Screening for HIV-specific T-cell responses using overlapping 15-mer peptide pools or optimized epitopes.
The IFN-y enzyme-linked immunospot (ELI-Spot) assay is often used to map HIV-specific CD8 T-cell responses. We compared overlapping 15-mer pools with optimized CD8 epitopes to screen ELISpot responses in HIV-infected individuals. The 15-mer pools detected responses to previously undefined epitopes, but often missed low-level responses to predefined epitopes, particularly when the epitope was central in the 15-mer, rather than at the N-terminus or C-terminus. These factors should be considered in the monitoring of HIV vaccine trials.


Identification of Autophagy-Inhibiting Factors of Mycobacterium tuberculosis by High-Throughput Loss-of-Function Screening.
The interaction of host cells with mycobacteria is complex and can lead to multiple outcomes ranging from bacterial clearance to progressive or latent infection. Autophagy is recognized as one component of host cell responses that has an essential role in innate and adaptive immunity to intracellular bacteria. Many microbes, including Mycobacterium tuberculosis, have evolved to evade or exploit autophagy, but the precise mechanisms and virulence factors are mostly unknown. Through a loss-of-function screening of an M. tuberculosis transposon mutant library, we identified 16 genes that contribute to autophagy inhibition, six of which encoded the PE/PPE protein family. Their expression in Mycobacterium smegmatis confirmed that these PE/PPE proteins inhibit autophagy and increase intracellular bacterial persistence or replication in infected cells. These effects were associated with increased mammalian target of rapamycin (mTOR) activity and also with decreased production of tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β). We also confirmed that the targeted deletion of the pe/ppe genes in M. tuberculosis resulted in enhanced autophagy and improved intracellular survival rates compared to those of wild-type bacteria in the infected macrophages. Differential expression of these PE/PPE proteins was observed in response to various stress conditions, suggesting that they may confer advantages to M. tuberculosis by modulating its interactions with host cells under various conditions. Our findings demonstrated that multiple M. tuberculosis PE/PPE proteins are involved in inhibiting autophagy during infection of host phagocytes and may provide strategic targets in developing therapeutics or vaccines against tuberculosis.


Solution-deposited organic-inorganic hybrid multilayer gate dielectrics. Design, synthesis, microstructures, and electrical properties with thin-film transistors.
We report here on the rational synthesis, processing, and dielectric properties of novel layer-by-layer organic/inorganic hybrid multilayer dielectric films enabled by polarizable π-electron phosphonic acid building blocks and ultrathin ZrO(2) layers. These new zirconia-based self-assembled nanodielectric (Zr-SAND) films (5-12 nm thick) are readily fabricated via solution processes under ambient atmosphere. Attractive Zr-SAND properties include amenability to accurate control of film thickness, large-area uniformity, well-defined nanostructure, exceptionally large electrical capacitance (up to 750 nF/cm(2)), excellent insulating properties (leakage current densities as low as 10(-7) A/cm(2)), and excellent thermal stability. Thin-film transistors (TFTs) fabricated with pentacene and PDIF-CN(2) as representative organic semiconductors and zinc-tin-oxide (Zn-Sn-O) as a representative inorganic semiconductor function well at low voltages (<±4.0 V). Furthermore, the TFT performance parameters of representative organic semiconductors deposited on Zr-SAND films, functionalized on the surface with various alkylphosphonic acid self-assembled monolayers, are investigated and shown to correlate closely with the alkylphosphonic acid chain dimensions.


Low sexual function is associated with menopausal status in mid-aged women with human immunodeficiency virus infection.
To assess the association between menopausal status and female sexual function among mid-aged women with human immunodeficiency virus (HIV) infection. We carried out a cross-sectional study of 221 sexually active HIV-infected women ages 40 to 59 years, based on a secondary analysis of a three-hospital survey in Lima, Perú. We classified menopausal status according to Stages of Reproductive Aging Workshop criteria (STRAW+10); this exposure variable was categorized as binary (non-postmenopausal and postmenopausal) and-for exploratory analysis-as multinomial (pre-, peri-, and postmenopausal). We defined low sexual function (LSF) using the 6-item Female Sexual Function Index (total score ≤19). Socio-demographic and clinical variables were assessed, including age, used highly active antiretroviral therapy scheme, disease duration, depressive symptoms, and co-morbidities. We performed Poisson generalized linear models with a robust variance to estimate 95% confidence interval (CI), crude prevalence ratios (cPRs), and adjusted prevalence ratios (aPRs) by epidemiological and statistical approaches using nonparametric method of bias-corrected and accelerated bootstrap resampling with 1,000 repetitions. Studied women had a median age of 47.0 years (interquartile range: 7.5); 25.3% were premenopausal, 25.8% were perimenopausal, and 48.9% were postmenopausal. Also, 64.3% had LSF. The frequency of LSF was 53.6% in non-postmenopausal and 75.0% in postmenopausal women. Postmenopausal status was associated with LSF in both the crude (cPR = 1.39; 95% CI: 1.13-1.71) and the adjusted regression models (aPR = 1.38; 95% CI: 1.12-1.71). HIV-infected postmenopausal women have a higher prevalence of LSF than those non-postmenopausal ones, even when adjusting for multiple potential confounders.


SU-E-J-195: Dosimetric Consequences of Metal Prosthesis in Prostate 3D CRT by Using Monte Carlo Dose Calculation Method.
To investigate dosimetric consequences of patient metal prosthesis in prostate radiotherapy, considering the body heterogeneity, metal artifacts, prostate daily displacements and target delineation. Three prostate patients with metal prosthesis were simulated with CT images and 3D ultrasound images. Conformal RT treatment plans were generated based on target volumes delineated on CT images without artifact correction (PTVa). The patients' treatments were aided with an ultrasound (US) localization system for daily PTV setup correction. We retrospectively reevaluated the delineation of PTVs by 1) using simulation CT images artifact-corrected by using an algorithm developed by M. Bazalova et al (2007) (PTVc) and 2) using the 3D ultrasound simulation images as guide (PTVus). Daily setup corrections to the PTVs were incorporated to calculate composite delivered dose by using XVMC simulation on the patient phantom derived from the artifact-corrected CT images. DVHs and dose distributions for different PTVs were then compared with the reference treatment plans (XVMC-calculated on the artifact-degraded CT images). The PTVa volume was the largest, about 1.3% larger than artifact-corrected PTVc and 5.2% larger than PTVus. Adapting artifact-corrected CT images can improve the DVH curves of PTVc and increase the D95% and V95% for PTVc by more than 5% while D50% and V50% for rectum and bladder are raised by up to 41.6%. DVH analysis on PTVa and PTVus shows a small difference in the changes of their DVH indices, less than 4% for the studied cases. The strike artifacts from metal prosthesis will increase the volume of PTVa, and affect the patient dose calculation. Original patient plan did not accurately predict the dose degradation. Artifact correction may be necessary for some cases having severe metal artifacts. Using US images to help delineating PTV makes a negligible clinical significance.


Design, fabrication, and performance of preferential-order volume grating waveguide couplers.
Both a nonfocusing and a focusing preferential-order volume grating waveguide coupler were designed, fabricated, and tested. These volume grating couplers are designed to outcouple a 633-nm wave guided in an adjacent polyimide waveguide film. The slanted-fringe volume gratings are recorded holographically by the interference of two 364-nm waves. The dynamics of the holographic photopolymer HRF600X001 are investigated in relation to the interaction with the guided wave. The fabricated couplers exhibited a preferential coupling of 98%, a spatial coupling rate of 3.6 mm(-1), and a coupling efficiency of 95%. The focusing grating coupler focused the outcoupled beam to a focal line with a full width at half-maximum of 10.49 microm located 25 mm above the grating.


High-Frequency Percussive Ventilation Facilitates Weaning from Extracorporeal Membrane Oxygenation in Adults.
Venoarterial extracorporeal membrane oxygenation (VA-ECMO) is an invaluable rescue therapy for patients suffering from cardiopulmonary arrest, but it is not without its drawbacks. There are cases where patients recover their cardiac function, yet they fail to wean to mechanical conventional ventilation (MCV). The use of high-frequency percussive ventilation (HFPV) has been described in patients with acute respiratory failure (RF) who fail MCV. We describe our experience with five patients who underwent VA-ECMO for cardiopulmonary arrest who were successfully weaned from VA-ECMO with HFPV after failure to wean with MCV. Weaning trials of HFPV a day before decannulation or at the time of separation from VA-ECMO were conducted. Primary endpoint data collected include pre- and post-HFPV partial pressures of oxygen (PaO2) and PaO2/FIO2 (P/F) ratios measured at 2 and 24 hours after institution of HFPV. Additional periprocedural data points were collected including length of time on ECMO, hospital stay, and survival to discharge. Four of five patients were placed on VA-ECMO subsequent to percutaneous coronary intervention. One patient had cardiac arrest secondary to RF. Mean PaO2 (44 ± 15.9 mmHg vs. 354 ± 149 mmHg, p < .01) and mean P/F ratio (44 ± 15.9 vs. 354 ± 149, p < .01) increased dramatically at 2 hours after the initiation of HFPV. The improvement in mean PaO2 and P/F ratio was durable at 24 hours whether or not the patient was returned to MCV (n = 3) or remained on HFPV (n = 2) (44 ± 15.9 mmHg vs. 131 ± 68.7 mmHg, p = .036 and 44 ± 15.9 vs. 169 ± 69.9, p < .01, respectively). Survival to discharge was 80%. The data presented suggest that HFPV may be used as a strategy to shorten time on ECMO, thereby reducing the negative effects of the ECMO circuit and improving its cost efficacy.


The "Subtile Aereal Spirit of Fountains": Mineral Waters and the History of Pneumatic Chemistry.
The standard history of pneumatic chemistry is dominated by a landmark-discoverers-type narrative stretching from Robert Boyle, through Stephen Hales, Joseph Black, and Joseph Priestley, to Antoine Lavoisier. This article challenges this view by demonstrating the importance of the study of mineral waters - and their "aerial component" - to the evolution of pneumatic chemistry, from around van Helmont to the period before Black (1640s-1750s). Among key figures examined are Joan Baptista van Helmont, Johann Joachim Becher, Robert Boyle, Friedrich Hoffmann, and William Brownrigg.


Surface functionalisation significantly changes the physical and electronic properties of carbon nano-dots.
Biomolecule functionalisation of carbon nano-dots (CDs) greatly enhances their biocompatibility and applicability, however, little is known about their molecular structure. Using an arsenal of spectroscopic and analytical techniques, we provide new insights into the physical and electronic structure of uncoated and glycan-functionalised CDs. Our studies reveal that surface functionalisation does not always result in a homogenous corona surrounding the core, and the choice of carbohydrate significantly affects the electronic structure of the surface CD states. Further, the average surface coverage of an ensemble of CDs can be probed via transient absorption spectroscopy. These findings have implications for CDs targeted at interactions with biological systems or local sensors.


Manifesto on small airway involvement and management in asthma and chronic obstructive pulmonary disease: an Interasma (Global Asthma Association - GAA) and World Allergy Organization (WAO) document endorsed by Allergic Rhinitis and its Impact on Asthma (ARIA) and Global Allergy and Asthma European Network (GA2LEN).
Evidence that enables us to identify, assess, and access the small airways in asthma and chronic obstructive pulmonary disease (COPD) has led INTERASMA (Global Asthma Association) and WAO to take a position on the role of the small airways in these diseases. Starting from an extensive literature review, both organizations developed, discussed, and approved the manifesto, which was subsequently approved and endorsed by the chairs of ARIA and GA2LEN. The manifesto describes the evidence gathered to date and defines and proposes issues on small airway involvement and management in asthma and COPD with the aim of challenging assumptions, fostering commitment, and bringing about change. The small airways (defined as those with an internal diameter <2 mm) are involved in the pathogenesis of asthma and COPD and are the major determinant of airflow obstruction in these diseases. Various tests are available for the assessment of the small airways, and their results must be integrated to confirm a diagnosis of small airway dysfunction. In asthma and COPD, the small airways play a key role in attempts to achieve disease control and better outcomes. Small-particle inhaled formulations (defined as those that, owing to their size [usually <2 μm], ensure more extensive deposition in the lung periphery than large molecules) have proved beneficial in patients with asthma and COPD, especially those in whom small airway involvement is predominant. Functional and biological tools capable of accurately assessing the lung periphery and more intensive use of currently available tools are necessary. In patients with suspected COPD or asthma, small airway involvement must be assessed using currently available tools. In patients with subotpimal disease control and/or functional or biological signs of disease activity, the role of small airway involvement should be assessed and treatment tailored. Therefore, the choice between large- and small-particle inhaled formulations must reflect the physician's considerations of disease features, phenotype, and response to previous therapy. This article is being co-published in Asthma Research and Practice and the World Allergy Organization Journal.


Gene expression signatures delineate biological and prognostic subgroups in peripheral T-cell lymphoma.
Peripheral T-cell lymphoma (PTCL) encompasses a heterogeneous group of neoplasms with generally poor clinical outcome. Currently 50% of PTCL cases are not classifiable: PTCL-not otherwise specified (NOS). Gene-expression profiles on 372 PTCL cases were analyzed and robust molecular classifiers and oncogenic pathways that reflect the pathobiology of tumor cells and their microenvironment were identified for major PTCL-entities, including 114 angioimmunoblastic T-cell lymphoma (AITL), 31 anaplastic lymphoma kinase (ALK)-positive and 48 ALK-negative anaplastic large cell lymphoma, 14 adult T-cell leukemia/lymphoma and 44 extranodal NK/T-cell lymphoma that were further separated into NK-cell and gdT-cell lymphomas. Thirty-seven percent of morphologically diagnosed PTCL-NOS cases were reclassified into other specific subtypes by molecular signatures. Reexamination, immunohistochemistry, and IDH2 mutation analysis in reclassified cases supported the validity of the reclassification. Two major molecular subgroups can be identified in the remaining PTCL-NOS cases characterized by high expression of either GATA3 (33%; 40/121) or TBX21 (49%; 59/121). The GATA3 subgroup was significantly associated with poor overall survival (P = .01). High expression of cytotoxic gene-signature within the TBX21 subgroup also showed poor clinical outcome (P = .05). In AITL, high expression of several signatures associated with the tumor microenvironment was significantly associated with outcome. A combined prognostic score was predictive of survival in an independent cohort (P = .004).


Drug-induced thrombocytopenia is associated with increased binding of IgG to platelets both in vivo and in vitro.
Thrombocytopenia is a common serious adverse effect of drug treatment. A variety of in vitro diagnostic techniques to confirm the diagnosis are available, but the majority lack sufficient sensitivity to detect all cases of drug-induced thrombocytopenia. We studied 19 patients with suspected drug-induced thrombocytopenia and demonstrated that platelet-associated IgG (PAIgG) was elevated in all at the time of thrombocytopenia, and PAIgG returned to normal levels as the thrombocytopenia resolved. In the majority of patients, the platelet count rapidly returned to normal after the drug was discontinued; however, in six patients, the thrombocytopenia persisted well beyond the period of time that the offending drug would be expected to be cleared from the blood. In 13 patients, serum obtained after recovery was used to identify the drug responsible for the thrombocytopenia in an in vitro assay. In all cases, the addition of the drug historically associated with the thrombocytopenic episode was associated with an increased binding of IgG to control platelets. For uncertain reasons, the concentration of drug required to increase the in vitro binding of IgG to test platelets was often more than the concentration usually achieved in vivo. Wider application of these techniques may provide better understanding of the clinical characteristics and mechanisms responsible for drug-induce thrombocytopenia.


Milk and dairy products: good or bad for human health? An assessment of the totality of scientific evidence.
There is scepticism about health effects of dairy products in the public, which is reflected in an increasing intake of plant-based drinks, for example, from soy, rice, almond, or oat. This review aimed to assess the scientific evidence mainly from meta-analyses of observational studies and randomised controlled trials, on dairy intake and risk of obesity, type 2 diabetes, cardiovascular disease, osteoporosis, cancer, and all-cause mortality. The most recent evidence suggested that intake of milk and dairy products was associated with reduced risk of childhood obesity. In adults, intake of dairy products was shown to improve body composition and facilitate weight loss during energy restriction. In addition, intake of milk and dairy products was associated with a neutral or reduced risk of type 2 diabetes and a reduced risk of cardiovascular disease, particularly stroke. Furthermore, the evidence suggested a beneficial effect of milk and dairy intake on bone mineral density but no association with risk of bone fracture. Among cancers, milk and dairy intake was inversely associated with colorectal cancer, bladder cancer, gastric cancer, and breast cancer, and not associated with risk of pancreatic cancer, ovarian cancer, or lung cancer, while the evidence for prostate cancer risk was inconsistent. Finally, consumption of milk and dairy products was not associated with all-cause mortality. Calcium-fortified plant-based drinks have been included as an alternative to dairy products in the nutrition recommendations in several countries. However, nutritionally, cow's milk and plant-based drinks are completely different foods, and an evidence-based conclusion on the health value of the plant-based drinks requires more studies in humans. The totality of available scientific evidence supports that intake of milk and dairy products contribute to meet nutrient recommendations, and may protect against the most prevalent chronic diseases, whereas very few adverse effects have been reported.


The effect of fibers on coagulation of casein-based enteral nutrition in an artificial gastric digestion model.
A serious complication seen in critically ill patients is the solidification of enteral nutrition causing gastrointestinal obstruction. It has been suggested that enteral nutrition enriched with insoluble fibers may increase the risk of this complication. Therefore, we investigate the effect of soluble and insoluble dietary fibers on the coagulation of a casein-based enteral nutrition in an artificial gastric digestion model. A 100% casein-based enteral nutrition was enriched with increasing concentrations of soluble fibers (acacia fiber, oligofructose and inulin) and insoluble fibers (soy polysaccharide, resistant starch and alpha cellulose). After digestion in an artificial gastric model, the chyme was poured over sequentially placed sieves, separating the coagulate into size fractions of larger than 2 mm, between 1 and 2 mm, and between 0.25 and 1 mm. Of these fractions we measured wet weight, dry weight and protein content. A significant effect on the fraction larger than 2 mm was considered to be clinically relevant. Addition of high concentrations soy polysaccharide and resistant starch to a casein-based enteral nutrition, did not alter the wet weight, whereas dry weight and protein content of the coagulate was significantly reduced. When high concentrations of soy polysaccharide and resistant starch are added to a 100% casein-based enteral nutrition, the coagulate consist of more water and less proteins, which may lead to an increased protein digestion and absorption in a clinical setting. The suggestion that insoluble fibers increase the risk of gastrointestinal obstruction in critically ill patients is not supported by these data.


Non-adherence to recommendations for further testing after outpatient CT and MRI.
Nearly 1 in every 5 outpatient visits ends with a request for a diagnostic imaging test, and imaging reports often contain recommendations for further testing. Little is known about adherence to recommendations for further testing after outpatient computed tomography (CT) and magnetic resonance imaging (MRI). We performed a retrospective cohort study linking provincial administrative data to a cross-sectional audit of 23,691 outpatient CT and MRI scans performed in 2005 in Ontario, Canada. After excluding patients who died (n=1031), were hospitalized (n=3030), or visited an emergency department (n=3660) within 180 days of the index CT/MRI scan, 15,970 CT/MRI scans were included. The primary outcome was adherence to recommendations for further testing within 180 days of an index CT/MRI scan. Further testing was recommended in 2027 of 15,970 (12.7%) index CT/MRI scan reports and was recommended most frequently after CT chest scans (593 of 2276 [26.1%]). From the 2027 scans in which further testing was recommended, we identified 2102 individual recommendations for a specific type of follow-up test and found that just over one third (37.6%) of these recommendations were followed at 180 days. Adherence was lower (32.3%) when patients had a visit to the referring physician within 180 days of the index CT/MRI scan, compared with when they had no such visit (50.5%; P <.001). Radiologists commonly recommend further testing after outpatient CT and MRI scanning. However, nearly two thirds of these recommendations are not followed. This suggests that substantial opportunities exist to improve the exchange of information between clinicians and radiologists and to advance the quality of outpatient care.


Microtubules in mammalian heart muscle.
Sections of adult mammalian cardiac muscles fixed at room temperature reveal numerous microtubules (24--28 nm in diameter) both near the nucleus and in the extra-myofibrillar space. Microtubules encircle the nucleus, are associated with the myofibrils in a helical arrangement, and form a network that runs transversely at the level of the I band and axially between the myofibrils. Microtubules are more numerous in muscle cells than previously recognized and may perform more than cytoskeletal function.


Antidepressants and Cdk inhibitors: releasing the brake on neurogenesis?
It is now clear that neurogenesis occurs in the brain of adult mammals. Many studies have attempted to establish relationships among neurogenesis, depression and the mechanism of action of antidepressant drugs. Therapeutic effects of antidepressants appear to be linked to increased neurogenesis in the hippocampus. Cdk inhibitors are expressed in multiple brain regions, presumably maintaining quiescence in differentiated neurons. Recently, the abundant expression of p21(Cip1) was found in neuroblasts and in newly developing neurons in the subgranular zone of the hippocampus, a region where adult neurogenesis occurs. Chronic treatment with the tricyclic antidepressant imipramine markedly decreased p21(Cip1) mRNA and protein levels and stimulated neurogenesis in this region. These results suggest that p21(Cip1) restrains neurogenesis in the hippocampus, and antidepressant-induced stimulation of neurogenesis might be a consequence of decreased p21(Cip1) expression, with the subsequent release of neuronal progenitor cells from the blockade of proliferation. These findings suggest the potential for new therapeutic strategies for the treatment of depression that target cell cycle proteins. However, there is a possibility that long-term stimulation of neurogenesis might exhaust the proliferation potentials of neuronal progenitors.


H. pylori receptor MHC class II contributes to the dynamic gastric epithelial apoptotic response.
To investigate the role of MHC class II in the modulation of gastric epithelial cell apoptosis induced by H pylori infection. After stimulating a human gastric epithelial cell line with bacteria or agonist antibodies specific for MHC class II and CD95, the quantitation of apoptotic and anti-apoptotic events, including caspase activation, BCL-2 activation, and FADD recruitment, was performed with a fluorometric assay, a cytometric bead array, and confocal microscopy, respectively. Pretreatment of N87 cells with the anti-MHC class II IgM antibody RFD1 resulted in a reduction in global caspase activation at 24 h of H pylori infection. When caspase 3 activation was specifically measured, crosslinking of MHC class II resulted in a marked reduced caspase activation, while simple ligation of MHC class II did not. Crosslinking of MHC class II also resulted in an increased activation of the anti-apoptosis molecule BCL-2 compared to simple ligation. Confocal microscope analysis demonstrated that the pretreatment of gastric epithelial cells with a crosslinking anti-MHC class II IgM blocked the recruitment of FADD to the cell surface. The results presented here demonstrate that the ability of MHC class II to modulate gastric epithelial apoptosis is at least partially dependent on its crosslinking. Furthermore, while previous research has demonstrated that MHC class II signaling can be pro-apoptotic during extended ligation, we have shown that the crosslinking of this molecule has anti-apoptotic effects during the earlier time points of H pylori infection. This effect is possibly mediated by the ability of MHC class II to modulate the activation of the pro-apoptotic receptor Fas by blocking the recruitment of the accessory molecule FADD, and this delay in apoptosis induction could allow for prolonged cytokine secretion by H pylori-infected gastric epithelial cells.


Whole-genome transcriptional profiling of Bradyrhizobium japonicum during chemoautotrophic growth.
Bradyrhizobium japonicum is a facultative chemoautotroph capable of utilizing hydrogen gas as an electron donor in a respiratory chain terminated by oxygen to provide energy for cellular processes and carbon dioxide assimilation via a reductive pentose phosphate pathway. A transcriptomic analysis of B. japonicum cultured chemoautotrophically identified 1,485 transcripts, representing 17.5% of the genome, as differentially expressed when compared to heterotrophic cultures. Genetic determinants required for hydrogen utilization and carbon fixation, including the uptake hydrogenase system and components of the Calvin-Benson-Bassham cycle, were strongly induced in chemoautotrophically cultured cells. A putative isocitrate lyase (aceA; blr2455) was among the most strongly upregulated genes, suggesting a role for the glyoxylate cycle during chemoautotrophic growth. Addition of arabinose to chemoautotrophic cultures of B. japonicum did not significantly alter transcript profiles. Furthermore, a subset of nitrogen fixation genes was moderately induced during chemoautotrophic growth. In order to specifically address the role of isocitrate lyase and nitrogenase in chemoautotrophic growth, we cultured aceA, nifD, and nifH mutants under chemoautotrophic conditions. Growth of each mutant was similar to that of the wild type, indicating that the glyoxylate bypass and nitrogenase activity are not essential components of chemoautotrophy in B. japonicum.


Ionic requirements for the active ileal bile salt transport system.
Taurocholate transport by everted ileal gut sacs was studied in physiological media containing graded amounts of sodium ions. Significant uphill transport of taurocholate was observed when the bulk of NaCl was replaced by osmotic equivalents of mannitol or choline chloride. Seventy-seven percent of control transport activity was observed when 36 milliequivalents per liter of Na+ were present in the incubation medium with mannitol acting as the isosmotic replacement, and 74% of the control transport was retained when 31 milliequivalents per liter of Na+ were present in the incubation medium with choline chloride acting as the osmotic replacement. Lowering the Na+ concentration to 19 milliequivalents per liter (i.e., 84% replacement of Na+) still allowed for 69% of the uphill transport observed in the control incubations. Taurodehydrocholate transport by ileal everted sacs was more sensitive to decreased Na+ concentrations; 29% of control transport was observed at 31 milliequivalents per liter of Na+. A kinetic analysis comparing the transport of taurocholate with taurodehydrocholate, the triketo analogue, at different concentrations of Na+ indicated that the apparent affinity of the transport system for Na+ is greater in the presence of taurocholate than in the presence of taurodehydrocholate. The ability of taurodehydrocholate to depress taurocholate transport is less in media of low Na+ concentration. Finally, in vivo intestinal perfusion studies demonstrated that the depression of taurocholate absorption, following Na+ removal, is reversible. These results are in agreement with the idea that Na+ has a physiological role in intestinal bile salt transport, and that the affinities of the anionic bile salt and the sodium cation for the transport system appear to be cooperative in that one enhances the binding of the other.


Experimental knee pain impairs submaximal force steadiness in isometric, eccentric, and concentric muscle actions.
Populations with knee joint damage, including arthritis, have noted impairments in the regulation of submaximal muscle force. It is difficult to determine the exact cause of such impairments given the joint pathology and associated neuromuscular adaptations. Experimental pain models that have been used to isolate the effects of pain on muscle force regulation have shown impaired force steadiness during acute pain. However, few studies have examined force regulation during dynamic contractions, and these findings have been inconsistent. The goal of the current study was to examine the effect of experimental knee joint pain on submaximal quadriceps force regulation during isometric and dynamic contractions. The study involved fifteen healthy participants. Participants were seated in an isokinetic dynamometer. Knee extensor force matching tasks were completed in isometric, eccentric, and concentric muscle contraction conditions. The target force was set to 10 % of maximum for each contraction type. Hypertonic saline was then injected into the infrapatella fat pad to generate acute joint pain. The force matching tasks were repeated during pain and once more 5 min after pain had subsided. Hypertonic saline resulted in knee pain with an average peak pain rating of 5.5 ± 2.1 (0-10 scale) that lasted for 18 ± 4 mins. Force steadiness significantly reduced during pain across all three muscle contraction conditions. There was a trend to increased force matching error during pain but this was not significant. Experimental knee pain leads to impaired quadriceps force steadiness during isometric, eccentric, and concentric contractions, providing further evidence that joint pain directly affects motor performance. Given the established relationship between submaximal muscle force steadiness and function, such an effect may be detrimental to the performance of tasks in daily life. In order to restore motor performance in people with painful arthritic conditions of the knee, it may be important to first manage their pain more effectively.


Insulin resistance and impaired glucose tolerance in overweight and obese Costa Rican schoolchildren.
Worldwide obesity has become an unprecedented public health challenge. In addition, a notable increase in the risk of insulin resistance and type 2 diabetes mellitus has emerged. In Costa Rica, there are no epidemiological data to establish the prevalence of type 2 diabetes in the pediatric population. However, information from the Endocrinology Department of the Children's National Hospital indicates an increased number of cases in the last 2 to 3 years. To determine the prevalence of insulin resistance and impaired glucose tolerance in overweight and obese schoolchildren. This cross-sectional study was conducted among 214 healthy 8- to 10-year-old children from urban schools of San José, Costa Rica. Anthropometric measurements and blood determinations of glucose, insulin, proinsulin, glycosylated hemoglobin, C-peptide, and leptin were performed. Indexes were calculated to assess insulin resistance. Information on social and lifestyle variables was obtained from questionnaires, and acanthosis nigricans was certified by a physician. Statistical analysis was performed with SPSS software for Windows, version 10.0. The prevalence of type 2 diabetes mellitus was very low (0.5%) in the studied population. However, hyperinsulinemia and impaired glucose tolerance were present in 20.6% and 6.5% of the subjects, respectively. On the basis of the Fasting Glucose-to-Insulin Resistance Ratio (FGIR), 46.7% of the children showed insulin resistance. Girls and obese children (body mass index > or = 95th percentile) were more likely to have higher serum insulin levels and insulin resistance than boys and overweight children (BMI > or = 85th percentile). Compared with the lowest quintile, children in the highest quintile of body-fat tissue had higher insulin resistance but had similar serum concentrations of glucose, C-peptide, and proinsulin. Positive family histories of type 2 diabetes mellitus and sedentarism (73.7% and 40.7%, respectively) were highly prevalent among overweight and obese children. The prevalence of impaired glucose tolerance and insulin resistance in obese children indicates a worrisome trend in the incidence of type 2 diabetes in Costa Rica. Strategies for weight reduction, obesity prevention, and promotion of healthy lifestyles are necessary to prevent the onset of type 2 diabetes during childhood and adolescence.


A randomized clinical trial of living donor nephrectomy: a plea for a differentiated appraisal of mini-open muscle splitting incision and hand-assisted laparoscopic donor nephrectomy.
A randomized controlled trial was designed to compare various outcome variables of the retroperitoneal mini-open muscle splitting incision (MSI) technique and the transperitoneal hand-assisted laparoscopic technique (HAL) in performing living donor nephrectomies. Fifty living kidney donors were randomized to MSI or HAL. Primary endpoint was pain experience scored on a visual analogue scale (VAS). After MSI living donors indicated lower median (range) VAS scores at rest than HAL living donors on postoperative day 2.5 [10 (0-44) vs. 15 (0-70), P = 0.043] and day 3 [7 (0-28) vs. 10 (0-91), P = 0.023] and lower VAS scores while coughing on postoperative day 3 [20 (0-73) vs. 42 (6-86), P = 0.001], day 7 [8 (0-66) vs. 33 (3-76), P < 0.001] and day 14 [2 (0-17) vs. 12 (0-51), P = 0.009]. The MSI technique also resulted in reduced morphine requirement, better scores on three domains of the RAND-36, reduced costs and reduced CRP and IL-6 levels. The HAL technique was superior in operating time and postoperative decrease of hemoglobin level. The MSI technique is superior to the HAL technique in performing living donor nephrectomies with regard to postoperative pain experience. This study reopens the discussion of the way to go in performing the living donor nephrectomy.


Phylogenetic barriers to horizontal transfer of antimicrobial peptide resistance genes in the human gut microbiota.
The human gut microbiota has adapted to the presence of antimicrobial peptides (AMPs), which are ancient components of immune defence. Despite its medical importance, it has remained unclear whether AMP resistance genes in the gut microbiome are available for genetic exchange between bacterial species. Here, we show that AMP resistance and antibiotic resistance genes differ in their mobilization patterns and functional compatibilities with new bacterial hosts. First, whereas AMP resistance genes are widespread in the gut microbiome, their rate of horizontal transfer is lower than that of antibiotic resistance genes. Second, gut microbiota culturing and functional metagenomics have revealed that AMP resistance genes originating from phylogenetically distant bacteria have only a limited potential to confer resistance in Escherichia coli, an intrinsically susceptible species. Taken together, functional compatibility with the new bacterial host emerges as a key factor limiting the genetic exchange of AMP resistance genes. Finally, our results suggest that AMPs induce highly specific changes in the composition of the human microbiota, with implications for disease risks.


Bayesian Intent Prediction in Object Tracking Using Bridging Distributions.
In several application areas, such as human computer interaction, surveillance and defence, determining the intent of a tracked object enables systems to aid the user/operator and facilitate effective, possibly automated, decision making. In this paper, we propose a probabilistic inference approach that permits the prediction, well in advance, of the intended destination of a tracked object and its future trajectory. Within the framework introduced here, the observed partial track of the object is modeled as being part of a Markov bridge terminating at its destination, since the target path, albeit random, must end at the intended endpoint. This captures the underlying long term dependencies in the trajectory, as dictated by the object intent. By determining the likelihood of the partial track being drawn from a particular constructed bridge, the probability of each of a number of possible destinations is evaluated. These bridges can also be employed to produce refined estimates of the latent system state (e.g., object position, velocity, etc.), predict its future values (up until reaching the designated endpoint) and estimate the time of arrival. This is shown to lead to a low complexity Kalman-filter-based implementation of the inference routine, where any linear Gaussian motion model, including the destination reverting ones, can be applied. Free hand pointing gestures data collected in an instrumented vehicle and synthetic trajectories of a vessel heading toward multiple possible harbors are utilized to demonstrate the effectiveness of the proposed approach.


Communication about serious illness care goals: a review and synthesis of best practices.
An understanding of patients' care goals in the context of a serious illness is an essential element of high-quality care, allowing clinicians to align the care provided with what is most important to the patient. Early discussions about goals of care are associated with better quality of life, reduced use of nonbeneficial medical care near death, enhanced goal-consistent care, positive family outcomes, and reduced costs. Existing evidence does not support the commonly held belief that communication about end-of-life issues increases patient distress. However, conversations about care goals are often conducted by physicians who do not know the patient, do not routinely address patients' nonmedical goals, and often fail to provide patients with sufficient information about prognosis to allow appropriate decisions; in addition, they tend to occur so late in the patient's illness that their impact on care processes is reduced. This article (1) reviews the evidence and describes best practices in conversations about serious illness care goals and (2) offers practical advice for clinicians and health care systems about developing a systematic approach to quality and timing of such communication to assure that each patient has a personalized serious illness care plan. Best practices in discussing goals of care include the following: sharing prognostic information, eliciting decision-making preferences, understanding fears and goals, exploring views on trade-offs and impaired function, and wishes for family involvement. Several interventions hold promise in systematizing conversations with patients about serious illness care goals: better education of physicians; systems to identify and trigger early discussions for appropriate patients; patient and family education; structured formats to guide discussions; dedicated, structured sections in the electronic health record for recording information; and continuous measurement. We conclude that communication about serious illness care goals is an intervention that should be systematically integrated into our clinical care structures and processes.


Habitat fragmentation leads to reduced pollinator visitation, fruit production and recruitment in urban mangrove forests.
Mangrove forests worldwide undergo anthropogenic fragmentation that may threaten their existence, and yet there have been few tests of the effects of fragmentation on demographic processes critical for mangrove regeneration. Predicting the effects of habitat fragmentation on mangroves is problematic as pollinators may move more freely across water than terrestrial habitat, and propagules can be widely dispersed by water. Here, within each of two estuaries, we compared pollinator diversity and activity, reproductive effort and output, and rates of recruitment for sets of three large (>1500 trees), medium (300-500) and small (<50) stands. As predicted, most measures of reproductive activity and success were inversely related to stand size with large stands typically producing significantly more and larger fruit, and significantly more seedlings. Most strikingly, we found the effect of fragmentation on the abundance of pollinators (honeybees), the production and quality of fruit and the survival rate of seedlings to be similar, showing significant reduction of recruitment in small stands. This study provides the first rigorous evidence that recruitment of mangroves, like for many terrestrial plants, is negatively impacted by habitat fragmentation. From a management perspective, we argue that in the short term our data imply the importance of conserving the largest possible stands. However, additional work is needed to determine (1) the proportion of recruits within small stands that originate within large stands, (2) how seedling performance varies with fruit size and genotype, and (3) how seedling size and performance vary with the abundance and diversity of pollen.


Best (but oft-forgotten) practices: the multiple problems of multiplicity-whether and how to correct for many statistical tests.
Testing many null hypotheses in a single study results in an increased probability of detecting a significant finding just by chance (the problem of multiplicity). Debates have raged over many years with regard to whether to correct for multiplicity and, if so, how it should be done. This article first discusses how multiple tests lead to an inflation of the α level, then explores the following different contexts in which multiplicity arises: testing for baseline differences in various types of studies, having >1 outcome variable, conducting statistical tests that produce >1 P value, taking multiple "peeks" at the data, and unplanned, post hoc analyses (i.e., "data dredging," "fishing expeditions," or "P-hacking"). It then discusses some of the methods that have been proposed for correcting for multiplicity, including single-step procedures (e.g., Bonferroni); multistep procedures, such as those of Holm, Hochberg, and Šidák; false discovery rate control; and resampling approaches. Note that these various approaches describe different aspects and are not necessarily mutually exclusive. For example, resampling methods could be used to control the false discovery rate or the family-wise error rate (as defined later in this article). However, the use of one of these approaches presupposes that we should correct for multiplicity, which is not universally accepted, and the article presents the arguments for and against such "correction." The final section brings together these threads and presents suggestions with regard to when it makes sense to apply the corrections and how to do so.


Prognostic value of auto-antibodies in the serum of Omani patients with gastric cancer.
To investigate the prevalence of a group of different autoantibodies, in Omani patients with gastric cancer, and to examine whether their presence correlates with clinical course of the disease. Ninety-three Omani patients with gastric cancer, and 100 gender-matched blood donors were investigated for the presence of 15 different auto-antibodies against nuclear antigens (ANA), extractible nuclear antigens (ENA), Scleroderma antigen (Scl-70), Sjogren syndrome antigen A/B (SSA/B), Smith antigen (Sm), ribonucleoprotein (RNP), Jo-1 antigen, double stranded DNA (ds-DNA), parietal cell antibodies (APCA), reticulin antibodies (ARA), smooth muscle antibodies (ASMA), proteinase 3 (PR3), myeloperoxidase (MPO), and mitochondria antibody (AMA). Antinuclear antigen were detected using human epithelial cells-2 (Hep-2 cells). Anti-dsDNA antibodies were measured using Crithidia lucilia slides; APCA, ARA, and ASMA were examined using mouse liver, kidney, and stomach sections. Other autoantibodies were detected using commercially available ELISA kits. Seventy-three out of the 93 patients with gastric cancer were divided into 4 groups (stages I to IV) according to disease severity. This study was conducted in the period of 2001-2005 in the Department of Microbiology and Immunology Laboratories of the College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Oman. Approximately 40% of the autoantibodies investigated were found to be significantly higher in patients with gastric cancer than in normal controls. These autoantibodies are ANA (57.3 versus 14%, p<0.0001), anti-ENA (38.7 versus 13.9%, p<0.01), anti-Scl-70 (29 versus 5%, p<0.001), ARA (19.8 versus 3.1%, p<0.0001), ASMA (72.9 versus 31.6%, p<0.01), and anti-PR3 (21.5 versus 5.3% p<0.01). Generally, the presence of auto-antibodies was more frequent in stage III and IV compared to stage I and II. However, some autoantibodies (ENA, SSA, Scl-70, and ASMA) were more common in stage II than stage IV. Auto-antibodies are more prevalent in the serum of patients with gastric cancer compared to healthy controls. Some of these auto-antibodies may prove to be important markers of prognostic values in patients with gastric cancer.


Towards delineating functions within the fasciola secreted cathepsin l protease family by integrating in vivo based sub-proteomics and phylogenetics.
fasciola hepatica, along with Fasciola gigantica, is the causative agent of fasciolosis, a foodborne zoonotic disease affecting grazing animals and humans worldwide. Pathology is directly related to the release of parasite proteins that facilitate establishment within the host. The dominant components of these excretory-secretory (ES) products are also the most promising vaccine candidates, the cathepsin L (Cat L) protease family. the sub-proteome of Cat L proteases from adult F. hepatica ES products derived from in vitro culture and in vivo from ovine host bile were compared by 2-DE. The individual Cat L proteases were identified by tandem mass spectrometry with the support of an in-house translated liver fluke EST database. The study reveals plasticity within the CL1 clade of Cat L proteases; highlighted by the identification of a novel isoform and CL1 sub-clade, resulting in a new Cat L phylogenetic analysis including representatives from other adult Cat L phylogenetic clades. Additionally, for the first time, mass spectrometry was shown to be sufficiently sensitive to reveal single amino acid polymorphisms in a resolved 2-DE protein spot derived from pooled population samples. we have investigated the sub-proteome at the population level of a vaccine target family using the Cat L proteases from F. hepatica as a case study. We have confirmed that F. hepatica exhibits more plasticity in the expression of the secreted CL1 clade of Cat L proteases at the protein level than previously realised. We recommend that superfamily based vaccine discovery programmes should screen parasite populations from different host populations and, if required, different host species via sub-proteomic assay in order to confirm the relative expression at the protein level prior to the vaccine development phase.


The safety and accuracy of ECG-guided PICC tip position verification applied in patients with atrial fibrillation.
Tip position verification of peripherally inserted central catheters (PICCs) is essential to the use of the catheter. Postprocedural chest X-ray as the "gold standard" practice for PICC tip confirmation can lead to a significant delay for patient IV therapy, cost more, and lead to radiation exposure for both patients and staffs. Intracavitary electrocardiogram (IC-ECG)-guided PICC placement which provides real-time tip confirmation during the insertion procedure has been widely used. However, safety and accuracy of ECG for abnormal surface ECG patients, such as patients with atrial fibrillation (AF), have not been reported. To determine the safety and accuracy of IC-ECG technique for PICC tip position verification among the patients with AF. A prospective cohort study was conducted in a teaching and tertiary referral hospital with more than 3,600 beds in Qingdao, People's Republic of China. Adult patients with diagnosis of AF who need a PICC for infusion from June 2015 to May 2017 were enrolled in the study. For every included patient with AF, ECG was used to detect the PICC tip position during catheterization and X-ray was done to confirm the tip position as the "gold standard" after PICC insertion. The effectiveness and accuracy of ECG-guided catheter tip positioning and chest X-ray confirmation were compared. Totally, 118 AF patients with 118 PICCs were enrolled (58 male and 60 female, age range 50-89 years old). There was no catheterization-related complication. When the catheter entered the lower 1/3 of superior vena cava, the amplitude of f wave reached the maximum. There was no statistical difference between X-ray PICC tip position verification and IC-ECG PICC tip position verification among patients with AF (χ2=1.31, P=0.232). Utilizing the cutoff point of f wave change ≥0.5 cm, a sensitivity of 0.94, a specificity of 0.71, a positive predictive value of 0.98, and a negative predictive value of 0.42 were observed. The area under the receiver operating characteristic curve was 0.909 (95% CI: 0.810-1.000). The ECG-guided technique represents a safe and accurate technique to verify the position of PICC tip in patients with AF and could potentially remove the requirement for postprocedural chest X-ray among the patients with AF.


Rapid changes in plasma potassium during a game of squash.
1. The game of squash has recently been associated with a high incidence of ventricular arrhythmias and sudden death. To investigate this further, plasma catecholamines and potassium (K+) were monitored during a game of squash in six normal volunteers. 2. No cardiac arrhythmias were seen in this study despite the subjects reaching maximum heart rates of 181 +/- 5 beats/min (mean +/- SEM). 3. During exercise, plasma K+ rose from 3.82 +/- 0.16 to 4.29 +/- 0.2 mmol/l, but after 90 s rest this fell to 3.68 +/- 0.28 mmol/l and after 180 s to 3.44 +/- 0.17 mmol/l. This rapid K+ shift could not be accounted for by generalized changes in venous acid-base status or by changes in venous plasma catecholamines. Although pretreatment with a beta 2-antagonist caused the overall plasma K+ levels to be higher, it had no significant effect on the fall in plasma K+ after exercise. 4. Such rapid K+ shifts after exercise might contribute to arrhythmogenesis in susceptible individuals. The precise mechanism of the fall in K+ after exercise remains undetermined, but it seems not to involve catecholamines stimulating beta 2-adrenoceptors and is more likely to be due to increased skeletal muscle blood flow and/or intracellular acidosis.


Impact of assertive community treatment on homeless persons with co-occurring severe psychiatric and substance use disorders.
This study evaluated the impact of an integrated (mental health/substance abuse) assertive community treatment program on homeless persons with serious mental and substance use disorders. High rates of retention in treatment, housing stability, and community tenure were attained, and all but the most severe substance users appeared to gain these benefits. While the intervention appears to be an effective means of retaining in services and monitoring such difficult-to-treat and costly populations, it did not yield high rates of abstinence and social benefits in severe users.


Comparison of herniorrhaphy versus clamping of umbilical hernias in horses: a retrospective study of 93 cases (1982-1994).
Many uncomplicated umbilical hernias have been managed successfully in foals by the application of a hernia clamp. Isolated reports of complications following clamp application have led some authors to suggest that it is an unsuitable method of treatment. Little information has been published comparing the complication rates associated with the use of hernia clamps and herniorrhaphy in the treatment of umbilical hernias. The purpose of this retrospective study was to report the characteristics of clinical cases of umbilical hernia and to compare the complication rates following these 2 treatment approaches. Information was collected from records or from owners to identify the occurrence of complications and owner satisfaction following treatment. Of 93 cases, 10 complicated and 18 uncomplicated hernias were treated by herniorrhaphy, 40 uncomplicated hernias were treated by clamping, 1 originally uncomplicated hernia was treated by both techniques, and 24 cases were untreated. Nineteen percent of uncomplicated hernias treated by herniorrhaphy, and 19% of those clamped developed minor complications. This study demonstrates that although minor complications may be associated with either technique, they generally do not result in significant morbidity.


Reversal of pathology in the entire brain of mucopolysaccharidosis type VII mice after lentivirus-mediated gene transfer.
Gene transfer vectors derived from human immunodeficiency virus (HIV-1) efficiently transduce nondividing cells and remain stably integrated in their genome. Long-term expression of reporter genes has been documented after intracerebral injection of these vectors. Using a HIV-based vector, we looked for a reversal of brain damage in the beta-glucuronidase-deficient mucopolysaccharidosis type VII mouse, an animal model of human lysosomal storage diseases. The vector suspension was injected stereotactically in the brain of 10-week-old animals, an age at which storage lesions are patent in glia, perivascular cells, and neurons. Either a single intrastriatal injection or multiple injections in both cerebral hemispheres and in the cerebellum were performed. Local tolerance, enzyme delivery, and correction of storage lesions were investigated by comprehensive analysis of serial sections of the entire brain of mice killed 6 or 16 weeks postinjection. Histochemical staining detected enzyme activity in widely distributed areas, the size of which increased with time. Clearance of lysosomal storage extended far beyond enzyme-positive areas. In mice receiving multiple injections of the vector, complete correction or significant reduction of the pathology was observed in every section, suggesting disease regression in the entire brain. These results may have implications for the treatment of neurological symptoms in lysosomal storage diseases.


Clinical and Genetic Risk Factors for Acute Incident Venous Thromboembolism in Ambulatory Patients With COVID-19.
The risk of venous thromboembolism (VTE) in ambulatory COVID-19 is controversial. In addition, the association of vaccination with COVID-19-related VTE and relevant clinical and genetic risk factors remain to be elucidated. To quantify the association between ambulatory COVID-19 and short-term risk of VTE, study the potential protective role of vaccination, and investigate clinical and genetic risk factors for post-COVID-19 VTE. This population-based cohort study of patients with COVID-19 from UK Biobank included participants with SARS-CoV-2 infection that was confirmed by a positive polymerase chain test reaction result between March 1, 2020, and September 3, 2021, who were then propensity score matched to COVID-19-naive people during the same period. Participants with a history of VTE who used antithrombotic drugs (1 year before index dates) or tested positive in hospital were excluded. First infection with SARS-CoV-2, age, sex, ethnicity, socioeconomic status, obesity, vaccination status, and inherited thrombophilia. The primary outcome was a composite VTE, including deep vein thrombosis or pulmonary embolism, which occurred 30 days after the infection. Hazard ratios (HRs) with 95% CIs were calculated using cause-specific Cox models. In 18 818 outpatients with COVID-19 (10 580 women [56.2%]; mean [SD] age, 64.3 [8.0] years) and 93 179 matched uninfected participants (52 177 women [56.0%]; mean [SD] age, 64.3 [7.9] years), the infection was associated with an increased risk of VTE in 30 days (incidence rate of 50.99 and 2.37 per 1000 person-years for infected and uninfected people, respectively; HR, 21.42; 95% CI, 12.63-36.31). However, risk was substantially attenuated among the fully vaccinated (HR, 5.95; 95% CI, 1.82-19.5; interaction P = .02). In patients with COVID-19, older age, male sex, and obesity were independently associated with higher risk, with adjusted HRs of 1.87 (95% CI, 1.50-2.33) per 10 years, 1.69 (95% CI, 1.30-2.19), and 1.83 (95% CI, 1.28-2.61), respectively. Further, inherited thrombophilia was associated with an HR of 2.05 (95% CI, 1.15-3.66) for post-COVID-19 VTE. In this population-based cohort study of patients with COVID-19, ambulatory COVID-19 was associated with a substantially increased risk of incident VTE, but this risk was greatly reduced in fully vaccinated people with breakthrough infection. Older age, male sex, and obesity were clinical risk factors for post-COVID-19 VTE; factor V Leiden thrombophilia was additionally associated with double the risk, comparable with the risk of 10-year aging. These findings may reinforce the need for vaccination, inform VTE risk stratification, and call for targeted VTE prophylaxis strategies for unvaccinated outpatients with COVID-19.


Vitamin D receptor expression and acid sphingomyelinase activity in prefrontal region of a learning animal model.
Contextual fear conditioning (CFC) paradigm is routinely used to study fear-based learning in animals and it provides a useful model for understanding fear and anxiety in human. In the present study, such model was used following the previously established CFC protocol, and immunohistochemistry, enzymatic activity and western blotting analysis approaches were used to identify the expression of acid sphingomyelinase (aSMase) and vitamin D receptor (VDR) in prefrontal region brain of rat. Results revealed an increase of aSMase activity in conditioned rats, suggesting an apoptotic condition in such animals. In addition, an increase of density and organization of axonal neurofilaments and of VDR expression has been observed in brain of conditioned rats, supporting an induction of growth and organization of new neurons in prefrontal regions, whose contribution to various aspects of contextual fear learning is still largely unknown.


The Economic Burden of Hospital-Acquired Clostridium difficile Infection: A Population-Based Matched Cohort Study.
BACKGROUND High-quality cost estimates for hospital-acquired Clostridium difficile infection (CDI) are vital evidence for healthcare policy and decision-making. OBJECTIVE To evaluate the costs attributable to hospital-acquired CDI from the healthcare payer perspective. METHODS We conducted a population-based propensity-score matched cohort study of incident hospitalized subjects diagnosed with CDI (those with the International Statistical Classification of Diseases and Related Health Problems, 10th Revision, Canada code A04.7) from January 1, 2003, through December 31, 2010, in Ontario, Canada. Infected subjects were matched to uninfected subjects (those without the code A04.7) on age, sex, comorbidities, geography, and other variables, and followed up through December 31, 2011. We stratified results by elective and nonelective admissions. The main study outcomes were up-to-3-year costs, which were evaluated in 2014 Canadian dollars. RESULTS We identified 28,308 infected subjects (mean annual incidence, 27.9 per 100,000 population, 3.3 per 1,000 admissions), with a mean age of 71.5 years (range, 0-107 years), 54.0% female, and 8.0% elective admissions. For elective admission subjects, cumulative mean attributable 1-, 2-, and 3-year costs adjusted for survival (undiscounted) were $32,151 (95% CI, $28,192-$36,005), $34,843 ($29,298-$40,027), and $37,171 ($30,364-$43,415), respectively. For nonelective admission subjects, the corresponding costs were $21,909 ($21,221-$22,609), $26,074 ($25,180-$27,014), and $29,944 ($28,873-$31,086), respectively. CONCLUSIONS Hospital-acquired CDI is associated with substantial healthcare costs. To the best of our knowledge, this study is the first CDI costing study to present longitudinal costs. New strategies may be warranted to mitigate this costly infectious disease. Infect Control Hosp Epidemiol 2016;37:1068-1078.


Hmo1 is required for TOR-dependent regulation of ribosomal protein gene transcription.
Ribosome biogenesis requires equimolar amounts of four rRNAs and all 79 ribosomal proteins (RP). Coordinated regulation of rRNA and RP synthesis by eukaryotic RNA polymerases (Pol) I, III, and II is a key requirement for growth control. Using a novel global genetic approach, we showed that the absence of Hmo1 becomes lethal when combined with mutations of components of either the RNA Pol II or Pol I transcription machineries, of specific RP, or of the TOR pathway. Hmo1 directly interacts with both the region transcribed by Pol I and a subset of RP gene promoters. Down-regulation of Hmo1 expression affects RP gene expression. Upon TORC1 inhibition, Hmo1 dissociates from ribosomal DNA (rDNA) and some RP gene promoters simultaneously. Finally, in the absence of Hmo1, TOR-dependent repression of RP genes is alleviated. Therefore, we show here that Saccharomyces cerevisiae Hmo1 is directly involved in coordinating rDNA transcription by Pol I and RP gene expression by Pol II under the control of the TOR pathway.


Molecular determinants for competitive inhibition of alpha4beta2 nicotinic acetylcholine receptors.
The Erythrina alkaloids erysodine and dihydro-beta-erythroidine (DHbetaE) are potent and selective competitive inhibitors of alpha4beta2 nicotinic acetylcholine receptors (nAChRs), but little is known about the molecular determinants of the sensitivity of this receptor subtype to inhibition by this class of antagonists. We addressed this issue by examining the effects of DHbetaE and a range of aromatic Erythrina alkaloids on [(3)H]cytisine binding and receptor function in conjunction with homology models of the alpha4beta2 nAChR, mutagenesis, and functional assays. The lactone group of DHbetaE and a hydroxyl group at position C-16 in aromatic Erythrina alkaloids were identified as major determinants of potency, which was decreased when the conserved residue Tyr126 in loop A of the alpha4 subunit was substituted by alanine. Sensitivity to inhibition was also decreased by substituting the conserved aromatic residues alpha4Trp182 (loop B), alpha4Tyr230 (loop C), and beta2Trp82 (loop D) and the nonconserved beta2Thr84; however, only alpha4Trp182 was predicted to contact bound antagonist, suggesting alpha4Tyr230, beta2Trp82, and beta2Thr84 contribute allosterically to the closed state elicited by bound antagonist. In addition, homology modeling predicted strong ionic interactions between the ammonium center of the Erythrina alkaloids and beta2Asp196, leading to the uncapping of loop C. Consistent with this, beta2D196A abolished sensitivity to inhibition by DHbetaE or erysodine but not by epierythratidine, which is not predicted to form ionic bonds with beta2Asp196. This residue is not conserved in subunits that comprise nAChRs with low sensitivity to inhibition by DHbetaE or erysodine, which highlights beta2Asp196 as a major determinant of the receptor selectivity of Erythrina alkaloids.


A theoretical and empirical modeling of anxiety integrated with RDoC and temporal dynamics.
The newly launched Research Domain Criteria (RDoC) emphasize specific mechanisms over diagnostic categories of psychopathology. In our view, RDoC provides a useful heuristic for mental health disorders, but does not capture the complexity of psychological data when proposed mechanisms are viewed as static entities. However, temporal and complex system dynamics may advance RDoC's utility. By investigating temporal patterns within trajectories and the interaction of complex networks, we propose that dynamic modeling provides comprehensive methods with which to investigate the etiopathology and maintenance of mental health disorders. We examine applications of dynamical systems to periphery physiology, an RDoC construct that has been widely used in psychological science. A review of the literature suggests methodological problems with aggregate and reductive models. We present a dynamical systems modeling of anxiety which suggests avenues for future biomarker research. This model appears congruent with RDoC and recent learning theory.


Organic Nanoflowers from a Wide Variety of Molecules Templated by a Hierarchical Supramolecular Scaffold.
Nanoflowers (NFs) are flowered-shaped particles with overall sizes or features in the nanoscale. Beyond their pleasing aesthetics, NFs have found a number of applications ranging from catalysis, to sensing, to drug delivery. Compared to inorganic based NFs, their organic and hybrid counterparts are relatively underdeveloped mostly because of the lack of a reliable and versatile method for their construction. We report here a method for constructing NFs from a wide variety of biologically relevant molecules (guests), ranging from small molecules, like doxorubicin, to biomacromolecules, like various proteins and plasmid DNA. The method relies on the encapsulation of the guests within a hierarchically structured particle made from supramolecular G-quadruplexes. The size and overall flexibility of the guests dictate the broad morphological features of the resulting NFs, specifically, small and rigid guests favor the formation of NFs with spiky petals, while large and/or flexible guests promote NFs with wide petals. The results from experiments using confocal fluorescence microscopy, and scanning electron microscopy provides the basis for the proposed mechanism for the NF formation.


ALS-linked mutant superoxide dismutase 1 (SOD1) alters mitochondrial protein composition and decreases protein import.
Mutations in superoxide dismutase 1 (SOD1) cause familial ALS. Mutant SOD1 preferentially associates with the cytoplasmic face of mitochondria from spinal cords of rats and mice expressing SOD1 mutations. Two-dimensional gels and multidimensional liquid chromatography, in combination with tandem mass spectrometry, revealed 33 proteins that were increased and 21 proteins that were decreased in SOD1(G93A) rat spinal cord mitochondria compared with SOD1(WT) spinal cord mitochondria. Analysis of this group of proteins revealed a higher-than-expected proportion involved in complex I and protein import pathways. Direct import assays revealed a 30% decrease in protein import only in spinal cord mitochondria, despite an increase in the mitochondrial import components TOM20, TOM22, and TOM40. Recombinant SOD1(G93A) or SOD1(G85R), but not SOD1(WT) or a Parkinson's disease-causing, misfolded α-synuclein(E46K) mutant, decreased protein import by >50% in nontransgenic mitochondria from spinal cord, but not from liver. Thus, altered mitochondrial protein content accompanied by selective decreases in protein import into spinal cord mitochondria comprises part of the mitochondrial damage arising from mutant SOD1.


MAPK phosphatase-1 contributes to trichostatin A inhibition of cyclooxygenase-2 expression in human umbilical vascular endothelial cells exposed to lipopolysaccharide.
Histone deacetylase (HDAC) inhibitors have emerged as a new class of antitumor agents because they were demonstrated to induce cell cycle arrest, promote cell apoptosis, and inhibit metastasis. Recently, HDAC inhibitors were also shown to exhibit pronounced anti-inflammatory properties. However, the underlying mechanism contributing to the suppression of inflammatory responses by HDAC inhibitors remains to be fully defined. In the present study, we explored the actions of trichostatin A (TSA), a potent HDAC inhibitor, on lipopolysaccharide (LPS)-induced cyclooxygenase (COX)-2 expression in human umbilical vascular endothelial cells (HUVECs). HUVECs were exposed to LPS in the absence or presence of TSA. COX-2 expression and signaling molecules (JNK, p38MAPK and c-jun) activated by LPS were assessed. The LPS-induced cox-2 messenger RNA and protein were markedly suppressed by TSA. TSA inhibited JNK and p38MAPK phosphorylation in cells exposed to LPS. Treatment of cells with a JNK signaling inhibitor (JNK inhibitor II) or a p38MAPK inhibitor (p38MAPK inhibitor III) markedly inhibited LPS-induced COX-2 expression. TSA suppression of JNK and p38MAPK phosphorylation and subsequent COX-2 expression were restored by selective inhibition of MKP-1 using MKP-1 siRNA. In addition, TSA caused an increase in MKP-1 phosphatase activity in HUVECs. In conclusion, TSA may cause MKP-1 activation to dephosphorylate JNK and p38MAPK, leading to the downregulation of COX-2 in HUVECs stimulated by LPS, a proinflammatory stimulus. MKP-1 contributes to TSA's protective actions in HUVECs exposed to LPS. The present study also supports the therapeutic value of TSA in treating inflammatory vascular diseases.


Transcriptomic Profiling of Sequential Tumors from Breast Cancer Patients Provides a Global View of Metastatic Expression Changes Following Endocrine Therapy.
Disease recurrence is a common problem in breast cancer and yet the mechanisms enabling tumor cells to evade therapy and colonize distant organs remain unclear. We sought to characterize global expression changes occurring with metastatic disease progression in the endocrine-resistant setting. Here, for the first time, RNAsequencing has been performed on matched primary, nodal, and liver metastatic tumors from tamoxifen-treated patients following disease progression. Expression of genes commonly elevated in the metastases of sequenced patients was subsequently examined in an extended matched patient cohort with metastatic disease from multiple sites. The impact of tamoxifen treatment on endocrine-resistant tumors in vivo was investigated in a xenograft model. The extent of patient heterogeneity at the gene level was striking. Less than 3% of the genes differentially expressed between sequential tumors were common to all patients. Larger divergence was observed between primary and liver tumors than between primary and nodal tumors, reflecting both the latency to disease progression and the genetic impact of intervening therapy. Furthermore, an endocrine-resistant in vivo mouse model demonstrated that tamoxifen treatment has the potential to drive disease progression and establish distant metastatic disease. Common functional pathways altered during metastatic, endocrine-resistant progression included extracellular matrix receptor interactions and focal adhesions. This novel global analysis highlights the influence of primary tumor biology in determining the transcriptomic profile of metastatic tumors, as well as the need for adaptations in cell-cell communications to facilitate successful tumor cell colonization of distant host organs.


Role of Pharmacogenetics in Adverse Drug Reactions: An Update towards Personalized Medicine.
Adverse drug reactions (ADRs) are an important and frequent cause of morbidity and mortality. ADR can be related to a variety of drugs, including anticonvulsants, anaesthetics, antibiotics, antiretroviral, anticancer, and antiarrhythmics, and can involve every organ or apparatus. The causes of ADRs are still poorly understood due to their clinical heterogeneity and complexity. In this scenario, genetic predisposition toward ADRs is an emerging issue, not only in anticancer chemotherapy, but also in many other fields of medicine, including hemolytic anemia due to glucose-6-phosphate dehydrogenase (G6PD) deficiency, aplastic anemia, porphyria, malignant hyperthermia, epidermal tissue necrosis (Lyell's Syndrome and Stevens-Johnson Syndrome), epilepsy, thyroid diseases, diabetes, Long QT and Brugada Syndromes. The role of genetic mutations in the ADRs pathogenesis has been shown either for dose-dependent or for dose-independent reactions. In this review, we present an update of the genetic background of ADRs, with phenotypic manifestations involving blood, muscles, heart, thyroid, liver, and skin disorders. This review aims to illustrate the growing usefulness of genetics both to prevent ADRs and to optimize the safe therapeutic use of many common drugs. In this prospective, ADRs could become an untoward "stress test," leading to new diagnosis of genetic-determined diseases. Thus, the wider use of pharmacogenetic testing in the work-up of ADRs will lead to new clinical diagnosis of previously unsuspected diseases and to improved safety and efficacy of therapies. Improving the genotype-phenotype correlation through new lab techniques and implementation of artificial intelligence in the future may lead to personalized medicine, able to predict ADR and consequently to choose the appropriate compound and dosage for each patient.


SFGD: a comprehensive platform for mining functional information from soybean transcriptome data and its use in identifying acyl-lipid metabolism pathways.
Soybean (Glycine max L.) is one of the world's most important leguminous crops producing high-quality protein and oil. Increasing the relative oil concentration in soybean seeds is many researchers' goal, but a complete analysis platform of functional annotation for the genes involved in the soybean acyl-lipid pathway is still lacking. Following the success of soybean whole-genome sequencing, functional annotation has become a major challenge for the scientific community. Whole-genome transcriptome analysis is a powerful way to predict genes with biological functions. It is essential to build a comprehensive analysis platform for integrating soybean whole-genome sequencing data, the available transcriptome data and protein information. This platform could also be used to identify acyl-lipid metabolism pathways. In this study, we describe our construction of the Soybean Functional Genomics Database (SFGD) using Generic Genome Browser (Gbrowse) as the core platform. We integrated microarray expression profiling with 255 samples from 14 groups' experiments and mRNA-seq data with 30 samples from four groups' experiments, including spatial and temporal transcriptome data for different soybean development stages and environmental stresses. The SFGD includes a gene co-expression regulatory network containing 23,267 genes and 1873 miRNA-target pairs, and a group of acyl-lipid pathways containing 221 enzymes and more than 1550 genes. The SFGD also provides some key analysis tools, i.e. BLAST search, expression pattern search and cis-element significance analysis, as well as gene ontology information search and single nucleotide polymorphism display. The SFGD is a comprehensive database integrating genome and transcriptome data, and also for soybean acyl-lipid metabolism pathways. It provides useful toolboxes for biologists to improve the accuracy and robustness of soybean functional genomics analysis, further improving understanding of gene regulatory networks for effective crop improvement. The SFGD is publically accessible at http://bioinformatics.cau.edu.cn/SFGD/, with all data available for downloading.


Assessment of modified gold surfaced titanium implants on skeletal fixation.
Noncemented implants are the primary choice for younger patients undergoing total hip replacements. However, the major concern in this group of patients regarding revision is the concern from wear particles, periimplant inflammation, and subsequently aseptic implant loosening. Macrophages have been shown to liberate gold ions through the process termed dissolucytosis. Furthermore, gold ions are known to act in an anti-inflammatory manner by inhibiting cellular NF-κB-DNA binding. The present study investigated whether partial coating of titanium implants could augment early osseointegration and increase mechanical fixation. Cylindrical porous coated Ti-6Al-4V implants partially coated with metallic gold were inserted in the proximal region of the humerus in ten canines and control implants without gold were inserted in contralateral humerus. Observation time was 4 weeks. Biomechanical push out tests and stereological histomorphometrical analyses showed no statistically significant differences in the two groups. The unchanged parameters are considered an improvement of the coating properties, as a previous complete gold-coated implant showed inferior mechanical fixation and reduced osseointegration compared to control titanium implants in a similar model. Since sufficient early mechanical fixation is achieved with this new coating, it is reasonable to investigate the implant further in long-term studies.


Fingerprinting the macro-organisation of pigment-protein complexes in plant thylakoid membranes in vivo by circular-dichroism spectroscopy.
Macro-organisation of the protein complexes in plant thylakoid membranes plays important roles in the regulation and fine-tuning of photosynthetic activity. These delicate structures might, however, undergo substantial changes during isolating the thylakoid membranes or during sample preparations, e.g., for electron microscopy. Circular-dichroism (CD) spectroscopy is a non-invasive technique which can thus be used on intact samples. Via excitonic and psi-type CD bands, respectively, it carries information on short-range excitonic pigment-pigment interactions and the macro-organisation (chiral macrodomains) of pigment-protein complexes (psi, polymer or salt-induced). In order to obtain more specific information on the origin of the major psi-type CD bands, at around (+)506, (-)674 and (+)690nm, we fingerprinted detached leaves and isolated thylakoid membranes of wild-type and mutant plants and also tested the effects of different environmental conditions in vivo. We show that (i) the chiral macrodomains disassemble upon mild detergent treatments, but not after crosslinking the protein complexes; (ii) in different wild-type leaves of dicotyledonous and monocotyledonous angiosperms the CD features are quite robust, displaying very similar excitonic and psi-type bands, suggesting similar protein composition and (macro-) organisation of photosystem II (PSII) supercomplexes in the grana; (iii) the main positive psi-type bands depend on light-harvesting protein II contents of the membranes; (iv) the (+)506nm band appears only in the presence of PSII-LHCII supercomplexes and does not depend on the xanthophyll composition of the membranes. Hence, CD spectroscopy can be used to detect different macro-domains in the thylakoid membranes with different outer antenna compositions in vivo.


Blood Glucose Prediction with Variance Estimation Using Recurrent Neural Networks.
Many factors affect blood glucose levels in type 1 diabetics, several of which vary largely both in magnitude and delay of the effect. Modern rapid-acting insulins generally have a peak time after 60-90 min, while carbohydrate intake can affect blood glucose levels more rapidly for high glycemic index foods, or slower for other carbohydrate sources. It is important to have good estimates of the development of glucose levels in the near future both for diabetic patients managing their insulin distribution manually, as well as for closed-loop systems making decisions about the distribution. Modern continuous glucose monitoring systems provide excellent sources of data to train machine learning models to predict future glucose levels. In this paper, we present an approach for predicting blood glucose levels for diabetics up to 1 h into the future. The approach is based on recurrent neural networks trained in an end-to-end fashion, requiring nothing but the glucose level history for the patient. Our approach obtains results that are comparable to the state of the art on the Ohio T1DM dataset for blood glucose level prediction. In addition to predicting the future glucose value, our model provides an estimate of its certainty, helping users to interpret the predicted levels. This is realized by training the recurrent neural network to parameterize a univariate Gaussian distribution over the output. The approach needs no feature engineering or data preprocessing and is computationally inexpensive. We evaluate our method using the standard root-mean-squared error (RMSE) metric, along with a blood glucose-specific metric called the surveillance error grid (SEG). We further study the properties of the distribution that is learned by the model, using experiments that determine the nature of the certainty estimate that the model is able to capture.


Triple-negative breast cancer frequency and type of BRCA mutation: Clues from Sardinia.
Germline mutations in BRCA1 or BRCA2 genes have been demonstrated to increase the risk of developing breast cancer. Among the prognostic factors currently used in clinical practice, the disease stage and the receptor status play a crucial role in the management of breast carcinoma. Triple-negative breast cancer (TNBC) has been classified as a disease subgroup that is negative for oestrogen, progesterone and HER2 receptor expression, and presents a poor prognosis. The present study investigated the correlation between BRCA1/2 mutations and TNBC status in a large series (n=726) of breast cancer patients from Sardinia. The BRCA mutation screening was performed on genomic DNA from peripheral blood samples by denaturing high-performance liquid chromatography analysis and automated DNA sequencing. Overall, 21/726 (2.9%) patients carried a germline mutation in BRCA1 or BRCA2. The TNBC phenotype was significantly associated with the BRCA1 mutations (P<0.001), whereas no association was found with the BRCA2 mutations (P=0.837). With respect to patient origin within Sardinia, a significant inverse distribution of mutations was found; BRCA1 and BRCA2 mutations represented 86 and 93% of the mutated cases in Southern and Middle-Northern Sardinia, respectively (P<0.001). Patients from the geographical area with BRCA1 mutation prevalence presented a TNBC incidence much higher than that observed in cases from the area with BRCA2 mutation prevalence (12 vs. 4%, respectively; P=0.037). These findings further confirmed that the occurrence of TNBC is significantly associated with the BRCA1 mutation carrier status and that a different 'genetic background' may have a phenotypic impact in the onset of breast cancer.


Voltage-dependent inhibition of recombinant NMDA receptor-mediated currents by 5-hydroxytryptamine.
The effect of 5-HT and related indolealkylamines on heteromeric recombinant NMDA receptors expressed in Xenopus oocytes was investigated using the two-electrode voltage-clamp recording technique. In the absence of external Mg(2+) ions, 5-HT inhibited NMDA receptor-mediated currents in a concentration-dependent manner. The inhibitory effect of 5-HT was independent of the NR1a and NR2 subunit combination. The inhibition of glutamate-evoked currents by 5-HT was use- and voltage-dependent. The voltage sensitivity of inhibition for NR1a+NR2 subunit combinations by 5-HT was similar, exhibiting an e-fold change per approximately 20 mV, indicating that 5-HT binds to a site deep within the membrane electric field. The inhibition of the open NMDA receptor by external Mg(2+) and 5-HT was not additive, suggesting competition between Mg(2+) and 5-HT for a binding site in the NMDA receptor channel. The concentration-dependence curves for 5-HT and 5-methoxytryptamine (5-MeOT) inhibition of NMDA receptor-mediated currents are shifted to the right in the presence of external Mg(2+). The related indolealkylamines inhibited glutamate-evoked currents with the following order of inhibitory potency: 5-MeOT=5-methyltryptamine>tryptamine>7-methyltryptamine>5-HT>>tryptophan=melatonin. Taken together, these data suggest that 5-HT and related compounds can attenuate glutamate-mediated excitatory synaptic responses and may provide a basis for drug treatment of excitoxic neurodegeneration.


"Hydroxychloroquine in patients with COVID-19: A Systematic Review and meta-analysis."
The role of hydroxychloroquine (HCQ) in the treatment of COVID-19 is not fully known. We studied the efficacy of HCQ compared to the control in COVID-19 subjects on - a. viral clearance measured by reverse transcriptase polymerase chain reaction (RT-PCR) and, b. death due to all cause. PubMed, Scopus, Cochrane and MedRxiv database were searched using the specific keywords up to April 30, 2020. Studies that met our objectives were assessed for the risk of bias applying various tools as indicated. Three studies each that reported the outcome of viral clearance by RT-PCR and death due to all cause, were meta-analyzed by applying inverse variance-weighted averages of logarithmic risk ratio (RR) using a random effects model. Heterogeneity and publication bias were assessed using the I2 statistic and funnel plots, respectively. Meta-analysis of 3 studies (n = 210) on viral clearance assessed by RT-PCR showed no benefit (RR, 1.05; 95% CI, 0.79 to 1.38; p = 0.74), although with a moderate heterogeneity (I2 = 61.7%, p = 0.07). While meta-analysis of 3 studies (n = 474) showed a significant increase in death with HCQ, compared to the control (RR, 2.17; 95% 1.32 to 3.57; p = 0.002), without any heterogeneity (I2 = 0.0%, p = 0.43). No benefit on viral clearance but a significant increase in mortality was observed with HCQ compared to control in patients with COVID-19.


The Mre11:Rad50 structure shows an ATP-dependent molecular clamp in DNA double-strand break repair.
The MR (Mre11 nuclease and Rad50 ABC ATPase) complex is an evolutionarily conserved sensor for DNA double-strand breaks, highly genotoxic lesions linked to cancer development. MR can recognize and process DNA ends even if they are blocked and misfolded. To reveal its mechanism, we determined the crystal structure of the catalytic head of Thermotoga maritima MR and analyzed ATP-dependent conformational changes. MR adopts an open form with a central Mre11 nuclease dimer and two peripheral Rad50 molecules, a form suited for sensing obstructed breaks. The Mre11 C-terminal helix-loop-helix domain binds Rad50 and attaches flexibly to the nuclease domain, enabling large conformational changes. ATP binding to the two Rad50 subunits induces a rotation of the Mre11 helix-loop-helix and Rad50 coiled-coil domains, creating a clamp conformation with increased DNA-binding activity. The results suggest that MR is an ATP-controlled transient molecular clamp at DNA double-strand breaks.


Point-of-care testing for haemoglobin A1c in remote Australian Indigenous communities improves timeliness of diabetes care.
In remote Australia timely access to pathology results and subsequent follow-up of patients for treatment is very challenging due to the long distances to the nearest laboratory. Point-of-care testing (POCT) offers a practical solution for pathology service provision in such remote communities. Since 2008, POCT for haemoglobin A1c (HbA1c) has been conducted in remote Northern Territory (NT) health centres for diabetes management of Indigenous patients through the national Quality Assurance in Aboriginal and Torres Strait Island Medical Services (QAAMS) Program. Point-of-care testing HbA1c results performed on Indigenous diabetes patients in the NT from July 2008 to April 2011 was accessed via the NT's electronic patient information system. Patients who had three or more HbA1c results performed by POCT across this period were assessed to determine their overall change in glycaemic control. An audit of 40 of these Indigenous diabetes patients (who exhibited a decrease in HbA1c levels of more than 1.5%) was undertaken to compare clinical and operational efficiency of POCT versus laboratory testing over an equivalent time period (15 months). No change in glycaemic control was observed when these patients received laboratory HbA1c testing prior to the introduction of POCT. Long turnaround times for receipt of results and follow-up consultation with patients were identified during this period, compared to immediate receipt and actioning of results using POCT. Frequency of HbA1c testing was higher with POCT than for the laboratory. This audit demonstrates that POCT can significantly improve the timeliness and clinical follow-up of pathology results in remote locations, while also reinforcing the clinical and cultural effectiveness of POCT and its critical role in assisting to improve diabetes management in Indigenous Australians.


Differential proteomics analysis of the analgesic effect of electroacupuncture intervention in the hippocampus following neuropathic pain in rats.
Evidence is building steadily on the effectiveness of acupuncture therapy in pain relief and repeated acupuncture-induced pain relief is accompanied by improvement of hippocampal neural synaptic plasticity. To further test the cellular and molecular changes underlying analgesic effect of acupuncture, the global change of acupuncture associated protein profiles in the hippocampus under neuropathic pain condition was profiled. The chronic constrictive injury (CCI) model was established by ligature of the unilateral sciatic nerve in adult Wistar rats. Rats were randomized into normal control (NC) group, CCI group, and CCI with electroacupuncture (EA) stimulation group. EA was applied to bilateral Zusanli (ST36) and Yanglingquan (GB34) in the EA group. Differentially expressed proteins in the hippocampus in the three groups were identified by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry. The functional clustering of the identified proteins was analyzed by Mascot software. After CCI, the thermal pain threshold of the affected hind footpad was decreased and was reversed gradually by 12 sessions of acupuncture treatment. Following EA, there were 19 hippocampal proteins identified with significant changes in expression (>2-fold), which are involved in metabolic, physiological, and cellular processes. The top three canonical pathways identified were "cysteine metabolism", "valine, leucine, and isoleucine degradation" and "mitogen-activated protein kinase (MAPK) signaling". These data suggest that the analgesic effect of EA is mediated by regulation of hippocampal proteins related to amino acid metabolism and activation of the MAPK signaling pathway.


Coumestrol inhibits autoantibody production through modulating Th1 response in experimental autoimmune thyroiditis.
Coumestrol is a common phytoestrogen found in plants and Chinese medicinal herbs. Its influences on experimental autoimmune thyroiditis (EAT) were investigated in this study. Female adult CBA/J mice were fed with drinking water containing 1% Tween80 only (Control group), 0.8 mg/l (L group) and 8 mg/l coumestrol (H group) from 6 to 15 weeks of age, respectively. Their serum coumestrol concentrations were determined by high performance liquid chromatography, which were undetectable, 43.70 ± 21.74 ng/ml and 135.07 ± 70.40 ng/ml, respectively. In addition, the mice (n = 14-16/group) were immunized twice with thyroglobulin (Tg) and Freund's adjuvant to induce EAT during the meantime. Although no overt changes in the extent of intrathyroidal mononuclear cell infiltration were shown in the two coumestrol-treated groups as compared with the controls, serum anti-Tg IgG2a, IgG3 and IgG1 titers, ratio of IgG2a to IgG1 and the percentage of T helper (Th)1 cells in the splenocytes were significantly reduced in the L group. Another consistent change was the significantly decreased expression of splenic IFN-γ mRNA after low dose of coumestrol exposure. Uterine weight was also markedly reduced in the mice of L group. These findings suggest that coumestrol treatment may have some beneficial actions against thyroid-specific autoantibody production in the development of autoimmune thyroiditis through suppression of Th1 response due to its anti-estrogenic activity.


Rapid determination of Escherichia coli O157:H7 lineage types and molecular subtypes by using comparative genomic fingerprinting.
In this study, variably absent or present (VAP) regions discovered through comparative genomics experiments were targeted for the development of a rapid, PCR-based method to subtype and fingerprint Escherichia coli O157:H7. Forty-four VAP loci were analyzed for discriminatory power among 79 E. coli O157:H7 strains of 13 phage types (PT). Twenty-three loci were found to maximize resolution among strains, generating 54 separate fingerprints, each of which contained strains of unique PT. Strains from the three previously identified major E. coli O157:H7 lineages, LSPA6-LI, LSPA6-LI/II, and LSPA6-LII, formed distinct branches on a dendrogram obtained by hierarchical clustering of comparative genomic fingerprinting (CGF) data. By contrast, pulsed-field gel electrophoresis (PFGE) typing generated 52 XbaI digestion profiles that were not unique to PT and did not cluster according to O157:H7 lineage. Our analysis identified a subpopulation comprised of 25 strains from a closed herd of cattle, all of which were of PT87 and formed a cluster distinct from all other E. coli O157:H7 strains examined. CGF found five related but unique fingerprints among the highly clonal herd strains, with two dominant subtypes characterized by a shift from the presence of locus fprn33 to its absence. CGF had equal resolution to PFGE typing but with greater specificity, generating fingerprints that were unique among phenotypically related E. coli O157:H7 lineages and PT. As a comparative genomics typing method that is amenable for use in high-throughput platforms, CGF may be a valuable tool in outbreak investigations and strain characterization.


Cadherin-based resistance to Bacillus thuringiensis cotton in hybrid strains of pink bollworm: fitness costs and incomplete resistance.
Recessive resistance to Bacillus thuringiensis (Bt) cotton, Gossypium hirsutum L., in laboratory-selected strains of pink bollworm, Pectinophora gossypiella (Saunders), is associated with three resistance alleles (r1, r2, and r3) of a cadherin gene. Previous experiments based on measurement of fitness components in Bt-resistant and Bt-susceptible strains revealed that fitness costs and incomplete resistance are associated with resistance. Here, we used two hybrid strains of pink bollworm, each containing a mixture of susceptible and resistant individuals, and polymerase chain reaction (PCR) amplifications to test the association between cadherin genotype and fitness components for individuals sharing a common genetic background. All survivors on Bt cotton had two r alleles, confirming that recessive cadherin alleles are tightly linked with resistance to Bt cotton. On non-Bt cotton, significantly greater developmental time for rr than ss larvae indicated a recessive fitness cost, but costs did not affect survival or pupal weight. Incomplete resistance was manifested as longer developmental time, lower survival, and smaller pupal weight in rr individuals developing on Bt cotton compared with non-Bt cotton. As in previous experiments, no significant variation in performance on Bt cotton was detected among rr genotypes. However, a meta-analysis of data from seven experiments revealed that survival on Bt cotton relative to non-Bt cotton was lower in r2r3 and higher in r1r2 compared with the other rr genotypes. Assessment of fitness components associated with cadherin genotypes in hybrid strains of pink bollworm confirms that recessive resistance to Bt cotton is associated with recessive fitness costs and incomplete resistance.