-for-age lung function as the lower limit of normal (ie, 5th percentile of distribution), and accounts for differences in age, sex, height, and ethnicity. Next, adjusted odds ratios and average attributable fractions for 10-y all-cause mortality were calculated, comparing FEV1 stages 2 and 3 against stage 1, expressed in L, % pred, and Z scores. The average attributable fraction estimates the proportion of deaths attributed to a predictor by combining the prevalence of the predictor with the relative risk of death conferred by that predictor. FEV1 stage 2 and 3 in L, % pred, and Z scores yielded similar adjusted odds ratios of death: 1.40-1.51 for stage 2 and 2.35-2.66 for stage 3. Conversely, FEV1 stages 2 and 3 in L, % pred, and Z scores differed in prevalence: 12.8-28.6% for stage 2 and 6.4-17.5% for stage 3, and also differed in the adjusted average attributable fraction for death: 3.2-6.4% for stage 2 and 4.5-9.1% for stage 3. In older persons, the proportion of deaths attributed to a reduced FEV1 is best stratified by Z score staging thresholds because these yield a similar relative risk of death but a more age- and sex-appropriate prevalence of FEV1 stage.


Experiences of self-stigmatization and parenting in Chinese mothers with severe mental illness.
Mental health stigma has serious ramifications on people with a severe mental illness (SMI). Stigma damages self-esteem, recovery outcomes, family relationships, socialization abilities, access to housing, and career prospects. The cultural tendencies of Chinese people have been shown to be associated with particularly high levels of stigmatization. These cultural tenets can result in high levels of self-stigma due to experiencing shame and a perceived need to keep mental illness a secret. Although there is a lack of existing evidence, it is possible that such experiences present unique challenges to Chinese mothers diagnosed with SMI when they parent their children. Therefore, this qualitative study explored the experiences of parenting and self-stigmatization of Chinese mothers with SMI. Individual semi-structured interviews were conducted with 15 mothers who were direct carers of their children aged under 18 and who were receiving community-based care in Hong Kong. Manual inductive thematic analysis was used to analyse the interview data. Three main themes related to self-stigmatization emerged from the interviews: (i) distancing and being distanced; (ii) doubting myself; and (iii) struggling for control. The experiences of self-stigmatization appeared to damage these mothers' self-efficacy, which may negatively affect their parenting self-esteem, tendency to seek professional help, and ability to manage their own mental health. Strategies to improve self-efficacy, including psychoeducation, and additional childcare support/resources are required for mothers with SMI so they can better manage and balance the demands of motherhood and their mental healthcare needs.


Nicotinamide improves NAD+ levels to protect against acetaminophen-induced acute liver injury in mice.
Acetaminophen (APAP) overdose causes acute liver injury (ALI). Nicotinamide adenine dinucleotide (NAD) is an essential coenzyme, and NAD+ is oxidized type which synthesized from nicotinamide (NAM). The present study aimed to investigate the role of NAD+ in ALI and protective property of NAM. The mice were subjected to different doses APAP. After 8 hours, the serum activities of alaninetransaminase (ALT) and aspartate aminotransferase (AST), the hepatic NAD+ level and nicotinamide phosphoribosyltransferase (NAMPT) expression were determined. Then, the mice were pretreated with NAM (800 mg/kg), the hepatoprotective effects and the key antioxidative molecules were evaluated. Our findings indicated that APAP resulted in remarkable NAD+ depletion in a dose-dependent manner accompanied by NAMPT downregulation, and NAM pretreatment significantly elevated the NAD+ decline due to upregulation of NAMPT. Moreover, the downregulated Kelch-like ECH-associated protein-1 (Keap1), upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) and its translocation activation after NAM administration were confirmed, which were in accordance with improved superoxide dismutase (SOD) and glutathione (GSH) levels. Finally, NAM dramatically exhibited hepatoprotective effects by reducing the liver index and necrotic area. This study has suggested that APAP impairs liver NAD+ level and NAM is able to improve hepatic NAD+ to activate antioxidant pathway against APAP-induced ALI.


Pyrrolizidine alkaloid toxicity in livestock: a paradigm for human poisoning?
Livestock poisoning, primarily liver damage, caused by consumption of plants containing 1,2-dehydropyrrolizidine ester alkaloids (dehydroPAs), and the corresponding N-oxides, is a relatively common occurrence worldwide. Because of the economic impact, extensive investigations of such episodes have been performed, particularly in Australia, South Africa the United States and, more recently, South America. Plant species most commonly involved are members of the families Boraginaceae, Asteraceae and Leguminosae. These may be native species that periodically flourish under particular climatic conditions or introduced species that thrive in the absence of natural control factors such as herbivory and competition. Contamination of grain crops with dehydroPA-producing plants has resulted in large-scale incidents of food poisoning in humans, with high morbidity and mortality, especially in Africa and in central and south Asia, with recent episodes in Afghanistan and possibly Ethiopia. Attention has recently focused on the potential for low levels of dehydroPAs to contaminate many food products in developed countries, possibly leading to progressive, chronic diseases that may not include overt hepatotoxicity. This overview examines the potential for better control of exposure and means of monitoring dehydroPA intake by extrapolation of knowledge gained from animal studies to the human situation.


Detecting antimicrobial resistance in Escherichia coli using benchtop attenuated total reflectance-Fourier transform infrared spectroscopy and machine learning.
The widespread dissemination of resistance to third-generation cephalosporins in the Enterobacterales through the production of extended-spectrum β-lactamase (ESBL) is considered a critical global crisis requiring urgent attention of clinicians and scientists alike. Rapid diagnostic methods that can identify microbial resistance profiles closer to the point of care are crucial to minimize the overuse of antimicrobial agents and improve patient outcomes. Although Fourier transform infrared (FTIR) microscopy has shown promise in distinguishing between bacterial species, the high cost and technical requirements of the IR microscope may limit broad clinical use. To address the practical needs of a clinical microbiology laboratory, here, we examine the ability of a lower cost portable benchtop attenuated total reflectance (ATR)-FTIR spectrometer to achieve antimicrobial resistance detection, using a simple, clinically aligned sampling protocol. The technical reproducibility was confirmed through multi-day analysis of an Escherichia coli type strain, which serves as quality control. We generated a dataset of 100 E. coli clinical bloodstream isolates with 63 ceftriaxone resistant blaCTX-M ESBL gene variant strains and developed a classifier for blaCTX-M genotype detection. After assessing 35 machine learning methods using the training set (n = 71), four methods were further optimised, and the best performing method was evaluated using the held-out testing set (n = 29). A tuned support vector machine model with a polynomial kernel, using the 700-1500 cm-1 range achieved a sensitivity of 89.2%, and specificity of 66.7% for detecting blaCTX-M in independent testing, approaching the reported performance of FTIR microscopy. With further algorithm improvement, these data suggest the potential deployment of a portable FTIR spectrometer as a rapid antimicrobial susceptibility prediction platform to enable the efficient use of antimicrobials.


Thermotropic phase behavior and headgroup interactions of the nonbilayer lipids phosphatidylethanolamine and monogalactosyldiacylglycerol in the dry state.
Although biological membranes are organized as lipid bilayers, they contain a substantial fraction of lipids that have a strong tendency to adopt a nonlamellar, most often inverted hexagonal (HII) phase. The polymorphic phase behavior of such nonbilayer lipids has been studied previously with a variety of methods in the fully hydrated state or at different degrees of dehydration. Here, we present a study of the thermotropic phase behavior of the nonbilayer lipids egg phosphatidylethanolamine (EPE) and monogalactosyldiacylglycerol (MGDG) with a focus on interactions between the lipid molecules in the interfacial and headgroup regions. Liposomes were investigated in the dry state by Fourier-transform Infrared (FTIR) spectroscopy and Differential Scanning Calorimetry (DSC). Dry EPE showed a gel to liquid-crystalline phase transition below 0°C and a liquid-crystalline to HII transition at 100°C. MGDG, on the other hand, was in the liquid-crystalline phase down to -30°C and showed a nonbilayer transition at about 85°C. Mixtures (1:1 by mass) with two different phosphatidylcholines (PC) formed bilayers with no evidence for nonbilayer transitions up to 120°C. FTIR spectroscopy revealed complex interactions between the nonbilayer lipids and PC. Strong H-bonding interactions occurred between the sugar headgroup of MGDG and the phosphate, carbonyl and choline groups of PC. Similarly, the ethanolamine moiety of EPE was H-bonded to the carbonyl and choline groups of PC and probably interacted through charge pairing with the phosphate group. This study provides a comprehensive characterization of dry membranes containing the two most important nonbilayer lipids (PE and MGDG) in living cells. These data will be of particular relevance for the analysis of interactions between membranes and low molecular weight solutes or soluble proteins that are presumably involved in cellular protection during anhydrobiosis.


Is adaptive management helping to solve fisheries problems?
Adaptive management has been widely recommended as a way to deal with extreme uncertainty in natural resource and environmental decision making. The core concept in adaptive management is that policy choices should be treated as deliberate, large-scale experiments; hence, policy choice should be treated at least partly as a problem of scientific experimental design. There have now been upwards of 100 case studies where attempts were made to apply adaptive management to issues ranging from restoration of endangered desert fish species to protection of the Great Barrier Reef. Most of these cases have been failures in the sense that no experimental management program was ever implemented, and there have been serious problems with monitoring programs in the handful of cases where an experimental plan was implemented. Most of the failures can be traced to three main institutional problems: i) lack of management resources for the expanded monitoring needed to carry out large-scale experiments; ii) unwillingness by decision makers to admit and embrace uncertainty in making policy choices; and iii) lack of leadership in the form of individuals willing to do all the hard work needed to plan and implement new and complex management programs.


Detecting clinically meaningful biomarkers with repeated measurements: An illustration with electronic health records.
Data sources with repeated measurements are an appealing resource to understand the relationship between changes in biological markers and risk of a clinical event. While longitudinal data present opportunities to observe changing risk over time, these analyses can be complicated if the measurement of clinical metrics is sparse and/or irregular, making typical statistical methods unsuitable. In this article, we use electronic health record (EHR) data as an example to present an analytic procedure to both create an analytic sample and analyze the data to detect clinically meaningful markers of acute myocardial infarction (MI). Using an EHR from a large national dialysis organization we abstracted the records of 64,318 individuals and identified 4769 people that had an MI during the study period. We describe a nested case-control design to sample appropriate controls and an analytic approach using regression splines. Fitting a mixed-model with truncated power splines we perform a series of goodness-of-fit tests to determine whether any of 11 regularly collected laboratory markers are useful clinical predictors. We test the clinical utility of each marker using an independent test set. The results suggest that EHR data can be easily used to detect markers of clinically acute events. Special software or analytic tools are not needed, even with irregular EHR data.


Calibration of measures for psychotherapy outcome studies.
Many findings in psychology, including those in psychotherapy, lack interpretability because measures are not in any meaningful metric. Measures need to be calibrated so as to endow them with inherent meaning. In particular, it is important to calibrate measures against behaviors and real events in people's lives. Approaches to calibrating measures include calibration against direct personal experience, against cross-experiential equivalents, and against cross-modal equivalents, to reflect empirically established behavioral implications as well as just noticeable differences in behavior or between people. Psychological measures may never be as closely calibrated as those for physical properties, but wider reporting of unstandardized regression equations would be of great help. Psychological measures also need to be calibrated against each other so as to permit easy transformations of values or substitutions of measures. Finally, greater standardization of measures is recommended so as to produce better bases for calibration.


Anomalous behaviour of a protein during SDS/PAGE corrected by chemical modification of carboxylic groups.
The 29,000-Mr Actinomadura R39 beta-lactamase exhibited a remarkably low electrophoretic mobility on SDS/PAGE, yielding an Mr value almost twice that computed from the corresponding gene sequence. We showed that chemical modification of the carboxylic groups of glutamic acid and aspartic acid residues restored a normal electrophoretic mobility and that the anomalous behaviour of that protein on SDS/PAGE was due to its very large negative charge at neutral pH. We also compared the behaviour of the same enzyme on gel filtration in the presence of SDS with those of other class A beta-lactamases (Mr approx. 30,000). These experiments suggested that the very low electrophoretic mobility of the Actinomadura R39 beta-lactamase upon SDS/PAGE was more probably due to a low degree of SDS binding rather than to an unusual shape of the SDS-protein complex.


[Implantable infusion devices. Experience of 360 cases].
Between january 1, 1983 and december 31, 1986, 360 implantable infusion devices were inserted for systemic chemotherapy. Most of the devices were Port A Cath systems. The main complications observed were thrombosis (18 cases, 5%) and infection (8 cases, 2.2%). Since one year the catheters are being introduced by subcutaneous puncture of the subclavian vein, the chambers being placed in a small lateral subclavicular cavity. Intra-operative radiography is necessary to minimize the risk of malposition and thrombosis. These systems, relayed by external pumps, enable chemotherapeutic regimens to be administered sequentially over a 24-hour period.


Activation of mitogen-activated protein kinase signaling and development of papillary thyroid carcinoma in thyroid-stimulating hormone receptor D633H knockin mice.
Nonautoimmune hyperthyroidism (NAH) is rare and occurs due to a constitutively activating thyroid stimulating hormone receptor (TSHR) mutation. In contrast to other thyroid nodules, no further evaluation for malignancy is recommended for hot thyroid nodules. In the first model for NAH in mice nearly all homozygous mice had developed papillary thyroid cancer by 12 months of age. To further evaluate these mice, whole exome sequencing and phosphoproteome analysis were employed in a further generation of mice to identify any other mutations potentially responsible and to identify the pathways involved in thyroid carcinoma development. Only three genes (Nrg1, Rrs1, Rasal2) were mutated in all mice examined, none of which were known primary drivers of papillary thyroid cancer development. Wild-type and homozygous TSHR D633H knockin mice showed distinct phosphoproteome profiles with an enrichment of altered phosphosites found in ERK/mitogen-activated protein kinase (MAPK) signaling. Most importantly, phosphosites with known downstream effects included BRAF p.S766, which forms an inhibitory site: a decrease of phosphorylation at this site suggests an increase in MEK/ERK pathway activation. The decreased phosphorylation at BRAF p.S766 would suggest decreased AMP-activated protein kinase (AMPK) signaling, which is supported by the decreased phosphorylation of STIM1 p.S257, a downstream AMPK target. The modified phosphoproteome profile of the homozygous mice in combination with human literature suggests a potential signaling pathway from constitutive TSHR signaling and cAMP activation to the activation of ERK/MAPK signaling. This is the first time that a specific mechanism has been identified for a possible involvement of TSH signaling in thyroid carcinoma development.


High identification rates of endogenous neuropeptides from mouse brain.
Mass spectrometry-based neuropeptidomics is one of the most powerful approaches for identification of endogenous neuropeptides in the brain. Until now, however, the identification rate of neuropeptides in neuropeptidomics is relatively low and this severely restricts insights into their biological function. In the present study, we developed a high accuracy mass spectrometry-based approach to enhance the identification rates of neuropeptides from brain tissue. Our integrated approach used mixing on column for loading aqueous and organic extracts to reduce the loss of peptides during sample treatment and used charge state-directed tandem mass spectrometry to increase the number of peptides subjected to high mass accuracy fragmentation. This approach allowed 206 peptides on average to be identified from a single mouse brain sample that was prepared using 15 μL of solutions per 1 mg of tissue. In total, we identified more than 500 endogenous peptides from mouse hypothalamus and whole brain samples. Our identification rate is about two to four times higher compared to previously reported studies conducted on mice or other species. The hydrophobic peptides, such as neuropeptide Y and galanin, could be presented and detected with hydrophilic peptides in the same LC-MS run, allowing a high coverage of peptide characterization over an organism. This will advance our understanding of the roles of diverse peptides and their links in the brain functions.


Adjuvant treatment for early ovarian cancer: a randomized phase III trial of intraperitoneal 32P or intravenous cyclophosphamide and cisplatin--a gynecologic oncology group study.
To conduct a prospective study of intraperitoneal radioactive chromic phosphate (32P) versus cyclophosphamide-cisplatin (CP) in women with early ovarian cancer at high risk for recurrence (International Federation of Gynecology and Obstetrics stage Ia or Ib grade 3 or Ic or stage II, no macroscopic residual disease) and to compare cumulative incidence of recurrence, overall survival, and relative toxicity. A total of 251 patients were randomly assigned to treatment with 32P or CP. Twenty-two (8.7%) were ineligible following centralized pathology review. Of the 229 patients included in the analysis, 110 received 32P, and 119 received CP. The cumulative incidence of recurrence at 10 years was 35% (95% CI, 27% to 45%) for patients receiving 32P and 28% (95% CI, 21% to 38%) for those receiving CP. Patients receiving CP had a recurrence rate 29% lower than that of those receiving 32P (P =.15, two-tail test). The death rate for patients treated with CP was 17% lower than that for patients treated with 32P (difference not significant). Combining both arms, the 10-year cumulative incidence of recurrence for all stage I patients was 27% (95% CI, 20% to 34%) compared with 44% (95% CI, 32% to 56%) for stage II patients (P =.01). Both regimens were reasonably well tolerated, but problems with inadequate distribution (7%) and small-bowel perforation (3%) make the otherwise less toxic 32P less acceptable. Although there are no statistically significant differences in survival, the lower cumulative recurrence seen with CP and complications of 32P administration make platinum-based combinations the preferred adjuvant therapy for early ovarian cancer patients at high-risk for recurrence.


Small-area geographic and socioeconomic inequalities in colorectal tumour detection in France.
The aim of this study was to assess the impact of area deprivation and primary care facilities on colorectal adenoma detection and on colorectal cancer (CRC) incidence in a French well-defined population before mass screening implementation. The study population included all patients aged 20 years or more living in Côte d'Or (France) with either colorectal adenoma or invasive CRC first diagnosed between 1995 and 2002 and who were identified from the Burgundy Digestive Cancer Registry and the Côte d'Or Polyp Registry. Area deprivation was assessed using the European deprivation index on the basis of the smallest French area available (Ilots Regroupés pour l'Information Statistique). Healthcare access was assessed using medical density of general practitioners (GPs) and road distance to the nearest GP and gastroenterologist. Bayesian regression analyses were used to estimate influential covariates on adenoma detection and CRC incidence rates. The results were expressed as relative risks (RRs) with their 95% credibility interval. In total, 5399 patients were diagnosed with at least one colorectal adenoma and 2125 with invasive incident CRC during the study period. Remoteness from GP [RR=0.71 (0.61-0.83)] and area deprivation [RR=0.98 (0.96-1.00)] independently reduced the probability of adenoma detection. CRC incidence was only slightly affected by GP medical density [RR=1.05 (1.01-1.08)] without any area deprivation effect [RR=0.99 (0.96-1.02)]. Distance to gastroenterologist had no impact on the rates of adenoma detection or CRC incidence. This study highlighted the prominent role of access to GPs in the detection of both colorectal adenomas and overall cancers. Deprivation had an impact only on adenoma detection.


Pre-employment screening and health management for safety forces-methods and techniques.
The purpose of this clinical report is to describe the process by which safety forces (police and fire) are tested prior to employment (pre-employment screening) and the periodic evaluations used to maintain such standards. The process began by testing 350 male police volunteers and developing a series of age stratified norms. These norms are used to compare incoming recruits such that candidates of a given age are compared to norms developed from police officers of the same age. The purposes of this report are to describe the clinical tests used in the pre-employment screening, to report the normative data and to describe the methods of comparing data obtained from applicants to the established norms. The proper management of high risk employees through pre-employment screening and intervention programs has been viewed as being necessary both for the individual employee in reducing injury and disease and for the municipality for cost saving purposes. J Orthop Sports Phys Ther 1990;11(9):398-401.


The LINC complex is essential for hearing.
Hereditary hearing loss is the most common sensory deficit. We determined that progressive high-frequency hearing loss in 2 families of Iraqi Jewish ancestry was due to homozygosity for the protein truncating mutation SYNE4 c.228delAT. SYNE4, a gene not previously associated with hearing loss, encodes nesprin-4 (NESP4), an outer nuclear membrane (ONM) protein expressed in the hair cells of the inner ear. The truncated NESP4 encoded by the families' mutation did not localize to the ONM. NESP4 and SUN domain-containing protein 1 (SUN1), which localizes to the inner nuclear membrane (INM), are part of the linker of nucleoskeleton and cytoskeleton (LINC) complex in the nuclear envelope. Mice lacking either Nesp4 or Sun1 were evaluated for hair cell defects and hearing loss. In both Nesp4-/- and Sun1-/- mice, OHCs formed normally, but degenerated as hearing matured, leading to progressive hearing loss. The nuclei of OHCs from mutant mice failed to maintain their basal localization, potentially affecting cell motility and hence the response to sound. These results demonstrate that the LINC complex is essential for viability and normal morphology of OHCs and suggest that the position of the nucleus in sensory epithelial cells is critical for maintenance of normal hearing.


Drosophila exoribonuclease nibbler is a tumor suppressor, acts within the RNAi machinery and is not enriched in the nuage during early oogenesis.
micro RNAs (miRNAs) are important regulators of many biological pathways. A plethora of steps are required to form, from a precursor, the mature miRNA that eventually acts on its target RNA to repress its expression or to inhibit translation. Recently, Drosophila nibbler (nbr) has been shown to be an important player in the maturation process of miRNA and piRNA. Nbr is an exoribonuclease which helps to shape the 3' end of miRNAs by trimming the 3' overhang to a final length. In contrast to previous reports on the localization of Nbr, we report that 1) Nbr is expressed only during a short time of oogenesis and appears ubiquitously localized within oocytes, and that 2) Nbr was is not enriched in the nuage where it was shown to be involved in piwi-mediated mechanisms. To date, there is little information available on the function of nbr for cellular and developmental processes. Due to the fact that nbr mutants are viable with minor deleterious effects, we used the GAL4/UAS over-expression system to define novel functions of nbr. We disclose hitherto unknown functions of nbr 1) as a tumor suppressor and 2) as a suppressor of RNAi. Finally, we confirm that nbr is a suppressor of transposon activity. Our data suggest that nbr exerts much more widespread functions than previously reported from trimming 3' ends of miRNAs only.


Case-control analysis of dietary folate and risk of bladder cancer.
Dietary folate, a water-soluble B vitamin found in a variety of fruits and vegetables, is of particular interest as a chemopreventive agent due to its role in DNA methylation and DNA synthesis and repair. We hypothesized that individuals with low folate intake would be at an increased risk for bladder cancer. Using an ongoing case-control study we assessed dietary folate in 409 incident bladder cancer patients and 451 healthy control subjects. A food-frequency questionnaire was used to estimate naturally occurring food folate (microg/kcal/day), dietary folate equivalents (DFE) from food sources (microg DFE/kcal/day), and DFE from all sources (microg DFE/kcal/day). Unconditional logistic regression analyses were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs). Bladder cancer patients reported a statistically significant lower intake of folate than control subjects for food folate and DFE from food sources (P < 0.001) but not for DFE from all sources (P = 0.061). In the highest quartile of food folate intake there was a 54% reduced risk for bladder cancer (OR = 0.46; 95% CI = 0.29-0.73) after adjusting for age, gender, ethnicity, smoking, and total energy intake. Similarly, the highest quartile of intake was associated with a 59% reduced risk for DFE from food sources (OR = 0.41; 95% CI = 0.26-0.65) and a 35% reduced risk for DFE from all sources (OR = 0.65; 95% CI = 0.42-1.00). In the joint-effects analyses using never smokers with high folate intake as the reference group (OR = 1.0), heavy smokers with low food folate intake had a 2.31-fold (95% CI = 1.11-4.82) increased risk, whereas heavy smokers with high folate intake had a reduced OR of 1.31 (95% CI = 0.53-3.26). Although the ORs were not statistically significant, light smokers and high folate intake exhibited a protective effect (OR = 0.62; 95% CI = 0.20-1.94), whereas an increased risk was observed for light smoking and low folate intake (OR = 1.41; 95% CI = 0.57-3.45). These patterns were consistent for the joint effects of smoking and DFE from food sources and DFE from all sources. In summary, high intake of dietary folate was associated with an overall decrease in bladder cancer risk. These data may have important implications for cancer prevention; however, large, hypothesis-driven, population-based clinical trials will be required to confirm these findings.


ERP measures of the effects of age and bilingualism on working memory performance.
Previous research has suggested that bilinguals may exhibit cognitive advantages over those who are monolingual, although conflicting results have been reported. This advantage may be heightened in older adults, because of age-related cognitive decline. However, the effects of bilingualism on working memory performance in older adults remain unknown. The current study uses electroencephalography to measure brain activity (event-related potential; ERP) differences between young and older monolinguals and bilinguals during a delayed matching-to-sample task. Although there were no effects of bilingualism in behavioral measures, differences were observed in electrophysiological measures. While no Age by Language interaction was observed, several main effects were identified. Compared to young adults, older adults exhibited smaller N2 amplitudes and larger P2 and P3b amplitudes in the medium and high load conditions. Older adults also displayed an increased slow wave amplitude that occurred in conjunction with increased reaction time. ERP differences during difficult tasks in older adults suggest the use of compensatory mechanisms to maintain similar performance to the young adults. Bilinguals exhibited smaller N2 and larger P2 and P3b amplitudes than monolinguals. ERP differences observed in bilinguals may reflect differences in cognitive processing. However, in the absence of performance differences between monolinguals and bilinguals, interpreting a bilingual advantage in working memory processing is difficult.


Measuring health-related quality of life in rheumatoid arthritis: validity, responsiveness and reliability of EuroQol (EQ-5D).
The EuroQol (EQ-5D) generic health index comprises a five-part questionnaire and a visual analogue self-rating scale. The questionnaire may be used as a health index to calculate a 'utility' value or as a health profile. The validity, reliability and responsiveness of EQ-5D were tested in 233 patients with rheumatoid arthritis stratified by functional class. EQ-5D demonstrated moderate to high correlations with measures of impairment and high correlations with disability measures. Stepwise regression models showed that EQ-5D utility values and visual analogue scores were explained best as a function of pain, disability, disease activity and mood (R2 approximately 70%), although other variables (side-effects, years of education) were required to explain the visual analogue scores. The EQ-5D health index and visual analogue scale are more responsive than any of the other measures, except pain and doctor-assessed disease activity. The reliability of the EQ-5D index and EQ-5D visual analogue scale is as good or better than that of all other instruments except the Health Assessment Questionnaire. Some patients with severe long-standing disease had health states which attracted utility values below zero, i.e. from a societal perspective they were regarded as being in states 'worse than death'. The practical and ethical implications of these utility valuations are discussed, and at present the utility values should be used and interpreted with caution. With this caveat, EQ-5D is simple to use, valid, responsive to change and sufficiently reliable for group comparisons. It is of potential use as an outcome measure in clinical trials, audit and health economic studies, but further work is required on its performance in other clinical contexts and on the interpretation of the utility values.


Smoking cessation is associated with lower rates of mood/anxiety and alcohol use disorders.
The psychological outcomes that accompany smoking cessation are not yet conclusive but positive outcomes could help to persuade quitting. We used data from the longitudinal National Epidemiological Study of Alcohol and Related Conditions. Logistic regression was used to examine associations between cigarette smoking reduction and Wave 2 status of addiction/mental health disorder among daily smokers at Wave 1, stratified by status of the diagnosis of interest at Wave 1. We adjusted for differences in baseline covariates between smokers with different levels of smoking reduction between Wave 1 and Wave 2 using propensity score regression adjustment. After adjusting for propensity scores and other mental health/addiction co-morbidities at Wave 2, among daily smokers who had current or lifetime history diagnosis of the outcome of interest at Wave 1, quitting by Wave 2 predicted a decreased risk of mood/anxiety disorder [adjusted odds ratio (aOR) 0.6, 95% confidence interval (CI) 0.4-0.9] and alcohol disorder (aOR 0.7, 95% CI 0.5-0.99) at Wave 2. Among daily smokers with no lifetime history diagnosis of the outcome of interest at Wave 1, quitting smoking by Wave 2 predicted a decreased risk of drug use disorder at Wave 2 (aOR 0.3, 95% CI 0.1-0.9). There is no support in our data for the concern that smoking cessation would result in smokers' increased risk of some mental disorders. To the contrary, our data suggest that smoking cessation is associated with risk reduction for mood/anxiety or alcohol use disorder, even among smokers who have had a pre-existing disorder.


Antiproliferative effects of interferons on human melanoma cells in the human tumor colony-forming assay.
The human tumor colony-forming assay (HTCFA) is an in vitro test that has been used to predict the activity of anticancer drugs against a patient's tumor. We utilized the assay to analyze the antiproliferative effects of seven interferons (IFNs) against 40 human melanomas to determine which IFN had the greatest antiproliferative activity in this drug-resistant tumor. IFNs studied included recombinant IFN-alpha 2; human lymphoblastoid IFN; IFN-alpha Cantell; native beta RPMI; two recombinant IFNs-beta; and recombinant IFN-gamma. Growth was sufficient [greater than 30 tumor colony-forming units (TCFU)/well] for assessing the antiproliferative effects of at least one IFN in 25 tumors (63%). A dose-response relationship was demonstrated by all IFNs in tumors in which some activity was observed (p less than or equal to 0.01). Individual melanomas differed in their sensitivities to the various IFNs. Overall, however, none of the IFNs was markedly more effective in antiproliferative effects than any other, although there was a trend toward IFN-beta ser having more potent antiproliferative properties when compared to IFN-alpha 2 (p = 0.055). Twelve of 13 tumors exposed to combinations of IFN-beta ser and IFN-gamma demonstrated a synergistic antiproliferative effect. In all but two of these, low concentrations of each IFN (less than or equal to 50 U/ml), when combined, resulted in 85-95% inhibition. As prolonged exposure to high concentrations of IFN are often not clinically tolerable, these data suggest that IFN combinations may be one way of achieving more clinically meaningful IFN doses, schedules, and regimens, provided antiproliferative effects are of importance in vivo.


Dementia knowledge assessment scale (DKAS): confirmatory factor analysis and comparative subscale scores among an international cohort.
Dementia is a life-limiting condition that is increasing in global prevalence in line with population ageing. In this context, it is necessary to accurately measure dementia knowledge across a spectrum of health professional and lay populations with the aim of informing targeted educational interventions and improving literacy, care, and support. Building on prior exploratory analysis, which informed the development of the preliminarily valid and reliable version of the Dementia Knowledge Assessment Scale (DKAS), a Confirmatory Factor Analysis (CFA) was performed to affirm construct validity and proposed subscales to further increase the measure's utility for academics and educators. A large, de novo sample of 3649 volunteer respondents to a dementia-related online course was recruited to evaluate the performance of the DKAS and its proposed subscales. Respondents represented diverse cohorts, including health professionals, students, and members of the general public. Analyses included CFA (using structural equation modelling), measures of internal consistency (α), and non-parametric tests of subscale correlation (Spearman Correlation) and score differences between cohorts (Kruskal-Wallis one-way analysis of variance). Findings of the CFA supported a 25-item, four-factor model for the DKAS with two items removed due to poor performance and one item moved between factors. The resultant model exhibited good reliability (α = .85; ω h = .87; overall scale), with acceptable subscale internal consistency (α ≥ .65; subscales). Subscales showed acceptable correlation without any indication of redundancy. Finally, total and DKAS subscale scores showed good discrimination between cohorts of respondents who would be anticipated to hold different levels of knowledge on the basis of education or experience related to dementia. The DKAS has been confirmed as a reliable and valid measure of dementia knowledge for diverse populations that is capable of elucidating knowledge characteristics across four coherent domains: 1) Causes and Characteristics, 2) Communication and Behaviour, 3) Care Considerations, and 4) Risks and Health Promotion. Importantly, the four confirmed subscales clearly distinguish between groups who might be expected to hold differing levels of knowledge about dementia, allowing for a fine-grained level of detail to be established when evaluating baseline understanding or knowledge change associated with educational intervention.


Synthesis of cytomegalovirus DNA is an antiviral target late in virus growth.
The mechanism of action of 9-(1,3-dihydroxypropoxymethyl)guanine (DHPG) and phosphonoformic acid (PFA) but not 5-fluorouridinedeoxyribose (FUdR), provides selective action against cytomegalovirus (CMV)-coded events and this was used to demonstrate that the synthesis of viral DNA was continuous during the extended phase of virus growth. The synthesis de novo of viral DNA was measured by restriction enzyme analysis after exposure to [32P]orthophosphate and its interruption by DHPG or PFA resulted in a cessation in the extrusion of infective virus from treated cells. The rate of decline in infectivity appeared to correspond to the failure of cells to maintain the synthesis of late proteins once DNA synthesis was blocked. Thus, regulation of late protein synthesis appeared to be linked to synthesis de novo of viral DNA even at late stages in CMV growth. The synthesis of the polyamines spermidine and spermine, considered obligatory for CMV growth, was unaffected by early or late inhibition of viral DNA and this showed that some virus-induced events were unaffected by the restriction on virus growth by DHPG. This provided evidence that polyamine biosynthesis was a target independent of viral DNA synthesis per se, which may be important in future considerations of combined drug therapies.


Hyperoxia depletes (6R)-5,6,7,8-tetrahydrobiopterin levels in the neonatal retina: implications for nitric oxide synthase function in retinopathy.
Retinopathy of prematurity is a sight-threatening complication of premature birth caused by nitro-oxidative insult to the developing retinal vasculature during therapeutic hyperoxia exposure and later ischemia-induced neovascularization on supplemental oxygen withdrawal. In the vasodegenerative phase, during hyperoxia, defective endothelial nitric oxide synthase (NOS) produces reactive oxygen and nitrogen free radicals rather than vasoprotective nitric oxide for unclear reasons. Crucially, normal NOS function depends on availability of the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4). Because BH4 synthesis is controlled enzymatically by GTP cyclohydrolase (GTPCH), we used GTPCH-depleted mice [hyperphenylalaninemia strain (hph1)] to investigate the impact of hyperoxia on BH4 bioavailability and retinal vascular pathology in the neonate. Hyperoxia decreased BH4 in retinas, lungs, and aortas in all experimental groups, resulting in a dose-dependent decrease in NOS activity and, in the wild-type group, elevated NOS-derived superoxide. Retinal dopamine levels were similarly diminished, consistent with the dependence of tyrosine hydroxylase on BH4. Despite greater depletion of BH4, the hph(+/-) and hph1(-/-) groups did not show exacerbated hyperoxia-induced vessel closure, but exhibited greater vascular protection and reduced progression to neovascular disease. This vasoprotective effect was independent of enhanced circulating vascular endothelial growth factor (VEGF), which was reduced by hyperoxia, but to local retinal ganglion cell layer-derived VEGF. In conclusion, a constitutively higher level of VEGF expression associated with retinal development protects GTPCH-deficient neonates from oxygen-induced vascular damage.


Comparison of ligand binding and conformational stability of human calmodulin with its homolog from the malaria parasite Plasmodium falciparum.
Calmodulin (CaM), the key calcium sensor of eukaryotic cells regulating a great number of target proteins, belongs to the most conserved proteins. We compared function and properties of CaMs from two evolutionarily distant species, the human (Homo sapiens) representing vertebrates, and the malaria parasite Plasmodium falciparum (Pf). The biophysical characterization revealed higher stability of Pf CaM attributed to the more stable C-terminal domain in both Ca2+ free and saturated states. In vitro binding and functional assays demonstrated that human and Pf CaM exhibit similar biochemical features involving small molecule inhibitor binding and target enzyme activation as illustrated by comparable affinities differing only within a factor of three. It has been reported that CaM antagonists proved to be antimalarials, so Pf CaM could be a potential target to combat malaria parasites. Indeed, we observed that phenotypically active compounds from the Malaria Box could show inhibitory action on Pf CaM, among them the most potent exhibited comparable inhibition to known antagonists of vertebrate CaM. However, based on the minor binding differences in Pf CaM to human CaM, we conclude that CaM is an unsuited target for human intervention against malaria, due to the likely interference with the host protein.


Matching variants for functional characterization of genetic variants.
Rapid and low-cost sequencing, as well as computer analysis, have facilitated the diagnosis of many genetic diseases, resulting in a substantial rise in the number of disease-associated genes. However, genetic diagnosis of many disorders remains problematic due to the lack of interpretation for many genetic variants, especially missenses, the infeasibility of high-throughput experiments on mammals, and the shortcomings of computational prediction technologies. Additionally, the available mutant databases are not well-utilized. Toward this end, we used Caenorhabditis elegans mutant resources to delineate the functions of eight missense variants (V444I, V517D, E610K, L732F, E817K, H873P, R1105K, and G1205E) and two stop codons (W937stop and Q1434stop), including several matching variants (MatchVar) with human in ciliopathy associated IFT-140 (also called CHE-11)//IFT140 (intraflagellar transport protein 140). Moreover, MatchVars carrying C. elegans mutants, including IFT-140(G680S) and IFT-140(P702A) for the human (G704S) (dbSNP: rs150745099) and P726A (dbSNP: rs1057518064 and a conflicting variation) were created using CRISPR/Cas9. IFT140 is a key component of IFT complex A (IFT-A), which is involved in the retrograde transport of IFT along cilia and the entrance of G protein-coupled receptors into cilia. Functional analysis of all 10 variants revealed that P702A and W937stop, but not others phenocopied the ciliary phenotypes (short cilia, IFT accumulations, mislocalization of membrane proteins, and cilia entry of nonciliary proteins) of the IFT-140 null mutant, indicating that both P702A and W937stop are phenotypic in C. elegans. Our functional data offered experimental support for interpreting human variants, by using ready-to-use mutants carrying MatchVars and generating MatchVars with CRISPR/Cas9.


Structural polymorphisms within a common powdery mildew effector scaffold as a driver of coevolution with cereal immune receptors.
In plants, host-pathogen coevolution often manifests in reciprocal, adaptive genetic changes through variations in host nucleotide-binding leucine-rich repeat immune receptors (NLRs) and virulence-promoting pathogen effectors. In grass powdery mildew (PM) fungi, an extreme expansion of a RNase-like effector family, termed RALPH, dominates the effector repertoire, with some members recognized as avirulence (AVR) effectors by cereal NLR receptors. We report the structures of the sequence-unrelated barley PM effectors AVRA6, AVRA7, and allelic AVRA10/AVRA22 variants, which are detected by highly sequence-related barley NLRs MLA6, MLA7, MLA10, and MLA22 and of wheat PM AVRPM2 detected by the unrelated wheat NLR PM2. The AVR effectors adopt a common scaffold, which is shared with the RNase T1/F1 family. We found striking variations in the number, position, and length of individual structural elements between RALPH AVRs, which is associated with a differentiation of RALPH effector subfamilies. We show that all RALPH AVRs tested have lost nuclease and synthetase activities of the RNase T1/F1 family and lack significant binding to RNA, implying that their virulence activities are associated with neo-functionalization events. Structure-guided mutagenesis identified six AVRA6 residues that are sufficient to turn a sequence-diverged member of the same RALPH subfamily into an effector specifically detected by MLA6. Similar structure-guided information for AVRA10 and AVRA22 indicates that MLA receptors detect largely distinct effector surface patches. Thus, coupling of sequence and structural polymorphisms within the RALPH scaffold of PMs facilitated escape from NLR recognition and potential acquisition of diverse virulence functions.