Long-term overall and disease-specific mortality associated with benign gynecologic surgery performed at different ages.
As bilateral salpingo-oophorectomy is frequently performed with hysterectomy for nonmalignant conditions, defining health outcomes associated with benign bilateral salpingo-oophorectomy performed at different ages is critical. We assessed mortality risk associated with benign total abdominal hysterectomy or bilateral salpingo-oophorectomy among 52,846 Breast Cancer Detection Demonstration Project follow-up study participants. Surgery and risk factor data were ascertained via baseline interview (1979-1986) and three questionnaires (1987-1998). During follow-up through December 2005 (mean, 22.1 y), 13,734 deaths were identified. We estimated hazard ratios (HRs) and 95% CIs for overall and disease-specific mortality for total abdominal hysterectomy or bilateral salpingo-oophorectomy performed by age 35, 40, 45, 50, or 55 years, compared with not having surgery, using landmark analyses and multivariable Cox regression. Undergoing bilateral salpingo-oophorectomy by age 35 years was associated with increased mortality risk (HR35 y, 1.20; 95% CI, 1.08-1.34), which decreased with age (HR40 y, 1.12; 95% CI, 1.04-1.21; HR45 y, 1.10; 95% CI, 1.03-1.17). Total abdominal hysterectomy alone performed by age 40 years was associated with increased mortality risk to a lesser extent (HR40 y, 1.08; 95% CI, 1.01-1.15). Analyses based on matched propensity scores related to having gynecologic surgery yielded similar results. Elevated mortality risks were largely attributable to noncancer causes. Benign gynecologic surgeries among young women are associated with increased mortality risk, which attenuates with age.


Expression of pp60c-src protein kinase in adult and fetal human tissue: high activities in some sarcomas and mammary carcinomas.
An endogenous protein of human cells pp60c-src, which is closely related to the product of the transforming gene of Rous sarcoma virus, pp60v-src, has been quantitated by measuring its enzymatic activity in an immunoglobulin G protein kinase assay. The influence of normal developmental processes on pp60c-src expression was assessed by comparative analysis of various adult and fetal human tissues. The maximal difference detected was a 2- to 3-fold-enhanced activity in fetal muscle compared with adult muscle. Organ-specific variations in the enzyme level were observed. Highest activity was found in brain, followed by kidney, lung, muscle, and connective tissue. Since overexpression of the cellular counterparts of viral-transforming genes may play a role in carcinogenesis, pp60c-src kinase was measured in nine spontaneous human sarcomas and 21 mammary carcinomas. Compared with the respective normal tissues and human diploid fibroblasts, 4- to 20-fold-enhanced activities were observed in one-third of the sarcomas and carcinomas. The remainder showed no or insignificantly elevated activity. The enzymes from normal and malignant tissues were indistinguishable from the virus-coded enzyme with respect to specificity for divalent cations and a predominance of phosphorylation in tyrosine. Patients carrying tumors with high pp60c-src protein kinase activity did not develop kinase-reactive antibodies against pp60c-src or pp60v-src.


Evolution and Adaptation of Wild Emmer Wheat Populations to Biotic and Abiotic Stresses.
The genetic bottlenecks associated with plant domestication and subsequent selection in man-made agroecosystems have limited the genetic diversity of modern crops and increased their vulnerability to environmental stresses. Wild emmer wheat, the tetraploid progenitor of domesticated wheat, distributed along a wide range of ecogeographical conditions in the Fertile Crescent, has valuable "left behind" adaptive diversity to multiple diseases and environmental stresses. The biotic and abiotic stress responses are conferred by series of genes and quantitative trait loci (QTLs) that control complex resistance pathways. The study of genetic diversity, genomic organization, expression profiles, protein structure and function of biotic and abiotic stress-resistance genes, and QTLs could shed light on the evolutionary history and adaptation mechanisms of wild emmer populations for their natural habitats. The continuous evolution and adaptation of wild emmer to the changing environment provide novel solutions that can contribute to safeguarding food for the rapidly growing human population.


Enhancing the effectiveness of self-administered videotape parent training for families with conduct-problem children.
Parents of 43 conduct-problem children, aged 3-8 years, were randomly assigned to one of two treatments: an individually self-administered video-tape modeling treatment (IVM) and IVM treatment plus therapist consultation (IVMC). Randomization also included a waiting-list control group (CON). Compared with the control group, both treatment groups of mothers reported significantly fewer child behavior problems, reduced stress levels, and less use of spanking. Home visit data indicated that both treatment groups exhibited significant behavioral changes. There were relatively few differences between the two treatment conditions. However, the IVMC children were significantly less deviant than the IVM children, suggesting that the IVMC (with therapist consultation) treatment was superior to self-administered treatment with no therapist involvement. The added benefits of therapist involvement are discussed.


Limited in vivo production of type I or type III interferon after infection of macaques with vaccine or wild-type strains of measles virus.
The innate immune response to viral infections often includes induction of types I and III interferons (IFNs) and production of antiviral proteins. Measles is a severe virus-induced rash disease, but in vitro studies suggest that in the absence of defective interfering RNAs, neither wild-type (WT) nor vaccine strains of measles virus (MeV) induce IFN. To determine whether IFN is produced in vivo, we studied tissues from macaques infected with vaccine or WT strains of MeV using quantitative reverse transcriptase-polymerase chain reaction to assess levels of IFN and IFN-stimulated gene (ISG) mRNAs and a flow cytometry-based bioassay to assess levels of biologically active IFN. There was little to no induction of type I IFN, type III IFN, Mx, or ISG56 mRNAs in monkeys infected with vaccine or WT MeV and no IFN detection by bioassay. Therefore, the innate responses to infection with vaccine or WT strains of MeV are not dependent on IFN production.


Gene expression patterns in the hippocampus and amygdala of endogenous depression and chronic stress models.
The etiology of depression is still poorly understood, but two major causative hypotheses have been put forth: the monoamine deficiency and the stress hypotheses of depression. We evaluate these hypotheses using animal models of endogenous depression and chronic stress. The endogenously depressed rat and its control strain were developed by bidirectional selective breeding from the Wistar-Kyoto (WKY) rat, an accepted model of major depressive disorder (MDD). The WKY More Immobile (WMI) substrain shows high immobility/despair-like behavior in the forced swim test (FST), while the control substrain, WKY Less Immobile (WLI), shows no depressive behavior in the FST. Chronic stress responses were investigated by using Brown Norway, Fischer 344, Lewis and WKY, genetically and behaviorally distinct strains of rats. Animals were either not stressed (NS) or exposed to chronic restraint stress (CRS). Genome-wide microarray analyses identified differentially expressed genes in hippocampi and amygdalae of the endogenous depression and the chronic stress models. No significant difference was observed in the expression of monoaminergic transmission-related genes in either model. Furthermore, very few genes showed overlapping changes in the WMI vs WLI and CRS vs NS comparisons, strongly suggesting divergence between endogenous depressive behavior- and chronic stress-related molecular mechanisms. Taken together, these results posit that although chronic stress may induce depressive behavior, its molecular underpinnings differ from those of endogenous depression in animals and possibly in humans, suggesting the need for different treatments. The identification of novel endogenous depression-related and chronic stress response genes suggests that unexplored molecular mechanisms could be targeted for the development of novel therapeutic agents.


Reconsolidation of a well-learned instrumental memory.
Once consolidated, memories are dynamic entities that go through phases of instability in order to be updated with new information, via a process of reconsolidation. The phenomenon of reconsolidation has been demonstrated in a wide variety of experimental paradigms. However, the memories underpinning instrumental behaviors are currently not believed to reconsolidate. We show that well-learned lever pressing in rats does undergo reconsolidation, which can be disrupted by systemic administration of the noncompetitive N-methyl-D-aspartate receptor (NMDAR) antagonist (+)-5-methyl-10,11-dihydro-SH-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) when administered prior to a switch to a variable, but not fixed, ratio schedule. Disruption of reconsolidation resulted in a reduction in long-term lever pressing performance and diminished the sensitivity of behavior to contingency change. Further investigation demonstrated that expression of the reconsolidation impairment was not affected by outcome value, implying a deficit in a stimulus-response (S-R) process. The ability to disrupt the performance of well-learned instrumental behaviors is potentially of great importance in the development of reconsolidation-based clinical treatments for conditions that involve compulsive seeking behaviors.


Circovirus Hepatitis Infection in Heart-Lung Transplant Patient, France.
In March 2022, a 61-year-old woman in France who had received a heart-lung transplant sought treatment with chronic hepatitis mainly characterized by increased liver enzymes. After ruling out common etiologies, we used metagenomic next-generation sequencing to analyze a liver biopsy sample and identified an unknown species of circovirus, tentatively named human circovirus 1 (HCirV-1). We found no other viral or bacterial sequences. HCirV-1 shared 70% amino acid identity with the closest known viral sequences. The viral genome was undetectable in blood samples from 2017-2019, then became detectable at low levels in September 2020 and peaked at very high titers (1010 genome copies/mL) in January 2022. In March 2022, we found >108 genome copies/g or mL in the liver and blood, concomitant with hepatic cytolysis. We detected HCirV-1 transcripts in 2% of hepatocytes, demonstrating viral replication and supporting the role of HCirV-1 in liver damage.


Outcome Definition Influences the Relationship Between Genetic Polymorphisms of ERCC1, ERCC2, SLC22A2 and Cisplatin Nephrotoxicity in Adult Testicular Cancer Patients.
Although previous research identified candidate genetic polymorphisms associated with cisplatin nephrotoxicity, varying outcome definitions potentially contributed to the variability in the effect size and direction of this relationship. We selected genetic variants that have been significantly associated with cisplatin-induced nephrotoxicity in more than one published study (SLC22A2 rs316019; ERCC1 rs11615 and rs3212986; ERCC2 rs1799793 and rs13181) and performed a replication analysis to confirm associations between these genetic polymorphisms and cisplatin nephrotoxicity using various outcome definitions. We included 282 germ cell testicular cancer patients treated with cisplatin from 2009-2014, aged >17 years recruited by the Canadian Pharmacogenomics Network for Drug Safety. Nephrotoxicity was defined using four grading tools: (1) Common Terminology Criteria for Adverse Events (CTCAE) v4.03 for acute kidney injury (AKI) or CTCAE-AKI; (2) adjusted cisplatin-induced AKI; (3) elevation of serum creatinine; and (4) reduction in the estimated glomerular filtration rate (eGFR). Significant associations were only found when using the CTCAE v4.03 definition: genotype CA of the ERCC1 rs3212986 was associated with decreased risk of cisplatin nephrotoxicity (ORadj = 0.24; 95% CI:0.08-0.70; p= 0.009) compared to genotype CC. In contrast, addition of allele A at SLC22A2 rs316019 was associated with increased risk (ORadj = 4.41; 95% CI:1.96-9.88; p < 0.001) while genotype AC was associated with a higher risk of cisplatin nephrotoxicity (ORadj = 5.06; 95% CI:1.69-15.16; p= 0.004) compared to genotype CC. Our study showed that different case definitions led to variability in the genetic risk ascertainment of cisplatin nephrotoxicity. Therefore, consensus on a set of clinically relevant outcome definitions that all such studies should follow is needed.


Effects of legume kernel fibres and citrus fibre on putative risk factors for colorectal cancer: a randomised, double-blind, crossover human intervention trial.
In some studies, high intake of dietary fibre has been associated with a lower risk of colorectal cancer. The present study aimed to compare physiological effects of three legume kernel fibres and citrus fibre on blood lipids (primary outcome: LDL cholesterol) and colonic health. Ninety-two subjects were recruited for the double-blind, controlled crossover trial. Seventy-eight participants were randomly divided into three groups. Following run-in, half the volunteers from each group consumed 25 g/d of a legume fibre, comprising blue lupin fibre, white lupin fibre, and soya fibre for two weeks. The other half received the same amount of citrus fibre (active comparator). The intervention was crossed within each group after two weeks wash-out. At the end of run-in and intervention, a quantitative faeces collection took place and fasting blood samples were drawn. Repeated measures ANOVA with the general linear model were applied to evaluate changes following interventions. Seventy-six subjects completed the study. Dietary fibre intake during all interventions was approximately twice the fibre intake at run-in. The lupin fibre supplementations increased daily faecal dry matter and faecal weight compared to run-in, representing an increase of 1.76 g faeces/g additional dietary fibre contributed by blue lupin and of 1.64 g faeces/g by white lupin, respectively. Both lupin interventions led to a significantly enhanced formation of short-chain fatty acids, and blue lupin fibre to a decrease in faecal pH compared to run-in (0.27 units, P < 0.01). Further, blue lupin increased primary bile acids-excretion (P = 0.02). All legume fibres reduced faecal concentrations of total and secondary bile acids (blue lupin: 16% white lupin: 24% soya: 16%). Blood lipids were not influenced by any intervention. No serious adverse effects were observed. The tested fibre preparations do not affect lipid metabolism through bile acid-binding in normocholesterolaemic subjects. However, particularly blue lupin kernel fibre improve colonic function and have beneficial effects on putative risk factors for colorectal cancer such as faecal mass, transit time, SCFA, faecal pH, and secondary bile acid concentration. Therefore, enhancing dietary fibre intake through blue lupin up to about 50 g/d can be recommended. NCT01036308.


Simulating future trends in urban stormwater quality for changing climate, urban land use and environmental controls.
The effects of climatic changes, progressing urbanization and improved environmental controls on the simulated urban stormwater quality in a northern Sweden community were studied. Future scenarios accounting for those changes were developed and their effects simulated with the Storm Water Management Model (SWMM). It was observed that the simulated stormwater quality was highly sensitive to the scenarios, mimicking progressing urbanization with varying catchment imperviousness and area. Thus, land use change was identified as one of the most influential factors and in some scenarios, urban growth caused changes in runoff quantity and quality exceeding those caused by a changing climate. Adaptation measures, including the reduction of directly connected impervious surfaces (DCIS) through the integration of more green spaces into the urban landscape, or disconnection of DCIS were effective in reducing runoff volume and pollutant loads. Furthermore, pollutant source control measures, including material substitution, were effective in reducing pollutant loads and significantly improving stormwater quality.


Intrapericardial minocycline sclerosis for malignant pericardial effusion.
To evaluate the effectiveness and safety of minocycline hydrochloride (minocycline) intrapericardially in patients with malignant pericardial effusion. Consecutive patients admitted to the hospital during a 32-month period received intrapericardial minocycline. A 900-bed university hospital. Fourteen consecutive patients with malignant pericardial effusion. Following percutaneous insertion of a pericardial drain, minocycline was administered at a dosage of 10 mg/kg every 48 h until fluid drainage stopped or until further therapy was deemed necessary. Complications associated with therapy, total minocycline requirements, immediate and late failure of therapy, and clinical and echocardiographic follow-up of at least 6 months. Mean amount of minocycline administered was 1.9 +/- 1.0g given in 2.4 divided doses. Total drainage time was 5.4 +/- 2.5 days. Recurrence of malignant pericardial effusion was seen in only 1 of 14 patients. Death occurred in 10 patients due to severe metastatic disease in all. Minocycline instillation was associated with severe chest pain in seven patients, and with ECG changes suggesting pericardial or subepicardial injury in two patients. (1) Intrapericardial minocycline instillation is very effective in preventing recurrence of malignant pericardial effusion. (2) Minocycline is irritative to the pericardium and may cause severe chest pain with transient ECG changes, suggesting pericardial or subepicardial injury.


PPARdelta is an APC-regulated target of nonsteroidal anti-inflammatory drugs.
PPARB was identified as a target of APC through the analysis of global gene expression profiles in human colorectal cancer (CRC) cells. PPARdelta expression was elevated in CRCs and repressed by APC in CRC cells. This repression was mediated by beta-catenin/Tcf-4-responsive elements in the PPARdelta promotor. The ability of PPARs to bind eicosanoids suggested that PPARdelta might be a target of chemopreventive non-steroidal anti-inflammatory drugs (NSAIDs). Reporters containing PPARdelta-responsive elements were repressed by the NSAID sulindac. Furthermore, sulindac was able to disrupt the ability of PPARdelta to bind its recognition sequences. These findings suggest that NSAIDs inhibit tumorigenesis through inhibition of PPARdelta, the gene for which is normally regulated by APC.


Atomistic folding simulations of the five-helix bundle protein λ(6−85).
Protein folding is a classic grand challenge that is relevant to numerous human diseases, such as protein misfolding diseases like Alzheimer's disease. Solving the folding problem will ultimately require a combination of theory, simulation, and experiment, with theory and simulation providing an atomically detailed picture of both the thermodynamics and kinetics of folding and experimental tests grounding these models in reality. However, theory and simulation generally fall orders of magnitude short of biologically relevant time scales. Here we report significant progress toward closing this gap: an atomistic model of the folding of an 80-residue fragment of the λ repressor protein with explicit solvent that captures dynamics on a 10 milliseconds time scale. In addition, we provide a number of predictions that warrant further experimental investigation. For example, our model's native state is a kinetic hub, and biexponential kinetics arises from the presence of many free-energy basins separated by barriers of different heights rather than a single low barrier along one reaction coordinate (the previously proposed incipient downhill folding scenario).


Large-Scale Exome-wide Association Analysis Identifies Loci for White Blood Cell Traits and Pleiotropy with Immune-Mediated Diseases.
White blood cells play diverse roles in innate and adaptive immunity. Genetic association analyses of phenotypic variation in circulating white blood cell (WBC) counts from large samples of otherwise healthy individuals can provide insights into genes and biologic pathways involved in production, differentiation, or clearance of particular WBC lineages (myeloid, lymphoid) and also potentially inform the genetic basis of autoimmune, allergic, and blood diseases. We performed an exome array-based meta-analysis of total WBC and subtype counts (neutrophils, monocytes, lymphocytes, basophils, and eosinophils) in a multi-ancestry discovery and replication sample of ∼157,622 individuals from 25 studies. We identified 16 common variants (8 of which were coding variants) associated with one or more WBC traits, the majority of which are pleiotropically associated with autoimmune diseases. Based on functional annotation, these loci included genes encoding surface markers of myeloid, lymphoid, or hematopoietic stem cell differentiation (CD69, CD33, CD87), transcription factors regulating lineage specification during hematopoiesis (ASXL1, IRF8, IKZF1, JMJD1C, ETS2-PSMG1), and molecules involved in neutrophil clearance/apoptosis (C10orf54, LTA), adhesion (TNXB), or centrosome and microtubule structure/function (KIF9, TUBD1). Together with recent reports of somatic ASXL1 mutations among individuals with idiopathic cytopenias or clonal hematopoiesis of undetermined significance, the identification of a common regulatory 3' UTR variant of ASXL1 suggests that both germline and somatic ASXL1 mutations contribute to lower blood counts in otherwise asymptomatic individuals. These association results shed light on genetic mechanisms that regulate circulating WBC counts and suggest a prominent shared genetic architecture with inflammatory and autoimmune diseases.


Temporal dynamics of eye movements are related to differences in scene complexity and clutter.
Recent research has begun to explore not just the spatial distribution of eye fixations but also the temporal dynamics of how we look at the world. In this investigation, we assess how scene characteristics contribute to these fixation dynamics. In a free-viewing task, participants viewed three scene types: fractal, landscape, and social scenes. We used a relatively new method, recurrence quantification analysis (RQA), to quantify eye movement dynamics. RQA revealed that eye movement dynamics were dependent on the scene type viewed. To understand the underlying cause for these differences we applied a technique known as fractal analysis and discovered that complexity and clutter are two scene characteristics that affect fixation dynamics, but only in scenes with meaningful content. Critically, scene primitives-revealed by saliency analysis-had no impact on performance. In addition, we explored how RQA differs from the first half of the trial to the second half, as well as the potential to investigate the precision of fixation targeting by changing RQA radius values. Collectively, our results suggest that eye movement dynamics result from top-down viewing strategies that vary according to the meaning of a scene and its associated visual complexity and clutter.


Enhancement of antibiotic activity by efflux inhibitors against multidrug resistant Mycobacterium tuberculosis clinical isolates from Brazil.
Drug resistant tuberculosis continues to increase and new approaches for its treatment are necessary. The identification of M. tuberculosis clinical isolates presenting efflux as part of their resistant phenotype has a major impact in tuberculosis treatment. In this work, we used a checkerboard procedure combined with the tetrazolium microplate-based assay (TEMA) to study single combinations between antituberculosis drugs and efflux inhibitors (EIs) against multidrug resistant M. tuberculosis clinical isolates using the fully susceptible strain H37Rv as reference. Efflux activity was studied on a real-time basis by a fluorometric method that uses ethidium bromide as efflux substrate. Quantification of efflux pump genes mRNA transcriptional levels were performed by RT-qPCR. The fractional inhibitory concentrations (FIC) indicated synergistic activity for the interactions between isoniazid, rifampicin, amikacin, ofloxacin, and ethidium bromide plus the EIs verapamil, thioridazine and chlorpromazine. The FICs ranged from 0.25, indicating a four-fold reduction on the MICs, to 0.015, 64-fold reduction. The detection of active efflux by real-time fluorometry showed that all strains presented intrinsic efflux activity that contributes to the overall resistance which can be inhibited in the presence of the EIs. The quantification of the mRNA levels of the most important efflux pump genes on these strains shows that they are intrinsically predisposed to expel toxic compounds as the exposure to subinhibitory concentrations of antibiotics were not necessary to increase the pump mRNA levels when compared with the non-exposed counterpart. The results obtained in this study confirm that the intrinsic efflux activity contributes to the overall resistance in multidrug resistant clinical isolates of M. tuberculosis and that the inhibition of efflux pumps by the EIs can enhance the clinical effect of antibiotics that are their substrates.


A taxonomy of epithelial human cancer and their metastases.
Microarray technology has allowed to molecularly characterize many different cancer sites. This technology has the potential to individualize therapy and to discover new drug targets. However, due to technological differences and issues in standardized sample collection no study has evaluated the molecular profile of epithelial human cancer in a large number of samples and tissues. Additionally, it has not yet been extensively investigated whether metastases resemble their tissue of origin or tissue of destination. We studied the expression profiles of a series of 1566 primary and 178 metastases by unsupervised hierarchical clustering. The clustering profile was subsequently investigated and correlated with clinico-pathological data. Statistical enrichment of clinico-pathological annotations of groups of samples was investigated using Fisher exact test. Gene set enrichment analysis (GSEA) and DAVID functional enrichment analysis were used to investigate the molecular pathways. Kaplan-Meier survival analysis and log-rank tests were used to investigate prognostic significance of gene signatures. Large clusters corresponding to breast, gastrointestinal, ovarian and kidney primary tissues emerged from the data. Chromophobe renal cell carcinoma clustered together with follicular differentiated thyroid carcinoma, which supports recent morphological descriptions of thyroid follicular carcinoma-like tumors in the kidney and suggests that they represent a subtype of chromophobe carcinoma. We also found an expression signature identifying primary tumors of squamous cell histology in multiple tissues. Next, a subset of ovarian tumors enriched with endometrioid histology clustered together with endometrium tumors, confirming that they share their etiopathogenesis, which strongly differs from serous ovarian tumors. In addition, the clustering of colon and breast tumors correlated with clinico-pathological characteristics. Moreover, a signature was developed based on our unsupervised clustering of breast tumors and this was predictive for disease-specific survival in three independent studies. Next, the metastases from ovarian, breast, lung and vulva cluster with their tissue of origin while metastases from colon showed a bimodal distribution. A significant part clusters with tissue of origin while the remaining tumors cluster with the tissue of destination. Our molecular taxonomy of epithelial human cancer indicates surprising correlations over tissues. This may have a significant impact on the classification of many cancer sites and may guide pathologists, both in research and daily practice. Moreover, these results based on unsupervised analysis yielded a signature predictive of clinical outcome in breast cancer. Additionally, we hypothesize that metastases from gastrointestinal origin either remember their tissue of origin or adapt to the tissue of destination. More specifically, colon metastases in the liver show strong evidence for such a bimodal tissue specific profile.


Detection and Characterization of a New Strain of Citrus Canker Bacteria from Key/Mexican Lime and Alemow in South Florida.
In the Wellington and Lake Worth areas of Palm Beach County, FL, citrus canker appeared on Key/Mexican lime (Citrus aurantiifolia) and alemow (C. macrophylla) trees over a period of about 6 to 7 years before detection, but nearby canker-susceptible citrus, such as grapefruit (C. × paradisi) and sweet orange (C. sinensis), were unaffected. Colonies of the causal bacterium, isolated from leaf, stem, and fruit lesions, appeared similar to the Asiatic group of strains of Xanthomonas axonopodis pv. citri (Xac-A) on the nutrient agar plate, but the growth on lima bean agar slants was less mucoid. The bacterium produced erumpent, pustule-like lesions of typical Asiatic citrus canker syndrome after inoculation into Key/Mexican lime, but brownish, flat, and necrotic lesions on the leaves of Duncan grapefruit, Madame Vinous sweet orange, sour orange (C. aurantium), citron (C. medica), Orlando tangelo (C. reticulata × C. × paradisi), and trifoliate orange (Poncirus trifoliata). The bacterium did not react with the Xac-A specific monoclonal antibody A1 using enzyme-linked immunosorbent assay (ELISA) and could not be detected by polymerase chain reaction (PCR)-based assays using primers selected for Xac-A. DNA reassociation analysis confirmed that the pathogen, designated as Xac-AW, was more closely related to Xac-A and Xac-A* strains than X. axonopodis pv. aurantifolii or the citrus bacterial spot pathogen (X. axonopodis pv. citrumelo). The strain can be easily differentiated from Xac-A and Xac-A* using ELISA, PCR-based tests, fatty acid analysis, pulsed-field gel electrophoresis of genomic DNA, and host specificity.


Experimental traumatic brain injury elevates brain prostaglandin E2 and thromboxane B2 levels in rats.
Prostaglandin E2 (PGE2) and thromboxane B2 (TxB2) levels were measured in rats following experimental traumatic brain injury. Rats (n = 36) were prepared for fluid percussion brain injury under pentobarbital anesthesia. Twenty-four hours later, rats were lightly anesthetized using methoxyflurane, injured (2.3 atm), and killed 5 or 15 min later. Twelve of the rats died before and are not included in the analyses. The following groups were used for data analysis: group I (n = 6) were sham-injured rats prepared for injury but not injured: group II (n = 6) were injured and killed 5 min later; group III (n = 12) were injured and killed 15 min posttrauma. Thirty seconds prior to sacrifice by decapitation into liquid nitrogen, all rats were injected with indomethacin (3 mg/kg, intravenously [IV]) to prevent postmortem PG synthesis. After sacrifice, brains were removed, weighed, and homogenized in a small quantity of phosphate buffer with indomethacin (50 micrograms/ml). PGE2 and TxB2 levels were determined using double-label radioimmunoassays. Posttraumatic convulsions were observed in 5 of 12 rats in group III and these rats were analyzed separately. PGE2 and TxB2 levels increased significantly (p less than 0.05) in both hemisphere and brainstem 5 min posttrauma. Fifteen minutes after injury, both PGE2 and TxB2 levels remained elevated but the levels were lower than at 5 min in the rats that did not exhibit posttraumatic seizures. This decrease in PG levels at 15 min was not observed in the rats that had seizures after injury and both PGE2 and TxB2 levels remained high in hemispheres and brainstem. Thus, fluid percussion brain injury results in substantial elevations in PGE2 and TxB2 levels and posttraumatic seizures exacerbate the observed increases.


Tuning the molecular order of C60-based self-assembled monolayers in field-effect transistors.
The control of order in organic semiconductor systems is crucial to achieve desired properties in electronic devices. We have studied the order in fullerene functionalized self-assembled monolayers by mixing the active molecules with supporting alkyl phosphonic acids of different chain length. By adjusting the length of the molecules, structural modifications of the alignment of the C60 head groups within the SAM can be tuned in a controlled way. These changes on the sub-nanometre scale were analysed by grazing incidence X-ray diffraction and X-ray reflectivity. To study the electron transport properties across these layers, self-assembled monolayer field-effect transistors (SAMFETs) were fabricated containing only the single fullerene monolayer as semiconductor. Electrical measurements revealed that a high 2D crystalline order is not the only important aspect. If the fullerene head groups are too confined by the supporting alkyl phosphonic acid molecules, defects in the crystalline C60 film, such as grain boundaries, start to strongly limit the charge transport properties. By close interpretation of the results of structural investigations and correlating them to the results of electrical characterization, an optimum chain length of the supporting alkyl phosphonic acids in the range of C10 was determined. With this study we show that minor changes in the order on the sub-nanometre scale, can strongly influence electronic properties of functional self-assembled monolayers.


Dynamics of recombination via conical intersection in a semiconductor nanocrystal.
Conical intersections are well known to introduce nonradiative decay pathways in molecules, but have only recently been implicated in nonradiative recombination processes in materials. Here we apply excited state ab initio molecular dynamics simulations based on a multireference description of the electronic structure to defective silicon nanocrystals up to 1.7 nm in diameter to search for accessible nonradiative recombination pathways. Dangling bond defects are found to induce conical intersections between the ground and first excited electronic states of five systems of various sizes. These defect-induced conical intersections are accessible at energies that are in the visible range (2.4-2.7 eV) and very weakly dependent on particle size. The dynamic simulations suggest that these intersections are accessed 40-60 fs after creation of a defect-localized excitation. This ultrafast recombination is attributed to the fact that Jahn-Teller distortion on the first excited state drives the defect directly towards a conical intersection with the ground electronic state.


Cilengitide modulates attachment and viability of human glioma cells, but not sensitivity to irradiation or temozolomide in vitro.
Cilengitide is a cyclic peptide antagonist of integrins alphavbeta3 and alphavbeta5 that is currently being evaluated as a novel therapeutic agent for recurrent and newly diagnosed glioblastoma. Its mode of action is thought to be mainly antiangiogenic but may include direct effects on tumor cells, notably on attachment, migration, invasion, and viability. In this study we found that, at clinically relevant concentrations, cilengitide (1-100 microM) induces detachment in some but not all glioma cell lines, while the effect on cell viability is modest. Detachment induced by cilengitide could not be predicted by the level of expression of the cilengitide target molecules, alphavbeta3 and alphavbeta5, at the cell surface. Glioma cell death induced by cilengitide was associated with the generation of caspase activity, but caspase activity was not required for cell death since ectopic expression of cytokine response modifier (crm)-A or coexposure to the broad-spectrum caspase inhibitor zVAD-fmk was not protective. Moreover, forced expression of the antiapoptotic protein marker Bcl-X(L) or altering the p53 status did not modulate cilengitide-induced cell death. No consistent effects of cilengitide on glioma cell migration or invasiveness were observed in vitro. Preliminary clinical results indicate a preferential benefit from cilengitide added to temozolomide-based radiochemotherapy in patients with O(6)-methylguanine DNA methyltransferase (MGMT) gene promoter methylation. Accordingly, we also examined whether the MGMT status determines glioma cell responses to cilengitide alone or in combination with temozolomide. Neither ectopic expression of MGMT in MGMT-negative cells nor silencing the MGMT gene in MGMT-positive cells altered glioma cell responses to cilengitide alone or to cilengitide in combination with temozolomide. These data suggest that the beneficial clinical effects derived from cilengitide in vivo may arise from altered perfusion, which promotes temozolomide delivery to glioma cells.


Multicolor observations of phobos with the viking lander cameras: evidence for a carbonaceous chondritic composition.
The reflectivity of Phobos has been determined in the spectral region from 0.4 to 1.1 micrometers from images taken with a Viking lander camera. The reflectivity curve is flat in this spectral interval and the geometric albedo equals 0.05 +/- 0.01. These results, together with Phobos's reflectivity spectrum in the ultraviolet, are compared with laboratory spectra of carbonaceous chondrites and basalts. The spectra of carbonaceous chondrites are consistent with the observations, whereas the basalt spectra are not. These findings raise the possibility that Phobos may be a captured object rather than a natural satellite of Mars.


Heterogeneous particle deaggregation and its implication for therapeutic aerosol performance.
Aerosolization performance of dry powder blends of drugs for the treatment of asthma or chronic obstructive pulmonary diseases have been reported in three previous articles. In vitro aerosolization was performed at defined shear stresses (0.624-13.143 N/m(2)). Formulations were characterized aerodynamically and powder aerosol deaggregation equations (PADE) and corresponding linear regression analyses for pharmaceutical aerosolization were applied. Particle deaggregation is the result of overcoming fundamental forces acting at the particle interface. A new method, PADE, describing dry powder formulation performance in a shear stress range has been developed which may allow a fundamental understanding of interparticulate and surface forces. The application of PADE predicts performance efficiency and reproducibility and supports rational design of dry powder formulations. The analogy of aerosol performance with surface molecular adsorption has important implications. Expressions describing surface adsorption were intended to allow elucidation of mechanisms involving surface heterogeneity, lateral interaction, and multilayer adsorption of a variety of materials. By using a similar expression for drug aerosolization performance, it is conceivable that an analogous mechanistic approach to the evaluation of particulate systems would be possible.


Forward assessment of 79 prehospital battlefield tourniquets used in the current war.
Battlefield tourniquet use can be lifesaving, but most reports are from hospitals with knowledge gaps remaining at the forward surgical team (FST). The quality of tourniquet applications in forward settings remain unknown. The purpose of this case series is to describe observations of tourniquet use at an FST in order to improve clinical performance. War casualties with tourniquet use presenting to an FST in Afghanistan in 2011 were observed. We identified appliers by training, device effectiveness, injury pattern, and clinical opportunities for improvement. Feedback was given to treating medics. Tourniquet applications (79) were performed by special operations combat medics (47, 59%), flight medics (17, 22%), combat medics (12, 15%), and general surgeons (3, 4%). Most tourniquets were Combat Application Tourniquets (71/79, 90%). With tourniquets in place upon arrival at the FST, most limbs (83%, 54/65) had palpable distal pulses present; 17% were pulseless (11/65). Of all tourniquets, the use was venous in 83% and arterial in 17%. In total, there were 14 arterial injuries, but only 5 had effective arterial tourniquets applied. Tourniquets are liberally applied to extremity injuries on the battlefield. 17% were arterial and 83% were venous tourniquets. When ongoing bleeding or distal pulses were appreciated, medics tightened tourniquets under surgeon supervision until distal pulses stopped. Medics were generally surprised at how tight a tourniquet must be to stop arterial flow ? convert a venous tourniquet into an arterial tourniquet. Implications for sustainment training should be considered with regard to this life-saving skill.


Transient prenatal Vitamin D deficiency is associated with hyperlocomotion in adult rats.
Rat experiments have shown that prenatal Vitamin D deficiency leads to altered neonatal brain morphology, cell density and neurotrophin expression. In the current study we examined the hypothesis that Vitamin D deficiency during early development alters adult behaviour even when there is an intervening period in which the animal receives normal Vitamin D in later development. Rats were conceived and born to Vitamin D deficient dams (Birth); conceived, born and weaned from Vitamin D deficient dams (Weaning); or deficient in Vitamin D from conception to 10 weeks of age (Life). Litters were standardized to three males and three females per litter. All rat offspring were rendered normocalcaemic with calcium supplemented water (2 mM) after weaning. Control animals were born to mothers fed a normal diet but subject to similar litter size and calcium supplementation. At 10 weeks all animals were tested on the holeboard test, elevated plus maze test, social interaction observation, acoustic startle response test, prepulse inhibition of the acoustic startle response and a forced swim test. Early Vitamin D deficiency (Birth group) enhanced locomotion in the holeboard test and increased activity in the elevated plus maze. Thus, transient prenatal Vitamin D deficiency induces hyperlocomotion in adulthood, without severe motor abnormalities.


Intraspecific diversity and ecological zonation in coral-algal symbiosis.
All reef-building corals are obligately associated with photosynthetic microalgal endosymbionts called zooxanthellae. Zooxanthella taxonomy has emphasized differences between species of hosts, but the possibility of ecologically significant zooxanthella diversity within hosts has been the subject of speculation for decades. Analysis of two dominant Caribbean corals showed that each associates with three taxa of zooxanthellae that exhibit zonation with depth--the primary environmental gradient for light-dependent marine organisms. Some colonies apparently host two taxa of symbionts in proportions that can vary across the colony. This common occurrence of polymorphic, habitat-specific symbioses challenges conventional understanding of the units of biodiversity but also illuminates many distinctive aspects of marine animal-algal associations. Habitat specificity provides ecological explanations for the previously documented poor concordance between host and symbiont phylogenies and the otherwise surprising lack of direct, maternal transmission of symbionts in many species of hosts. Polymorphic symbioses may underlie the conspicuous and enigmatic variability characteristic of responses to environmental stress (e.g., coral "bleaching") and contribute importantly to the phenomenon of photoadaptation.


Substance use outcomes of patients served by a large US implementation of Screening, Brief Intervention and Referral to Treatment (SBIRT).
To estimate changes in the substance use behaviors of patients who received services as part of the US Substance Abuse and Mental Health Services Administration's (SAMHSA) Screening, Brief Intervention and Referral to Treatment (SBIRT) grant program. We use a pre-post design and performance monitoring data collected by SBIRT organizations. For a sample of 17 575 patients, we compare pre-SBIRT substance use with substance use 6 months after receipt of SBIRT services. SBIRT's correlation with changes in substance use was estimated using generalized linear mixed models to account for the clustering of patients within health-care facility and US state. From pre- to post-SBIRT we found large and statistically significant decreases for almost every measure of substance use. Model-adjusted means indicate that the prevalence of alcohol use was lower 6 months later by 35.6%, heavy drinking by 43.4% and illicit drug use by 75.8%. Greater intervention intensity was associated with larger decreases in substance use. The study design does not support causal conclusions and estimated decreases in reported substance use are due, at least in part, to a well-known set of confounders and natural substance use patterns that may be unrelated to any particular SBIRT intervention. Compared with previously published findings on the Screening, Brief Intervention and Referral to Treatment grant program, our estimates of substance use reduction were smaller, but still consistently large in absolute magnitude and within ranges of estimates from past trials of Screening, Brief Intervention and Referral to Treatment.


The role of phylogeny and ecology in shaping morphology in 21 genera and 127 species of Australo-Papuan myobatrachid frogs.
Body shape is predicted to differ among species for functional reasons and in relation to environmental niche and phylogenetic history. We quantified morphological differences in shape and size among 98.5% of the 129 species and all 21 genera of the Australo-Papuan endemic myobatrachid frogs to test the hypothesis that habitat type predicts body shape in this radiation. We tested this hypothesis in a phylogenetic context at two taxonomic levels: across the entire radiation and within the four largest genera. Thirty-four external measurements were taken on 623 museum specimens representing 127 species. Data for seven key environmental variables relevant to anurans were assembled for all Australian-distributed species based on species' distributions and 131,306 locality records. The Australo-Papuan myobatrachid radiation showed high diversity in adult body size, ranging from minute (15 mm snout-vent length) to very large species (92 mm), and shape, particularly sin relative limb length. Five main morphological and environmental summary variables displayed strong phylogenetic signal. There was no clear relationship between body size and environmental niche, and this result persisted following phylogenetic correction. For most species, there was a better match between environment/habitat and body shape, but this relationship did not persist following phylogenetic correction. At a broad level, species fell into three broad groups based on environmental niche and body shape: 1) species in wet habitats with relatively long limbs, 2) species in arid environments with relatively short limbs (many of which are forward or backward burrowers) and 3) habitat generalist species with a conservative body shape. However, these patterns were not repeated within the four largest genera - Crinia, Limnodynastes, Pseudophryne and Uperoleia. Each of these genera displayed a highly conservative anuran body shape, yet individual species were distributed across the full spectrum of Australian environments. Our results suggest that phylogenetic legacy is important in the evolution of body size and shape in Australian anurans, but also that the conservative body plan of many frogs works well in a wide variety of habitats.


All-optical extinction-ratio enhancement of a 160 GHz pulse train by a saturable-absorber vertical microcavity.
A vertical-access passive all-optical gate has been used to improve the extinction ratio of a 160 GHz pico-second pulse train at 1555 nm. An extinction ratio enhancement of 6 dB is observed within an 8 nm bandwidth. Such a device is a promising candidate for low-cost all optical reamplication and reshaping (2R) regeneration at 160 Gbits/s.


Origin of orientation-dependent asymmetries in vestibulo-ocular reflexes evoked by caloric stimulation.
A caloric stimulus evokes primarily a horizontal vestibulo-ocular reflex (VOR) when subjects are in a supine or prone orientation with the horizontal semicircular canal plane oriented vertically. In both monkeys and humans, the magnitude of VOR eye movements is greater in the supine than in the prone orientation, indicating that some factor or factors, other than the conventionally accepted convective stimulation of the horizontal canals, contributes to the generation of the VOR. We used long-duration caloric irrigations and mathematical models of canal-otolith interactions to investigate factors contributing to the prone/supine asymmetry. Binaural caloric irrigations were applied for 7.5 or 9.5 min with subjects in a null orientation with horizontal canals in the earth-horizontal plane (control trial), or with the subject's pitch orientation periodically changing between null, supine, and prone positions with each orientation held for 30 s (caloric step trial). The control trial responses identified a small response attributable to a direct thermal effect on vestibular afferent activity that accounted for only 15% of the observed prone/supine asymmetry. We show that the gravito-inertial force resolution hypothesis for sensory integration of canal and otolith information predicts that the central processing of canal and otolith information produces an internal estimate of motion that includes both a rotational motion component and a linear acceleration component. These components evoke a horizontal angular VOR and linear VOR, which combine additively in the supine orientation, but subtract in the prone orientation, thus accounting for the majority of the observed prone/supine asymmetry.


Variable Selection in Semiparametric Regression Modeling.
In this paper, we are concerned with how to select significant variables in semiparametric modeling. Variable selection for semiparametric regression models consists of two components: model selection for nonparametric components and select significant variables for parametric portion. Thus, it is much more challenging than that for parametric models such as linear models and generalized linear models because traditional variable selection procedures including stepwise regression and the best subset selection require model selection to nonparametric components for each submodel. This leads to very heavy computational burden. In this paper, we propose a class of variable selection procedures for semiparametric regression models using nonconcave penalized likelihood. The newly proposed procedures are distinguished from the traditional ones in that they delete insignificant variables and estimate the coefficients of significant variables simultaneously. This allows us to establish the sampling properties of the resulting estimate. We first establish the rate of convergence of the resulting estimate. With proper choices of penalty functions and regularization parameters, we then establish the asymptotic normality of the resulting estimate, and further demonstrate that the proposed procedures perform as well as an oracle procedure. Semiparametric generalized likelihood ratio test is proposed to select significant variables in the nonparametric component. We investigate the asymptotic behavior of the proposed test and demonstrate its limiting null distribution follows a chi-squared distribution, which is independent of the nuisance parameters. Extensive Monte Carlo simulation studies are conducted to examine the finite sample performance of the proposed variable selection procedures.


Quantum phase transitions of hard-core bosons in background potentials.
We study the zero temperature phase diagram of hard-core bosons in two dimensions subjected to three types of background potentials: staggered, uniform, and random. In all three cases there is a quantum phase transition from a superfluid (at small potential) to a normal phase (at large potential), but with different universality classes. As expected, the staggered case belongs to the XY universality, while the uniform potential induces a mean field transition. The disorder driven transition is clearly different from both; in particular, we find z approximately 1.4, nu approximately 1, and beta approximately 0.6.


Influence of luminal diameters on flow surveillance of hemodialysis grafts: insights from a mathematical model.
Randomized controlled trials have not shown that surveillance of graft blood flow (Q) prolongs graft life. Because luminal diameters affect flow resistance, this study examined whether the influence of diameters on Q can explain the limitations of surveillance. Inflow artery and outflow vein diameters were determined from duplex ultrasound studies of 94 patients. These diameters were applied to a mathematical model for determination of how they affect the relation between Q and stenosis. Also determined was the correlation between Q (by ultrasound dilution) and diameters, stenosis, and mean arterial pressure in 88 patients. Artery and vein diameters varied widely between patients, but arteries generally were narrower than veins. The model predicts that the relation between Q and stenosis is sigmoid: as stenosis progresses, Q initially remains unchanged but then rapidly decreases. A narrower artery increases flow resistance, causing a longer delay followed by a more rapid reduction in Q. In a multiple regression analysis of data from patients, Q correlated with artery and vein diameters, sum of largest stenoses from each circuit segment, and mean arterial pressure (R = 0.689, P < 0.001). This study helps to explain why Q surveillance predicts thrombosis in some patients but not others. Luminal diameters control the relation between Q and stenosis, and these diameters vary widely. During progressive stenosis, the delay and then rapid reduction in Q may impair recognition of low Q before thrombosis occurs. Surveillance outcomes might be improved by taking frequent measurements so that there is no delay in discovering that Q has decreased.


Purification and partial characterization of feline pepsinogen.
To purify and partially characterize feline pepsinogen (fPG) from the gastric mucosa and compare fPG with PGs of other species. Stomachs of 6 cats. A crude protein extract was prepared from the gastric mucosa of feline stomachs. Feline PG A was purified by ammonium sulfate precipitation, weak-anion-exchange chromatography, size-exclusion chromatography, and strong-anion exchange chromatography. Partial characterization consisted of estimation of molecular weights (MWs) and isoelectric points, N-terminal amino acid sequencing, and investigation of susceptibility to pepstatin inhibition. Several fPG A-group isoforms were identified. The MWs of the isoforms ranged from 37,000 to 44,820. Isoelectric points were all < pH 3.0. The proteolytic activity of the activated PGs was inhibited completely by pepstatin in a range of equimolar to 10-fold molar excess. The specific absorbance of fPG A was 1.29. The N-terminal amino acid sequence of the first 25 residues of the predominant fPG A7 had 75%, 72%, 64%, and 56% homology with PG A of dogs, rabbits, cattle, and humans, respectively. Sequences of 4 other fPG A-group isoforms were similar to fPG A7. All isoforms were immunologically cross-reactive with sheep anti-fPG A7 antiserum. PG A is the only identified type of PG in cats and, similar to pg in other species, comprises multiple isoforms. The availability of fPG A may be used to facilitate the development of an immunoassay to quantify serum fPG A as a potential marker for gastric disorders in cats.


Enhanced receptor-mediated endocytosis and cytotoxicity of a folic acid-desacetylvinblastine monohydrazide conjugate in a pemetrexed-resistant cell line lacking folate-specific facilitative carriers but with increased folate receptor expression.
The reduced folate carrier (RFC), proton-coupled folate transporter (PCFT), and folate receptors (FR) are folate-specific transporters. Antifolates currently in the clinic, such as pemetrexed, methotrexate, and pralatrexate, are transported into tumor cells primarily via RFC. Folic acid conjugated to cytotoxics, a new class of antineoplastics, are transported into cells via FR-mediated endocytosis. To better define the role of PCFT in antifolate resistance, a methotrexate-resistant cell line, M160-8, was selected from a HeLa subline in which the RFC gene was deleted and PCFT was highly overexpressed. These cells were cross-resistant to pemetrexed. PCFT function and the PCFT mRNA level in M160-8 cells were barely detectable, and FR-α function and mRNA level were increased as compared with the parent cells. While pemetrexed rapidly associated with FR and was internalized within endosomes in M160-8 cells, consistent with FR-mediated transport, subsequent pemetrexed and (6S)-5-formyltetrahydrofolate export into the cytosol was markedly impaired. In contrast, M160-8 cells were collaterally sensitive to EC0905, a folic acid-desacetylvinblastine monohydrazide conjugate also transported by FR-mediated endocytosis. However, in this case a sulfhydryl bond is cleaved to release the lipophilic cytotoxic moiety into the endosome, which passively diffuses out of the endosome into the cytosol. Hence, resistance to pemetrexed in M160-8 cells was due to entrapment of the drug within the endosome due to the absence of PCFT under conditions in which the FR cycling function was intact.


Neuronal excitability after water intoxication in young rats.
Our previous experiments with animal models revealed that water intoxication induces brain oedema and opens plasma membranes. Present study is aimed to determine whether the standard method of hyperhydration can influence cerebral microenvironment also in young rats. Neuronal functions were tested by standard electrical cortical stimulation. Hyperhydration was induced by administration of distilled water (DW) intraperitoneally. Three groups of young rats were used: 12, 25, and 35-day-old. Cortical excitability was tested 19 to 20 hours after DW administration by electrical stimulation of the sensorimotor cortex with intensity necessary to elicit cortical afterdischarges (AD). Water content in the brain was estimated by dry/wet ratio and value of natremia by standard biochemical examination. Control animals of the same age groups were tested in the same way, only they did not receive DW. Brain water content in hyperhydrated animals was smaller than in controls in all studied age groups. Natremia was the same (normal) in both the hyperhydrated and control animals aged 25 days. Excitability of cortical neurons in young hyperhydrated animals was significantly inhibited in comparison to the same age groups of controls. Hyperhydration induced in young rats (12, 25, 35-day-old) had different effects than in adults. Absence of hyponatremia, lower water content in the brain and significant inhibition of cortical excitability can be explained on the basis of ontogenetically dependent aquaporine expression (AQP 4) and different activity of ionic membrane transporters.


Visuo-proprioceptive interactions during adaptation of the human reach.
We examined whether visual and proprioceptive estimates of transient (midreach) target capture errors contribute to motor adaptation according to the probabilistic rules of information integration used for perception. Healthy adult humans grasped and moved a robotic handle between targets in the horizontal plane while the robot generated springlike loads that varied unpredictably from trial to trial. For some trials, a visual cursor faithfully tracked hand motion. In others, the handle's position was locked and subjects viewed motion of a point-mass cursor driven by hand forces. In yet other trials, cursor feedback was dissociated from hand motion or altogether eliminated. We used time- and frequency-domain analyses to characterize how sensorimotor memories influence performance on subsequent reaches. When the senses were used separately, subjects were better at rejecting physical disturbances applied to the hand than virtual disturbances applied to the cursor. In part, this observation reflected differences in how participants used sensorimotor memories to adapt to perturbations when performance feedback was limited to only proprioceptive or visual information channels. When both vision and proprioception were available to guide movement, subjects processed memories in a manner indistinguishable from the vision-only condition, regardless of whether the cursor tracked the hand faithfully or whether we experimentally dissociated motions of the hand and cursor. This was true even though, on average, perceptual uncertainty in the proprioceptive estimation of movement extent exceeded that of visual estimation by just 47%. In contrast to perceptual tasks wherein vision and proprioception both contribute to an optimal estimate of limb state, our findings support a switched-input, multisensory model of predictive load compensation wherein visual feedback of transient performance errors overwhelmingly dominates proprioception in determining adaptive reach performance.


Bioactivation of carbamazepine in the rat in vivo. Evidence for the formation of reactive arene oxide(s).
The metabolism of carbamazepine (CBZ) and its major metabolite in humans, carbamazepine 10,11-epoxide (CBZ-E), was examined in the rat in vivo. Particular emphasis was placed on the identification of dihydrohydroxythio adducts, which are detoxication products of reactive arene oxide intermediates. Anesthetized and cannulated male Wistar rats were administered [3H]CBZ (25 micrograms.kg-1 or 25 mg.kg-1) or [3H]CBZ-E (25 micrograms.kg-1 or 25 mg.kg-1) intravenously and bile and urine collected for 5 hr. Less than 8% of drug was excreted in the urine for each dosing regimen. Biliary excretion accounted for 73.7 +/- 6.2 and 41.8 +/- 6.2% (mean +/- SD, N = 4) of administered CBZ (25 micrograms.kg-1 and 25 mg.kg-1, respectively) and 47.6 (N = 2) and 28.1 +/- 6.0% of administered CBZ-E (25 micrograms.kg-1 and 25 mg.kg-1, respectively). The major route of metabolism of both CBZ and CBZ-E was N-glucuronidation. In rats given CBZ (25 mg.kg-1), the N-glucuronide of the parent compound accounted for 12.6 +/- 2.6% of the dose, whereas CBZ-E N-glucuronide accounted for 12.3 +/- 3.8% of the dose. At the lower dose of 25 micrograms.kg-1, these accounted for 18.6 +/- 3.0 and 36.7 +/- 4.7% of the dose, respectively. Similarly, for rats given CBZ-E (25 micrograms.kg-1), the N-glucuronide of the parent compound was the major metabolite, accounting for 19.1 +/- 4.5% of the dose. O-glucuronides were relatively minor metabolites of both drugs. Glutathione adducts were identified in the bile of both groups of animals. Although these adducts were relatively minor metabolites of CBZ-E (1.8% of the dose), they were more substantial products of the metabolism of CBZ. Three isometric glutathionyl dihydrohydroxy-CBZ adducts were identified by LC/MS. They collectively accounted for 5.8 +/- 0.9% of the dose. In conclusion, we have provided evidence, in rats, for the generation of a reactive arene oxide species from CBZ. If not adequately detoxified, via conjugation with glutathione, this has the potential to initiate cellular damage. In humans, a similar mechanism may be involved in CBZ-associated hypersensitivity.


Protection from secondary dengue virus infection in a mouse model reveals the role of serotype cross-reactive B and T cells.
The four dengue virus (DENV) serotypes cause dengue fever and dengue hemorrhagic fever/dengue shock syndrome. Although severe disease has been associated with heterotypic secondary DENV infection, most secondary DENV infections are asymptomatic or result in classic DF. The role of cross-reactive immunity in mediating cross-protection against secondary heterotypic DENV infection is not well understood. DENV infection of IFN-α/β and IFN-γ receptor-deficient (AG129) mice reproduces key features of human disease. We previously demonstrated a role in cross-protection for pre-existing cross-reactive Abs, maintained by long-lived plasma cells. In this study, we use a sequential infection model, infecting AG129 mice with DENV-1, followed by DENV-2 6-8 wk later. We find that increased DENV-specific avidity during acute secondary heterotypic infection is mediated by cross-reactive memory B cells, as evidenced by increased numbers of DENV-1-specific cells by ELISPOT and higher avidity against DENV-1 of supernatants from polyclonally stimulated splenocytes isolated from mice experiencing secondary DENV-2 infection. However, increased DENV-specific avidity is not associated with increased DENV-specific neutralization, which appears to be mediated by naive B cells. Adoptive transfer of DENV-1-immune B and T cells into naive mice prior to secondary DENV-2 infection delayed mortality. Mice depleted of T cells developed signs of disease, but recovered after secondary DENV infection. Overall, we found that protective cross-reactive Abs are secreted by both long-lived plasma cells and memory B cells and that both cross-reactive B cells and T cells provide protection against a secondary heterotypic DENV infection. Understanding the protective immunity that develops naturally against DENV infection may help design future vaccines.


Modeling mitochondrial bioenergetics with integrated volume dynamics.
Mathematical models of mitochondrial bioenergetics provide powerful analytical tools to help interpret experimental data and facilitate experimental design for elucidating the supporting biochemical and physical processes. As a next step towards constructing a complete physiologically faithful mitochondrial bioenergetics model, a mathematical model was developed targeting the cardiac mitochondrial bioenergetic based upon previous efforts, and corroborated using both transient and steady state data. The model consists of several modified rate functions of mitochondrial bioenergetics, integrated calcium dynamics and a detailed description of the K(+)-cycle and its effect on mitochondrial bioenergetics and matrix volume regulation. Model simulations were used to fit 42 adjustable parameters to four independent experimental data sets consisting of 32 data curves. During the model development, a certain network topology had to be in place and some assumptions about uncertain or unobserved experimental factors and conditions were explicitly constrained in order to faithfully reproduce all the data sets. These realizations are discussed, and their necessity helps contribute to the collective understanding of the mitochondrial bioenergetics.


Demonstration of sustained sinus and atrial re-entry as a mechanism of paroxysmal supraventricular tachycardia.
Electrophysiological studies in five patients with documented (4) or suspected (1) paroxysmal supraventricular tachycardia (PSVT), suggested sinus or atrial reentrance (SR or AR). Two of the patients had preexcitation, three had evidence of atrial enlargement, and all had organic heart disease. The following observations supported a diagnosis of SR and AR; 1) induction of sustained PSVT with atrial extrastimulus technique allowing definition of an echo zone; 2) induction of sustained PSVT during constant rapid atrial pacing at a rate less than that producing A-V nodal Wenckebach periods, or producing normalization of QRS complex in patients with pre-excitation: 3) P waves preceding each QRS during PSVT with an A-H interval appropriate for the rate of the PSVT; 4) antegrade P wave morphology during PSVT, a normal high to low sequence of right atrial activation (SR), or P wave morphology and atrial activation sequence different from sinus (AR); 5) lack of correlation of PSVT induction with critical A-H interval. The rates of induced sustained PSVT ranged from 114 to 143 beats/min, and were similar to those observed during spontaneous episodes of PSVT in the four patients. PSVT could be terminated with critically timed extra-stimuli or carotid massage. In conclusion, SR and AR appear to be mechanisms of spontaneous PSVT in man. Rates of SR and AR PSVT tend to be relatively slow.


Diet in dermatology: present perspectives.
Many nutrients are essential for life, and an adequate amount of nutrients in the diet is necessary for providing energy, building and maintaining body organs, and for various metabolic processes. The role of food in the induction of various skin disorders and skin diseases leading to nutritional deficiencies is well known. The photo-protective potential of antioxidants, the effects of micronutrient supplementation on the skin immune system, and the modulating effects of fatty acids on skin disorders are well documented. Skin diseases due to nutritional deficiencies, the dietary role in skin immunity and various skin diseases, and the role of antioxidants and other supplements in skin health have been reviewed.


Anticipating divine protection? Reminders of god can increase nonmoral risk taking.
Religiosity and participation in religious activities have been linked with decreased risky behavior. In the current research, we hypothesized that exposure to the concept of God can actually increase people's willingness to engage in certain types of risks. Across seven studies, reminders of God increased risk taking in nonmoral domains. This effect was mediated by the perceived danger of a risky option and emerged more strongly among individuals who perceive God as a reliable source of safety and protection than among those who do not. Moreover, in an eighth study, when participants were first reminded of God and then took a risk that produced negative consequences (i.e., when divine protection failed to materialize), participants reported feeling more negatively toward God than did participants in the same situation who were not first reminded of God. This research contributes to an understanding of the divergent effects that distinct components of religion can exert on behavior.


Thermal stability of intralipid optical phantoms.
We investigated the stability of light transmission through Intralipid-based optical phantoms in the wavelength range of 400-950 nm at temperatures between 35 and 70 °C. Optical phantoms are materials commonly used to simulate the light scattering and absorption properties of biological materials. These simulations require the phantom to be optically stable. We demonstrate that the scattering properties of Intralipid remain stable at higher temperatures, varying less than 0.5%. We also present results that show this is not the case for absorption below 700 nm at 35 and 70 °C, with greater instability at 70 °C. For example, at 500 nm, the light intensity transmitted through 15 mm of Intralipid dropped 39% over 12 h. We demonstrate that oxidation of fatty acids in Intralipid could account for this effect and show, by flushing the system continuously with nitrogen gas, the instability is reduced.


Pr is a member of a restricted class of σ70-dependent promoters that lack a recognizable -10 element.
The Pr promoter is the first verified member of a class of bacterial σ(70)-promoters that only possess a single match to consensus within its -10 element. In its native context, the activity of this promoter determines the ability of Pseudomonas putida CF600 to degrade phenolic compounds, which provides proof-of-principle for the significance of such promoters. Lack of identity within the -10 element leads to non-detection of Pr-like promoters by current search engines, because of their bias for detection of the -10 motif. Here, we report a mutagenesis analysis of Pr that reveals strict sequence requirements for its activity that includes an essential -15 element and preservation of non-consensus bases within its -35 and -10 elements. We found that highly similar promoters control plasmid- and chromosomally- encoded phenol degradative systems in various Pseudomonads. However, using a purpose-designed promoter-search algorithm and activity analysis of potential candidate promoters, no bona fide Pr-like promoter could be found in the entire genome of P. putida KT2440. Hence, Pr-like σ(70)-promoters, which have the potential to be a widely distributed class of previously unrecognized promoters, are in fact highly restricted and remain in a class of their own.


Antigen-primed CD8+ T cells can mediate resistance, preventing allogeneic marrow engraftment in the simultaneous absence of perforin-, CD95L-, TNFR1-, and TRAIL-dependent killing.
Engraftment failure following allogeneic bone marrow (BM) transplantation is of clinical concern particularly involving T-cell-depleted inoculum and transplantations for aplastic anemia. Immune resistance by lymphoid and natural killer (NK) populations with "barrier" function is well established. Major histocompatibility complex (MHC)-identical marrow allografts were examined to investigate effector pathways in non-NK-mediated resistance. Barrier function was examined in cytotoxic normal and deficient B6 (H-2(b)) recipients primed to donor minor histocompatibility antigen (MiHA) prior to BM transplantation. Host resistance was sensitively evaluated by colony-forming unit (CFU) assays to directly assess for donor progenitor cell (PC) and peripheral chimerism. B6 host CD8(+) T cells but not CD4(+) or NK1.1(+) cells effected rejection of primitive (CFU-HPP [high-proliferative potential]) and lineage-committed (CFU-IL3/GM [interleukin 3/granulocyte macrophage]) allogeneic donor progenitors. To address complementation by the cytotoxic pathways existing in singly deficient (perforin or FasL) recipients, cytotoxically double (perforin plus FasL) deficient (cdd) recipients were used. Resistance in B6-cdd recipients was comparable to that of wild-type B6 recipients and was also dependent on CD8(+) T cells. A "triple" cytotoxic deficient model, involving transplantation of TNFR1(-/-) (tumor necrosis factor receptor 1) progenitor grafts did not diminish the ability of B6-cdd recipients to reject allografts. Finally, injection of anti-TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) monoclonal antibody (mAb) in B6-cdd recipients also failed to inhibit rejection of TNFR1(-/-) marrow grafts. In total, these studies demonstrate that CD8(+) host T cells can effectively resist MHC-matched MiHA-mismatched donor PCs via alternative effector pathway(s) independent of perforin-, FasL-, TNFR-1-, and TRAIL-dependent cytotoxicity. Therefore, inhibition of these effector pathways in sensitized recipients is unlikely to result in stem cell engraftment following PC allografts.


High-efficiency subwavelength diffractive optical element in GaAs for 975 nm.
We have fabricated subwavelength diffractive optical elements with binary phase profiles for operation at 975 nm. The individual surface-relief features of the elements are smaller than the wavelength of light in the material. By modulating the size and spacing of the features we form artificial, gradient, effective index-of-refraction surfaces. The blazed transmission gratings were designed with rigorous coupled-wave analysis and fabricated by direct-write electron-beam lithography and reactive ion-beam etching in GaAs. The gratings have minimum features 63 nm wide. Transmission measurements show 85% diffraction efficiency into the first order.


Insights into the origin of high activity and stability of catalysts derived from bulky, electron-rich monophosphinobiaryl ligands in the Pd-catalyzed C-N bond formation.
A comparative kinetic examination of catalyst systems based on several monophosphinobiaryl ligands is reported. The bulk of the phosphine ligand controls the catalytic activity and the rate of catalyst activation with the catalyst based on 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl providing the greatest activity and fastest activation. In the case where catalyst activation is slow (i.e., use of the smaller ligands such as 2-dicyclohexylphosphino-2'-methylbiphenyl in combination with Pd(OAc)2) stirring the amine with the catalyst/base mixture prior to the commencement of the reaction increases the reaction rate along with the rate of catalyst activation. Kinetic isotope effects established that the catalyst activation process occurs through a beta-hydride elimination pathway.


Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy.
Recording light-microscopy images of large, nontransparent specimens, such as developing multicellular organisms, is complicated by decreased contrast resulting from light scattering. Early zebrafish development can be captured by standard light-sheet microscopy, but new imaging strategies are required to obtain high-quality data of late development or of less transparent organisms. We combined digital scanned laser light-sheet fluorescence microscopy with incoherent structured-illumination microscopy (DSLM-SI) and created structured-illumination patterns with continuously adjustable frequencies. Our method discriminates the specimen-related scattered background from signal fluorescence, thereby removing out-of-focus light and optimizing the contrast of in-focus structures. DSLM-SI provides rapid control of the illumination pattern, exceptional imaging quality and high imaging speeds. We performed long-term imaging of zebrafish development for 58 h and fast multiple-view imaging of early Drosophila melanogaster development. We reconstructed cell positions over time from the Drosophila DSLM-SI data and created a fly digital embryo.


The influence of different deep-sea coral habitats on sediment macrofaunal community structure and function.
Deep-sea corals can create a highly complex, three-dimensional structure that facilitates sediment accumulation and influences adjacent sediment environments through altered hydrodynamic regimes. Infaunal communities adjacent to different coral types, including reef-building scleractinian corals and individual colonies of octocorals, are known to exhibit higher macrofaunal densities and distinct community structure when compared to non-coral soft-sediment communities. However, the coral types have different morphologies, which may modify the adjacent sediment communities in discrete ways. Here we address: (1) how infaunal communities and their associated sediment geochemistry compare among deep-sea coral types (Lophelia pertusa, Madrepora oculata, and octocorals) and (2) do infaunal communities adjacent to coral habitats exhibit typical regional and depth-related patterns observed in the Gulf of Mexico (GOM). Sediment push cores were collected to assess diversity, composition, numerical abundance, and functional traits of macrofauna (>300 µm) across 450 kilometers in the GOM at depths ranging from 263-1,095 m. Macrofaunal density was highest in L. pertusa habitats, but similar between M. oculata and octocorals habitats. Density overall exhibited a unimodal relationship with depth, with maximum densities between 600 and 800 m. Diversity and evenness were highest in octocoral habitats; however, there was no relationship between diversity and depth. Infaunal assemblages and functional traits differed among coral habitats, with L. pertusa habitats the most distinct from both M. oculata and octocorals. These patterns could relate to differences in sediment geochemistry as L. pertusa habitats contained high organic carbon content but low proportions of mud compared to both M. oculata and octocoral habitats. Distance-based linear modeling revealed depth, mud content, and organic carbon as the primary factors in driving coral infaunal community structure, while geographic location (longitude) was the primary factor in functional trait composition, highlighting both the location and ecological differences of L. pertusa habitats from other coral habitats. Enhanced habitat structural complexity associated with L. pertusa and differences in localized hydrodynamic flow may contribute to the dissimilarities in the communities found among the coral types. Our results suggest a decoupling for infaunal coral communities from the typical depth-related density and diversity patterns present throughout soft-sediment habitats in the GOM, highlighting the importance of deep-sea corals in structuring unique communities in the nearby benthos.


Effects of abiotic factors on the phylogenetic diversity of bacterial communities in acidic thermal springs.
Acidic thermal springs offer ideal environments for studying processes underlying extremophile microbial diversity. We used a carefully designed comparative analysis of acidic thermal springs in Yellowstone National Park to determine how abiotic factors (chemistry and temperature) shape acidophile microbial communities. Small-subunit rRNA gene sequences were PCR amplified, cloned, and sequenced, by using evolutionarily conserved bacterium-specific primers, directly from environmental DNA extracted from Amphitheater Springs and Roaring Mountain sediment samples. Energy-dispersive X-ray spectroscopy, X-ray diffraction, and colorimetric assays were used to analyze sediment chemistry, while an optical emission spectrometer was used to evaluate water chemistry and electronic probes were used to measure the pH, temperature, and E(h) of the spring waters. Phylogenetic-statistical analyses found exceptionally strong correlations between bacterial community composition and sediment mineral chemistry, followed by weaker but significant correlations with temperature gradients. For example, sulfur-rich sediment samples contained a high diversity of uncultured organisms related to Hydrogenobaculum spp., while iron-rich sediments were dominated by uncultured organisms related to a diverse array of gram-positive iron oxidizers. A detailed analysis of redox chemistry indicated that the available energy sources and electron acceptors were sufficient to support the metabolic potential of Hydrogenobaculum spp. and iron oxidizers, respectively. Principal-component analysis found that two factors explained 95% of the genetic diversity, with most of the variance attributable to mineral chemistry and a smaller fraction attributable to temperature.


Age-related endothelial dysfunction : potential implications for pharmacotherapy.
Aging per se is associated with abnormalities of the vascular wall linked to both structural and functional changes that can take place at the level of the extracellular matrix, the vascular smooth muscle and the endothelium of blood vessels. Endothelial dysfunction is generally defined as a decrease in the capacity of the endothelium to dilate blood vessels in response to physical and chemical stimuli. It is one of the characteristic changes that occur with age, independently of other known cardiovascular risk factors. This may account in part for the increased incidence of cardiovascular events in elderly people that can be reversed by restoring endothelial function. A better understanding of the mechanisms involved and the aetiopathogenesis of this process will help in the search for new therapeutic agents.Age-dependent alteration of endothelium-dependent relaxation seems to be a widespread phenomenon both in conductance and resistance arteries from several species. In the course of aging, there is an alteration in the equilibrium between relaxing and contracting factors released by the endothelium. Hence, there is a progressive reduction in the participation of nitric oxide and endothelium-derived hyperpolarising factor associated with increased participation of oxygen-derived free radicals and cyclo-oxygenase-derived prostanoids. Also, the endothelin-1 and angiotensin II pathways may play a role in age-related endothelial dysfunction. The use of drugs acting at different levels of these signalling cascades, including antioxidant therapy, lipid-lowering drugs and estrogens, seems to be promising.


Hypovitaminosis D in Delirium: a Retrospective Cross-sectional Study.
As vitamin D may have a neuroprotective effect, the authors studied the association of biomarkers of vitamin D status and delirium to see if low vitamin D status was common in delirium cases. Biochemical measures of vitamin D (25-hydroxyvitamin D [25-OHD]) and calcium metabolism were used in this retrospective cross-sectional analysis of adult in-patients with delirium, admitted at three Canadian academic hospitals from January 2011 to July 2012. Primary outcome was to determine estimates of the prevalence of hypovitaminosis D in this group in whom vitamin D was checked. Seventy-one (5.8%) out of 1,232 delirium inpatients had their vitamin D measured. Thirty-nine (55%) showed vitamin D insufficiency (25-OHD of 25-75 nmol/L) and 8 (11%) showed vitamin D deficiency (25-OHD < 25 nmol/L). Mean serum 25-OHD levels were lower in males (57.1±7.7 nmol/L) than in females (78.2±6.1 nmol/L), p = .01, even when controlled for age and season. Men were younger than the women (74.4±2.3 vs. 82.4±1.7, p = .005). Mean age was 78.7±1.5 years, and 33 (47%) were male. Although vitamin D is rarely checked during delirium workup and/or management, high rates of hypovitaminosis D were found to be common in the delirium in-patients in whom it was checked. Larger studies would be needed to estimate the prevalence of hypovitaminosis D in delirium and whether hypovitaminosis D plays a role in the pathogenesis of delirium.


Socioeconomic status inequalities in low-grade inflammation during childhood.
Family socioeconomic status (SES) is an important source of child health disparities in the USA. Chronic stress is one way SES may impact children's physiology with implications for later health inequalities. These processes may work differently across childhood due to differences in exposure and susceptibility to stressors at different ages. We assess associations between family SES and one biomarker of chronic stress exposure-low-grade inflammation detected by elevated C reactive protein (CRP)-and evaluate differences in the associations by child age. We used nationally representative data from the National Health and Nutrition Examination Survey and Tobit regression models to estimate SES associations with CRP and the moderating effects of age for children age 2-18 years. Our sample was limited to CRP ≤10 mg/l to focus on low-grade inflammation (N=13 165). Children whose parent had less than a high school degree had 35% higher CRP than those with a college graduate parent; and, poor children had 24% higher CRP than those with high family income, net of controls. When children's body mass index was accounted for, low education and poverty associations were reduced to 19% and 15%, respectively. Child age interactions were negative and significant for both parental education and family income. This study provides new evidence that SES is associated with low-grade inflammation in children, and that these associations may be particularly strong during early and mid-childhood. Future research should further our understanding of stressors related to low family SES that may lead to immune system dysregulation during childhood.


Incidence of and Factors Influencing Femoral Neck Shortening in Elderly Patients After Fracture Fixation with Multiple Cancellous Screws.
BACKGROUND To study the incidence of and factors influencing "neck shortening" in elderly patients treated for femoral neck fractures using multiple cancellous screws. MATERIAL AND METHODS Of the 197 femoral neck fracture cases treated via closed reduction and cancellous screws fixation from January 2006 to February 2010, 110 were followed up. Patient age, gender, operative time, implantation method, reduction quality, fracture type, bone mineral density, loading time, length of hospital stay, and Harris hip score 12 months after operation were recorded. The patients were divided into two groups (shortening and non-shortening) based on their X-ray performance during follow-up. The healing rates and Harris hip scores of the two groups were compared, and the influencing factors of femoral neck shortening were analyzed. RESULTS Of the 110 cases followed up, 94.5% (104/110) were healed and neck shortening occurred in 41.8% (46/110) within 12.5 months (mean) after treatment. The Harris hip score of the shortening group was lower than that of the non-shortening group (78±17 vs. 86±23, p=0.048). The fracture healing rates of the two groups were not significantly different (p=0.068). The factors influencing neck shortening were significantly correlated with bone mineral density, patient age, gender, and type of fracture. CONCLUSIONS The incidence of neck shortening in elderly patients treated for femoral neck fracture using cancellous screws was high. Bone mineral density, patient age, gender, and type of fracture were the influencing factors of neck shortening.


Crystal structure of the human odorant binding protein, OBPIIa.
Human odorant-binding protein, OBPIIa , is expressed by nasal epithelia to facilitate transport of hydrophobic odorant molecules across the aqueous mucus. Here, we report its crystallographic analysis at 2.6 Å resolution. OBPIIa is a monomeric protein that exhibits the classical lipocalin fold with a conserved eight-stranded β-barrel harboring a remarkably large hydrophobic pocket. Basic residues within the four loops that shape the entrance to this ligand-binding site evoke a positive electrostatic potential. Human OBPIIa shows distinct features compared with other mammalian OBPs, including a potentially reactive Cys side chain within its pocket similar to human tear lipocalin.


The spacer peptide between human immunodeficiency virus capsid and nucleocapsid proteins is essential for ordered assembly and viral infectivity.
Morphogenesis of retroviruses involves ordered assembly of the structural Gag- and Gag-Pol polyproteins, with subsequent budding from the plasma membrane and proteolytic cleavage by the viral proteinase (PR). Two cleavage sites exist between the capsid (CA) and nucleocapsid (NC) domains of the human immunodeficiency virus (HIV) type 1 Gag polyprotein which are separated by a 14-amino-acid spacer peptide of unknown function. To analyze the role of the two cleavage sites and the spacer peptide, both sites were individually mutated and a deletion mutation that precisely removes the spacer peptide was constructed. Following transfection of proviral DNA carrying the point mutations, mutant polyproteins were synthesized and assembled like wild-type polyprotein, and release of particles was not significantly altered. Both mutations abolished cleavage at the respective site and reduced or abolished viral infectivity. Deletion of the spacer peptide severely affected ordered assembly and reduced particle release. The extracellular particles that were released exhibited normal density but were heterogeneous in size. Electron micrographs revealed large electron-dense plaques underneath the plasma membrane of transfected cells which appeared like confluent ribonucleoprotein complexes arrested early in the budding process. Extracellular particles exhibited very aberrant and heterogeneous morphology and were incapable of inducing viral spread. These particles may correspond to membrane vesicles sequestered by the rigid structures underneath the cell membrane and not released by a regular budding process.


Upregulation of REG Ialpha accelerates tumor progression in pancreatic cancer with diabetes.
Diabetes is now generally accepted as a crucial event in the process of pancreatic cancer (PaC). However, molecular mechanisms underlying the relationship between diabetes and PaC are not fully understood. Regenerating gene (REG) Ialpha is a growth factor affecting pancreatic islet beta cells, and it has been shown to be involved in the carcinogenesis in gastrointestinal tract. It is rational to speculate that REG Ialpha plays a potential role in the pathogenesis of PaC with diabetes. The aim of this study was to evaluate the REG Ialpha protein expression profile in PaC with and without diabetes, and define the contribution of REG Ialpha on PaC development. We found that REG Ialpha protein preferentially expressed in cancerous tissues of PaC patients with diabetes by Western blot. REG Ialpha positive cancer cells in PaC with diabetes (n = 38) was significantly higher than that in subjects without diabetes (n = 42, p < 0.05) by immunohistochemical analysis. Furthermore, we found that overexpression of REG Ialpha protein in PaC cell lines resulted in accelerated cell proliferation and consequently tumor growth, both in vitro and in vivo. The findings suggest that REG Ialpha may act as one of the tumor promoter and contribute to the aggressive nature of PaC, especially in the subpopulation with diabetes. This study would shed new insights for understanding the molecular mechanisms underlying the link between diabetes and PaC.


American crows as carriers of vancomycin-resistant enterococci with vanA gene.
We studied the vanA-carrying vancomycin-resistant enterococci (VRE) isolated from American crows in the United States during the winter 2011/2012. Faecal samples from crows were cultured selectively for VRE and characterized. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to examine epidemiological relationships of vanA-containing VRE. Isolates were tested in vitro for their ability to horizontally transfer the vancomycin resistance trait. VRE with the vanA gene were found in 15 (2.5%) of 590 crows samples, from which we obtained 22 different isolates. Enterococcal species were Enterococcus faecium (14) and E. faecalis (8). One, two and 19 isolates originated from Kansas, New York State and Massachusetts, respectively. Based on MLST analysis, E. faecium isolates were grouped as ST18 (6 isolates), ST555 (2), and novel types ST749 (1), ST750 (3), ST751 (1), ST752 (1). Enterococcus faecalis isolates belonged to ST6 (1), ST16 (3) and ST179 (4). All isolates were able to transfer the vancomycin resistance trait via filter mating with very high transfer range. Clinically important enterococci with the vanA gene occur in faeces of wild American crows throughout the United States. These migrating birds may contribute to the dissemination of VRE in environment over large distances. [Correction added after first online publication on 06 August 2013: The number of E. faecium ST752 isolate is now amended to '1', consistent with that shown in the 'Results' section and Figure 2.].


Human endometrial epithelial telomerase is important for epithelial proliferation and glandular formation with potential implications in endometriosis.
How does regulation of telomerase activity (TA) in human endometrial epithelial cells (EEC) by ovarian hormones impact on telomere lengths (TL) and cell proliferation? Healthy endometrial epithelial cell proliferation is characterized by high TA and endometrial TL changes according to the ovarian hormone cycle, with shortest TL observed in the progesterone dominant mid-secretory phase, when TA is lowest, implicating progesterone in the negative regulation of TA and TL. Critical shortening of telomeres may result in permanent cell cycle arrest while the enzyme telomerase maintains telomere length (TL) and replicative capacity of cells. Telomerase expression and activity change in the human endometrium with the ovarian hormone cycle, however the effect of this on endometrial TL and cell growth is not known. A prospective observational study, which included endometrial and blood samples collected from 196 women. We studied endometrial samples from five different groups of women. Endometrial and matched blood TL and circulating steroid hormones were studied in samples collected from 85 women (Group 1). Fresh epithelial and stromal cell isolation and culture in vitro for TL and TA was done on endometrial biopsies collected from a further 74 healthy women not on hormonal therapy (Group 2) and from 5 women on medroxyprogesterone acetate (MPA) for contraception (Group 3). The epithelial TL and telomerase protein expression was examined in active, peritoneal, ectopic endometriotic and matched uterine (eutopic) endometrial samples collected from 10 women with endometriosis (Group 4); the in vivo effect of mifepristone on telomerase protein expression by immunohistochemistry (IHC) was examined in endometrium from 22 healthy women in mid-secretory phase before (n = 8), and after administering 200 mg mifepristone (n = 14) (Group 5). TA was measured by telomere repeat amplification protocol (TRAP) assay; TL by qPCR, and Q-FISH; cell proliferation was assessed by immunoblotting of histone H3 and 3D-culture to assess the ability of EECs to form spheroids; telomerase reverse transcriptase protein levels and Ki-67 (proliferative index) were assessed with IHC. Endometrial TLs correlated negatively with serum progesterone levels (n = 58, r = -0.54) and were significantly longer than corresponding blood TLs (4893 ± 929 bp versus 3955 ± 557 bp, P = 0.002) suggesting a tissue-specific regulation. High TA and short TLs were observed in proliferating EECs in vivo and in vitro. During the progesterone dominant mid-secretory phase endometrial TL were significantly shorter compared with the proliferative phase (P = 0.0002). Progestagen treatment suppressed EEC TA in vivo and reduced endometrial TA in explant (P = 0.01) and in vitro cultures (P = 0.02) compared with untreated cells. Mifepristone (progesterone receptor antagonist) increased telomerase protein levels in vivo (P < 0.05). In 2D culture, Imetelstat inhibited EEC TA (P = 0.03), proliferation (P = 0.009) and in 3D culture disrupted endometrial glandular architecture (P = 0.03). The in vitro telomerase inhibition data were tested in a mono-cellular system for a short-term. Further confirmation of the results in an in vivo model is necessary. The women in group 2 included a high proportion of women although with a regular menstrual cycle, with an increased BMI (>25) therefore this may affect extrapolation of data to other groups. The observed effects of telomerase inhibition in vitro on epithelial cell proliferation, suggest that telomerase might be an attractive target in developing new therapies for proliferative disorders of the endometrium, such as endometriosis.