Influence of Environmental Drivers and Potential Interactions on the Distribution of Microbial Communities From Three Permanently Stratified Antarctic Lakes.
The McMurdo Dry Valley (MDV) lakes represent unique habitats in the microbial world. Perennial ice covers protect liquid water columns from either significant allochthonous inputs or seasonal mixing, resulting in centuries of stable biogeochemistry. Extreme environmental conditions including low seasonal photosynthetically active radiation (PAR), near freezing temperatures, and oligotrophy have precluded higher trophic levels from the food webs. Despite these limitations, diverse microbial life flourishes in the stratified water columns, including Archaea, bacteria, fungi, protists, and viruses. While a few recent studies have applied next generation sequencing, a thorough understanding of the MDV lake microbial diversity and community structure is currently lacking. Here we used Illumina MiSeq sequencing of the 16S and 18S rRNA genes combined with a microscopic survey of key eukaryotes to compare the community structure and potential interactions among the bacterial and eukaryal communities within the water columns of Lakes Bonney (east and west lobes, ELB, and WLB, respectively) and Fryxell (FRX). Communities were distinct between the upper, oxic layers and the dark, anoxic waters, particularly among the bacterial communities residing in WLB and FRX. Both eukaryal and bacterial community structure was influenced by different biogeochemical parameters in the oxic and anoxic zones. Bacteria formed complex interaction networks which were lake-specific. Several eukaryotes exhibit potential interactions with bacteria in ELB and WLB, while interactions between these groups in the more productive FRX were relatively rare.


Induction of the mitochondrial permeability transition in cultured astrocytes by glutamine.
Ammonia is a toxin that has been strongly implicated in the pathogenesis of hepatic encephalopathy (HE), and astrocytes appear to be the principal target of ammonia toxicity. Glutamine, a byproduct of ammonia metabolism, has been implicated in some of the deleterious effects of ammonia on the CNS. We have recently shown that ammonia induces the mitochondrial permeability transition (MPT) in cultured astrocytes, but not in neurons. We therefore determined whether glutamine is also capable of inducing the MPT in cultured astrocytes. Astrocytes were treated with glutamine (4.5 mM) for various time periods and the MPT was assessed by changes in 2-deoxyglucose (2-DG) mitochondrial permeability, calcein fluorescence assay, and by changes in cyclosporin A (CsA)-sensitive inner mitochondrial membrane potential (deltapsi(m)) using the potentiometric dye, JC-1. Astrocytes treated with glutamine significantly increased 2-DG permeability (120%, P<0.01), decreased mitochondrial calcein fluorescence, and concomitantly dissipated the deltapsi(m). All of these effects were blocked by CsA. These data indicate that glutamine induces the MPT in cultured astrocytes. The induction of the MPT by glutamine in astrocytes, and the subsequent development of mitochondrial dysfunction, may partially explain the deleterious affects of glutamine on the CNS in the setting of hyperammonemia.


Spatial patterning of supermarkets and fast food outlets with respect to neighborhood characteristics.
A large body of literature has reported differences in exposure to environments supporting either healthy (e.g. supermarkets) or unhealthy (e.g. fast food outlets) dietary choices by neighborhood characteristics. We explored the associations of both supermarkets and fast food outlets availability with neighborhood characteristics, and clustering of these two outlet types in a largely rural state. Compared to block groups without a supermarket, those with a supermarket had a significantly higher income, higher housing value, larger population with high school education and above, lower minority population and lower population living below poverty even after controlling for urbanicity and population density of census block groups. Surprisingly, a similar relationship was found for block groups with and without fast food outlets. This was due to spatial co-occurrence and clustering of fast food outlets around supermarket locations. Hence, future studies exploring the associations of food environment with diet or diet-related health outcome should concurrently examine all aspects of food environment (healthy and unhealthy).


IMAGING OF PIGMENT EPITHELIAL DETACHMENTS WITH OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY.
To investigate the utility of optical coherence tomography angiography (OCTA) for detecting pathologic vascularization within pigment epithelial detachments (PEDs). This was a retrospective, cross-sectional, consecutive case series. Multimodal imaging (structural OCT, fluorescein, and indocyanine green angiography) was used as the gold standard to classify PEDs as nonvascularized or vascularized. Optical coherence tomography angiography imaging of the PED was subsequently and independently evaluated to classify PEDs as vascularized or nonvascularized. Specifically, OCTA images were evaluated for the presence of abnormal flow on cross-sectional OCTA and the presence of a vascular complex on en face OCTA. Comparisons between OCTA and the gold standard were determined. Sixty-four eyes of 49 patients were evaluated. A total of 18 eyes were classified as nonvascularized PED, and 46 eyes were classified as vascularized PED using the gold standard. Optical coherence tomography angiography was found to have a sensitivity of 76%, specificity of 61%, positive predictive value of 83%, and negative predictive value of 50% for detecting vascularized PEDs. False positive cases in the nonvascularized PED group were due to projection or flow artifacts from hyperreflective material overlying the PED. False negative cases were seen in eyes with minimal exudation on structural OCT and also those manifesting retinal pigment epithelial tears. Our proposed two-step approach of OCTA interpretation, first using cross-sectional OCTA and then en face OCTA, may allow the detection of vascularization within PEDs and, in some cases, reduce the need for conventional angiography. Increased awareness about potential artifacts and limitations of OCTA may help clinicians interpret OCTA more accurately.


Specificity of the DNA Mismatch Repair System (MMR) and Mutagenesis Bias in Bacteria.
The mutation rate of an organism is influenced by the interaction of evolutionary forces such as natural selection and genetic drift. However, the mutation spectrum (i.e., the frequency distribution of different types of mutations) can be heavily influenced by DNA repair. Using mutation-accumulation lines of the extremophile bacterium Deinococcus radiodurans ΔmutS1 and the model soil bacterium Pseudomonas fluorescens wild-type and MMR- (Methyl-dependent Mismatch Repair-deficient) strains, we report the mutational features of these two important bacteria. We find that P. fluorescens has one of the highest MMR repair efficiencies among tested bacteria. We also discover that MMR of D. radiodurans preferentially repairs deletions, contrary to all other bacteria examined. We then, for the first time, quantify genome-wide efficiency and specificity of MMR in repairing different genomic regions and mutation types, by evaluating the P. fluorescens and D. radiodurans mutation data sets, along with previously reported ones of Bacillus subtilis subsp. subtilis, Escherichia coli, Vibrio cholerae, and V. fischeri. MMR in all six bacteria shares two general features: 1) repair efficiency is influenced by the neighboring base composition for both transitions and transversions, not limited to transversions as previously reported; and 2) MMR only recognizes indels <4 bp in length. This study demonstrates the power of mutation accumulation lines in quantifying DNA repair and mutagenesis patterns.


Metabolic shift underlies recovery in reversible infantile respiratory chain deficiency.
Reversible infantile respiratory chain deficiency (RIRCD) is a rare mitochondrial myopathy leading to severe metabolic disturbances in infants, which recover spontaneously after 6-months of age. RIRCD is associated with the homoplasmic m.14674T>C mitochondrial DNA mutation; however, only ~ 1/100 carriers develop the disease. We studied 27 affected and 15 unaffected individuals from 19 families and found additional heterozygous mutations in nuclear genes interacting with mt-tRNAGlu including EARS2 and TRMU in the majority of affected individuals, but not in healthy carriers of m.14674T>C, supporting a digenic inheritance. Our transcriptomic and proteomic analysis of patient muscle suggests a stepwise mechanism where first, the integrated stress response associated with increased FGF21 and GDF15 expression enhances the metabolism modulated by serine biosynthesis, one carbon metabolism, TCA lipid oxidation and amino acid availability, while in the second step mTOR activation leads to increased mitochondrial biogenesis. Our data suggest that the spontaneous recovery in infants with digenic mutations may be modulated by the above described changes. Similar mechanisms may explain the variable penetrance and tissue specificity of other mtDNA mutations and highlight the potential role of amino acids in improving mitochondrial disease.


In vitro biological activities and fatty acid profiles of Pistacia terebinthus fruits and Pistacia khinjuk seeds.
This study reports in vitro anticholinesterase, antioxidant and antimicrobial effects of the n-hexane, dichloromethane, ethanol and ethanol-water extracts prepared from Pistacia terebinthus L. fruits and Pistacia khinjuk Stocks seeds as well as their total phenolic and flavonoid contents, and fatty acid compositions. Ethanol and ethanol-water extracts of both species exhibited higher anticholinesterase activity than galanthamine. Among ABTS, DPPH and CUPRAC assays, the highest antioxidant capacity of the extracts was found in the last one. P. terebinthus ethanol extract being rich in flavonoid content showed the best cupric reducing effect. All extracts possessed no antimicrobial activity. The main fatty acid in P. terebinthus fruits (52.52%) and P. khinjuk seeds (59.44%) was found to be oleic acid. Our results indicate that P. terebinthus fruits and P. khinjuk seeds could be a good source of anticholinesterase compounds, and could be phytochemically investigated.


Dosimetry for quantitative analysis of the effects of low-dose ionizing radiation in radiation therapy patients.
We have developed and validated a practical approach to identifying the location on the skin surface that will receive a prespecified biopsy dose (ranging down to 1 cGy) in support of in vivo biological dosimetry in humans. This represents a significant technical challenge since the sites lie on the patient's surface outside the radiation fields. The PEREGRINE Monte Carlo simulation system was used to model radiation dose delivery, and TLDs were used for validation on phantoms and for confirmation during patient treatment. In the developmental studies, the Monte Carlo simulations consistently underestimated the dose at the biopsy site by approximately 15% (of the local dose) for a realistic treatment configuration, most likely due to lack of detail in the simulation of the linear accelerator outside the main beam line. Using a single, thickness-independent correction factor for the clinical calculations, the average of 36 measurements for the predicted 1-cGy point was 0.985 cGy (standard deviation: 0.110 cGy) despite patient breathing motion and other real-world challenges. Since the 10-cGy point is situated in the region of high-dose gradient at the edge of the field, patient motion had a greater effect, and the six measured points averaged 5.90 cGy (standard deviation: 1.01 cGy), a difference that is equivalent to approximately a 6-mm shift on the patient's surface.


Sequence variation and disease in the wake of the draft human genome.
The sequencing phase of the human genome project will soon be over. In its wake, repertoires of sequence polymorphisms among the human population are being sampled and a battery of functional genomics projects, from gene and protein expression studies to whole proteome interaction experiments, are generating vast quantities of data. Now that the data, or the means to generate data, are available it is the application of this information in enhancing our understanding of biology that represents the next formidable challenge. Two prominent issues should be considered. First, existing data must be analysed using the best methods available. The prediction of enzymatic activity for bestrophin, whose gene is mutated in Best macular dystrophy, is described in this review. This is an example of the experimentally testable hypotheses that can result from such detailed and exhaustive analyses. Secondly, the torrents of data from high-throughput studies will need to be made more accessible to all using web-based resources that integrate and digest complementary data types. The internet sites that showcase the human genome sequence are blazing a new trail. Ultimately, the success of genome sequencing and functional genomics will be measured not by the quantity and accuracy of raw data generated, but how rapidly they can be harnessed to span the divide between genotype and phenotype.


Multimodel ensembles of wheat growth: many models are better than one.
Crop models of crop growth are increasingly used to quantify the impact of global changes due to climate or crop management. Therefore, accuracy of simulation results is a major concern. Studies with ensembles of crop models can give valuable information about model accuracy and uncertainty, but such studies are difficult to organize and have only recently begun. We report on the largest ensemble study to date, of 27 wheat models tested in four contrasting locations for their accuracy in simulating multiple crop growth and yield variables. The relative error averaged over models was 24-38% for the different end-of-season variables including grain yield (GY) and grain protein concentration (GPC). There was little relation between error of a model for GY or GPC and error for in-season variables. Thus, most models did not arrive at accurate simulations of GY and GPC by accurately simulating preceding growth dynamics. Ensemble simulations, taking either the mean (e-mean) or median (e-median) of simulated values, gave better estimates than any individual model when all variables were considered. Compared to individual models, e-median ranked first in simulating measured GY and third in GPC. The error of e-mean and e-median declined with an increasing number of ensemble members, with little decrease beyond 10 models. We conclude that multimodel ensembles can be used to create new estimators with improved accuracy and consistency in simulating growth dynamics. We argue that these results are applicable to other crop species, and hypothesize that they apply more generally to ecological system models.


Peptide processing and targeting in the neuronal secretory pathway.
The abdominal ganglion of the marine mollusk Aplysia contains a pair of identified neuronal clusters, the bag cells, which control egg laying by means of a number of unique regulatory mechanisms. Each neuron in the bag cell clusters synthesizes several peptides derived from a single prohormone and packages them into separate vesicles. These vesicles are then differentially localized in specific neuronal processes, thus segregating peptides destined for autocrine and hormonal release sites. Therefore in this system, protein trafficking through the secretory pathway organizes multiple peptide neurochemical messengers to efficiently regulate simple behaviors.


Expanding the Dynamic Range of Fluorescence Assays through Single-Molecule Counting and Intensity Calibration.
Surface capture assays can measure fluorescently labeled analytes across a 1000-fold concentration range and at the sub-nanomolar level, but many biological molecules exhibit 1,000,000-fold variations in abundance down to the femtomolar level. The goal of this work is to expand the dynamic range of fluorescence assays by using imaging to combine molecular counting with single-molecule calibration of ensemble intensities. We evaluate optical limits imposed by surface-captured fluorescent labels, compare performances of different fluorophore classes, and use detector acquisition parameters to span wide ranges of fluorescence irradiance. We find that the fluorescent protein phycoerythrin provides uniquely suitable properties with exceptionally intense and homogeneous single-fluorophore brightness that can overcome arbitrary spot detection threshold biases. Major limitations imposed by nonspecifically bound fluorophores were then overcome using rolling circle amplification to densely label cancer-associated miRNA biomarkers, allowing accurate single-molecule detection and calibration across nearly 5 orders of magnitude of concentration with a detection limit of 29 fM. These imaging and molecular counting strategies can be widely applied to expand the limit of detection and dynamic range of a variety of surface fluorescence assays.


A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin.
The dystrophin-glycoprotein complex was tested for interaction with several components of the extracellular matrix as well as actin. The 156-kD dystrophin-associated glycoprotein (156-kD dystroglycan) specifically bound laminin in a calcium-dependent manner and was inhibited by NaCl (IC50 = 250 mM) but was not affected by 1,000-fold (wt/wt) excesses of lactose, IKVAV, or YIGSR peptides. Laminin binding was inhibited by heparin (IC50 = 100 micrograms/ml), suggesting that one of the heparin-binding domains of laminin is involved in binding dystroglycan while negatively charged oligosaccharide moieties on dystroglycan were found to be necessary for its laminin-binding activity. No interaction between any component of the dystrophin-glycoprotein complex and fibronectin, collagen I, collagen IV, entactin, or heparan sulfate proteoglycan was detected by 125I-protein overlay and/or extracellular matrix protein-Sepharose precipitation. In addition, laminin-Sepharose quantitatively precipitated purified dystrophin-glycoprotein complex, demonstrating that the laminin-binding site is accessible when dystroglycan is associated with the complex. Dystroglycan of nonmuscle tissues also bound laminin. However, the other proteins of the striated muscle dystrophin-glycoprotein complex appear to be absent, antigenically dissimilar or less tightly associated with dystroglycan in nonmuscle tissues. Finally, we show that the dystrophin-glycoprotein complex cosediments with F-actin but does not bind calcium or calmodulin. Our results support a role for the striated muscle dystrophin-glycoprotein complex in linking the actin-based cytoskeleton with the extracellular matrix. Furthermore, our results suggest that dystrophin and dystroglycan may play substantially different functional roles in nonmuscle tissues.


The activating NKG2D ligand MHC class I-related chain A transfers from target cells to NK cells in a manner that allows functional consequences.
Recently, it has become apparent that surface proteins commonly transfer between immune cells in contact. Inhibitory receptors and ligands exchange between cells during NK cell surveillance and we report here that NK cells also acquire activating ligands from target cells. Specifically, the stress-inducible activating ligand for NKG2D, MHC class I-related chain A (MICA), transferred to NK cells upon conjugation with MICA-expressing target cells. Acquisition of MICA from target cells was dependent on cell contact and occurred after accumulation of MICA at the immunological synapse. Moreover, transfer of MICA was facilitated by specific molecular recognition via NKG2D and augmented by Src kinase signaling. Importantly, MICA associated with its new host NK cell membrane in an orientation that allowed engagement with NKG2D in trans and indeed could down-regulate NKG2D in subsequent homotypic interactions with other NK cells. MICA captured from target cells could subsequently transfer between NK cells and, more importantly, NK cell degranulation was triggered in such NK cell-NK cell interactions. Thus, NK cells can influence other NK cells with proteins acquired from target cells and our data specifically suggest that NK cells could lyse other NK cells upon recognition of activating ligands acquired from target cells. This mechanism could constitute an important function for immunoregulation of NK cell activity.


Accurate molecular detection of non-small cell lung cancer metastases in mediastinal lymph nodes sampled by endoscopic ultrasound-guided needle aspiration.
The recurrence of disease after the complete resection of early stage non-small cell lung cancer (NSCLC) indicates that undetected metastases were present at the time of surgery. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) is a highly sensitive technique for detecting rare gene transcripts that may indicate the presence of cancer cells, and endoscopic ultrasound (EUS)-guided fine-needle aspiration (FNA) is a minimally invasive technique for the nonoperative sampling of mediastinal lymph nodes. The aim of this study was to determine whether these two techniques could enhance the preoperative detection of occult metastases. Patients with NSCLC were evaluated with chest CT and positron emission tomography scans. Those patients without evidence of metastases (87 patients) underwent EUS-guided FNA. Lymph nodes from levels 2, 4, 5, 7, 8, and 9 were sampled and evaluated by standard cytopathology and real-time RT-PCR. Normal control FNA specimens were obtained from patients without cancer who were undergoing EUS for benign disease (17 control specimens). For each sample, messenger RNA was extracted and real-time RT-PCR was used to quantitate the expression of six lung cancer-associated genes (ie, CEA, CK19, KS1/4, lunx, muc1, and PDEF) relative to the expression of an internal control gene (beta(2)-microglobulin). Clinical thresholds of marker positivity were set at 100% specificity, as determined by the receiver operating characteristic curve analysis. Of the cytology-positive lymph nodes (27 lymph nodes), the expression of the KS1/4 gene was above its respective clinical threshold in 25 of 27 samples (93%), making this the most sensitive marker for the detection of metastatic NSCLC. At least one of the six lung cancer-associated genes was overexpressed in 18 of 61 cytology-negative patients (30%), of which KS1/4 was overexpressed in 15 of 61 patients (25%). Based on the high accuracy of EUS-guided FNA/RT-PCR, we predict that some of the patients in the cytology-negative/marker-positive category will have high NSCLC recurrence rates. Among the genes used in our marker panel, KS1/4 appears particularly useful for the detection of overt or occult metastatic disease.


Nasopharyngeal swabs vs. saliva sampling for SARS-CoV-2 detection: A cross-sectional survey of acceptability for caregivers and children after experiencing both methods.
Saliva sampling is a promising alternative to nasopharyngeal swabs for SARS-CoV-2 testing, but acceptability data is lacking. We characterize the acceptability of saliva sampling and nasopharyngeal swabs for primary decision makers and their children after experiencing both testing modalities. We administered a cross-sectional survey to participants aged 6-to-17 years and their primary decision makers at an Ottawa community COVID-19 testing centre in March 2021. Included were participants meeting local guidelines for testing. Excluded were those identified prior to participation as having inability to complete the consent, sampling, or survey process. Acceptability in multiple hypothetical scenarios was rated using a 5-point Likert scale. Pain was measured using the Faces Pain Scale-Revised (FPS-R). Preference for testing was assessed with direct binary questions. 48 participants and 48 primary decision makers completed the survey. Nasopharyngeal swab acceptability differed between scenarios, ranging 79% [95%CI: 66, 88] to 100% [95%CI: 95, 100]; saliva sampling acceptability was similar across scenarios, ranging 92% [95%CI: 82, 97] to 98% [95%CI: 89, 99]. 58% of youth described significant pain with nasopharyngeal swabbing, versus none with saliva sampling. 90% of children prefer saliva sampling. 66% of primary decision makers would prefer nasopharyngeal swabbing if it were 10% more sensitive. Though youth prefer saliva sampling over nasopharyngeal swabs, primary decision makers present for testing remain highly accepting of both. Acceptance of nasopharyngeal swabs, however, varies with the testing indication and is influenced by perceived test accuracy. Understanding factors that influence sampling acceptance will inform more successful testing strategies.


Elevated depression symptoms, antidepressant medicine use, and risk of developing diabetes during the diabetes prevention program.
To assess the association between elevated depression symptoms or antidepressant medicine use on entry to the Diabetes Prevention Program (DPP) and during the study and the risk of developing diabetes during the study. DPP participants (n = 3,187) in three treatment arms (intensive lifestyle [ILS], metformin [MET], and placebo [PLB]) completed the Beck Depression Inventory (BDI) and reported their use of antidepressant medication at randomization and throughout the study (average duration in study 3.2 years). When other factors associated with the risk of developing diabetes were controlled, elevated BDI scores at baseline or during the study were not associated with diabetes risk in any arm. Baseline antidepressant use was associated with diabetes risk in the PLB (hazard ratio 2.25 [95% CI 1.38-3.66]) and ILS (3.48 [1.93-6.28]) arms. Continuous antidepressant use during the study (compared with no use) was also associated with diabetes risk in the same arms (PLB 2.60 [1.37-4.94]; ILS 3.39 [1.61-7.13]), as was intermittent antidepressant use during the study in the ILS arm (2.07 [1.18-3.62]). Among MET arm participants, antidepressant use was not associated with developing diabetes. A strong and statistically significant association between antidepressant use and diabetes risk in the PLB and ILS arms was not accounted for by measured confounders or mediators. If future research finds that antidepressant use independently predicts diabetes risk, efforts to minimize the negative effects of antidepressant agents on glycemic control should be pursued.


The Role of Psychological Factors in Judo: A Systematic Review.
(1) Background: Psychological parameters are relevant in the practice of judo. Previous studies have shown that parameters such as anxiety or motivation can have a negative or positive impact on the athlete's performance and general well-being, depending on the athlete's perception. This systematic review aimed to summarize the studies examining the influence of various psychological parameters on well-being and performance in judo athletes; (2) Methods: We followed preferred reporting elements for systematic reviews and meta-analyses. We searched the Web of Science database for studies that explained the role of these parameters in elite athletes. Of the 286 articles initially identified, 17 met our eligibility criteria and were included in the review. In total, we analyzed data from 721 judo athletes; (3) Results: The studies found have demonstrated the impact of various psychological parameters during high-level performance and how these parameters can influence and lead an athlete to win or lose a competition. The feelings of tension, anger, anxiety, and nervousness were significantly increased in athletes who were facing defeat, while a decrease in the same segments and an increase in motivation among athletes who were experiencing better performance was observed. Further research under standardized conditions is needed to better understand the effects of these parameters on judo athletes; (4) Conclusions: Considering the athlete's psychological state can affect performance, and it is therefore important to monitor and train these factors.


Rivaroxaban: an oral direct factor Xa inhibitor for the prevention of thromboembolism.
Rivaroxaban is a novel oral direct factor Xa inhibitor. It is currently awaiting approval from the US Food and Drug Administration as a prophylaxis for venous thromboembolism in total knee and hip replacements. Based on phase II and III trials, rivaroxaban has been shown to be as efficacious, if not more, as traditional antithrombotic therapy with similar safety profiles. The Regulation of Coagulation in Major Orthopedic Surgery Reducing the Risk of Deep Vein Thrombosis and Pulmonary Embolism (RECORD) phase III trials have shown rivaroxaban 10 mg once daily to be superior to the low molecular weight heparin, enoxaparin, when used for prophylaxis of venous thromboembolism in orthopedic surgeries. Rivaroxaban has been shown to have tolerable adverse effects and a low potential for drug-drug or drug-food interactions. It has the major advantages of once daily oral dosing and no required laboratory monitoring, giving it the potential to replace current antithrombotics in the market today.


Development of tailored real-time RT-PCR assays for the detection and differentiation of serotype O, A and Asia-1 foot-and-mouth disease virus lineages circulating in the Middle East.
Rapid and accurate diagnosis is essential for effective control of foot-and-mouth disease (FMD). In countries where FMD is endemic, identification of the serotypes of the causative virus strains is important for vaccine selection and tracing the source of outbreaks. In this study, real-time reverse transcription polymerase chain reaction (rRT-PCR) assays using primer/probe sets designed from the VP1 coding region of the virus genomes were developed for the specific detection of serotype O, A and Asia-1 FMD viruses (FMDVs) circulating in the Middle East. These assays were evaluated using representative field samples of serotype O strains belonging exclusively to the PanAsia-2 lineage, serotype A strains of the Iran-05 lineage and serotype Asia-1 viruses from three relevant sub-groups. When RNA extracted from archival and contemporary field strains was tested using one- or two-step rRT-PCR assays, all three primer/probe sets detected the RNA from homotypic viruses and no cross-reactivity was observed with heterotypic viruses. Similar results were obtained using both single- and multiplex assay formats. Using plasmid standards, the minimum detection level of these tests was found to be lower than two copies. The results illustrate the potential of tailored rRT-PCR tools for the detection and categorization of viruses circulating in the Middle East belonging to distinct subgroups of serotypes O, A and Asia-1. These assays can also overcome the problem of serotyping samples which are found positive by the generic rRT-PCR diagnostic assays but negative by virus isolation and antigen-detection ELISA which would otherwise have to be serotyped by nucleotide sequencing. A similar approach could be used to develop serotyping assays for FMDV strains circulating in other regions of the world.


Crustal Layer of Seismic Velocity 6.9 to 7.6 Kilometers per Second under the Deep Oceans.
Refraction measurements made in the deep ocean between the Marshall and Hawaiian islands reveal a layer of seismic velocity 7.3 kilometers per second between the 6.8 kilometer per second oceanic crustal layer and the mantle. This layer, normally masked as a second arrival, is revealed by continuous air gun refraction data. The layer may be widespread in the deep oceans.


High incidence of hepatitis C virus reinfection in a cohort of injecting drug users.
An estimated 170 million people worldwide carry the hepatitis C virus (HCV), and in more developed countries the prevalence and incidence of HCV is particularly high among injecting drug users (IDUs). Spontaneous clearance of HCV infection and reinfection is well recognized but the level of protection against further infection conferred by HCV infection and clearance remains uncertain. We conducted a prospective study of HCV infection in IDUs recruited in Melbourne, Australia, using a much shorter median testing interval than in previous studies. Incidences of naive infection and reinfection were calculated by the person-year method and Cox proportional hazards regression used to adjust for covariates. A significantly higher HCV incidence rate was measured in previously infected IDUs (46.8% per year) compared with HCV-naive IDUs (15.5% per year). The hazard ratio for previously infected IDUs compared to HCV-naive IDUs, after adjustment for time-dependent covariates, was 2.54 (95% confidence interval, 1.11-5.78, P > |z| < 0.05). Viral persistence after reinfection appeared similar to that following naive infection. Our data suggest that HCV infection in IDUs is more likely following prior infection and clearance than in HCV-naive individuals, implying no increased immunity against further infection. This result has important implications for the future development of an HCV vaccine.


Identifying strategies to improve access to credible and relevant information for public health professionals: a qualitative study.
Movement towards evidence-based practices in many fields suggests that public health (PH) challenges may be better addressed if credible information about health risks and effective PH practices is readily available. However, research has shown that many PH information needs are unmet. In addition to reviewing relevant literature, this study performed a comprehensive review of existing information resources and collected data from two representative PH groups, focusing on identifying current practices, expressed information needs, and ideal systems for information access. Nineteen individual interviews were conducted among employees of two domains in a state health department--communicable disease control and community health promotion. Subsequent focus groups gathered additional data on preferences for methods of information access and delivery as well as information format and content. Qualitative methods were used to identify themes in the interview and focus group transcripts. Informants expressed similar needs for improved information access including single portal access with a good search engine; automatic notification regarding newly available information; access to best practice information in many areas of interest that extend beyond biomedical subject matter; improved access to grey literature as well as to more systematic reviews, summaries, and full-text articles; better methods for indexing, filtering, and searching for information; and effective ways to archive information accessed. Informants expressed a preference for improving systems with which they were already familiar such as PubMed and listservs rather than introducing new systems of information organization and delivery. A hypothetical ideal model for information organization and delivery was developed based on informants' stated information needs and preferred means of delivery. Features of the model were endorsed by the subjects who reviewed it. Many critical information needs of PH practitioners are not being met efficiently or at all. We propose a dual strategy of: 1) promoting incremental improvements in existing information delivery systems based on the expressed preferences of the PH users of the systems and 2) the concurrent development and rigorous evaluation of new models of information organization and delivery that draw on successful resources already operating to deliver information to clinical medical practitioners.


Characterization of pRGO1, a plasmid from Propionibacterium acidipropionici, and its use for development of a host-vector system in propionibacteria.
The complete nucleotide sequence of pRGO1, a cryptic plasmid from Propionibacterium acidipropionici E214, was determined. pRGO1 is 6, 868 bp long, and its G+C content is 65.0%. Frame analysis of the sequence revealed six open reading frames, which were designated Orf1 to Orf6. The deduced amino acid sequences of Orf1 and Orf2 showed extensive similarities to an initiator of plasmid replication, the Rep protein, of various plasmids of gram-positive bacteria. The amino acid sequence of the putative translation product of orf3 exhibited a high degree of similarity to the amino acid sequences of DNA invertase in several bacteria. For the putative translation products of orf4, orf5, and orf6, on the other hand, no homologous sequences were found. The function of these open reading frames was studied by deletion analysis. A shuttle vector, pPK705, was constructed for shuttling between Escherichia coli and a Propionibacterium strain containing orf1 (repA), orf2 (repB), orf5, and orf6 from pRGO1, pUC18, and the hygromycin B-resistant gene as a drug marker. Shuttle vector pPK705 successfully transformed Propionibacterium freudenreichii subsp. shermanii IFO12426 by electroporation at an efficiency of 8 x 10(6) CFU/microg of DNA under optimized conditions. Transformation of various species of propionibacteria with pPK705 was also performed at efficiencies of about 10(4) to 10(7) CFU/microg of DNA. The vector was stably maintained in strains of P. freudenreichii subsp. shermanii, P. freudenreichii, P. pentosaceum, and P. freudenreichii subsp. freudenreichii grown under nonselective conditions. Successful manipulation of a host-vector system in propionibacteria should facilitate genetic studies and lead to creation of genes that are useful industrially.


Late-preterm birth and lifetime socioeconomic attainments: the Helsinki birth cohort study.
We examined if those born late-preterm (at 34 to 36 weeks of gestation) differed from those born at term in their maximum attained lifetime socioeconomic position (SEP) across the adult years up to 56 to 66 years, and in intergenerational social mobility from childhood parental SEP to own attained SEP. Participants were 8993 Finnish men and women of the Helsinki Birth Cohort Study born between 1934 and 1944. Gestational age was extracted from hospital birth records and socioeconomic attainments from Finnish National Census. Compared with those born at term, those born late-preterm were more likely to be manual workers, have a basic or upper secondary level of education, belong to the lowest third based on their incomes, and less likely to belong to the highest third based on their incomes. Late-preterm individuals were also less likely to be upwardly mobile and more likely to be downwardly mobile; they were less likely to have higher occupations and more likely to have lower occupations than their fathers. They were also less likely to be upwardly mobile if incomes were used as the outcome of own attained SEP, and men were more likely to be downwardly mobile if education was used as the outcome of own attained SEP. This study demonstrates that there are considerable long-term socioeconomic disadvantages associated with late-preterm birth, which are not explained by the parent-of-origin SEP.


A new method for the determination of alpha1-protease inhibitor (alpha1-antitrypsin) phenotypes based on the formation of alpha1-protease inhibitor allele product-elastase complexes.
Up until now it has been assumed that the protease-binding property of alpha1-protease inhibitor (alpha1PI) was destroyed by acid starch gel electrophoresis (pH 4.9). Analyses on acid starch gel blocks for pH and conductivity changes during and following a typical electrophoretic run showed that it was unlikely that the separating alpha1PI would be exposed to pH values lower than 6.2, and that the allele products, following the passage of the buffer front, were in an environment of constant pH(6.3), extremely low conductivity and high field strength. These results strongly suggested the likelihood that alpha1-PI would be chemically and physically unchanged as a result of exposure to acid starch gel electrophoresis. In order to test this likelihood, human serum was electrophoretically separated in acid starch gel and following electrophoresis, was immersed in 0.1 M diethylbarbiturate buffer, pH 8.6, containing 20 mug/ml of pancreatic elastase. The pH-adjusted (8.15) and elastase-impregnated starch gel layer was superimposed on hemoglobin-agar for 2.5 h at 37 degrees C followed by immersion of the hemoglobin-agar layer in 1% NaCl overnight, distilled water for 2 h, drying under filter paper and staining. The results showed zones of undigested hemoglobin indicating, unequivocally, that the separated alpha1PI allele products are capable of forming complexes with proteases and that alpha1PI is not inactivated following exposure to acid starch gel electrophoresis. Densitometric analysis of the transparent stained zones on a clear agar gel background offers an alternative to analysis of the acid starch gel-separated zones by antigen-antibody crossed electrophoresis and as such is suitable for identification of alpha1-protease inhibitor phenotypes. Further, the method is specific for alpha1PI and a densitometric scan provides direct information relative to the protease-binding capacity of the sample as well as the contribution of each alpha1PI allele product to that capacity.


Variation in outcome and ranking of hospitals: an analysis from the Swedish knee arthroplasty register.
Hospital-specific variation in outcome is generally considered to be an important source of information for clinical improvement. We have measured the magnitude of this variation. We determined the revision risk in 37,642 cemented primary total knee arthroplasties inserted as a result of osteoarthritis from 1993 through 2002 at 93 hospitals in Sweden. We used 2 essentially different methods to estimate risk of revision: a fixed-effects model (Cox's proportional hazards model) and a random-effects model (shared gamma frailty model). The 2 models ranked hospitals differently. As expected, the fixed-effects model provided more dispersed estimates of hospital-specific revision rates. In contrast to the random-effects model, chance events can easily cause overly optimistic or pessimistic outcomes in the fixed-effects model. Although the revision risk varied significantly between hospitals, the overall revision risk was still low. Assessment of variation in outcome is an important instrument in the continuing effort to improve clinical care. However, regarding revision rate after knee arthroplasty, we do not believe that such analyses necessarily provide valid information on the current quality of care. We question their value as information source for seeking personal healthcare.


[Cutaneous leishmaniasis in France: towards the end of injectable therapy?].
Today no drug likely to be efficient on the majority of human-infecting species, well tolerated, and easy to administer is available for the treatment of human cutaneous leishmaniasis. But recent progress has been made. Efficient against visceral leishmaniasis, orally administered miltefosine may supplant pentavalent antimonials for the treatment of cutaneous leishmaniasis acquired in the New World. Right now, the reference treatment is still parenteral pentavalent antimonials 20 mg Sbv/kg/d for a duration that may probably be reduced from 20 to 10 days. The benefit/risk ratio of pentamidine still compares well with that of pentavalent antimonials for the treatment of lesions due to species belonging to the L. panamensis/L. guyanensis/L. shawi group. Pentamidine, which is easier to handle than antimonials, remains the reference treatment for cases from areas where these species predominate. Oral fluconazole is an improvement, readily available for cases from L. major foci. If its efficacy is confirmed in other foci and against other species, mechanisms will have to be implemented to make this therapeutic improvement affordable to poor patients in endemic countries. The development of an efficient and well tolerated topical treatment is still warranted. A new formulation of aminosidine is currently under evaluation. One can hope that the treatment of cutaneous leishmaniasis will soon become simpler, both for patients and doctors. For the benefits of this simplification to be rapidly affordable to all patients, the pharmaceutical and clinical research outlay must be maintained.


Oral health-related quality of life of children and adolescents with and without migration background in Germany.
To compare oral health-related quality of life (OHRQoL) in children and adolescents with and without migration background, and to assess whether potential differences in OHRQoL can be sufficiently explained by oral health characteristics. A consecutive sample of 112 children and adolescents was recruited in a German university-based orthodontic clinic, and a convenience sample of 313 children and adolescents of German public schools was enrolled in the study (total N = 425, age range 7-17 years). However, 29 participants were excluded due to insufficient information regarding migration background. Accordingly, the non-migrant group consisted of 262 participants (61.6%). For children with migration background, two groups were classified: (i) one parent born in a foreign country (N = 41, 9.6%, single-sided migration background), and (ii) both parents and/or child born in a foreign country ( N= 93, 21.9%, double-sided migration background). OHRQoL was assessed using the German 19-item version of the Child Oral Health Impact Profile (COHIP-G19). Additionally, physical oral health of 269 children with classified migration background was determined in a dental examination. Overall, OHRQoL was significantly lower in the group with double-sided migration background indicated by lower COHIP-G19 summary scores (mean: 58.6 points) than in the group with single-sided migration background (mean: 63.3 points) or the non-migrant group (mean: 63.2 points). Likewise, the summary scores of the subscale "oral health well-being" and the subscale "social/emotional, school, and self-image" were also lower in the double-sided migrant group than in the other two groups. Linear regression analysis showed an association between double-sided migration background and impaired OHRQoL, even after statistically controlling for demographic, socioeconomic, and oral health characteristics. Children and adolescents with double-sided migration background have poorer OHRQoL than comparably aged migrants with single-sided migration background or non-migrations. Between-group differences in OHRQoL could not be sufficiently explained by effects of socioeconomic status or physical oral health characteristics. Thus, other methodological, cultural, or immigration-related factors might also play an important role for the observed effects.


Gut microbial succession follows acute secretory diarrhea in humans.
Disability after childhood diarrhea is an important burden on global productivity. Recent studies suggest that gut bacterial communities influence how humans recover from infectious diarrhea, but we still lack extensive data and mechanistic hypotheses for how these bacterial communities respond to diarrheal disease and its treatment. Here, we report that after Vibrio cholerae infection, the human gut microbiota undergoes an orderly and reproducible succession that features transient reversals in relative levels of enteric Bacteroides and Prevotella. Elements of this succession may be a common feature in microbiota recovery from acute secretory diarrhea, as we observed similar successional dynamics after enterotoxigenic Escherichia coli (ETEC) infection. Our metagenomic analyses suggest that multiple mechanisms drive microbial succession after cholera, including bacterial dispersal properties, changing enteric oxygen and carbohydrate levels, and phage dynamics. Thus, gut microbiota recovery after cholera may be predictable at the level of community structure but is driven by a complex set of temporally varying ecological processes. Our findings suggest opportunities for diagnostics and therapies targeting the gut microbiota in humans recovering from infectious diarrhea. Disability after diarrhea is a major burden on public health in the developing world. Gut bacteria may affect this recovery, but it remains incompletely understood how resident microbes in the digestive tract respond to diarrheal illness. Here, we observed an orderly and reproducible succession of gut bacterial groups after cholera in humans. Genomic analyses associated the succession with bacterial dispersal in food, an altered microbial environment, and changing phage levels. Our findings suggest that it may one day be feasible to manage resident bacterial populations in the gut after infectious diarrhea.


Prognosis for vision in amblyopia after the loss of the good eye.
Two hundred three cases of unilateral amblyopia with the loss of the good eye included 59 taken from literature and 144 obtained from a questionnaire sent to ophthalmologists. The improvement noted in both sources was quite different: 47.5% v 28.5%. Because the cases from the questionnaire constituted a much more random selection than those from the literature, the former serve as a basis for discussion and conclusions. In 17.4% of the cases improvement was obtained without treatment. This result is closely linked to the existence of foveal fixation (93.7%). In 11.1% of the cases, the improvement was obtained with pleoptic treatment. The important prognostic factors were the existence of foveal fixation and the loss of the good eye. Pleoptic treatment was valuable in our sample, especially for patients with eccentric fixation.


When does age-related cognitive decline begin?
Cross-sectional comparisons have consistently revealed that increased age is associated with lower levels of cognitive performance, even in the range from 18 to 60 years of age. However, the validity of cross-sectional comparisons of cognitive functioning in young and middle-aged adults has been questioned because of the discrepant age trends found in longitudinal and cross-sectional analyses. The results of the current project suggest that a major factor contributing to the discrepancy is the masking of age-related declines in longitudinal comparisons by large positive effects associated with prior test experience. Results from three methods of estimating retest effects in this project, together with results from studies comparing non-human animals raised in constant environments and from studies examining neurobiological variables not susceptible to retest effects, converge on a conclusion that some aspects of age-related cognitive decline begin in healthy educated adults when they are in their 20s and 30s.


Botrytis cinerea Loss and Restoration of Virulence during In Vitro Culture Follows Flux in Global DNA Methylation.
Pathogenic fungi can lose virulence after protracted periods of culture, but little is known of the underlying mechanisms. Here, we present the first analysis of DNA methylation flux at a single-base resolution for the plant pathogen B. cinerea and identify differentially methylated genes/genomic regions associated with virulence erosion during in vitro culture. Cultures were maintained for eight months, with subcultures and virulence testing every month. Methylation-sensitive amplified polymorphisms were performed at monthly intervals to characterise global changes to the pathogen's genome during culture and also on DNA from mycelium inoculated onto Arabidopsis thaliana after eight months in culture. Characterisation of culture-induced epialleles was assessed by whole-genome re-sequencing and whole-genome bisulfite sequencing. Virulence declined with time in culture and recovered after inoculation on A. thaliana. Variation detected by methylation-sensitive amplified polymorphisms followed virulence changes during culture. Whole-genome (bisulfite) sequencing showed marked changes in global and local methylation during culture but no significant genetic changes. We imply that virulence is a non-essential plastic character that is at least partly modified by the changing levels of DNA methylation during culture. We hypothesise that changing DNA methylation during culture may be responsible for the high virulence/low virulence transition in B. cinerea and speculate that this may offer fresh opportunities to control pathogen virulence.


Human insula activation reflects risk prediction errors as well as risk.
Understanding how organisms deal with probabilistic stimulus-reward associations has been advanced by a convergence between reinforcement learning models and primate physiology, which demonstrated that the brain encodes a reward prediction error signal. However, organisms must also predict the level of risk associated with reward forecasts, monitor the errors in those risk predictions, and update these in light of new information. Risk prediction serves a dual purpose: (1) to guide choice in risk-sensitive organisms and (2) to modulate learning of uncertain rewards. To date, it is not known whether or how the brain accomplishes risk prediction. Using functional imaging during a simple gambling task in which we constantly changed risk, we show that an early-onset activation in the human insula correlates significantly with risk prediction error and that its time course is consistent with a role in rapid updating. Additionally, we show that activation previously associated with general uncertainty emerges with a delay consistent with a role in risk prediction. The activations correlating with risk prediction and risk prediction errors are the analogy for risk of activations correlating with reward prediction and reward prediction errors for reward expectation. As such, our findings indicate that our understanding of the neural basis of reward anticipation under uncertainty needs to be expanded to include risk prediction.


Weight-bearing asymmetries during Sit-To-Stand in patients with mild-to-moderate hip osteoarthritis.
The Sit-To-Stand (STS) transition is a mechanically demanding task that may pose particular challenges for individuals with lower limb osteoarthritis (OA). Biomechanical features of STS have been investigated in patients with OA, but not in patients with early stage hip OA. The purpose of this study was to explore inter-limb weight-bearing asymmetries (WBA) and selected kinematic and kinetic variables during STS in patients with mild-to-moderate hip OA compared with healthy controls. Twenty-one hip OA patients and 23 controls were included in the study. Sagittal and frontal plane kinematic and kinetic data were collected using an eight-camera motion analysis system synchronized with two force plates embedded in the floor. There were no distinctive biomechanical alterations in sagittal or frontal plane kinematics or kinetics, movement time, or time to reach peak ground reaction force (GRF) in hip OA patients compared with controls. However, the hip OA patients revealed a distinct pattern of WBA compared with the controls, in unloading their involved limb by 18.4% at peak GRF. These findings indicate that patients with early stage hip OA are not yet forced into a stereotypical movement strategy for STS; however, the observed pattern of WBA requires clinical attention.


The EQUATOR Network and reporting guidelines: Helping to achieve high standards in reporting health research studies.
Poorly reported research seriously undermines the usability of reported findings and misleads clinicians, researchers, policy makers and, ultimately, patients. Guidelines for reporting health research are available but they are not widely used. The EQUATOR Network is an international initiative that aims to systematically tackle the problems of poor reporting. The main goals of the EQUATOR Network are to improve the clarity, completeness and transparency of scientific publications by providing resources and education relating to the reporting of health research and assisting in the development, dissemination and implementation of robust reporting guidelines.


Meningioangiomatosis: A review of the variable manifestations and complex pathophysiology.
Meningioangiomatosis (MA) is a rare, complex and heterogeneous disease of meningovascular proliferation that is found primarily in the leptomeninges and cerebral cortex but can involve subcortical white matter and other brain regions such as the cerebellum and deep gray matter. MA may be found in pediatric or adult populations and may be sporadic or neurofibromatosis-associated. The presentation of MA is highly variable: it may be associated with other neurological diseases; clinically presents on a spectrum from asymptomatic to seizures or focal deficits; radiologically presents with multifocal, tumor-like, or cystic lesions, or may appear normal; and pathologically may have cellular or vascular predominance. In this article, we review the various manifestations of MA including neurofibromatosis-associated MA, multifocal MA, cystic MA, and MA associated with meningioma, other brain tumors, focal cortical dysplasia, neurodegenerative changes, and post-radiation changes. The treatment of MA is also reviewed. While the pathogenesis of MA remains elusive, we discuss the proposed theories such as developmental, dysplastic, hamartomatous or reactive ethology in given variants. It is important for physicians to be aware of MA as more research on this complex entity is needed and timely diagnosis may benefit outcomes in patients with MA.


Mechanisms of epileptogenesis in pediatric epileptic syndromes: Rasmussen encephalitis, infantile spasms, and febrile infection-related epilepsy syndrome (FIRES).
The mechanisms of epileptogenesis in pediatric epileptic syndromes are diverse, and may involve disturbances of neurodevelopmental trajectories, synaptic homeostasis, and cortical connectivity, which may occur during brain development, early infancy, or childhood. Although genetic or structural/metabolic factors are frequently associated with age-specific epileptic syndromes, such as infantile spasms and West syndrome, other syndromes may be determined by the effect of immunopathogenic mechanisms or energy-dependent processes in response to environmental challenges, such as infections or fever in normally-developed children during early or late childhood. Immune-mediated mechanisms have been suggested in selected pediatric epileptic syndromes in which acute and rapidly progressive encephalopathies preceded by fever and/or infections, such as febrile infection-related epilepsy syndrome, or in chronic progressive encephalopathies, such as Rasmussen encephalitis. A definite involvement of adaptive and innate immune mechanisms driven by cytotoxic CD8(+) T lymphocytes and neuroglial responses has been demonstrated in Rasmussen encephalitis, although the triggering factor of these responses remains unknown. Although the beneficial response to steroids and adrenocorticotropic hormone of infantile spasms, or preceding fever or infection in FIRES, may support a potential role of neuroinflammation as pathogenic factor, no definite demonstration of such involvement has been achieved, and genetic or metabolic factors are suspected. A major challenge for the future is discovering pathogenic mechanisms and etiological factors that facilitate the introduction of novel targets for drug intervention aimed at interfering with the disease mechanisms, therefore providing putative disease-modifying treatments in these pediatric epileptic syndromes.


Upregulation of microglia in drug users with and without pre-symptomatic HIV infection.
It is generally thought that infection of the central nervous system (CNS) by HIV-1 can occur early, even around the time of seroconversion, and evidence from animal studies supports this. However, the mode and timing of viral entry remain poorly understood since there have been comparatively few studies of the early neuropathology of HIV infection. In this study, samples of frontal and temporal lobes, and basal ganglia, were selected from 12 HIV-positive drug users who had been infected for 4-130 months before death, 10 HIV-negative drug users and 10 non-drug using controls, all age and sex matched. Routine and immunocytochemical staining showed that leptomeningeal and perivascular lymphocytic infiltrate was upregulated in HIV-infected cases compared with the two control groups, and choroid plexitis was confined to the HIV-positive subjects, suggesting an association with viral infection. In contrast, CD68-positive microglia were enhanced in both HIV- positive and HIV-negative drug users, considerably above the baseline seen in normal controls. However, there was no statistical difference between the three groups in relation to astrocytes. Screening and competitive polymerase chain reaction (PCR) undertaken on multiple samples including brain tissue, choroid plexus and leptomeninges from four of the HIV-positive subjects and one control case showed that the pro-viral burden was never more than 13 copies/microg DNA and was negative in multiple samples from one HIV-positive case and one control case. All the basal ganglia samples were PCR-negative. This study has not revealed any t spots' of viral load in brain tissue, choroid plexus or meninges, either early or late in the course of pre-symptomatic HIV infection. Drug use alone is associated with significant upregulation of microglia and this may predispose to HIV infection of the nervous system in drug users.


The twin-arginine translocation pathway in α-proteobacteria is functionally preserved irrespective of genomic and regulatory divergence.
The twin-arginine translocation (Tat) pathway exports fully folded proteins out of the cytoplasm of Gram-negative and Gram-positive bacteria. Although much progress has been made in unraveling the molecular mechanism and biochemical characterization of the Tat system, little is known concerning its functionality and biological role to confer adaptive skills, symbiosis or pathogenesis in the α-proteobacteria class. A comparative genomic analysis in the α-proteobacteria class confirmed the presence of tatA, tatB, and tatC genes in almost all genomes, but significant variations in gene synteny and rearrangements were found in the order Rickettsiales with respect to the typically described operon organization. Transcription of tat genes was confirmed for Anaplasma marginale str. St. Maries and Brucella abortus 2308, two α-proteobacteria with full and partial intracellular lifestyles, respectively. The tat genes of A. marginale are scattered throughout the genome, in contrast to the more generalized operon organization. Particularly, tatA showed an approximately 20-fold increase in mRNA levels relative to tatB and tatC. We showed Tat functionality in B. abortus 2308 for the first time, and confirmed conservation of functionality in A. marginale. We present the first experimental description of the Tat system in the Anaplasmataceae and Brucellaceae families. In particular, in A. marginale Tat functionality is conserved despite operon splitting as a consequence of genome rearrangements. Further studies will be required to understand how the proper stoichiometry of the Tat protein complex and its biological role are achieved. In addition, the predicted substrates might be the evidence of role of the Tat translocation system in the transition process from a free-living to a parasitic lifestyle in these α-proteobacteria.


Rotationally symmetric construction optics for a waveguide focusing grating.
Waveguide grating couplers have been under study as a method of focusing laser diode radiation on the surface of an optical data storage device. In this letter, we describe a simple method of holographically constructing a waveguide grating coupler at visible wavelengths which may be used with collimated infrared laser radiation to form high numerical aperture, diffraction limited beams. The design method includes the necessary compensation aberrations to offset the effects of differences between construction and end-use wavelengths.


Periconceptional Folic Acid Supplementation and Infant Risk of Congenital Heart Defects in Norway 1999-2009.
The birth prevalence of congenital heart defects (CHDs) has decreased in Canada and Europe. Recommended intake of folic acid in pregnancy is a suggestive risk-reducing factor for CHDs. We investigated the association between periconceptional intake of folic acid supplements and infant risk of CHDs. Information on maternal intake of folic acid supplements before and during pregnancy in the Medical Birth Registry of Norway 1999-2009 was updated with information on CHD diagnoses from national health registers and the Cardiovascular Diseases in Norway Project. The association between folic acid intake and infant risk of CHD was estimated as relative risk (RR) with binomial log linear regression. Among 517 784 non-chromosomal singleton births, 6200 children were identified with CHD and 1153 with severe CHD. For all births, 18.4% of the mothers initiated folic acid supplements before pregnancy and 31.6% during pregnancy. The adjusted RR for severe CHD was 0.99 [95% confidence interval [CI] 0.86, 1.13] comparing periconceptional intake of folic acid with no intake. Specifically, RR for conotruncal defects was 0.99 [95% CI 0.80, 1.22], atrioventricular septal defects 1.19 [95% CI 0.78, 1.81], left ventricular outflow tract obstructions 1.02 [95% CI 0.78, 1.32], and right ventricular outflow tract obstructions 0.97 [95% CI 0.72, 1.29]. Birth prevalence of septal defects was higher in the group exposed to folic acid supplements with RR 1.19 [95% CI 1.10, 1.30]. Periconceptional folic acid supplement use showed no association with severe CHDs in the newborn. An unexpected association with an increased risk of septal defects warrants further investigation.


A surgical review of the priority claims attributed to Abraham Groves (1847-1935).
The practice of surgery had changed little over millennia when Abraham Groves and William Osler attended medical school together in Toronto, Ontario. The invention of anesthesia sparked such rapid development that by the time of Groves' and Osler's deaths, surgical practice resembled the current model. Several priority claims have been attributed to Groves' life in surgery, including aseptic surgery (1874), suprapubic lithotomy (1878), appendectomy (1883), surgical gloves (1885) and cancer radiotherapy (1903). These claims arise from an autobiography written by Groves at the age of 87 years in 1934. The purpose of this paper is to assess these priority claims from a modern surgical perspective. We did a systematic search of contemporary (1873-1934) and modern journals for articles by or about Groves. We searched relevant archives and museums. We reviewed the 1934 autobiography, notes held by descendants, reminiscences by contemporaries and collateral information. We assessed the information not only for priority but also for the development of organized surgical thought. Groves published frequently throughout his career; thus far we have located 36 papers, almost all of which were published in Canadian journals. He spoke regularly at regional meetings in Ontario. Many medical students apprenticed with him (including his brother, son and grandson), he established a hospital and he founded a school of nursing. His contemporaries published complimentary reminiscences, but no correspondence with his classmate, William Osler, is known. Groves' priority claims for aseptic surgery, suprapubic lithotomy and radiotherapy are supported by contemporary publications. Groves independently developed an organized surgical system that remains valid today. Priority claims for appendectomy and the use of surgical gloves are entirely consistent with that system. Although Groves' impact was reduced by his location and the limited circulation of the journals in which he wrote, he demonstrated a systematic understanding of modern surgery well ahead of his contemporaries.


The shape of the distal femur: a geometrical study using MRI.
We scanned 25 left knees in healthy human subjects using MRI. Multiplanar reconstruction software was used to take measurements of the inferior and posterior facets of the femoral condyles and the trochlea. A 'basic circle' can be defined which, in the sagittal plane, fits the posterior and inferior facets of the lateral condyle, the posterior facet of the medial condyle and the floor of the groove of the trochlea. It also approximately fits both condyles in the coronal plane (inferior facets) and the axial plane (posterior facets). The circle fitting the inferior facet of the medial condyle in the sagittal plane was consistently 35% larger than the other circles and was termed the 'medial inferior circle'. There were strong correlations between the radii of the circles, the relative positions of the centres of the condyles, the width of the condyles, the total knee width and skeletal measurements including height. There was poor correlation between the radii of the circles and the position of the trochlea relative to the condyles. In summary, the condyles are approximately spherical except for the inferior facet medially, which has a larger radius in the sagittal plane. The size and position of the condyles are consistent and change with the size of the person. However, the position of the trochlea is variable even though its radius is similar to that of the condyles. This information has implications for understanding anterior knee pain and for the design of knee replacements.


Biases, Barriers, and Possible Solutions: Steps Towards Addressing Autism Researchers Under-Engagement with Racially, Ethnically, and Socioeconomically Diverse Communities.
Autistic individuals who are also people of color or from lower socioeconomic strata are historically underrepresented in research. Lack of representation in autism research has contributed to health and healthcare disparities. Reducing these disparities will require culturally competent research that is relevant to under-resourced communities as well as collecting large nationally representative samples, or samples in which traditionally disenfranchised groups are over-represented. To achieve these goals, a diverse group of culturally competent researchers must partner with and gain the trust of communities to identify and eliminate barriers to participating in research. We suggest community-academic partnerships as one promising approach that results in high-quality research built on cultural competency, respect, and shared decision making.


The California Hotspots Project: identifying regions of rapid diversification of mammals.
The high rate of anthropogenic impact on natural systems mandates protection of the evolutionary processes that generate and sustain biological diversity. Environmental drivers of diversification include spatial heterogeneity of abiotic and biotic agents of divergent selection, features that suppress gene flow, and climatic or geological processes that open new niche space. To explore how well such proxies perform as surrogates for conservation planning, we need first to map areas with rapid diversification -'evolutionary hotspots'. Here we combine estimates of range size and divergence time to map spatial patterns of neo-endemism for mammals of California, a global biodiversity hotspot. Neo-endemism is explored at two scales: (i) endemic species, weighted by the inverse of range size and mtDNA sequence divergence from sisters; and (ii) as a surrogate for spatial patterns of phenotypic divergence, endemic subspecies, again using inverse-weighting of range size. The species-level analysis revealed foci of narrowly endemic, young taxa in the central Sierra Nevada, northern and central coast, and Tehachapi and Peninsular Ranges. The subspecies endemism-richness analysis supported the last four areas as hotspots for diversification, but also highlighted additional coastal areas (Monterey to north of San Francisco Bay) and the Inyo Valley to the east. We suggest these hotspots reflect the major processes shaping mammal neo-endemism: steep environmental gradients, biotic admixture areas, and areas with recent geological/climate change. Anthropogenic changes to both environment and land use will have direct impacts on regions of rapid divergence. However, despite widespread changes to land cover in California, the majority of the hotspots identified here occur in areas with relatively intact ecological landscapes. The geographical scope of conserving evolutionary process is beyond the scale of any single agency or nongovernmental organization. Choosing which land to closely protect and/or purchase will always require close coordination between agencies.


Understanding the implications of the breast cancer pathology report: a case study.
Case Study A.K. is a 50-year-old Caucasian female who was recently diagnosed with breast cancer and is presenting for an opinion regarding adjuvant therapy following a right segmental mastectomy and sentinel lymph node biopsy. The advanced practitioner (AP) in the outpatient oncology treatment center will be expected to participate in the discussion regarding the patient's care plan. In her review of the final pathology, the AP notes that the breast tumor is infiltrating ductal carcinoma, the most common type of invasive breast cancer. It starts in the cells that line the milk ducts in the breast, grows outside the ducts, and often spreads to the lymph nodes. Infiltrating ductal carcinoma represents 65% to 85% of all breast cancer cases (College of American Pathologists, 2011). The breast tumor measures 1.2 × 0.9 × 1.0 cm(3) (T1), is estrogen receptor positive with an H score of 280, is progesterone receptor negative with an H score of 0, and is HER2 equivocal 2+ by immunohistochemistry with a fluorescence in situ hybridization ratio of 1.9 with a copy number of 5. The Ki-67 proliferation rate is 60%, and the nuclear grade is 2/3, with a Nottingham score of 5/9. The surgical margins from the segmental mastectomy are clear by 0.4 mm. There is lymphovascular invasion present. Of two sentinel lymph nodes, none is positive (N0). There has been no workup for metastatic disease. Additionally, the AP notes that the patient is premenopausal and that A.K.'s family history is positive for a maternal aunt diagnosed with a stage I breast cancer at age 75. What is the recommended plan of care for A.K.?


Acyl-chain mismatch driven superlattice arrangements in DPPC/DLPC/cholesterol bilayers.
Fluorescence and infrared spectroscopy and cholesterol oxidase activity were employed to investigate the effect of phosphatidylcholine (PC) acyl chain length mismatch on the lateral organizations of lipids in liquid-ordered dipalmitoyl-PC/dilauroyl-PC/cholesterol (DPPC/DLPC/CHOL) bilayers. Plots of steady-state fluorescence emission anisotropy of diphenylhexatriene (DPH) labeled PC (DPH-PC) embedded in the DPPC/DLPC/CHOL bilayers revealed significant peaks at several DPPC mole fractions (Y(DPPC)) when the cholesterol mole fraction (X(CHOL)) was fixed to particular values. Analogously, the DPH-PC anisotropy peaked at several critical X(CHOL)'s when Y(DPPC) was fixed. Acyl chain C-H and C horizontal lineO vibrational peak frequencies of native PC as well as the activity of cholesterol oxidase also revealed dips and peaks at similar Y(DPPC)'s. Importantly, most of the observed peaks/dips coincide with the critical mole fractions predicted by the Superlattice (SL) model. A three-dimensional map of DPH-PC anisotropy versus composition in the range 0.32 <or= X(CHOL) <or= 0.50; 0.54 <or= Y(DPPC) <or= 0.72 revealed a prominent peak at (X(CHOL), Y(DPPC)) approximately (0.42, 0.64). This suggests a simultaneous presence of two different types of superlattices, one where cholesterol is the quest molecule in a PC host lattice and another where DPPC is the guest in the DLPC host lattice. Time-resolved measurements of DPH-PC fluorescence indicated the existence of an ordered, rotationally hindered environment of acyl chains at that "critical" composition consistent with the existence of SL arrangements. We propose that beside CHOL/PC superlattices, DPPC, and DLPC as well tend to adopt regular SL-like lateral distributions relative to each other, presumably because the less hydrophobic DLPC molecule is slightly displaced toward the aqueous phase, thus allowing more room and mobility for the head groups of both DPPC and DLPC as well as for the acyl chain tails of DPPC. The parallel presence of two kinds of superlattices, that is, CHOL/PC-SL and DPPC/DLPC-SL as demonstrated here, has intriguing implications regarding lipid homeostasis of eukaryote membranes.


Molybdenum at high pressure and temperature: melting from another solid phase.
The Gibbs free energies of bcc and fcc Mo are calculated from first principles in the quasiharmonic approximation in the pressure range from 350 to 850 GPa at room temperatures up to 7500 K. It is found that Mo, stable in the bcc phase at low temperatures, has lower free energy in the fcc structure than in the bcc phase at elevated temperatures. Our density-functional-theory-based molecular dynamics simulations demonstrate that fcc melts at higher than bcc temperatures above 1.5 Mbar. Our calculated melting temperatures and bcc-fcc boundary are consistent with the Mo Hugoniot sound speed measurements. We find that melting occurs at temperatures significantly above the bcc-fcc boundary. This suggests an explanation of the recent diamond anvil cell experiments, which find a phase boundary in the vicinity of our extrapolated bcc-fcc boundary.


Long-lived charge-separated states in ligand-stabilized silver clusters.
Recently developed synthesis methods allow for the production of atomically monodisperse clusters of silver atoms stabilized in solution by aromatic thiol ligands, which exhibit intense absorption peaks throughout the visible and near-IR spectral regions. Here we investigated the time-dependent optical properties of these clusters. We observed two kinetic processes following ultrafast laser excitation of any of the absorption peaks: a rapid decay, with a time constant of 1 ps or less, and a slow decay, with a time constant that can be longer than 300 ns. Both time constants decrease as the polarity of the solvent increases, indicating that the two processes correspond to the formation and recombination, respectively, of a charge-separated state. The long lifetime of this state and the broad optical absorption spectrum mean that the ligand-stabilized silver clusters are promising materials for solar energy harvesting.


Development of an oral operant nicotine/ethanol co-use model in alcohol-preferring (p) rats.
Alcohol abuse is frequently associated with nicotine (Nic) use. The current experiments were conducted to establish an oral operant ethanol + Nic (EtOH + Nic) co-use model and to characterize some aspects of EtOH + Nic co-use. Rats were allowed to choose between EtOH alone or EtOH + Nic solutions. Additionally, alcohol-preferring (P) rats were allowed to concurrently self-administer 3 distinct EtOH solutions (10, 20, and 30%) with varying amounts of Nic (0.07, 0.14, or 0.21 mg/ml) under operant conditions. P rats were also allowed to concurrently self-administer 2 distinct amounts of Nic (0.07 and 0.14 mg/ml) added to saccharin (Sacc; 0.025%) solutions. During acquisition, P rats responded for the EtOH + Nic solutions at the same level as for EtOH alone, and responding for EtOH + Nic solutions was present throughout all drinking conditions. P rats also readily maintained stable self-administration behaviors for Nic + Sacc solutions. The results demonstrated that P rats readily acquired and maintained stable self-administration behaviors for EtOH + 0.07 and EtOH + 0.14 mg/ml Nic solutions. Self-administration of EtOH + 0.21 mg/ml Nic was established in only 50% of the subjects. P rats readily expressed seeking behaviors for the EtOH + Nic solutions and reacquired EtOH + Nic self-administration during relapse testing. In addition, tail blood samples indicated that EtOH + Nic co-use resulted in pharmacologically relevant levels of both EtOH and Nic in the blood. Overall, the results indicate that P rats readily consume EtOH + Nic solutions concurrently in the presence of EtOH alone, express drug-seeking behaviors, and will concurrently consume physiologically relevant levels of both drugs. These results support the idea that this oral operant EtOH + Nic co-use model would be suitable for studying the development of co-abuse and the consequences of long-term chronic co-abuse.


"Complementing" toll signaling.
Studies of signal transduction are often focused on dissecting the cellular response to a single stimulus that activates a single receptor. These types of studies laid the foundation for our current understanding of signaling, as well as the generation of countless arrow-containing models in today's textbooks. Implicit in most models is the suggestion that the arrows emanating from an activated receptor represent the core signaling pathways that are always activated by a given receptor, thus leading to a core cellular response. In nature, however, it is likely that no signaling pathway is activated in isolation. Rather, cells often respond to multiple stimuli simultaneously, and the cellular response may be the result of several signaling pathways. A new study attempts to model such conditions in vitro and reveals that when macrophages encounter bacteria, two signal transduction pathways interact in a way that profoundly alters the cellular response to infection.


The structurally related auxin and melatonin tryptophan-derivatives and their roles in Arabidopsis thaliana and in the human malaria parasite Plasmodium falciparum.
Indole compounds are involved in a range of functions in many organisms. In the human malaria parasite Plasmodium falciparum, melatonin and other tryptophan derivatives are able to modulate its intraerythrocytic cycle, increasing the schizont population as well as parasitemia, likely through ubiquitin-proteasome system (UPS) gene regulation. In plants, melatonin regulates root development, in a similar way to that described for indoleacetic acid, suggesting that melatonin and indoleacetic acid could co-participate in some physiological processes due to structural similarities. In the present work, we evaluate whether the chemical structure similarity found in indoleacetic acid and melatonin can lead to similar effects in Arabidopsis thaliana lateral root formation and P. falciparum cell cycle modulation, as well as in the UPS of gene regulation, by qRT-PCR. Our data show that P. falciparum is not able to respond to indoleacetic acid either in the modulation of the intraerythrocytic cycle or in the gene regulation mediated by the UPS as observed for melatonin. The similarities of these indole compounds are not sufficient to confer synergistic functions in P. falciparum cell cycle modulation, but could interplay in A. thaliana lateral root formation.


Early inner retinal astrocyte dysfunction during diabetes and development of hypoxia, retinal stress, and neuronal functional loss.
Neuronal and glial alterations precede the overt vascular change that characterizes diabetic retinopathy. Because retinal astrocytes modulate neuronal and vascular function, this study investigated the time course of astrocyte, Müller cell, and neuronal change during diabetes to determine whether astrocytes may play an early role in diabetic retinopathy. Sprague-Dawley rats were rendered diabetic via streptozotocin and neuronal and glial changes were assessed after 2-10 weeks. Astrocyte change was investigated using connexin-26 immunolabeling, whereas connexin-26 and -43 gene expressions were quantified using real-time PCR. Hypoxia was measured by pimonidazole labeling and the expression of hypoxia-inducible factor-1 alpha (HIF-1α) was quantified using Western blot. Müller cell gliosis was assessed by glial fibrillary acidic protein immunolabeling and retinal function assessed using the electroretinogram. Astrocyte connexin-26 and -43 gene and protein expression decreased after 4 weeks of diabetes, before significant astrocyte loss. At the same time, the retina became hypoxic, with increased HIF-1α expression and pimonidazole labeling in the ganglion cell layer. This coincided with a decrease in ganglion cell function. After 6 weeks of diabetes, Müller cell gliosis became more evident and there were additional functional deficits in photoreceptoral and amacrine cell responses. These findings suggest that early changes in astrocytes are coincident with inner retinal hypoxia and ganglion cell functional deficits, whereas Müller cell gliosis and more extensive decreases in neuronal function occur later. Astrocytes may play an early and key role in changes in retinal vasculature and inner retinal dysfunction in diabetes.


A study of different fabrics to increase radar cross section of humans.
This purpose of the study was to increase the visibility on radar for unprotected pedestrians with the aid of conducting fabric. The experiment comprised measurements of four types of fabric to determine the radio frequency properties, such as radar cross section (RCS) for the vehicle radar frequency 77 GHz and transmission (shielding) in the frequency range 3-18 GHz. Two different thicknesses of polypyrrole (PPy) nonvowen fabric were tested and one thickness for 30 % and 40 % stainless steel fabrics respectively. A jacket with the thinner nonvowen material and one with 40 % steel were tested and compared to an unmodified jacket in the RCS measurement. The measurement showed an increase in RCS of 4 dB for the jacket with the 40 % steel lining compared to the unmodified jacket. The transmission measurement was aimed at determining the fabric with the highest transmission of an incoming radio wave. The 30 % steel fabric and the two thicknesses of the nonvowen fabrics were tested. One practical application is for example the use of radar reflective material in search and rescue (SAR) clothes. The study showed that the 30 % steel fabric was the best candidate for further RCS measurements.


Eye-safe lidar system for pesticide spray drift measurement.
Spray drift is one of the main sources of pesticide contamination. For this reason, an accurate understanding of this phenomenon is necessary in order to limit its effects. Nowadays, spray drift is usually studied by using in situ collectors which only allow time-integrated sampling of specific points of the pesticide clouds. Previous research has demonstrated that the light detection and ranging (lidar) technique can be an alternative for spray drift monitoring. This technique enables remote measurement of pesticide clouds with high temporal and distance resolution. Despite these advantages, the fact that no lidar instrument suitable for such an application is presently available has appreciably limited its practical use. This work presents the first eye-safe lidar system specifically designed for the monitoring of pesticide clouds. Parameter design of this system is carried out via signal-to-noise ratio simulations. The instrument is based on a 3-mJ pulse-energy erbium-doped glass laser, an 80-mm diameter telescope, an APD optoelectronic receiver and optomechanically adjustable components. In first test measurements, the lidar system has been able to measure a topographic target located over 2 km away. The instrument has also been used in spray drift studies, demonstrating its capability to monitor the temporal and distance evolution of several pesticide clouds emitted by air-assisted sprayers at distances between 50 and 100 m.


Second-line mitoxantrone, etoposide, and cytarabine for acute myeloid leukemia: a single-center experience.
The majority of patients with acute myeloid leukemia (AML) will require second-line chemotherapy for either relapsed or refractory disease. Currently, only allogeneic hematopoietic cell transplantation (HCT) offers a curative option in this setting and no preferred regimen has been established. The reported efficacy of second-line regimens is widely disparate, thus limiting informed clinical decision making. A retrospective review of 77 patients receiving therapy between 2001 and 2008 with relapsed, 42, and refractory, 35, AML was performed to determine overall response rate and survival following mitoxantrone (8 mg/m(2)/day), etoposide (100 mg/m(2)/day), and cytarabine (1,000 mg/m(2)/day) chemotherapy administered over 5 days. Among 77 patients (median age of 54 years and 64% intermediate risk karyotype) with median follow-up of 153 days, 18% achieved a complete response and 8% a morphologic leukemia-free state. Fifty-seven (74%) experienced treatment failure, 10 of whom achieved a remission after additional therapy. Median overall survival (OS) was 6.8 months. Among patients achieving a response, 50% received consolidation with allogeneic HCT, autologous HCT (5%), or consolidation chemotherapy alone (45%). A nonsignificant trend in overall response (50%, 27%, and 23.8%) and median OS (8.3, 6.8, and 4.7 months) was observed by cytogenetic stratification into favorable, intermediate, and unfavorable risk. Patients with refractory versus relapsed disease had similar overall responses (20% and 31%, P = 0.41) and median OS (5.3 and 7.6 months, P = 0.36). Despite risk stratification by the European Prognostic Index, our series demonstrates inferior rates of response and survival, illustrating the limited activity of this regimen in our cohort.


Canine infectious respiratory disease: New insights into the etiology and epidemiology of associated pathogens.
Canine infectious respiratory disease (CIRD) is a syndrome where multiple viral and bacterial pathogens are involved sequentially or synergistically to cause illness. There is limited information regarding the prevalence of pathogens related to CIRD in the United States as well as the role of co-infections in the pathogenesis of the syndrome. We aimed to conduct a comprehensive etiologic and epidemiologic study of multiple CIRD agents in a diverse dog population using molecular methods and statistical modeling analyses. In addition, a novel probe-based multiplex real-time PCR was developed to simultaneously detect and differentiate two species of Mycoplasma (M. canis and M. cynos). Canine adenovirus, canine distemper virus, canine parainfluenza virus, coronavirus, influenza A virus (H3N2 and H3N8), Bordetella bronchiseptica, M. canis, M. cynos and Streptococcus equi subsp. zooepidemicus were investigated in specimens from clinically ill and asymptomatic dogs received at the Athens Veterinary Diagnostic Laboratory. Results showed low occurrence of classical CIRD agents such as B. bronchiseptica, canine adenovirus and distemper virus, while highlighting the potential role of emerging bacteria such as M. canis and M. cynos. Statistical modeling analyses of CIRD pathogens emphasized the impact of co-infections on the severity of clinical presentation, and showed that host factors, such as animal age, are the most important predictors of disease severity. This study provides new insights into the current understanding of the prevalence and role of co-infections with selected viruses and bacteria in the etiology of CIRD, while underscoring the importance of molecular diagnosis and vaccination against this disease.


Defense of Elevated Body Weight Setpoint in Diet-Induced Obese Rats on Low Energy Diet Is Mediated by Loss of Melanocortin Sensitivity in the Paraventricular Hypothalamic Nucleus.
Some animals and humans fed a high-energy diet (HED) are diet-resistant (DR), remaining as lean as individuals who were naïve to HED. Other individuals become obese during HED exposure and subsequently defend the obese weight (Diet-Induced Obesity- Defenders, DIO-D) even when subsequently maintained on a low-energy diet. We hypothesized that the body weight setpoint of the DIO-D phenotype resides in the hypothalamic paraventricular nucleus (PVN), where anorexigenic melanocortins, including melanotan II (MTII), increase presynaptic GABA release, and the orexigenic neuropeptide Y (NPY) inhibits it. After prolonged return to low-energy diet, GABA inputs to PVN neurons from DIO-D rats exhibited highly attenuated responses to MTII compared with those from DR and HED-naïve rats. In DIO-D rats, melanocortin-4 receptor expression was significantly reduced in dorsomedial hypothalamus, a major source of GABA input to PVN. Unlike melanocortin responses, NPY actions in PVN of DIO-D rats were unchanged, but were reduced in neurons of the ventromedial hypothalamic nucleus; in PVN of DR rats, NPY responses were paradoxically increased. MTII-sensitivity was restored in DIO-D rats by several weeks' refeeding with HED. The loss of melanocortin sensitivity restricted to PVN of DIO-D animals, and its restoration upon prolonged refeeding with HED suggest that their melanocortin systems retain the ability to up- and downregulate around their elevated body weight setpoint in response to longer-term changes in dietary energy density. These properties are consistent with a mechanism of body weight setpoint.


Multi-copy expression and fed-batch production of Rhodotorula araucariae epoxide hydrolase in Yarrowia lipolytica.
Epoxide hydrolases (EHs) of fungal origin have the ability to catalyze the enantioselective hydrolysis of epoxides to their corresponding diols. However, wild type fungal EHs are limited in substrate range and enantioselectivity. Additionally, the production of fungal epoxide hydrolase (EH) by wild-type strains is typically very low. In the present study, the EH-encoding gene from Rhodotorula araucariae was functionally expressed in Yarrowia lipolytica, under the control of a growth phase inducible hp4d promoter, in a multi-copy expression cassette. The transformation experiments yielded a positive transformant, with a final EH activity of 220 U/g dw in shake-flask cultures. Evaluation of this transformant in batch fermentations resulted in approximately 7-fold improvement in EH activity over the flask scale. Different constant specific feed rates were tested in fed-batch fermentations, resulting in an EH activity of 1,750 U/g dw at a specific feed rate of approximately 0.1 g/g/h, in comparison to enzyme production levels of 0.3 U/g dw for the wild type R. araucariae and 52 U/g dw for an Escherichia coli recombinant strain expressing the same gene. The expression of EH in Y. lipolytica using a multi-copy cassette demonstrates potential for commercial application.


Safety and effectiveness of a prothrombin complex concentrate in approved and off-label indications.
To evaluate the effectiveness and safety of prothrombin complex concentrates (PCCs) in approved and off-label indications. PCCs are approved for the urgent reversal of vitamin K antagonists (VKAs). Data concerning the efficacy, safety and dosing for off-label indications are limited, but they are included in massive bleeding protocols. This was a retrospective review of cases treated with four-factor PCCs (4F-PCCs) between January 2009 and 2016. Efficacy end-points include: (i) VKA reversal efficacy assessed by international normalised ratio (INR) normalisation (<1·5) and (ii) clinical efficacy as bleeding cessation and/or decreased number of transfused blood components and 24-h mortality in bleeding coagulopathy. The safety end-point is the incidence of thromboembolic events. A total of 328 patients were included (51·8% male, median age 78 years old). Indications were as follows: VKA reversal (66·6%), bleeding coagulopathy (30·5%) and direct anticoagulant (DOAC) reversal due to bleeding (2·5%). VKA reversal was effective in 97·1% of patients, and 76·5% demonstrated complete reversal (INR < 1·5); only 34·3% patients needed hemoderivatives. Prior to emergency procedures, PCCs achieved global responses in 83% of patients, with no bleeding complication during intervention. DOAC reversal was effective in 88·9% of patients. Bleeding cessation was associated with the dose administered (P = 0·002). In coagulopathy bleeding, haemorrhage cessation, established by the International Society of Thrombosis and Haemostais (ISTH) definition, occurred in 56·7% of massive bleeding events and in 42·5% of other coagulopathies; 24-h mortality was 30%, mainly related to active bleeding. Ten thrombotic episodes were observed (3·1%). 4F-PCC was effective as adjuvant treatment with an acceptable safety profile, not only for the emergent reversal of VKAs but also for refractory coagulopathy associated with major bleeding.


A COL4A3 gene mutation and post-transplant anti-alpha 3(IV) collagen alloantibodies in Alport syndrome.
The X-linked Alport syndrome is associated with mutations and deletions in COL4A5 gene, one of six genes which constitute the alpha-chains of type IV collagen in basement membranes. The autosomal recessive form of Alport syndrome is characterized by mutations and deletions in the COL4A3 and COL4A4 genes. A fraction of Alport patients who undergo renal transplantation develop anti-glomerular basement membrane (GBM) nephritis, which results in loss of the renal allograft function. Recently, the target for alloantibodies from an X-linked Alport patient with complete COL4A5 gene deletion was determined to be the alpha 3 chain of type IV collagen. The present study characterized the post-transplant alloantibodies from an autosomal recessive Alport patient with anti-GBM glomerulonephritis and a COL4A3 gene mutation which predicted a loss of 85% of the alpha 3(IV) NC1 domain. The specificity of these new antibodies were studied using glomerular basement membrane constituents and recombinant type IV collagen domains. The results establish the target for the alloantibodies from an autosomal recessive Alport patient with COL4A3 deletion as principally the alpha 3(IV) collagen chain, similar to the post-transplant alloantibodies from X-linked Alport patients with COL4A5 gene deletions. The absence of alpha 3(IV) chain in the GBM of patients with both these forms of Alport syndrome, due either to a failure of synthesis or a failure of assembly, presumably leads to a loss of immunologic tolerance for the alpha 3(IV) NC1 domain in transplanted allografts.