World Health Organization recommendations are often strong based on low confidence in effect estimates.
Expert guideline panelists are sometimes reluctant to offer weak/conditional/contingent recommendations. Grading of Recommendations Assessment, Development and Evaluation (GRADE) guidance warns against strong recommendations when confidence in effect estimates is low or very low, suggesting that such recommendations may seldom be justified. We aim to characterize the classification of strength of recommendations and confidence in estimates in World Health Organization (WHO) guidelines that used the GRADE approach and graded both strength and confidence (GRADEd). We reviewed all WHO guidelines (January 2007 to December 2012), identified those that were GRADEd, and, in these, examined the classifications of strong and weak and associated confidence in estimates (high, moderate, low, and very low). We identified 116 WHO guidelines in which 43 (37%) were GRADEd and had 456 recommendations, of which 289 (63.4%) were strong and 167 (36.6%) were conditional/weak. Of the 289 strong recommendations, 95 (33.0%) were based on evidence warranting low confidence in estimates and 65 (22.5%) on evidence warranting very low confidence in estimates (55.5% strong recommendations overall based on low or very low confidence in estimates). Strong recommendations based on low or very low confidence estimates are very frequently made in WHO guidelines. Further study to determine the reasons for such high uncertainty recommendations is warranted.


Identifying barriers to accrual in radiation oncology randomized trials.
Data about factors driving accrual to radiation oncology trials are limited. In oncology, 30%-40% of trials are considered unsuccessful, many because of poor accrual. The goal of the present study was to inform the design of future trials by evaluating the effects of institutional, clinician, and patient factors on accrual rates to a randomized radiation oncology trial. Investigators participating in sabr-comet (NCT01446744), a randomized phase ii trial open in Canada, Europe, and Australia that is evaluating the role of stereotactic ablative radiotherapy (sabr) in oligometastatic disease, were invited to complete a survey about factors affecting accrual. Institutional ethics approval was obtained. The primary endpoint was the annual accrual rate per institution. Univariable and multivariable linear regression analyses were used to identify factors predictive of annual accrual rates. On univariable linear regression analysis, off-trial availability of sabr (p = 0.014) and equipoise of the referring physician (p = 0.014) were found to be predictive of annual accrual rates. The annual accrual rates were lower when centres offered sabr for oligometastases off-trial (median: 3.7 patients vs. 8.4 patients enrolled) and when referring physicians felt that, compared with having equipoise, sabr was beneficial (median: 4.8 patients vs. 8.4 patients enrolled). Multivariable analysis identified perceived level of equipoise of the referring physician to be predictive of the annual accrual rate (p = 0.023). The level of equipoise of referring physicians might play a key role in accrual to radiation oncology randomized controlled trials. Efforts to communicate with and educate referring physicians might therefore be beneficial for improving trial accrual rates.


Estimating the cost of interventions to improve water, sanitation and hygiene in healthcare facilities across India.
Despite increasing utilisation of institutional healthcare in India, many healthcare facilities (HCFs) lack access to basic water, sanitation and hygiene (WASH) services. WASH services protect patients by improving infection prevention and control (IPC), which in turn can reduce the burden of healthcare-associated infections (HAIs). However, data on the cost of implementing WASH interventions in Indian HCFs are limited. We surveyed 32 HCFs across India, varying in size, type and setting to obtain the direct costs of providing improved water supply, sanitation and IPC-supporting infrastructure. We calculated the average costs of WASH interventions and the number of HCFs nationwide requiring investments in WASH to estimate the financial cost of improving WASH across India's public healthcare system over 1 year. Improving WASH across India's public healthcare sector and sustaining services among upgraded facilities for 1 year would cost US$354 million in capital costs and US$289 million in recurrent costs from the provider perspective. The most costly interventions were those on water (US$238 million), linen reprocessing (US$112 million) and sanitation (US$104 million), while the least costly were interventions on hand hygiene (US$52 million), medical device reprocessing (US$56 million) and environmental surface cleaning (US$80 million). Overall, investments in rural HCFs would account for 64.4% of total costs, of which 52.3% would go towards primary health centres. Improving IPC in Indian public HCFs can aid in the prevention of HAIs to reduce the spread of antimicrobial resistance. Although WASH is a necessary component of IPC, coverage remains low in HCFs in India. Using ex-post costs, our results estimate the investment levels needed to improve WASH across the Indian public healthcare system and provide a basis for policymakers to support IPC-related National Action Plan activities for antimicrobial resistance through investments in WASH.


Potential for dramatic improvement in sequence alignment against structures of remote homologous proteins by extracting structural information from multiple structure alignment.
A novel method has been developed for acquiring the correct alignment of a query sequence against remotely homologous proteins by extracting structural information from profiles of multiple structure alignment. A systematic search algorithm combined with a group of score functions based on sequence information and structural information has been introduced in this procedure. A limited number of top solutions (15,000) with high scores were selected as candidates for further examination. On a test-set comprising 301 proteins from 75 protein families with sequence identity less than 30%, the proportion of proteins with completely correct alignment as first candidate was improved to 39.8% by our method, whereas the typical performance of existing sequence-based alignment methods was only between 16.1% and 22.7%. Furthermore, multiple candidates for possible alignment were provided in our approach, which dramatically increased the possibility of finding correct alignment, such that completely correct alignments were found amongst the top-ranked 1000 candidates in 88.3% of the proteins. With the assistance of a sequence database, completely correct alignment solutions were achieved amongst the top 1000 candidates in 94.3% of the proteins. From such a limited number of candidates, it would become possible to identify more correct alignment using a more time-consuming but more powerful method with more detailed structural information, such as side-chain packing and energy minimization, etc. The results indicate that the novel alignment strategy could be helpful for extending the application of highly reliable methods for fold identification and homology modeling to a huge number of homologous proteins of low sequence similarity. Details of the methods, together with the results and implications for future development are presented.


Opioid prescribing and risk mitigation implementation in the management of acute pain: Results from The National Dental Practice-Based Research Network.
Minimal information exists regarding the consistency and correlates of dentists' implementation of risk mitigation strategies when prescribing opioids, including risk screening, prescription drug monitoring program (PDMP) use, and patient education. The authors conducted a Web-based, cross-sectional survey among practicing dentist members of The National Dental Practice-Based Research Network. The authors used the survey to assess pain management prescribing practices and risk mitigation implementation. The authors linked survey data with network enrollment questionnaire data to include practitioner demographic and practice characteristics. A total of 822 dentists completed the survey. A minority of dentists reported prescribing opioids only (11%) or opioids in combination with a recommendation for nonsteroidal anti-inflammatory drugs or acetaminophen (18%) to one-half or more of their patients needing management of acute pain. Higher levels of opioid prescribing were associated significantly with less consistent implementation of PDMP use (r = -0.20) and patient education (r = -0.11). Most dentists reported infrequent PDMP use and counseling patients regarding risks, storage, and disposal of opioids. Higher frequency of opioid prescribing was associated with less consistent risk mitigation implementation. When opioid prescribing is indicated, risk of misuse and diversion may be mitigated by consistent PDMP use and provision of patient education. Dental education in this arena is needed.


Probing charge transfer dynamics in a single iron tetraphenylporphyrin dyad adsorbed on an insulating surface.
Although the dynamics of charge transfer (CT) processes can be probed with ultimate lifetime resolution, the inability to control CT at the nanoscale is one of the most important roadblocks to revealing some of its deep fundamental aspects. In this work, we present an investigation of CT dynamics in a single iron tetraphenylporphyrin (Fe-TPP) donor/acceptor dyad adsorbed on a CaF2/Si(100) insulating surface. The tip of a scanning tunneling microscope (STM) is used to create local ionic states in one fragment of the dyad. The CT process is monitored by imaging subsequent changes in the neighbor acceptor molecule and its efficiency is mapped, revealing the influence of the initial excited state in the donor molecule. In the validation of the experiments, simulations based on density functional theory show that holes have a higher donor-acceptor CT rate compared to electrons and highlight a noticeable initial state dependence on the CT process. We leverage the unprecedented spatial resolution achieved in our experiments to show that the CT process in the dyad is governed via molecule-molecule coherent tunneling with negligible surface-mediated character.


Application of genome editing technologies to the study and treatment of hematological disease.
Genome editing technologies have advanced significantly over the past few years, providing a fast and effective tool to precisely manipulate the genome at specific locations. The three commonly used genome editing technologies are Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Cas9 (CRISPR/Cas9) system. ZFNs and TALENs consist of endonucleases fused to a DNA-binding domain, while the CRISPR/Cas9 system uses guide RNAs to target the bacterial Cas9 endonuclease to the desired genomic location. The double-strand breaks made by these endonucleases are repaired in the cells either by non-homologous end joining, resulting in the introduction of insertions/deletions, or, if a repair template is provided, by homology directed repair. The ZFNs, TALENs and CRISPR/Cas9 systems take advantage of these repair mechanisms for targeted genome modification and have been successfully used to manipulate the genome in human cells. These genome editing tools can be used to investigate gene function, to discover new therapeutic targets, and to develop disease models. Moreover, these genome editing technologies have great potential in gene therapy. Here, we review the latest advances in the application of genome editing technology to the study and treatment of hematological disorders.


Diagnostic Accuracy of Endoscopic Trimodal Imaging and Chromoendoscopy for Lesion Characterization in Ulcerative Colitis.
During surveillance colonoscopy of patients with long-standing ulcerative colitis [UC], a variety of dysplastic and non-dysplastic lesions are detected. The aim of this study was to address the diagnostic accuracy of endoscopic characterization of endoscopic trimodal imaging [ETMI] and chromoendoscopy [CE]. ETMI includes the combination of autofluorescence imaging [AFI], narrow band imaging [NBI] and white light endoscopy [WLE]. This is a pre-specified additional analysis of a multi-centre, randomized controlled trial that compared AFI with CE for dysplasia detection in 210 patients with long-standing UC [FIND-UC trial]. In the AFI arm, endoscopists used the ETMI system to record AFI colour, Kudo pit pattern using NBI and WLE for lesion characterization. For AFI, purple colour and ambiguous colour combined with pit pattern type III-V on NBI was considered dysplastic. Kudo pit pattern was described in the CE arm. For pit pattern description using NBI and CE, type III-V was considered dysplastic. Histology was the reference standard. In total, 52 dysplastic and 255 non-dysplastic lesions were detected. Overall sensitivity for real-time prediction of dysplasia was 76.9% (95% confidence interval [CI] 46.2-95.0) for ETMI, and 81.6% [95% CI 65.7-92.3] for CE. Overall negative predictive value [NPV] for ETMI was 96.9% [95% CI 92.0-98.8] and 94.7% [90.2-97.2] for CE. Sensitivity for endoscopic differentiation of dysplastic lesions detected during surveillance of patients with long-standing UC seems limited using ETMI and CE. Future research is warranted as the high NPV indicates that these techniques are valuable for the exclusion of dysplastic lesions [NTR4062].


Echo-Doppler determinants of outcomes in patients with unoperated significant mitral regurgitation in current era.
One-half of patients with severe symptomatic mitral regurgitation (MR) do not undergo surgery due to comorbidities. We evaluated prognosticators of outcomes in patients with unoperated significant MR. In this observational study, we retrospectively evaluated medical records of 75 consecutive patients with unoperated significant MR. All-cause mortality was 39% at 5 years. Non-survivors (n=29) versus survivors (n=46) were: older (77±9.8 vs 68±14, p=0.006), had higher New York Heart Association (NYHA) class (2.7±0.8 vs 2.3±0.8, p=0.037), higher brain natriuretic peptide (1157±717 vs 427±502 pg/mL, p=0.024, n=18), more coronary artery disease (61% vs 35%, p=0.031), more frequent left ventricular ejection fraction <50% (20.7% vs 4.3%, p=0.026), more functional MR (41% vs 22%, p=0.069), higher mitral E/E(') (12.7±4.6 vs 9.8±4, p=0.008), higher pulmonary artery systolic pressure (PASP; 52.6±18.7 vs 36.7±14, p <0.001), more ≥3+ tricuspid regurgitation (28% vs 4%, p=0.005) and more right ventricular dysfunction (26% vs 6%, p=0.035). Significant predictors of 5-year mortality were PASP (p=0.001) and E/E(') (p=0.011) using multivariate regression analysis. Patients with unoperated significant MR have high mortality. Elevated PASP and mitral E/E(') were the most significant predictors of 5-year survival in patients with unoperated significant MR. Current American College of Cardiology (ACC)/American Heart Association (AHA) guidelines provide a limited incorporation of echo-Doppler parameters in the preoperative risk stratification of patients with severe MR.


Extreme Modification of the Tetrapod Forelimb in a Triassic Diapsid Reptile.
The tetrapod forelimb is one of the most versatile structures in vertebrate evolution, having been co-opted for an enormous array of functions. However, the structural relationships between the bones of the forelimb have remained largely unchanged throughout the 375 million year history of Tetrapoda, with a radius and ulna made up of elongate, paralleling shafts contacting a series of shorter carpal bones. These features are consistent across nearly all known tetrapods, suggesting that the morphospace encompassed by these taxa is limited by some sort of constraint(s). Here, we report on a series of three-dimensionally preserved fossils of the small-bodied (<1 m) Late Triassic diapsid reptile Drepanosaurus, from the Chinle Formation of New Mexico, USA, which dramatically diverge from this pattern. Along with the crushed type specimen from Italy, these specimens have a flattened, crescent-shaped ulna with a long axis perpendicular to that of the radius and hyperelongate, shaft-like carpal bones contacting the ulna that are proximodistally longer than the radius. The second digit supports a massive, hooked claw. This condition has similarities to living "hook-and-pull" digging mammals and demonstrates that specialized, modern ecological roles had developed during the Triassic Period, over 200 million years ago. The forelimb bones in Drepanosaurus represent previously unknown morphologies for a tetrapod and, thus, a dramatic expansion of known tetrapod forelimb morphospace.


Location of the serine residue involved in the linkage between the terminal protein and the DNA of phage phi 29.
B. subtilis phage phi 29 has a terminal protein, p3, covalently linked to the 5' ends of the DNA through a phosphodiester bond between a serine residue and 5'-dAMP. This protein acts as a primer in DNA replication by forming an initiation complex with the 5'-terminal nucleotide dAMP. The amino acid sequence of the terminal protein, deduced from the nucleotide sequence of gene 3, showed the presence of 18 serine residues in a total of 266 amino acids. In this paper we have identified the serine involved in the linkage with the DNA as the residue 232, located close to the C-terminus of the molecule. This result was obtained by amino acid analysis of the peptide that remains linked to the DNA after proteinase K digestion of the terminal protein-phi 29 DNA complex and automated Edman degradation of the corresponding [125I]-labeled tryptic peptide. Prediction of the secondary structure of the terminal protein suggested that the serine residue involved in the linkage with the DNA is placed in a beta-turn, probably located on the external part of the molecule, as indicated by hydropathic values.


The stability and unexpected chemistry of oxide clusters.
Using evolutionary structure prediction and ab initio thermodynamics, we determine stable compositions and structures of small CemOn and FemOn clusters at realistic temperatures and oxygen pressures. We use second energy differences as the criterion determining clusters of particular stability ("magic" clusters), whereas HOMO-LUMO gaps are used to gauge chemical inertness - i.e. the ability of a cluster to survive in a complex chemical environment. We find that, similar to atomic nuclei (which are clusters made of neutrons and protons), compositional space of two-component clusters also has ridges and islands of stability, surrounded by sea of instability. Long ridges of stability correspond to stoichiometric compositions - e.g., (CeO2)k, (Ce2O3)k, (FeO)k, (Fe2O3)k and (Fe3O4)k series of clusters, while "islands of stability" can have very unexpected compositions. For example, at room temperature and ambient atmosphere, superoxidized Fe4O8 clusters will be dominant among the Fe4On clusters. We emphasize that stability is dictated not only by closed geometric and electronic shells, but also by magnetism.


Dissecting the regulatory landscape of the Abd-B gene of the bithorax complex.
The three homeotic genes of the bithorax complex (BX-C), Ubx, abd-A and Abd-B control the identity of the posterior thorax and all abdominal segments. Large segment-specific cis-regulatory regions control the expression of Ubx, abd-A or Abd-B in each of the segments. These segment-specific cis-regulatory regions span the whole 300 kb of the BX-C and are arranged on the chromosome in the same order as the segments they specify. Experiments with lacZ reporter constructs revealed the existence of several types of regulatory elements in each of the cis-regulatory regions. These include initiation elements, maintenance elements, cell type- or tissue-specific enhancers, chromatin insulators and the promoter targeting sequence. In this paper, we extend the analysis of regulatory elements within the BX-C by describing a series of internal deficiencies that affect the Abd-B regulatory region. Many of the elements uncovered by these deficiencies are further verified in transgenic reporter assays. Our results highlight four key features of the iab-5, iab-6 and iab-7 cis-regulatory region of Abd-B. First, the whole Abd-B region is modular by nature and can be divided into discrete functional domains. Second, each domain seems to control specifically the level of Abd-B expression in only one parasegment. Third, each domain is itself modular and made up of a similar set of definable regulatory elements. And finally, the activity of each domain is absolutely dependent on the presence of an initiator element.


Is blood pleurodesis effective for determining the cessation of persistent air leak?
A best evidence topic in thoracic surgery was written according to a structured protocol. The question addressed whether blood pleurodesis is effective for cessation of persistent air leak (PAL). Altogether more than 43 papers were found using the reported search, of which 10 represented the best evidence to answer the clinical question. The authors, journal, date and country of publication, patient group studied, study type, relevant outcomes and results of these papers are tabulated. We conclude that autologous blood pleurodesis has superior outcomes when compared with conservative management for treatment of postoperative PAL. In addition, for PAL causing pneumothorax, blood pleurodesis [optimal volume 100 ml (from two studies)] should be considered in patients who are unsuitable for surgery, talc pleurodesis is ineffective or not viable (including cases complicated by acute respiratory distress syndrome) and a prompt resolution is required. Some 70-81% of patients treated for postoperative air leak resolved within 12 h and 95-100% within 48 h vs. a mean of 3-6.3 days (from two studies) with simple drainage. Resolution of pneumothorax with blood pleurodesis was also significantly shorter (P<0.01). Overall success rates (from all studies) were 92.7% (n=133) from patients having undergone pulmonary surgery (76.6% in one injection, n=111), and 91.7% (n=109) of patients with pneumothorax. Recurrence rates were between 0 and 29% compared with 35-41% for simple drainage, although one controlled study in which the recurrence rate was improved from 16% in controls to 0% in the blood pleurodesis group (at 12-48 months). Minor complication (empyema/fever/pleural effusion) rates varied between studies (0-18%), although they show reduced incidence in line with improving technique over time. A controlled study looking at acute respiratory distress syndrome complicated by pneumothorax showed a significant reduction in mortality (odds ratio 0.6), time to cessation of air leak (P<0.01), weaning time (P<0.01) and intensive treatment unit (ITU) stay (P<0.01) whilst another randomized control study showed significant reduction in hospital stay following pulmonary resection (P<0.001).


Biosynthesis of hemiketal eicosanoids by cross-over of the 5-lipoxygenase and cyclooxygenase-2 pathways.
The prostaglandin and leukotriene families of lipid mediators are formed via two distinct biosynthetic pathways that are initiated by the oxygenation of arachidonic acid by either cyclooxygenase-2 (COX-2) or 5-lipoxygenase (5-LOX), respectively. The 5-LOX product 5S-hydroxyeicosatetraenoic acid, however, can also serve as an efficient substrate for COX-2, forming a bicyclic diendoperoxide with structural similarities to the arachidonic acid-derived prostaglandin endoperoxide PGH(2) [Schneider C, et al. (2006) J Am Chem Soc 128:720-721]. Here we identify two cyclic hemiketal (HK) eicosanoids, HKD(2) and HKE(2), as the major nonenzymatic rearrangement products of the diendoperoxide using liquid chromatography-mass spectrometry analyses as well as UV and NMR spectroscopy. HKD(2) and HKE(2) are furoketals formed by spontaneous cyclization of their respective 8,9-dioxo-5S,11R,12S,15S-tetrahydroxy- or 11,12-dioxo-5S,8S,9S,15S-tetrahydroxy-eicosadi-6E,13E-enoic acid precursors, resulting from opening of the 9S,11R- and 8S,12S-peroxide rings of the diendoperoxide. Furthermore, the diendoperoxide is an efficient substrate for the hematopoietic type of prostaglandin D synthase resulting in formation of HKD(2), equivalent to the enzymatic transformation of PGH(2) to PGD(2). HKD(2) and HKE(2) were formed in human blood leukocytes activated with bacterial lipopolysaccharide and calcium ionophore A23187, and biosynthesis was blocked by inhibitors of 5-LOX or COX-2. HKD(2) and HKE(2) stimulated migration and tubulogenesis of microvascular endothelial cells, implicating a proangiogenic role of the hemiketals in inflammatory sites that involve expression of 5-LOX and COX-2. Identification of the highly oxygenated hemiketal eicosanoids provides evidence for a previously unrecognized biosynthetic cross-over of the 5-LOX and COX-2 pathways.


Frequency domain analysis of noise in simple gene circuits.
Recent advances in single cell methods have spurred progress in quantifying and analyzing stochastic fluctuations, or noise, in genetic networks. Many of these studies have focused on identifying the sources of noise and quantifying its magnitude, and at the same time, paying less attention to the frequency content of the noise. We have developed a frequency domain approach to extract the information contained in the frequency content of the noise. In this article we review our work in this area and extend it to explicitly consider sources of extrinsic and intrinsic noise. First we review applications of the frequency domain approach to several simple circuits, including a constitutively expressed gene, a gene regulated by transitions in its operator state, and a negatively autoregulated gene. We then review our recent experimental study, in which time-lapse microscopy was used to measure noise in the expression of green fluorescent protein in individual cells. The results demonstrate how changes in rate constants within the gene circuit are reflected in the spectral content of the noise in a manner consistent with the predictions derived through frequency domain analysis. The experimental results confirm our earlier theoretical prediction that negative autoregulation not only reduces the magnitude of the noise but shifts its content out to higher frequency. Finally, we develop a frequency domain model of gene expression that explicitly accounts for extrinsic noise at the transcriptional and translational levels. We apply the model to interpret a shift in the autocorrelation function of green fluorescent protein induced by perturbations of the translational process as a shift in the frequency spectrum of extrinsic noise and a decrease in its weighting relative to intrinsic noise.


A mouse model of Down syndrome trisomic for all human chromosome 21 syntenic regions.
Down syndrome (DS) is caused by the presence of an extra copy of human chromosome 21 (Hsa21) and is the most common genetic cause for developmental cognitive disability. The regions on Hsa21 are syntenically conserved with three regions located on mouse chromosome 10 (Mmu10), Mmu16 and Mmu17. In this report, we describe a new mouse model for DS that carries duplications spanning the entire Hsa21 syntenic regions on all three mouse chromosomes. This mouse mutant exhibits DS-related neurological defects, including impaired cognitive behaviors, reduced hippocampal long-term potentiation and hydrocephalus. These results suggest that when all the mouse orthologs of the Hsa21 genes are triplicated, an abnormal cognitively relevant phenotype is the final outcome of the elevated expressions of these orthologs as well as all the possible functional interactions among themselves and/or with other mouse genes. Because of its desirable genotype and phenotype, this mutant may have the potential to serve as one of the reference models for further understanding the developmental cognitive disability associated with DS and may also be used for developing novel therapeutic interventions for this clinical manifestation of the disorder.


Preventative services offered by veterinarians on sheep farms in England and Wales: Opinions and drivers for proactive flock health planning.
Recent independent UK government reports and studies have highlighted the importance, but lack, of flock health services provided by veterinarians. Qualitative interviews were analysed by thematic analysis to construct belief statements to understand veterinarians' opinions on preventative advice and drivers for current services to sheep farmers. A postal questionnaire was sent to 515 sheep practices registered with the Royal College of Veterinary Surgeon (RCVS) in England and Wales in 2012 to gather quantitative data on these belief statements and to gather demographic information and current services provided by the veterinarian. Exploratory factor analysis with heuristic approaches was conducted on the respondents' belief statements to identify common factors of veterinarian beliefs. Three main factors were identified: motivation for proactiveness, perceived capability to offer preventative services and perceived opportunity to deliver these services. A beta regression model was built to identify the factors significantly associated with the time veterinarians spent in an advisory role. The relative proportion of time increased by 10% (1.01-1.19), 16% (1.03-1.30) and 29% (CI: 1.09-1.53) for each unit increase in score for factor 1 motivation, factor 2 capability and factor 3 opportunity respectively, indicating that these latent factors explained time veterinarians spent in an advisory role with sheep clients. There was a significant correlation between these factors suggesting influence of the associated beliefs between factors. This study provides insight into the nature and drivers of veterinarians' current behaviour and beliefs. These results could be further tested in behaviour intervention studies and help in designing efficient strategies aiming at promoting proactive health services offered by veterinarians on sheep farms in England and Wales.


A Novel Cinnamon-Related Natural Product with Pim-1 Inhibitory Activity Inhibits Leukemia and Skin Cancer.
The Pim-1 kinase regulates cell survival, proliferation, and differentiation and is overexpressed frequently in many malignancies, including leukemia and skin cancer. In this study, we used kinase profiling analysis to demonstrate that 2'-hydroxycinnamicaldehyde (2'-HCA), a compound found in cinnamon, specifically inhibits Pim-1 activity. Cocrystallography studies determined the hydrogen bonding pattern between 2'-HCA and Pim-1. Notably, 2'-HCA binding altered the apo kinase structure in a manner that shielded the ligand from solvent, thereby acting as a gatekeeper loop. Biologically, 2'-HCA inhibited the growth of human erythroleukemia or squamous epidermoid carcinoma cells by inducing apoptosis. The compound was also effective as a chemopreventive agent against EGF-mediated neoplastic transformation. Finally, 2'-HCA potently suppressed the growth of mouse xenografts representing human leukemia or skin cancer. Overall, our results offered preclinical proof of concept for 2'-HCA as a potent anticancer principle arising from direct targeting of the Pim-1 kinase.


Elevated level of DNA damage and impaired repair of oxidative DNA damage in patients with recurrent depressive disorder.
Depressive disorder (DD), including recurrent DD (rDD), is a severe psychological disease, which affects a large percentage of the world population. Although pathogenesis of the disease is not known, a growing body of evidence shows that inflammation together with oxidative stress may contribute to development of DD. Since reactive oxygen species produced during stress may damage DNA, we wanted to evaluate the extent of DNA damage and efficiency of DNA repair in patients with depression. We measured and compared the extent of endogenous DNA damage--single- and double-strand breaks, alkali-labile sites, and oxidative damage of the pyrimidines and purines--in peripheral blood mononuclear cells isolated from rDD patients (n=40) and healthy controls (n=46) using comet assay. We also measured DNA damage evoked by hydrogen peroxide and monitored changes in DNA damage during repair incubation. We found an increased number DNA breaks, alkali-labile sites, and oxidative modification of DNA bases in the patients compared to the controls. Exposure to hydrogen peroxide evoked the same increased damage in both groups. Examination of the repair kinetics of both groups revealed that the lesions were more efficiently repaired in the controls than in the patients. For the first time we showed that patients with depression, compared with non-depresses individuals, had more DNA breaks, alkali-labile sites, and oxidative DNA damage, and that those lesions may be accumulated by impairments of the DNA repair systems. More studies must be conducted to elucidate the role of DNA damage and repair in depression.


Functional consequences of DECTIN-1 early stop codon polymorphism Y238X in rheumatoid arthritis.
Dectin-1, a pattern recognition receptor expressed by the innate immune system, is known to be a major receptor inducing Th17-type adaptive immune responses that have been demonstrated to mediate autoimmunity. In this study, dectin-1 mRNA and protein expression, as well as the recently characterized DECTIN-1 Y238X early stop codon polymorphism, were studied in relation to rheumatoid arthritis (RA) susceptibility and severity. Dectin-1 mRNA expression was measured in synovial tissue specimens of RA, osteoarthritis (OA), and nonrheumatic patients. Dectin-1 protein expression and localization were assessed in RA synovial tissue specimens. Macrophages from individuals with different DECTIN-1 genotypes were examined for differences in cytokine responses on dectin-1 stimulation. Furthermore, clinical parameters of inflammation and bone destruction of 262 RA patients were correlated with the presence of the DECTIN-1 Y238X polymorphism. Evaluation of dectin-1 mRNA expression in synovial tissue biopsies revealed an increased expression in RA specimens, compared with biopsies from OA and nonrheumatic patients. Accordingly, dectin-1 protein expression in RA synovial tissue biopsies was moderate to high, especially on macrophage-like cells. Cytokine production capacity of macrophages bearing the DECTIN-1 Y238X polymorphism was demonstrated to be impaired on dectin-1 stimulation. However, the presence of the DECTIN-1 Y238X polymorphism was not associated with RA susceptibility or disease severity. Although expression of dectin-1 was high in synovial tissue of RA patients, and reduced cytokine production was observed in macrophages of individuals bearing the DECTIN-1 Y238X polymorphism, loss of one functional allele of DECTIN-1 is not associated with either susceptibility to or severity of RA.


MicroRNA Microarray-Based Identification of Involvement of miR-155 and miR-19a in Development of Oral Lichen Planus (OLP) by Modulating Th1/Th2 Balance via Targeting eNOS and Toll-Like Receptor 2 (TLR2).
BACKGROUND A wide range of microRNAs (miRNAs) have been shown to play a significant role in disease regulation. The objective of this study was to explore the role of miR-155 and miR-19a in the regulation of oral lichen planus (OLP). MATERIAL AND METHODS Microarray assay, real-time PCR, Western blot assay, computational analysis, luciferase assay, ELISA, and immunohistochemistry analysis were carried out to investigate the role of miR-155 and miR-19a in OLP. RESULTS According to microarray assay and real-time PCR results, the expression of miR-155 was most significantly decreased among the 16 candidate miRNAs in the OLP group, whereas the expression of miR-19a was most significantly increased. MiR-155 and miR-19a directly targeted endothelial nitric oxide synthase (eNOS) and TLR2, respectively, since only the cells co-transfected with miR-155/wild-type eNOS 3'UTR or cells co-transfected with miR-19a/wild-type TLR2 3'UTR exhibited decreased luciferase activity. In addition, the expression of TLR2 was highly upregulated in OLP, whereas the expression of eNOS was significantly downregulated. A negative correlation was found between miR-19a and TLR2 mRNA, with a coefficient value of -0.40. Similarly, a negative correlation was found between miR-155 and eNOS mRNA, with a coefficient value of -0.54. A lower level of NO, IL-4, IL-5, and IL-10 was observed in OLP, which was also accompanied by a higher level of TNF-α and IFN-γ. Finally, the upregulation in miR-155 directly decreased the expression of eNOS and further inhibited the production of NO. Downregulation of miR-19a directly increased the expression of TLR2. The inhibition of NO production and the enhancement in TLR2 expression synergistically increased the production of TNF-α and IFN-γ, while decreasing the levels of IL-4, IL-5, and IL-10. CONCLUSIONS In this study, the peripheral blood mononuclear cells (PBMCs) from subjects with or without OLP were collected and their gene expression profiles were compared. It was found that OLP changed the expression profile of miR-155 and miR-19a, which in turn directly affected the production of eNOS and TLR2, respectively. In addition, by synergistically inducing an imbalance between Th1 and Th2, the simultaneous deregulation of miR-155/eNOS and miR-19a/TLR2 was responsible for an elevated risk of OLP.


End-of-Life Co-residence of Older Parents and Their Sons in Rural China.
This study examined how intergenerational exchanges with sons and daughters predicted older parents' likelihood of co-residing with a son prior to death in a rural area of China's Anhui Province. Our investigation drew on theories of contingent co-residence, modernization, and social exchange, conceptualizing co-residence as having practical and symbolic importance in rural Chinese culture. The sample included 470 older parents, reported as deceased during 2001-2009, and their posthumous informants. We used logistic regression to assess intergenerational support and cohesion as predictors of co-residence with a son just prior to death. Older parents who provided instrumental support to, and received instrumental support from, sons and had better emotional relationships with sons were more likely than their counterparts to co-reside with a son at the end of life. Living with sons demonstrates filial piety for older parents at the end of life, but its realization is sensitive to intergenerational transactions.


Fluoxetine inhibits the extracellular signal regulated kinase pathway and suppresses growth of cancer cells.
Fluoxetine (FLX) is a widely prescribed antidepressant. Concerns were raised about the potential impact of FLX on cancer growth, because FLX was shown to promote development of breast cancer in rodents. Here we studied the effect of FLX on tumor growth in lung (A549), colon (HT29), neuroblastoma (SKNAS), medulloblastoma/rhabdomyosarcoma (TE671), astrocytoma (MOGGCCM) and breast (T47D) cancer cells and explored potential mechanisms of its action. In our study, FLX reduced growth of cancer cells in vitro in a concentration dependent manner. The antiproliferative effect of FLX was already evident after 24 hours exposure and more pronounced at 96 hours. We demonstrate that FLX inhibits phosphorylation of ERK1/2 kinases in a time and concentration-dependent manner, followed by reduced phosphorylation of transcription factor c-Myc in A549 and HT29 cells. After treatment with FLX, A549 and HT29 cells demonstrated concentration-dependent decrease in the expression of c-fos, c-jun, cyclin A, cyclin D1, and increased expression of p21(waf1) and p53 genes, which resulted in slowing of the cell cycle progression. We suggest that these changes could be responsible for observed inhibition of cancer cell proliferation during FLX treatment in vitro.


Dialysis frequency versus dialysis time, that is the question.
We reviewed a number of prospective randomized and multiple retrospective cohort studies of different dialysis prescriptions: longer dialysis time, at a frequency of at least three times a week, or a frequency of daily hemodialysis with a shorter dialysis time. Interestingly, the retrospective analyses have generally found significant survival benefits in the intensive dialysis groups, whereas more modest effects were observed in the prospective randomized controlled trials. The reason for this discrepancy may be related to the retrospective nature of the studies and possible selection bias; for example, the patients who were prescribed more frequent dialysis may have had more difficulties with volume control or high blood pressure. In contrast, the randomized controlled trials of increased dialysis frequency, which have shown indirect and modest benefits in complex coprimary end points, have small sample sizes and are plagued with difficulties in recruitment and compliance with the randomly allocated more frequent dialysis. This review, which attempts to balance the potential benefits of more frequent dialysis with the burden on the patient's lifestyle, an increased risk of access malfunction, as well as societal costs of such intensive dialysis prescriptions, concludes in favor of the conventional three times per week dialysis (at a minimum) but at longer dialysis times than is currently prescribed based on the Kt/Vurea metric alone.


A family of genes located on four separate 32-kilobase circular plasmids in Borrelia burgdorferi B31.
We have identified four loci in Borrelia burgdorferi B31 that contain open reading frames capable of encoding six proteins that are related to the antigenic proteins OspE and OspF. We have designated these proteins Erp, for OspEF-related protein, and named their respective genes erp. The erpA and erpB genes are linked, as are erpC and erpD, and the pairs probably constitute two operons. The erpG and erpH genes appear to be monocistronic. The ErpA and ErpC proteins are expressed by B. burgdorferi B31 in culture and are recognized by a polyclonal antiserum raised against the OspE protein of B. burgdorferi N40. The four erp loci are each located on different 32-kb circular plasmids that contain additional DNA sequences that are homologous to each other and to an 8.3-kb circular plasmid of B. burgdorferi sensu lato Ip2l. All four 32-kb plasmids can be maintained within a single bacterium, which may provide a model for the study of plasmid replication and segregation in B. burgdorferi.


Compound heterozygosity for loss-of-function lysyl-tRNA synthetase mutations in a patient with peripheral neuropathy.
Charcot-Marie-Tooth (CMT) disease comprises a genetically and clinically heterogeneous group of peripheral nerve disorders characterized by impaired distal motor and sensory function. Mutations in three genes encoding aminoacyl-tRNA synthetases (ARSs) have been implicated in CMT disease primarily associated with an axonal pathology. ARSs are ubiquitously expressed, essential enzymes responsible for charging tRNA molecules with their cognate amino acids. To further explore the role of ARSs in CMT disease, we performed a large-scale mutation screen of the 37 human ARS genes in a cohort of 355 patients with a phenotype consistent with CMT. Here we describe three variants (p.Leu133His, p.Tyr173SerfsX7, and p.Ile302Met) in the lysyl-tRNA synthetase (KARS) gene in two patients from this cohort. Functional analyses revealed that two of these mutations (p.Leu133His and p.Tyr173SerfsX7) severely affect enzyme activity. Interestingly, both functional variants were found in a single patient with CMT disease and additional neurological and non-neurological sequelae. Based on these data, KARS becomes the fourth ARS gene associated with CMT disease, indicating that this family of enzymes is specifically critical for axon function.


Oligomeric states in sodium ion-dependent regulation of cyanobacterial histidine kinase-2.
Two-component signal transduction systems (TCSs) consist of sensor histidine kinases and response regulators. TCSs mediate adaptation to environmental changes in bacteria, plants, fungi and protists. Histidine kinase 2 (Hik2) is a sensor histidine kinase found in all known cyanobacteria and as chloroplast sensor kinase in eukaryotic algae and plants. Sodium ions have been shown to inhibit the autophosphorylation activity of Hik2 that precedes phosphoryl transfer to response regulators, but the mechanism of inhibition has not been determined. We report on the mechanism of Hik2 activation and inactivation probed by chemical cross-linking and size exclusion chromatography together with direct visualisation of the kinase using negative-stain transmission electron microscopy of single particles. We show that the functional form of Hik2 is a higher-order oligomer such as a hexamer or octamer. Increased NaCl concentration converts the active hexamer into an inactive tetramer. The action of NaCl appears to be confined to the Hik2 kinase domain.


Metal levels in the bones and livers of globally threatened marbled teal and white-headed duck from El Hondo, Spain.
El Hondo is a key area for marbled teal and white-headed duck. We present Pb, Cu, Zn, Se, and As data for bone and liver in birds found dead between 1996 and 2001. Several metals were higher in adult white-headed ducks than in marbled teal. They were higher in female than in male white-headed ducks, and did not differ with sex in marbled teal, but did by age. Lead in liver of adults was influenced by Pb shot ingestion, which was detected in 21% of marbled teal and in 71% of white-headed duck. No marbled teal had liver levels indicative of Pb poisoning, while 86% of white-headed ducks did. Selenium, Zn, and Cu were elevated in 13%, 7%, and 39% of birds, respectively. Whilst Pb shot poses the greatest threat to these species, further work should assess exposure via plants, invertebrates, water, and sediments for other metals, and investigate possible sub-lethal effects.


Waist circumference and metabolic syndrome: the risk for silent coronary artery disease in males.
Waist circumference (WC) is a component used to define metabolic syndrome. However, its role as an independent predictor of silent coronary artery disease (CAD), above its contribution to metabolic syndrome, remains unknown. Male veterans without known CAD, undergoing cardiac stress testing for indications other than typical angina or its equivalent, were evaluated for the presence of silent CAD. High WC and metabolic syndrome were defined per the revised National Cholesterol Education Program (NCEP-R) and the International Diabetes Federation (IDF) criteria. Data on 1,071 patients (age 61±11 years) were analyzed retrospectively. On multivariable logistic regression analysis [odds ratio (OR), 95% confidence interval (CI), P value), a WC ≥94 cm (1.42, 1.04-1.93; P=0.026), metabolic syndrome by NCEP-R (1.73, 1.29-2.33; P<0.0001), and metabolic syndrome by IDF (1.57, 1.17-2.11; P=0.003) were independent predictors of silent CAD. When comparing patients meeting criteria for metabolic syndrome defined by either NCEP-R or IDF, the prevalence of silent CAD was not statistically different (P=0.86). The prevalence of silent CAD associated with a high WC was not inferior to that seen between silent CAD and metabolic syndrome as defined by either criterion. Last, among patients with metabolic syndrome defined by NCEP-R, those with a high WC as a defining component of metabolic syndrome had a higher prevalence of silent CAD (30% vs. 20%; P=0.026). A WC ≥94 cm in males is independently associated with an increased prevalence of silent CAD. In patients with metabolic syndrome, this prevalence is increased by the presence of high WC.


Hole mobility in porphyrin- and porphyrin-fullerene electropolymers.
Charge transport within films of several new types of electropolymerized porphyrin and porphyrin-fullerene dyad polymers was studied in order to obtain information on the suitability of these organic semiconductors for applications in solar energy conversion, sensor devices, etc. The films, prepared by electropolymerization on a conductive substrate, were immersed in acetonitrile and studied using chronocoulometric and cyclic voltammetric electrochemical methods. The charge diffusion coefficients were found to be dependent upon the electrolytic medium. Electrolyte anion size plays a significant role in determining the rate of migration of charge through the polymers, demonstrating that migration of positive charge is accompanied by migration of negative counterions. Bulkier anions markedly decrease the charge diffusion coefficient. This strong dependence suggests that anion mobility is the rate-limiting process for diffusional charge transport within the porphyrin polymer films and that the largest rates obtained are lower limits to the intrinsic cation mobility. With electrolytes containing the relatively small perchlorate anion, charge diffusion coefficients of the porphyrin polymers were similar to those reported for polyaniline under acidic conditions. The charge diffusion coefficient for a zinc porphyrin polymer was found to decrease 2 orders of magnitude in the presence of pyridine, suggesting that metal-containing porphyrins polymer films may have sensor applications. Cation (hole) mobilities previously reported in the literature for porphyrin-containing polymers with chemical structures quite different from those investigated here were much smaller than those found for the polymers in this study, but further investigation suggests that the differences are due to choice of electrode size and material.


Transgenerational influence of parental morphine exposure on pain perception, anxiety-like behavior and passive avoidance memory among male and female offspring of Wistar rats.
Accumulating evidence suggests that epigenetic mechanisms play an important role in the formation and maintenance of memory within the brain. Moreover, the effect of parental drug-exposure before gestation on behavioral state of offspring has been little studied. The main objective of the current study is to evaluate the effect of parental morphine exposure on avoidance memory, morphine preference and anxiety-like behavior of offspring. The total of 32 males and 32 females were used for mating. The animals were treated with morphine. The offspring according to their parental morphine treatment was divided into four groups (n=16) including paternally treated, maternally treated, both of parents treated and naïve animals. The pain perception, anxiety-like behavior, and avoidance memory were evaluated in the offspring. In the current study, the total of 256 offspring was used for the experiments (4 tasks × 4 groups of offspring × 8 female offspring × 8 male offspring). The finding revealed that the avoidance memory and visceral pain were reduced significantly in male and female offspring with at least one morphine-treated parent. Moreover, anxiety-like behavior was reduced significantly in the male offspring with at least one morphine-treated parent. While anxiety-like behavior was increased significantly in female offspring that were treated by morphine either maternally or both of parents. The data revealed that the endogenous opioid system may be altered in the offspring of morphine-treated parent(s), and epigenetic role could be important. However, analysis of variance signified the important role of maternal inheritance.


Palonosetron for the prevention of chemotherapy-induced nausea and vomiting: approval and efficacy.
Chemotherapy-induced nausea and vomiting (CINV) is associated with a significant deterioration in quality of life. The emetogenicity of the chemotherapeutic agents, repeated chemotherapy cycles, and patient characteristics (female gender, younger age, low alcohol consumption, history of motion sickness) are the major risk factors for CINV. This review provides a detailed description of palonosetron, a second-generation 5-hydroxytryptamine 3 (5-HT(3)) receptor antagonist. The chemistry and pharmacology of palonosetron are described, as well as the initial and recent clinical trials. Palonosetron has a longer half-life and a higher binding affinity than the first-generation 5-HT(3) receptor antagonists. Palonosetron has been approved for the prevention of acute CINV in patients receiving either moderately or highly emetogenic chemotherapy and for the prevention of delayed CINV in patients receiving moderately emetogenic chemotherapy. In recent studies, compared to the first-generation 5-HT(3) receptor antagonists, palonosetron in combination with dexamethasone demonstrated better control of delayed CINV in patients receiving highly emetogenic chemotherapy. There were no clinically relevant adverse reactions reported in the palonosetron clinical trials which were different from the common reactions reported for the 5-HT(3) receptor antagonist class. Due to its efficacy in controlling both acute and delayed CINV, palonosetron may be very effective in the clinical setting of multiple-day chemotherapy and bone marrow transplantation.


Predictive factors for failure of nonoperative management in perforated appendicitis.
Identifying patients on admission with perforated appendicitis who have phlegmon or abscess initially selected for but likely to fail nonoperative management may avoid delays in definitive treatment. Patients older than 15 years presenting to a university tertiary care hospital with perforated appendicitis and abscess or phlegmon and planned nonoperative management were reviewed. Comorbidities, clinical findings, laboratory markers, radiographic findings, and nonsurgical treatments associated with failure of nonoperative management were recorded. Eighty-nine patients were identified, and 69 were managed successfully to discharge without operation. Length of stay was greater in the failure group (11 days vs. 5 days, p = 0.001), and intensive care unit care was more common (10% vs. 0%, p = 0.049). On univariate and multivariate analyses, smoking (odds ratio [OR], 13.20; 95% confidence interval [CI], 1.13-142; p = 0.039), tachycardia (OR, 4.93; 95% CI, 1.21-20.06; p = 0.026), and generalized abdominal tenderness (OR, 5.52; 95% CI, 1.40-21.73; p = 0.015) were associated with failure of nonoperative management. On computed tomographic scan, the failure group had higher rates of abscess (75% vs. 55%, p = 0.110), and their abscesses were more likely smaller than 50 mm (OR, 2.83; 95% CI, 1.01-7.92; p = 0.043). Patients with perforated appendicitis and phlegmon or abscess who smoke or present with tachycardia, generalized abdominal tenderness, and abscesses smaller than 50 mm are more likely to fail nonoperative management and should be considered for early operation. These findings should be validated prospectively. Therapeutic study, level III.


Airway remodelling assessed by sputum and high-resolution computed tomography in asthma and COPD.
It is not known whether sputum elastase, metalloproteinase (MMP)-9 and tissue-inhibitor metalloproteinase (TIMP)-1 are related to structural changes of the airways, as assessed by high-resolution computed tomography (HRCT) scan. The relationships between these markers and the magnitude of structural changes of the airways in asthma and chronic obstructive pulmonary disease (COPD) were assessed. Induced sputum and HRCT scan were performed in 30 asthmatics (14 mild and 16 severe) and in 12 patients with COPD. A greater extent of HRCT scan abnormalities was found in COPD than in severe and mild asthmatics. HRCT scan abnormalities correlated with the degree of airway obstruction in COPD and in severe asthma. HRCT scan abnormalities also correlated with the levels of sputum elastase both in COPD and in severe asthma. HRCT scan abnormalities were associated with sputum MMP-9/TIMP-1 ratio in mild asthma, severe asthma and COPD. In conclusion, this study demonstrates that sputum elastase and the metalloproteinase-9/tissue-inhibitor metalloproteinase-1 ratio are associated with the magnitude of high-resolution computed tomography scan abnormalities of the airways in asthma and chronic obstructive pulmonary disease, and suggests that the levels of these markers reflect the extent of structural changes of the airways.


Analysis of bio-obtainable endocrine disrupting metals in river water and sediment, sewage influent/effluent, sludge, leachate, and concentrated leachate, in the irish midlands shannon catchment.
The application of an acid digestion and subsequent solid-phase extraction (SPE) procedure were implemented as preliminary treatments prior to quantifying the levels of potentially endocrine disrupting metals (EDMs) in a variety of solid and liquid matrices. These included (solid) river sediment, leachate sediment and sewage sludge and also (liquid) river water, landfill leachate, concentrated leachate, sewage influent, and sewage effluent, sampled in the Irish Midlands. The total concentrations of cobalt (Co), cadmium (Cd), copper (Cu), chromium (Cr), nickel (Ni), lead (Pb), zinc (Zn), and manganese (Mn), after extraction and preconcentration, were determined by atomic absorption spectroscopy (AAS). Mercury (Hg) in sediment and sludge was determined using cold-vapour atomic fluorescence spectroscopy (AFS). For sewage sludge maximum values (mg/kg(dw)) of 4700 Ni, 1642 Mn, 100.0 Cd, 3400 Zn, 36.70 Co, 750.0 Pb, 485.8 Cr, and 1003 Cu were determined whilst in leachate sediment, maximum values (mg/kg(dw)) of 32.10 Ni, 815.0 Mn, 32.78 Cd, 230.3 Zn, 26.73 Co, 3525 Pb, 124.9 Cr, and 50.13 Cu were found. Over several months, the data showed elevated levels in sewage influents, effluents, and sludges compared to a battery of adjacent river water samples and corresponding sediments. There was a definite trend for target values for sediments to be exceeded, while intervention values were only exceeded for cadmium. Overall the pattern in terms of concentration was sewage > leachate > river matrices. A nonparametric assessment of the effect of sewage treatment method on median metal levels in sludge revealed statistically significant differences at the 95% level of confidence for Co, Cr, and Hg and at the 90% level of confidence for Cd.


Anaerobic activation of the entire denitrification pathway in Pseudomonas aeruginosa requires Anr, an analog of Fnr.
The Pseudomonas aeruginosa gene anr, which encodes a structural and functional analog of the anaerobic regulator Fnr in Escherichia coli, was mapped to the SpeI fragment R, which is at about 59 min on the genomic map of P. aeruginosa PAO1. Wild-type P. aeruginosa PAO1 grew under anaerobic conditions with nitrate, nitrite, and nitrous oxide as alternative electron acceptors. An anr deletion mutant, PAO6261, was constructed. It was unable to grow with these alternative electron acceptors; however, its ability to denitrify was restored upon the introduction of the wild-type anr gene. In addition, the activities of two enzymes in the denitrification pathway, nitrite reductase and nitric oxide reductase, were not detectable under oxygen-limiting conditions in strain PAO6261 but were restored when complemented with the anr+ gene. These results indicate that the anr gene product plays a key role in anaerobically activating the entire denitrification pathway.


A Memetic Algorithm for Periodic Capacitated Arc Routing Problem.
This paper investigates the Periodic Capacitated Arc Routing Problem (PCARP), which is often encountered in the waste collection application. PCARP is an extension of the well-known Capacitated Arc Routing Problem (CARP) from a single period to a multi-period horizon. PCARP is a hierarchical optimization problem which has a primary objective (minimizing the number of vehicles ) and a secondary objective (minimizing the total cost ). An important factor that makes PCARP challenging is that its primary objective is little affected by existing operators and thus difficult to improve. We propose a new Memetic Algorithm (MA) for solving PCARP. The MA adopts a new solution representation scheme and a novel crossover operator. Most importantly, a Route-Merging (RM) procedure is devised and embedded in the algorithm to tackle the insensitive objective . The MA with RM (MARM) has been compared with existing meta-heuristic approaches on two PCARP benchmark sets and a real-world data set. The experimental results show that MARM obtained better solutions than the compared algorithms in much less time, and even updated the best known solutions of all the benchmark instances. Further study reveals that the RM procedure plays a key role in the superior performance of MARM.


Anti-inflammatory and analgesic effects of egg yolk: a comparison between organic and machine made.
Yolk composition may be influenced by the bird's dietary intake. Quality of egg yolk from different sources is also varied. Especially egg yolk from organic sources, those prepared by the hen and duck were naturally fed. Traditional medicine users in north of Iran believe the oil extracted by direct heat from egg yolk has analgesic activity. In this work anti-inflammatory and antinociceptive activities of organic and machine made hen yolk and duck yolk were evaluated. Three various sources of egg yolk (organic hens, duck and machine made) were chosen. Each yolk was extracted by two methods, solvent extraction (hexane) and direct heating. In order to investigate the analgesic and anti-inflammatory activity, carageenan, hot plate and writhing tests were carried out. The organic hen and duck oil extracted by direct heat method showed the highest activity compared to other types. Organic sources may contain some special components which are not existed in machine made eggs. Oil samples obtained by direct heat from three different kinds of egg yolk showed significant anti-inflammatory activity at 100-300 mg/kg compared to controls (p < 0.001). All samples indicated analgesic properties at all doses in writhing test. The highest activity was seen at 300 mg/kg of organic hen egg yolk oil prepared by heat (HO) and hexane extract of duck egg yolk (ED) which was similar to 5 mg/kg morphine (p > 0.001). In hot plate test all of the oil samples did not show significant difference with controls. Hexane extract of organic egg yolk (EO) (300 mg/kg) and ED (200 and 300 mg/kg) showed significant analgesic activity in hot plate test. This study confirms anti-inflammatory and analgesic scheme of yolk. We can also claim environmental and dietary condition is very important in yolk contents. In order to improve life quality of human beings and better efficiency for egg yolk components we suggest considering the way of feeding for poultry. We can propose organic yolk oil prepared by direct heat as an alternative remedy for inflammatory joint conditions.


Genome characterization of lipid-containing marine bacteriophage PM2 by transposon insertion mutagenesis.
Bacteriophage PM2 presently is the only member of the Corticoviridae family. The virion consists of a protein-rich lipid vesicle, which is surrounded by an icosahedral protein capsid. The lipid vesicle encloses a supercoiled circular double-stranded DNA genome of 10,079 bp. PM2 belongs to the marine phage community and is known to infect two gram-negative Pseudoalteromonas species. In this study, we present a characterization of the PM2 genome made using the in vitro transposon insertion mutagenesis approach. Analysis of 101 insertion mutants yielded information on the essential and dispensable regions of the PM2 genome and led to the identification of several new genes. A number of lysis-deficient mutants as well as mutants displaying delayed- and/or incomplete-lysis phenotypes were identified. This enabled us to identify novel lysis-associated genes with no resemblance to those previously described from other bacteriophage systems. Nonessential genome regions are discussed in the context of PM2 genome evolution.


Prevalence of the mutational pattern E44D/A and/or V118I in the reverse transcriptase (RT) gene of HIV-1 in relation to treatment with nucleoside analogue RT inhibitors.
It has been reported that a new pattern of mutations, E44D/A and/or V118I, in the reverse transcriptase (RT) gene of HIV-1 confers a moderate level of resistance to lamivudine in the absence of the M184V mutation. The prevalence of this mutational pattern was studied in HIV-1 isolates obtained from 280 patients. These mutations were not identified in the RT sequences from 23 antiretroviral-naive patients but were detected in 82 (31.9%) of the 257 RT sequences obtained from nucleoside reverse transcriptase inhibitors (NRTI)-experienced patients. Mutation at codon 44 was identified in 41 patients (7 mutations E44A and 34 mutations E44D), mutation V118I was identified in 73 patients and a combination of mutations at codons 44 and 118 was found in 32 patients. Multivariate analysis showed an association between the E44D/A and/or V118I mutational pattern and the RT mutations D67N, T69D, L210W, and T215Y/F. No relationship was observed between this mutational pattern and the lamivudine-specific resistance mutation M184V. The prevalence of these mutations increased significantly with the number of drug regimens experienced and a prevalence of 42.4% was observed in patients who had received >or= 4 antiretroviral regimens. A relationship was found between the E44D/A and/or V118I mutational pattern and experience with didanosine or stavudine but not with lamivudine. The results suggest that the development of the E44D/A and/or V118I mutational pattern is frequent in patients treated with NRTIs. Thymidine analogues and didanosine, but not lamivudine, could promote the development of these mutations.


The effect of precision of molecular orbital descriptors on toxicity modeling of selected pyridines.
The response-surface approach to QSARs attempts to model toxic potency of diverse groups of chemicals while avoiding problems associated with the identification of the mechanism of toxic action or specific chemical class often associated with other approaches. However, while hydrophobicity-dependent, simple regression QSARs derived for congeneric series of organic compounds typically have coefficients of determination greater than 0.90, more heterogeneous multiple regression QSARs exhibit typically 10-15% more unexplained variability. One difference between these approaches is the use of a quantum chemical (QC) descriptor, particularly molecular orbital (MO) energy values such as the energy of the lowest unoccupied molecular orbital (E(LUMO)). The reduced statistical fit exhibited by QSAR models, which include these QC-MO descriptors, could be a result of the variability inherent in the calculation of these descriptors. The present investigation with a structurally and mechanistically diverse set of pyridines revealed that variability is associated with the calculation of the MO descriptor E(LUMO) both between selected Hamiltonians and selected software packages. However, this variability in no way affects the statistical significance of QSARs for toxicity using these values. Specifically, the E(LUMO) values calculated with the PM3 and AM1 Hamiltonians in the two software packages were highly related. There was no relationship between molecular complexity or chemical reactivity and increased differences in individual ELUMO values as described by the standard errors of the mean. Although nine appeared to be the number of calculations, which best minimizes the standard error in energy values relative to computational costs; this minimization did not alter the statistics of the QSARs derived with single vs. mean E(LUMO) values. While the energy of the highest occupied molecular orbital (E(HOMO)) values were not used in the modeling of toxicity, a comparison of these values revealed greater variability between the Hamiltonians and software packages than observed for ELUMO values. Examination of the magnitudes of standard error of the E(HOMO) values in connection to structural features or reactivity likewise revealed no trends.


Cardiovascular reactivity patterns and pathways to hypertension: a multivariate cluster analysis.
Substantial evidence links exaggerated mental stress induced blood pressure reactivity to future hypertension, but the results for heart rate reactivity are less clear. For this reason multivariate cluster analysis was carried out to examine the relationship between heart rate and blood pressure reactivity patterns and hypertension in a large prospective cohort (age range 55-60 years). Four clusters emerged with statistically different systolic and diastolic blood pressure and heart rate reactivity patterns. Cluster 1 was characterised by a relatively exaggerated blood pressure and heart rate response while the blood pressure and heart rate responses of cluster 2 were relatively modest and in line with the sample mean. Cluster 3 was characterised by blunted cardiovascular stress reactivity across all variables and cluster 4, by an exaggerated blood pressure response and modest heart rate response. Membership to cluster 4 conferred an increased risk of hypertension at 5-year follow-up (hazard ratio=2.98 (95% CI: 1.50-5.90), P<0.01) that survived adjustment for a host of potential confounding variables. These results suggest that the cardiac reactivity plays a potentially important role in the link between blood pressure reactivity and hypertension and support the use of multivariate approaches to stress psychophysiology.


Dosage transmission disequilibrium test (dTDT) for linkage and association detection.
Both linkage and association studies have been successfully applied to identify disease susceptibility genes with genetic markers such as microsatellites and Single Nucleotide Polymorphisms (SNPs). As one of the traditional family-based studies, the Transmission/Disequilibrium Test (TDT) measures the over-transmission of an allele in a trio from its heterozygous parents to the affected offspring and can be potentially useful to identify genetic determinants for complex disorders. However, there is reduced information when complete trio information is unavailable. In this study, we developed a novel approach to "infer" the transmission of SNPs by combining both the linkage and association data, which uses microsatellite markers from families informative for linkage together with SNP markers from the offspring who are genotyped for both linkage and a Genome-Wide Association Study (GWAS). We generalized the traditional TDT to process these inferred dosage probabilities, which we name as the dosage-TDT (dTDT). For evaluation purpose, we developed a simulation procedure to assess its operating characteristics. We applied the dTDT to the simulated data and documented the power of the dTDT under a number of different realistic scenarios. Finally, we applied our methods to a family study of alcohol dependence (COGA) and performed individual genotyping on complete families for the top signals. One SNP (rs4903712 on chromosome 14) remained significant after correcting for multiple testing Methods developed in this study can be adapted to other platforms and will have widespread applicability in genomic research when case-control GWAS data are collected in families with existing linkage data.


Interhospital variation in reperfusion rates following endovascular treatment for acute ischemic stroke.
Patients who have successful reperfusion following endovascular therapy for acute ischemic stroke have improved clinical outcomes. We sought to determine if the chance of successful reperfusion differs among hospitals, and if hospital site is an independent predictor of reperfusion. Nine hospitals recruited patients in the Diffusion and Perfusion Imaging Evaluation for Understanding Stroke Evolution Study 2 (DEFUSE 2), a prospective cohort study of endovascular stroke treatment conducted between 2008 and 2011. Patients were included for analysis if they had a baseline Thrombolysis in Cerebral Infarction (TICI) score of 0 or 1. Successful reperfusion was defined as a TICI reperfusion score of 2b or 3 at completion of the procedure. Collaterals were assessed using the Collateral Flow Grading System and were dichotomized as poor (0-2) or good (3-4). The association between hospital site and successful reperfusion was first assessed in an unadjusted analysis and subsequently in a multivariate analysis that adjusted for predictors of successful reperfusion. 36 of 89 patients (40%) achieved successful reperfusion. The rate of reperfusion varied from 0% to 77% among hospitals in the univariate analysis (χ(2) p<0.001) but hospital site did not remain as an independent predictor of reperfusion in multivariate analysis (p=0.81) after adjustment for the presence of good collaterals (p<0.01) and use of the Merci retriever (p<0.05). Reperfusion rates vary among hospitals, which may be related to differences in treatment protocols and patient characteristics. Additional studies are needed to identify all of the factors that underlie this variability as this could lead to strategies that reduce interhospital variability in reperfusion rates and improve clinical outcomes.


The use of neoadjuvant larotrectinib in the management of children with locally advanced TRK fusion sarcomas.
The highly selective oral tropomyosin receptor kinase (TRK) inhibitor larotrectinib has demonstrated significant activity in adult and pediatric TRK fusion cancers. In the current study, the authors describe the clinical course of children with locally advanced TRK fusion sarcoma who were treated preoperatively with larotrectinib and underwent subsequent surgical resection. A total of 24 children were treated on a pediatric phase 1 trial of larotrectinib (ClinicalTrials.gov identifier NCT02637687). Five children who had a documented TRK fusion sarcoma and underwent surgical resection were included in the current analysis. Tumor response (Response Evaluation Criteria In Solid Tumors [RECIST] version 1.1) and surgical outcomes were collected prospectively. A total of 5 patients (median age, 2 years; range, 0.4-12 years) had locally advanced infantile fibrosarcoma (3 patients) or soft-tissue sarcoma (2 patients). Four patients had disease that was refractory to standard therapy. All 5 patients achieved a partial response to larotrectinib by version 1.1 of RECIST and underwent surgical resection after a median of 6 cycles (range, 4-9 cycles) of treatment. Surgical resections were R0 (negative resection margins with no tumor at the inked resection margin) in 3 patients, R1 (microscopic residual tumor at the resection margin) in 1 patient, and R2 (macroscopic residual tumor at the resection margin) in 1 patient. Three patients achieved complete (2 patients) or near-complete (>98% treatment effect; 1 patient) pathologic responses. These patients remained in follow-up and were no longer receiving larotrectinib for a minimum of 7 to 15 months postoperatively. Two patients had viable tumor at the time of surgical resection and positive resection margins and continued to receive adjuvant larotrectinib. No patients experienced postoperative complications or wound healing issues. Children with locally advanced TRK fusion sarcomas may proceed to surgical resection after treatment with the selective TRK inhibitor larotrectinib, thereby sparing them the potentially significant morbidity noted with current approaches. These results support the evaluation of larotrectinib as presurgical therapy in children with newly diagnosed TRK fusion sarcomas.


Tailor-made ezrin actin binding domain to probe its interaction with actin in-vitro.
Ezrin, a member of the ERM (Ezrin/Radixin/Moesin) protein family, is an Actin-plasma membrane linker protein mediating cellular integrity and function. In-vivo study of such interactions is a complex task due to the presence of a large number of endogenous binding partners for both Ezrin and Actin. Further, C-terminal actin binding capacity of the full length Ezrin is naturally shielded by its N-terminal, and only rendered active in the presence of Phosphatidylinositol bisphosphate (PIP2) or phosphorylation at the C-terminal threonine. Here, we demonstrate a strategy for the design, expression and purification of constructs, combining the Ezrin C-terminal actin binding domain, with functional elements such as fusion tags and fluorescence tags to facilitate purification and fluorescence microscopy based studies. For the first time, internal His tag was employed for purification of Ezrin actin binding domain based on in-silico modeling. The functionality (Ezrin-actin interaction) of these constructs was successfully demonstrated by using Total Internal Reflection Fluorescence Microscopy. This design can be extended to other members of the ERM family as well.


Advantages of a modified scoring method for the Rush Video-Based Tic Rating Scale.
Previously, we published a video-based objective rating scale of tics that met reliability and validity criteria for measurement of five domains of tic disability. In the original form, the scale's metric properties did not permit internal comparison of each of the five domains of impairment and did not provide a total score for use as a primary outcome measure. In this study, we retained the original scale and videotape protocol but tested whether a modified scoring system corrected these limitations. The new scoring method rated assigned tic data to ratings of 0-4 on five disability categories: number of body areas, frequency of motor tics, frequency of phonic tics, severity of motor tics, and severity of phonic tics. The sums of these ratings yielded a total score of overall tic disability (0-20). In a series of 31 patients with Gilles de la Tourette syndrome, we assessed Spearman correlation coefficients for the old and new scoring systems as well as the correlation of the new ratings with the objectively derived sections of the Yale Global Tic Severity Scale (YGTSS), another valid and reliable scale used in clinical practice and research. For each domain, the rank order for the scores on the original scale was well retained in the new scores. Likewise, for each domain, ranking with the new scoring system correlated well with scores on the comparable objective item from the YGTSS. The new total score accurately captured the rank order of the combined five domains from the original scale and correlated well with the total objective motor plus phonic tic score from the YGTSS and the YGTSS Tourette Syndrome Overall Impairment Rating. These data demonstrate that the modified videotape-based scoring system retains the essential information gathered in the original Rush scale. The modification provides comparisons among the five assessed domains and a total objectively based disability score that can be used as a single outcome measure for assessing tic disability.


Delta9-tetrahydrocannabivarin as a marker for the ingestion of marijuana versus Marinol: results of a clinical study.
Delta9-tetrahydrocannabinol (THC), the main psychologically active ingredient of the cannabis plant (marijuana), has been prepared synthetically and used as the bulk active ingredient of Marinol, which was approved by the FDA for the control of nausea and vomiting in cancer patients receiving chemotherapy and as an appetite stimulant for AIDS patients. Because the natural and the synthetic THC are identical in all respects, it is impossible to determine the source of the urinary metabolite of THC, 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH), in a urine specimen provided in a drug-testing program. Over the last few years there has been a need to determine whether a marijuana positive drug test is the result of the ingestion of marijuana (or a related product) or whether it results from the sole use of Marinol. We have previously proposed the use of delta9-tetrahydrocannabivarin (THCV, the C3 homologue of THC) as a marker for the ingestion of marijuana (or a related product) because THCV is a natural component of most cannabis products along with THC and does not exist in Marinol. We have also reported that THCV is metabolized by human hepatocytes to 11-nor-delta9-tetrahydrocannabivarin-9-carboxylic acid (THCV-COOH); therefore, the presence of the latter in a urine specimen would indicate that the donor must have used marijuana or a related product (with or without Marinol). In this study, we provide clinical data showing that THCV-COOH is detected in urine specimens collected from human subjects only after the ingestion of marijuana and not after the ingestion of Marinol (whether the latter is ingested orally or by smoking). Four subjects (male and female) participated in the study in a three-session, within-subject, crossover design. The sessions were conducted at one-week intervals. Each subject received, in separate sessions and in randomized order, an oral dose of Marinol (15 mg), a smoked dose of THC (16.88 mg) in a placebo marijuana cigarette, or a smoked dose of marijuana (2.11% THC and 0.12% THCV). Urine samples were collected and vital signs were monitored every 2 h for a 6-h period following drug administration. Subjects were then transported home, were given sample collection containers and logbooks, and were instructed to record at home the volume and time of every urine collection for 24 h, and once a day for the remainder of a week (6 days). Subjects were also instructed to freeze the urine samples until the next session. All urine samples were analyzed by GC-MS for THC-COOH and THCV-COOH using solid-phase extraction and derivatization procedure on RapidTrace and TBDMS as the derivative. The method had a limit of detection of 1.0 ng/mL and 1.0 ng/mL for THCV-COOH and THC-COOH, respectively.


Bone turnover markers in the management of postmenopausal osteoporosis.
Osteoporosis is the most common cause of fragility fractures. Bone remodelling is essential for repairing damaged areas within bone to preserve bone strength and for assisting in mineral homeostases. In young adults, bone remodelling is usually balanced with approximately as much bone replaced as is removed during each remodelling cycle. However, when remodelling becomes accelerated in combination with an imbalance that favours bone resorption over formation, such as during menopause, precipitous losses in bone mass occur. Bone turnover markers (BTMs) measure the rate of bone remodelling allowing for a dynamic assessment of skeletal status and hold promise in identifying those at highest risk of rapid bone loss and subsequent fracture. Further, the use of BTMs to monitor individuals administered osteoporosis therapy is attractive as monitoring anti-fracture efficacy with bone mineral density has significant limitations. This review details remodelling biology, pre-analytical and analytical sources of variability for BTMs, describes the most commonly used resorption and formation markers, and offers some guidelines for their use and interpretation in the laboratory and the clinic.


Beam shaping using Gaussian beam modes.
A beam shaping method is presented where a diffractive optical element (DOE) is designed by optimizing the complex mode coefficient weights of a set of Gaussian beam modes. This method is compared with the more standard unidirectional approach. Differential evolution is used for the optimization in both the unidirectional and Gaussian beam mode optimization methods. For the particular transforms carried out, the Gaussian beam mode set optimization (GBMSO) approach achieved more optimal solutions. The GBMSO approach is extended to design DOEs that control the amplitude distribution of a beam at multiple planes, rather than at just a single plane (i.e., the far field).


The short form of the revised almost perfect scale.
We created a shorter and more refined item set from the Almost Perfect Scale-Revised (APS-R; Slaney, Mobley, Trippi, Ashby, & Johnson, 1996; Slaney, Rice, Mobley, Trippi, & Ashby, 2001) to measure 2 major dimensions of perfectionism: standards (high performance expectations) and discrepancy (self-critical performance evaluations). In Study 1, after testing the internal structure of the measure (N = 749), a subset of the current APS-R items was derived (Short Almost Perfect Scale [SAPS]) that possessed good psychometric features, such as strong item-factor loadings, score reliability, measurement invariance between women and men, and criterion-related validity through associations with neuroticism, conscientiousness, academic performance, and depression. Controlling for neuroticism and conscientiousness, factor mixture modeling supported a 2-factor, 3-class model of perfectionism, and results were consistent with labeling the classes as nonperfectionists and adaptive and maladaptive perfectionists. Measurement results were cross-validated in a separate sample (N = 335). Study 2 also provided substantial evidence for the convergent, discriminant, and criterion-related validity of SAPS scores. Both studies supported the SAPS as a brief and psychometrically strong measure of major perfectionism factors and classes of perfectionists.


Epidemiology of ankle arthritis: report of a consecutive series of 639 patients from a tertiary orthopaedic center.
The purpose of our study was to identify the cause of symptomatic ankle arthritis in a consecutive series of patients presenting in a tertiary care setting. Between 1991 and 2004, 639 patients with Kellgren grade 3 or 4 ankle arthritis presented to the University of Iowa Orthopaedic Foot and Ankle Surgery service. The cause of the arthritis was determined based on medical history, physical examination, and imaging studies. To get a sense of the relative prevalence of the etiologies of lower extremity arthritis in our setting, we evaluated the cause of arthritis of all new patients presenting to the University of Iowa Orthopaedic Department from 1999-2004 with arthritis of the ankle, to those with arthritis of the hip or knee during one year. Of the 639 arthritic ankles, 445 (70%) were post-traumatic, 76 (12%) were rheumatoid disease and 46 (7%) were idiopathic (primary osteoarthritis). The post-traumatic ankle arthritis patients were most commonly associated with past rotational ankle fractures. The majority of ankle arthritis is associated with previous trauma, whereas the primary cause of knee or hip arthritis is idiopathic. Unique strategies to prevent or treat post-traumatic ankle arthritis are needed.


Scintigraphic assessment of patellar vascularity in total knee replacement surgeries following lateral release.
For a total knee replacement (TKR) to function well, the patella must track centrally in the trochlear groove. Lateral retinacular release (LR) is performed as an integral step in TKR to avoid maltracking. Patellar resurfacing and infra patellar fat pad excision are other small surgical procedures performed during TKR that can also easily deprive the patella of its vascularity. A three phase bone scan helps in the assessment of patellar vascularity. Literature review reveals a variable association (10-56%) of LR and patellar hypovascularity in Caucasians. LR release done in TKR is known to compromise patellar viability, while its extent and severity is still debated. Our prospective study was undertaken to evaluate the effects of LR on patella along with other variables like patellar resurfacement, fat pad excision, patelloplasty on knees by 99mTechnetium labeled methylene diphosphonate (99mTc MDP) three phase bone scintigraphy (TBS). 45 TKRs were done between Jan 05 and Jan 06. Of them, 15 patients (21 knees) of primary TKR were considered prospectively. Patients undergoing uni/bilateral TKR due to symptomatic knee osteoarthritis were only selected for this study. Pre- and immediate postoperative TBS was done with modified positioning of knee joints (adducted, externally rotated and flexed to 30°) to visualize patellae away from knee joint, which otherwise gets superimposed on the femoral condyles. A follow-up TBS at 8-10 weeks was done in patients showing immediate post-op patellar hypovascularity. 12/21 knees (57%) needed LR. Of these 12 knees, 8 (66%) showed hypovascularity; whereas 9 knees with no LR, 1 knee showed hypovascularity. All 9 knees (8 post LR+1 without LR) with hypovascularity underwent TBS again at 8-10 weeks and were found to show improvement in their vascularity status in all cases. Our study showed a higher incidence of LR in our population (57%). Association of LR and patellar ischemia was significant, about 16 times the risk. MDP bone scintigraphy is the only objective tool to quantify patellar vascularity. Overall 42.8% knees post-TKR had a transient patellar ischemia, and 38% of them were following LR. All of them recovered at 8-10 weeks postoperatively.


A biphenyl inhibitor of eIF4E targeting an internal binding site enables the design of cell-permeable PROTAC-degraders.
The eukaryotic translation initiation factor 4E (eIF4E) is the master regulator of cap-dependent protein synthesis. Overexpression of eIF4E is implicated in diseases such as cancer, where dysregulation of oncogenic protein translation is frequently observed. eIF4E has been an attractive target for cancer treatment. Here we report a high-resolution X-ray crystal structure of eIF4E in complex with a novel inhibitor (i4EG-BiP) that targets an internal binding site, in contrast to the previously described inhibitor, 4EGI-1, which binds to the surface. We demonstrate that i4EG-BiP is able to displace the scaffold protein eIF4G and inhibit the proliferation of cancer cells. We provide insights into how i4EG-BiP is able to inhibit cap-dependent translation by increasing the eIF4E-4E-BP1 interaction while diminishing the interaction of eIF4E with eIF4G. Leveraging structural details, we designed proteolysis targeted chimeras (PROTACs) derived from 4EGI-1 and i4EG-BiP and characterized these on biochemical and cellular levels. We were able to design PROTACs capable of binding eIF4E and successfully engaging Cereblon, which targets proteins for proteolysis. However, these initial PROTACs did not successfully stimulate degradation of eIF4E, possibly due to competitive effects from 4E-BP1 binding. Our results highlight challenges of targeted proteasomal degradation of eIF4E that must be addressed by future efforts.


Characterizing early child growth patterns of height-for-age in an urban slum cohort of Bangladesh with functional principal component analysis.
Early childhood is a critical stage of physical and cognitive growth that forms the foundation of future wellbeing. Stunted growth is presented in one of every 4 children worldwide and contributes to developmental impairment and under-five mortality. Better understanding of early growth patterns should allow for early detection and intervention in malnutrition. We aimed to characterize early child growth patterns and quantify the change of growth curves from the World Health Organization (WHO) Child Growth Standards. In a cohort of 626 Bangladesh children, longitudinal height-for-age z-scores (HAZ) were modelled over the first 24 months of life using functional principal component analysis (FPCA). Deviation of individual growth from the WHO standards was quantified based on the leading functional principal components (FPCs), and growth faltering was detected as it occurred. The risk factors associated with growth faltering were identified in a linear regression. Ninety-eight percent of temporal variation in growth trajectories over the first 24 months of life was captured by two leading FPCs (FPC1 for overall growth and FPC2 for change in growth trajectory). A derived index, adj-FPC2, quantified the change in growth trajectory (i.e., growth faltering) relative to the WHO standards. In addition to HAZ at birth, significant risk factors associated with growth faltering in boys included duration of breastfeeding, family size and income and in girls maternal weight and water source. The underlying growth patterns of HAZ in the first 2 years of life were delineated with FPCA, and the deviations from the WHO standards were quantified from the two leading FPCs. The adj-FPC2 score provided a meaningful measure of growth faltering in the first 2 years of life, which enabled us to identify the risk factors associated with poor growth that would have otherwise been missed. Understanding faltering patterns and associated risk factors are important in the development of effective intervention strategies to improve childhood growth globally. ClinicalTrials.gov Identifier: NCT02734264 , registered 22 March, 2016.


Drug-inducible, dendritic cell-based genetic immunization.
Determining the mechanism of Ag loading of Langerhans cells (LC) for genetic immunization (GI) is complicated by the inability to distinguish between the response generated by direct transfection of LC from that due to exogenous uptake. To unravel this mechanism, we examined the impact of gene gun treatment on LC with respect to their activation and migration from skin, transgene expression, and ability to initiate humoral and cellular immune responses upon transfer to naive mice. To assess responses generated by direct LC transfection, an RU486-inducible expression system was used as a GI vector. In vitro skin organ cultures were developed from gene gun immunized mouse ear specimens to obtain LC. Gene gun treatment markedly augmented (3-fold) LC migration from ear skin, and these LC expressed the transgene at RNA and protein levels. Transfer of 2 x 10(5) migratory cells resulted in identical cellular responses to, but 10-fold lower humoral responses than, standard GI. Using an RU486-inducible system, we were able to measure responses generated by directly transfected LC. Our results indicate that direct transfection is a predominant pathway for LC Ag loading. The ability to regulate transgene expression with inducible DC-based vaccines demonstrates a new level of immunological control.


Vascular access for hemodialysis and cardiovascular complications.
Native arterio-venous fistula (AVF), is the first choice of the vascular access for hemodialysis treatment. In comparison with other vascular accesses such as prosthetic arterio-venous graft or permanent central venous catheter, AVF has shown a significant reduction in cardiovascular mortality. It needs, of course, an appropriate blood flow that provides a flow of 400-800 mL/min in the distal seat and a flow of 800-1500 ml/min in the proximal seat. As a consequence, the creation of AVF may be associated with changes in blood flow, pulmonary pressure and cardiac output. All these haemodynamic changes may not have any cardiac consequences, or they could lead to congestive heart disease particularly when the blood flow of AVF is greater than 2000 ml/min. For this reason, the patient's cardiac status should determine the choice of dialysis and the type of vascular access. Cardiac function monitoring is essential, particularly when dealing with patients at high risk for congestive heart disease and AVF in the proximal seat. Vascular access reduction surgery may be effective in some cases, otherwise a suitable alternative could be that of closing AVF. However, the likely side effects that can be generated by closing arterio-venous AVF such as a sudden increase of venous peripheral systemic should be taken into account.


Radiofrequency ablation of the great saphenous vein, comparing one versus two treatment cycles for the proximal vein segment.
To evaluate the results of radiofrequency ablation (RFA) of the great saphenous vein (GSV) using one versus two 20 s energy cycle treatment in the proximal 7 cm segment of the GSV. All patients who underwent RFA of the GSV from 1 May 2013 to 30 September 2013 in eight of our vein centers were included. Duplex ultrasound scans (DUSs) were performed prior to treatment on all patients and 2-3 days, and 1 month after procedure. Demographic data, GSV diameters, and other relevant data were recorded. Clinical, Etiologic, Anatomic, Pathologic (CEAP) classification and Venous Clinical Severity Scores (VCSSs) were determined prior to ablation and one month later. Patients who developed endovenous heat induced thrombosis (EHIT) were followed till resolution. A total of 205 patients had one cycle treatment (group A) and 204 had two cycle treatment (group B). The two groups were comparable in their demography, CEAP classification, and VCSS scores. The rate of failure of ablation and incidence of EHIT were also not significantly different. The incidence of complications was low, <5% in both groups and all were minor. Two cycle treatment of the proximal GSV for vein ablation does not improve the success rate of vein closure in the short term, compared to one cycle treatment. It also does not increase the risks of DVT, EHIT, major bleeding, and other complications. However, we do not know at what diameter two cycles may be superior to one cycle.


Discovery of a chemical probe for PRDM9.
PRDM9 is a PR domain containing protein which trimethylates histone 3 on lysine 4 and 36. Its normal expression is restricted to germ cells and attenuation of its activity results in altered meiotic gene transcription, impairment of double-stranded breaks and pairing between homologous chromosomes. There is growing evidence for a role of aberrant expression of PRDM9 in oncogenesis and genome instability. Here we report the discovery of MRK-740, a potent (IC50: 80 ± 16 nM), selective and cell-active PRDM9 inhibitor (Chemical Probe). MRK-740 binds in the substrate-binding pocket, with unusually extensive interactions with the cofactor S-adenosylmethionine (SAM), conferring SAM-dependent substrate-competitive inhibition. In cells, MRK-740 specifically and directly inhibits H3K4 methylation at endogenous PRDM9 target loci, whereas the closely related inactive control compound, MRK-740-NC, does not. The discovery of MRK-740 as a chemical probe for the PRDM subfamily of methyltransferases highlights the potential for exploiting SAM in targeting SAM-dependent methyltransferases.