A "push-pull" mechanism for heterolytic o-o bond cleavage in hydroperoxo manganese porphyrins.
A water-soluble manganese porphyrin, 5,10,15,20-tetrakis-(1,3-dimethylimidazolium-2-yl)porphyrinatomanganese(III) (Mn(III)TDMImP) is shown to react with H(2)O(2) to generate a relatively stable dioxomanganese(V) porphyrin complex (a compound I analog). Stopped-flow kinetic studies revealed Michaelis Menton-type saturation kinetics for H(2)O(2). The visible spectrum of a compound 0 type intermediate, assigned as Mn(III)(OH)(OOH)TDMImP, can be directly observed under saturating H(2)O(2) conditions (Soret band at 428 nm and Q bands at 545 and 578 nm). The rate-determining O-O heterolysis step was found to have a very small activation enthalpy (ΔH(≠) = 4.2 ± 0.2 kcal mol(-1)) and a large, negative activation entropy (ΔS(≠) = -36 ± 1 cal mol(-1) K(-1)). The O-O bond cleavage reaction was pH independent at 8.8 < pH < 10.4 with a first-order rate constant of 66 ± 12 s(-1). These observations indicate that the O-O bond in Mn(III)(OH)(OOH)TDMImP is cleaved via a concerted "push-pull" mechanism. In the transition state, the axial (proximal) (-)OH is partially deprotonated ("push"), while the terminal oxygen in (-)OOH is partially protonated ("pull") as a water molecule is released to the medium. This mechanism is reminiscent of O-O bond cleavage in heme enzymes, such as peroxidases and cytochrome P450, and similar to the fast, reversible O-Br bond breaking and forming reaction mediated by similar manganese porphyrins. The small enthalpy of activation suggests that this O-O bond cleavage could also be made reversible.


Sex Differences in Attributions to Positive and Negative Sexual Scenarios in Men and Women With and Without Sexual Problems: Reconsidering Stereotypes.
People with sexual problems are more likely to attribute negative sexual experiences to themselves, in contrast to sexually functional individuals who attribute negative sexual experiences to external factors such as the circumstance or partner. We investigated attribution patterns in 820 men and 753 women, some of whom reported an orgasmic problem, to assess differences between the sexes and those with and without an orgasmic difficulty. Specifically, using an Internet-based approach, we compared attribution responses to four sexual scenarios, one representing a positive sexual experience and three representing negative sexual experiences. Women were more likely to attribute positive outcomes to their partner than men. Women were also more likely to attribute negative outcomes to themselves than men, but they more readily blamed their partner and circumstances for negative outcomes than men as well. Those with orgasmic problems were less willing to take credit for positive outcomes and more willing to accept blame for negative outcomes. Interaction effects between sex and orgasmic problems further highlighted differences between men's and women's attribution patterns. These results are interpreted in the context of traditional notions that men's attributions tend to be more self-serving and women's attributions more self-derogatory.


QTc and anti-tuberculosis drugs: a perfect storm or a tempest in a teacup? Review of evidence and a risk assessment.

SUMMARY
Multidrug-resistant (MDR) and extensively drug-resistant tuberculosis (XDR-TB) are global concerns, with stagnant treatment success rates of roughly 54% and 30%, respectively. Despite adverse events associated with several DR-TB drugs, newly developed drugs and shorter regimens are bringing hope; recent concern has focused on drugs that prolong the corrected QT interval (QTc). QTc prolongation is a risk factor for torsades de pointe (TdP), a potentially lethal cardiac arrhythmia. While QTc prolongation is used in research as a surrogate marker for drug safety, the correlation between QTc and TdP is not perfect and depends on additional risk factors. The electrocardiogram (ECG) monitoring that has been recommended when new drugs are used has created alarm among clinicians and National Tuberculosis Programmes (NTPs). ECG monitoring is often challenging in high-burden settings where treatment alternatives are limited. According to a review of studies, the prevalence of sudden death directly attributable to TdP by QTc-prolonging DR-TB drugs is likely less than 1%. The risk of death from an ineffective MDR-TB/XDR-TB regimen thus far exceeds the risk of death from arrhythmia. In patients with QTc prolongation who develop cardiac events, other significant risk factors in addition to the drugs themselves are nearly always present. Clinicians and NTPs should be aware of and manage all possible circumstances that may trigger an arrhythmia (hypopotassaemia and human immunodeficiency virus infection are probably the most frequent in DR-TB patients). We present the limited but growing evidence on QTc prolongation and DR-TB management and propose a clinical approach to achieve an optimal balance between access to life-saving drugs and patient safety.


External adjustment of unmeasured confounders in a case-control study of benzodiazepine use and cancer risk.
Previous studies have reported diverging results on the association between benzodiazepine use and cancer risk. We investigated this association in a matched case-control study including incident cancer cases during 2002-2009 in the Danish Cancer Registry (n = 94 923) and age- and sex-matched (1:8) population controls (n = 759 334). Long-term benzodiazepine use was defined as ≥500 defined daily doses 1-5 years prior to the index date. We implemented propensity score (PS) calibration using external information on confounders available from a survey of the Danish population. Two PSs were used: The error-prone PS using register-based confounders and the calibrated PS based on both register- and survey-based confounders, retrieved from the Health Interview Survey. Register-based data showed that cancer cases had more diagnoses, higher comorbidity score and more co-medication then population controls. Survey-based data showed lower self-rated health, more self-reported diseases, and more smokers as well as subjects with sedentary lifestyle among benzodiazepine users. By PS calibration, the odds ratio for cancer overall associated with benzodiazepine use decreased from 1.16 to 1.09 (95% confidence interval 1.00-1.19) and for smoking-related cancers from 1.20 to 1.10 (95% confidence interval 1.00-1.21). We conclude that the increased risk observed in the solely register-based study could partly be attributed to unmeasured confounding.


Continuous invasive hemodynamic monitoring using steerable guide catheter to optimize mitraclip transcatheter mitral valve repair: A multicenter, proof-of-concept study.
We report our multicenter experience on continuous hemodynamic monitoring using exclusively the steerable guide catheter (SGC) during MitraClip repair. Left atrial pressure (LAP) and V-wave are useful to evaluate MitraClip repair but no simple method of continuous monitoring exists. From 11/2016 to 8/2017, 74 patients from four centers with symptomatic moderate-severe to severe mitral regurgitation (MR), underwent MitraClip NT repair with continuous hemodynamic monitoring via the SGC. Real-time LAP/V-wave changes were compared with transesophageal echocardiography (TEE). When mitral stenosis was suspected, transmitral gradients were verified by invasive hemodynamics. Clinical and echocardiographic outcomes were determined. Mean age was 78 ± 10 years and STS score 9.1 ± 11.0%. Pathology included leaflet prolapse/flail (45%), restriction (35%), and mixed (20%). Number of clips averaged 1.7 ± 0.7 per case. There was a significant reduction in LAP (21 ± 10 to 15 ± 7 mmHg, P < 0.0001) and V-wave(37 ± 19 to 24 ± 10 mmHg, P < 0.0001) post MitraClip, but the decrease was less in patients with atrial fibrillation (P < 0.05). Transmitral gradient significantly increased from 2.0 ± 1.2 to 4.0 ± 1.7 mmHg (P < 0.0001). Paradoxical increases in LAP and V-wave despite MR reduction were observed in three cases requiring MitraClip repositioning or retrieval to avoid stenosis. Follow-up averaged 5.0 ± 2.9 months and was 100% complete. KCCQ improvement was significant and MR reduction to <1+ was 67% and <2+ was 93% at 30 days. Continuous hemodynamic monitoring using the SGC complements TEE to assess and optimize MitraClip repair in real-time. Further validation is necessary but this feature may be part of future MitraClip and other transcatheter mitral repair systems.


Do European Union countries adequately address the healthcare needs of adolescents in the area of sexual reproductive health and rights?
Adolescent sexual and reproductive health and rights (SRHR) are of particular relevance given their potential short-term or long-term health consequences. This study evaluates recommendations and policies regarding access to care in this area in 31 European countries (European Union (EU) plus Iceland, Norway and Switzerland). As part of the EU funded Models of Child Health Appraised project, data were gathered using a 43-item questionnaire sent to experts responsible for collecting information in each country. Ten countries have not developed any formal policy or recommendation that guarantee the respect of confidentiality and the possibility of consulting a physician without parents knowing. Nearly half of the countries do not have centres specialised in adolescent healthcare, tackling comprehensive health issues or focusing specifically on SRH. Access to emergency contraception and information regarding pregnancy, including testing, is easy in most countries. However, oral contraception is delivered free of charge in only 10 countries. Twenty-three countries do not meet current standards in terms of providing policy-based pregnancy care, and only 13 have set up special programmes for pregnant adolescents. In only seven countries can adolescents definitely have their pregnancy terminated without their parents knowing (and in another seven countries in selected situations). The provision and availability of adolescent-friendly SRHR care are far from optimal in around half of the surveyed countries. These results call for the review and implementation of policies, specialised healthcare centres and training initiatives for primary care providers.


Identification of trichloroethanol visualized proteins from two-dimensional polyacrylamide gels by mass spectrometry.
Proteins visualized by 2,2,2-trichloroethanol (TCE) on two-dimensional electrophoresis gels are efficiently identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and MS/MS. In a previous study, a method was developed that placed TCE in the polyacrylamide gel so that protein bands can be visualized without staining in less than 5 min. A visible fluorophore is generated by reaction of TCE with tryptophan that allows for protein visualization. In this study, MALDI-TOF MS and LC-MS/MS are used to identify randomly selected Escherichia coli proteins. The identification of TCE visualized proteins is compared to the identification of Coomassie brilliant blue (CBB) stained proteins from two-dimensional gel electrophoresis of E. coli proteins. This study demonstrated that TCE visualized proteins are compatible with protein identification by MALDI-TOF peptide mass fingerprinting. For 10 randomly selected spots, TCE visualization lead to statistically significant identification of 5 proteins and CBB visualization lead to identification of 6 proteins. TCE visualized proteins are also shown to be well suited for protein identification using LC-MS/MS. In 16 spots selected for MS/MS analysis, TCE samples lead to the identification of 79 peptides; while CBB samples lead to the identification of 65 peptides. TCE samples also supported the identification of more proteins. The low stoichiometry of labeling of tryptophan residues does not require inclusion of this modification for database searches. In addition to being a rapid visualization technique compatible with MS, TCE visualization utilizes rapid washing conditions for sample preparation of proteins spots excised from polyacrylamide gels.


A toolbox facilitating stable transfection of Eimeria species.
Stable transfection of Eimeria species has been difficult to achieve because of the obligate requirement for in vivo amplification and selection of the parasites. Strategies to generate and stabilise populations of transfected Eimeria tenella are described here, together with the identification of optimal parameters for the transfection process. A series of plasmids expressing selectable markers, including a panel of fluorescent reporter genes and a mutant Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (DHFR-TSm2m3) gene that confers resistance to pyrimethamine, were electroporated into sporozoites of the E. tenella Wisconsin strain and stabilised by selective passage through chickens. Very high transfection efficiencies of up to 25% sporozoites in transient transfection and up to 9% oocysts following a single round of in vivo selection were achieved. Crucial factors include the use of very freshly harvested parasites with the AMAXA nucleofection system (program U33 in a cytomix-buffered reaction) and linearised plasmid DNA. The use of a restriction enzyme mediated integration (REMI) protocol boosted overall efficiency and elevated insertion rate per genome. Successful development of methods to generate and isolate stable populations of transfected Eimeria parasites will now stimulate rapid expansion of reverse genetic studies in this important coccidian.


Analysis of peptide-protein binding using amino acid descriptors: prediction and experimental verification for human histocompatibility complex HLA-A0201.
Amino acid descriptors are often used in quantitative structure-activity relationship (QSAR) analysis of proteins and peptides. In the present study, descriptors were used to characterize peptides binding to the human MHC allele HLA-A0201. Two sets of amino acid descriptors were chosen: 93 descriptors taken from the amino acid descriptor database AAindex and the z descriptors defined by Wold and Sandberg. Variable selection techniques (SIMCA, genetic algorithm, and GOLPE) were applied to remove redundant descriptors. Our results indicate that QSAR models generated using five z descriptors had the highest predictivity and explained variance (q2 between 0.6 and 0.7 and r2 between 0.6 and 0.9). Further to the QSAR analysis, 15 peptides were synthesized and tested using a T2 stabilization assay. All peptides bound to HLA-A0201 well, and four peptides were identified as high-affinity binders.


Cyclin E ablation in the mouse.
E type cyclins (E1 and E2) are believed to drive cell entry into the S phase. It is widely assumed that the two E type cyclins are critically required for proliferation of all cell types. Here, we demonstrate that E type cyclins are largely dispensable for mouse development. However, endoreplication of trophoblast giant cells and megakaryocytes is severely impaired in the absence of cyclin E. Cyclin E-deficient cells proliferate actively under conditions of continuous cell cycling but are unable to reenter the cell cycle from the quiescent G(0) state. Molecular analyses revealed that cells lacking cyclin E fail to normally incorporate MCM proteins into DNA replication origins during G(0)-->S progression. We also found that cyclin E-deficient cells are relatively resistant to oncogenic transformation. These findings define a molecular function for E type cyclins in cell cycle reentry and reveal a differential requirement for cyclin E in normal versus oncogenic proliferation.


Genome-Wide Analysis of C2H2 Zinc-Finger Family Transcription Factors and Their Responses to Abiotic Stresses in Poplar (Populus trichocarpa).
C2H2 zinc-finger (C2H2-ZF) proteins are a large gene family in plants that participate in various aspects of normal plant growth and development, as well as in biotic and abiotic stress responses. To date, no overall analysis incorporating evolutionary history and expression profiling of the C2H2-ZF gene family in model tree species poplar (Populus trichocarpa) has been reported. Here, we identified 109 full-length C2H2-ZF genes in P. trichocarpa, and classified them into four groups, based on phylogenetic analysis. The 109 C2H2-ZF genes were distributed unequally on 19 P. trichocarpa linkage groups (LGs), with 39 segmental duplication events, indicating that segmental duplication has been important in the expansion of the C2H2-ZF gene family. Promoter cis-element analysis indicated that most of the C2H2-ZF genes contain phytohormone or abiotic stress-related cis-elements. The expression patterns of C2H2-ZF genes, based on heatmap analysis, suggested that C2H2-ZF genes are involved in tissue and organ development, especially root and floral development. Expression analysis based on quantitative real-time reverse transcription polymerase chain reaction indicated that C2H2-ZF genes are significantly involved in drought, heat and salt response, possibly via different mechanisms. This study provides a thorough overview of the P. trichocarpa C2H2-ZF gene family and presents a new perspective on the evolution of this gene family. In particular, some C2H2-ZF genes may be involved in environmental stress tolerance regulation. PtrZFP2, 19 and 95 showed high expression levels in leaves and/or roots under environmental stresses. Additionally, this study provided a solid foundation for studying the biological roles of C2H2-ZF genes in Populus growth and development. These results form the basis for further investigation of the roles of these candidate genes and for future genetic engineering and gene functional studies in Populus.


Neuronal cell differentiation of human neuroblastoma cells by retinoic acid plus herbimycin A.
We investigated the effect of retinoic acid (RA) and herbimycin A (herb-A) on cell growth, cell differentiation, and colony formation of human neuroblastoma cell lines. The neuroblastoma line SK-N-SH expressed both neuroblast and nonneuronal phenotypes, whereas its subclone SH-SY5Y and the Kelly cell line were predominantly neuroblastic. Both herb-A and RA, given alone, moderately reduced cell growth and colony formation of the neuroblastic cell lines. Growth curve analyses with SK-N-SH suggested that herb-A greatly reduced the number of initially growing cells, whereas RA slightly enhanced initial cell growth. Morphological changes were determined with the use of rhodaminephalloidin staining of actin. Retinoic acid caused an increase in the fraction of neuroblast cell in SK-N-SH, and conversely of nonneuronal cells in SH-SY5Y and Kelly cell lines. Both drugs also caused partial differentiation towards a neuronal phenotype, and herb-A induced selective lysis of nonneuronal cells of SK-N-SH. Because of their discrepant effects, RA (10 microM) and herb-A (236 nM) were tested in combination at a concentration that had only moderate effects when given alone. The combination further reduced cell growth and colony formation and dramatically enhanced differentiation towards a neuronal morphology. The Kelly cell line with amplified N-myc genome, which correlates with clinical progression of neuroblastoma, was also sensitive to RA plus herb-A. These results recommend the combination of RA and herb-A for differentiation therapy of neuroblastoma.


Metabolic syndrome is associated with reduced flow mediated dilation independent of obesity status.
Data suggest that metabolic health status, incorporating components of metabolic syndrome (MetS), predicts cardiovascular disease (CVD) risk better than BMI. This study explored the association of MetS and obesity with endothelial function, a prognostic risk factor for incident CVD. Forty-four participants were phenotyped according to BMI as non-obese vs obese (<30 or >30 kg/m2) and according to the International Diabetes Federation criteria of MetS: ≤2 criteria MetS (MetS-) vs ≥3 criteria MetS (MetS+); (1.)non-obese MetS- vs (2.) non-obese MetS+ and (3.) obese MetS- vs (4.) obese MetS+. Flow-mediated dilation (FMD), body composition including liver fat (MRI and spectroscopy), dietary intake, intensities of habitual physical activity and cardio-respiratory fitness were determined. Variables were analysed using a one-factor between-groups ANOVA and linear regression; mean (95% CI) are presented. Individuals with MetS+ displayed lower FMD than those with MetS-. For non-obese individuals mean difference between MetS+ and MetS- was 4.1% ((1.0, 7.3); P = 0.004) and obese individuals had a mean difference between MetS+ and MetS- of 6.2% ((3.1, 9.2); P < 0.001). Although there was no association between BMI and FMD (P = 0.27), an increased number of MetS components was associated with a lower FMD (P = 0.005), and after adjustment for age and sex, 19.7% of the variance of FMD was explained by MetS, whereas only 1.1% was explained by BMI. In this study cohort, components of MetS, rather than obesity per se, contribute to reduced FMD, which suggests a reduced bioavailability of nitric oxide and thus increased risk of CVD.


Hypermethylation can selectively silence individual p16ink4A alleles in neoplasia.
Inactivation of p16ink4A and other tumor suppressor genes has been associated with promoter region hypermethylation in neoplasia. However, direct proof for aberrant DNA methylation as an independent event for loss of gene function has been difficult to obtain. We addressed this question in the colon carcinoma cell line HCT116, which contains one allele of p16ink4A with a coding region frameshift mutation and one wild-type allele. Neither allele contains a mutation in the proximal promoter region. The promoter of the wild-type allele, but not the mutant allele, is hypermethylated, and only the mutant allele is expressed. Transcription from the methylated/wild-type allele was restored after cell treatment with the demethylating agent 5-aza-2'-deoxycytidine. Thus, in neoplastic cells, stable allele-specific loss of transcription may arise from aberrant methylation of a nonmutated promoter region, identifying hypermethylation as a direct mechanism for tumor suppressor gene inactivation.


Location of enhancers is essential for the imprinting of H19 and Igf2 genes.
Genomic imprinting is the process in mammals by which gamete-specific epigenetic modifications establish the differential expression of the two alleles of a gene. The tightly linked H19 and Igf2 genes are expressed in tissues of endodermal and mesodermal origin, with H19 expressed from the maternal chromosome and Igf2 expressed from the paternal chromosome. A model has been proposed to explain the reciprocal imprinting of these genes; in this model, expression of the genes is governed by competition between their promoters for a common set of enhancers. An extra set of enhancers might be predicted to relieve the competition, thereby eliminating imprinting. Here we tested this prediction by generating mice with a duplication of the endoderm-specific enhancers. The normally silent Igf2 gene on the maternal chromosome was expressed in liver, consistent with relief from competition. We then generated a maternal chromosome containing a single set of enhancers located equidistant from 1gf2 and H19; the direction of the imprint was reversed. Thus, the location of the enhancers determines the outcome of competition in liver, and the strength of the H19 promoter is not sufficient to silence Igf2.


Prophylaxis of heterotopic ossification in patients sedated after polytrauma : medical and ethical considerations.
Heterotopic ossification (HO) often follows acetabular fractures after multitrauma. Irradiation is a mean for prophylaxis. We established a standard procedure in our hospital for patients under sedation, when obtaining informed consent for HO prophylaxis is impossible. We reviewed current scientific evidence, calculated the risks of radiation and presented the ethical and legal framework. The subject was scrutinised by an interdisciplinary panel. Irradiation is the most effective means for prophylaxis and has few adverse effects in adult patients with fractures of the acetabulum. The lifetime risk of radiation-induced cancer or infertility are insignificant. Informed consent for irradiation should be obtained before operation whenever possible. When this cannot be done prophylaxis can be postponed for a maximum of 3 days in order to obtain consent. If the patient is not able to communicate within this period, prophylactic irradiation should be given after consulting the relatives. The patient must be informed as soon as possible.


UV-B affects photosynthesis, ROS production and motility of the freshwater flagellate, Euglena agilis Carter.
The effects of ultraviolet B (UV-B; 295-320 nm) radiation on certain vital physiological (photosynthesis), biochemical (production of reactive oxygen species - ROS) and behavioral (motility and orientation) characteristics were investigated in the unicellular photoautotroph, Euglena agilis Carter. The photosynthetic performance of E. agilis was recorded after exposure of between 15 and 60 min followed by a period of recovery lasting 6-24h under dim light (5-10 μmol photons m(-2) s(-1)). The maximum quantum yield of PS II (F(v)/F(m)) was reduced to 65% and 14% of initial values immediately following 15 and 30 min UV-B exposure, but recovered to 100 and 86% of the initials, respectively. Values of rETR(max) in E. agilis exposed to 15 min UV-B were similar to those of the initials, but a 30 min UV exposure resulted in 75% reduction of rETR(max) with only a 43% recovery as compared with the initial after 24h recovery. After a 60 min UV-B exposure, there were no Chl a fluorescence signals, and hence no F(v)/F(m) or rETR(max). A UV dose-dependent increase in DCFH-DA fluorescence was found in E. agilis cells, reflecting an increase in ROS production. After exposures to UV-B for between 15 and 60 min, the percentages of motile cells in the population decreased to 76, 39 and 15%, respectively. Following 24h in dim light, the percentage of motile cells increased to between 66% and 95% of the initial value. The velocity of non-irradiated cells was 60 μm s(-1), which decreased to 16-35 μm s(-1) immediately following exposure for 15-60 min. After periods of time in dim light (6, 12 and 24h) velocities had recovered to between 44 and 81% of the initial value. In untreated controls, the r-value was 0.23, indicating random movement of E. agilis, but it increased to 0.35 and 0.72 after exposure to UV-B for 30 and 60 min, respectively. There was a tendency towards vertical downward movement of cells proportional to the duration of exposure. The compactness of E. agilis decreased from 2.9 in controls to 1.8-2.3 in cells treated with UV-B although significant recovery followed. UV-B dose-dependent interaction between photosynthetic activity, ROS production and movement is discussed in terms of a UV-protective mechanism in E. agilis.


Arithmetic mismatch negativity and numerical magnitude processing in number matching.
This study examined the relationship of the arithmetic mismatch negativity (AMN) and the semantic evaluation of numerical magnitude. The first question was whether the AMN was sensitive to the incongruity in numerical information per se, or rather, to the violation of strategic expectations. The second question was whether the numerical distance effect could appear independently of the AMN. Event-related potentials (ERPs) were recorded while participants decided whether two digits were matching or non-matching in terms of physical similarity. The AMN was enhanced in matching trials presented infrequently relative to non-matching trials presented frequently. The numerical distance effect was found over posterior sites during a 92 ms long interval (236-328 ms) but appeared independently of the AMN. It was not the incongruity in numerical information per se, but rather, the violation of strategic expectations that elicited the AMN. The numerical distance effect might only temporally coincide with the AMN and did not form an inherent part of it.


Genetic population of Plasmodium knowlesi during pre-malaria elimination in Thailand.
Thailand is committed to eliminating malaria by 2024. From 2013 to 2020, the total number of malaria cases have decreased, from 37,741 to 4474 (an 88.1% reduction). However, infections with Plasmodium knowlesi, a monkey malarial pathogen that can also infect humans, have been increasingly observed. This study focused on the molecular analysis of P. knowlesi parasites causing malaria in Thailand. Under Thailand's integrated Drug Efficacy Surveillance (iDES), which includes drug-resistance monitoring as part of routine case-based surveillance and responses, specimens were collected from malaria patients (n = 966) between 2018 and 2020. Thirty-one mono P. knowlesi infections (3.1%), most of which were from eastern and southern Thailand, were observed and confirmed by nested PCR assay and DNA sequencing. To evaluate whether these pathogens were from different lineages, cluster analysis based on seven microsatellite genotyping markers and the merozoite surface protein 1 (pkmsp1) gene was carried out. The P. knowlesi pyrimethamine resistance gene dihydrofolate reductase (pkdhfr) was sequenced and homology modelling was constructed. The results of analysing the seven microsatellite markers and pkmsp1 sequence demonstrated that P. knowlesi parasites from eastern Thailand were of the same lineage as those isolated in Cambodia, while the parasites causing malaria in southern Thailand were the same lineage as those isolated from Malaysia. The sequencing results for the pkdhfr genes indicated the presence of two mutations, Arg34Leu and a deletion at position 105. On analysis with homology modelling, the two mutations were not associated with anti-malarial drug resistance. This report compared the genetic populations of P. knowlesi parasites in Thailand from 2018 to 2020 and have shown similar lineages as those isolated in Cambodia and Malaysia of P. knowlesi infection in Thailand and demonstrated that the P. knowlesi parasites were of the same lineages as those isolated in Cambodia and Malaysia. The parasites were also shown to be sensitive to pyrimethamine.


Surgical treatment of meningiomas located in the rolandic area: the role of navigated transcranial magnetic stimulation for preoperative planning, surgical strategy, and prediction of arachnoidal cleavage and motor outcome.
Surgical treatment of convexity meningiomas is usually considered a low-risk procedure. Nevertheless, the risk of postoperative motor deficits is higher (7.1%-24.7% of all cases) for lesions located in the rolandic region, especially when an arachnoidal cleavage plane with the motor pathway is not identifiable. The authors analyzed the possible role of navigated transcranial magnetic stimulation (nTMS) for planning resection of rolandic meningiomas and predicting the presence or lack of an intraoperative arachnoidal cleavage plane as well as the postoperative motor outcome. Clinical data were retrospectively collected from surgical cases involving patients affected by convexity, parasagittal, or falx meningiomas involving the rolandic region, who received preoperative nTMS mapping of the motor cortex (M1) and nTMS-based diffusion tensor imaging (DTI) fiber tracking of the corticospinal tract before surgery at 2 different neurosurgical centers. Surgeons' self-reported evaluation of the impact of nTMS-based mapping on surgical strategy was analyzed. Moreover, the nTMS mapping accuracy was evaluated in comparison with intraoperative neurophysiological mapping (IONM). Lastly, we assessed the role of nTMS as well as other pre- and intraoperative parameters for predicting the patients' motor outcome and the presence or absence of an intraoperative arachnoidal cleavage plane. Forty-seven patients were included in this study. The nTMS-based planning was considered useful in 89.3% of cases, and a change of the surgical strategy was observed in 42.5% of cases. The agreement of nTMS-based planning and IONM-based strategy in 35 patients was 94.2%. A new permanent motor deficit occurred in 8.5% of cases (4 of 47). A higher resting motor threshold (RMT) and the lack of an intraoperative arachnoidal cleavage plane were the only independent predictors of a poor motor outcome (p = 0.04 and p = 0.02, respectively). Moreover, a higher RMT and perilesional edema also predicted the lack of an arachnoidal cleavage plane (p = 0.01 and p = 0.03, respectively). Preoperative motor status, T2 cleft sign, contrast-enhancement pattern, and tumor volume had no predictive value. nTMS-based motor mapping is a useful tool for presurgical assessment of rolandic meningiomas, especially when a clear cleavage plane with M1 is not present. Moreover, the RMT can indicate the presence or absence of an intraoperative cleavage plane and predict the motor outcome, thereby helping to identify high-risk patients before surgery.


Peripheral blood dendritic cells in children with acute Plasmodium falciparum malaria.
The importance of dendritic cells (DCs) for the initiation and regulation of immune responses not only to foreign organisms but also to the self has raised considerable interest in the qualitative and quantitative analysis of these cells in various human diseases. Plasmodium falciparum malaria is characterized by the poor induction of long-lasting protective immune responses. This study, therefore, investigated the percentage of peripheral blood DCs as lineage marker-negative and HLA-DR(+) or CD83(+) cells in healthy children and in children suffering from acute malaria in Kilifi, Kenya. Comparable percentages of CD83(+) DCs were found in peripheral blood of healthy children and children with malaria. However, the percentage of HLA-DR(+) peripheral blood DCs was significantly reduced in children with malaria. The results suggest that a proportion of peripheral blood DCs may be functionally impaired due to the low expression of HLA-DR on their surface.


The Application of GeneXpert MTB/RIF for Smear-Negative TB Diagnosis as a Fee-Paying Service at a South Asian General Hospital.
The GeneXpert MTB/RIF assay (Xpert) is a novel automated diagnostic tool for tuberculosis but its optimal placement in the healthcare system has not been determined. The objective of this study was to determine the possibility of additional case detection for pulmonary tuberculosis (PTB) by offering Xpert to smear-negative patients in a low-HIV burden setting with no Mycobacterium tuberculosis (M.tb.) culture facilities. Patients routinely presenting with symptoms suggestive of PTB with negative smears were offered single Xpert test on a fee-paying basis. Data were retrospectively reviewed to determine case detection in patients tested from February to December 2013. Symptoms associated with a positive test were analysed to determine if refinement of clinical criteria would reduce unnecessary testing. 258 smear-negative patients were included and M.tb. was detected in 55 (21.32%, n = 55/258). Using standard clinical assessment for selection, testing 5 patients detected one case of smear-negative PTB. These results demonstrate that fee-paying Xpert service in low-income setting can increase TB case confirmation substantially and further systematic studies of health economic implications should be conducted to determine optimal implementation models to increase access to Xpert in low- and middle-income countries.


Optical torques guiding cell motility.
The main mechanism responsible for cell motility is the stochastic generation and breakup of actin filaments forming the cytoskeleton. However, the role of environmental factors in the migration and differentiation of cells is yet to be fully understood. Here we demonstrate that polarized optical fields can exert controllable torques on the actin network and therefore influence the treadmilling process responsible for cells motility. Through systematic experiments and stochastic modeling we demonstrate that actively guiding the dynamics of large groups of cells is possible in a noninvasive manner.


The reliability of the twelve-item general health questionnaire (GHQ-12) under realistic assumptions.
The twelve-item General Health Questionnaire (GHQ-12) was developed to screen for non-specific psychiatric morbidity. It has been widely validated and found to be reliable. These validation studies have assumed that the GHQ-12 is one-dimensional and free of response bias, but recent evidence suggests that neither of these assumptions may be correct, threatening its utility as a screening instrument. Further uncertainty arises because of the multiplicity of scoring methods of the GHQ-12. This study set out to establish the best fitting model for the GHQ-12 for three scoring methods (Likert, GHQ and C-GHQ) and to calculate the degree of measurement error under these more realistic assumptions. GHQ-12 data were obtained from the Health Survey for England 2004 cohort (n = 3705). Structural equation modelling was used to assess the fit of [1] the one-dimensional model [2] the current 'best fit' three-dimensional model and [3] a one-dimensional model with response bias. Three different scoring methods were assessed for each model. The best fitting model was assessed for reliability, standard error of measurement and discrimination. The best fitting model was one-dimensional with response bias on the negatively phrased items, suggesting that previous GHQ-12 factor structures were artifacts of the analysis method. The reliability of this model was over-estimated by Cronbach's Alpha for all scoring methods: 0.90 (Likert method), 0.90 (GHQ method) and 0.75 (C-GHQ). More realistic estimates of reliability were 0.73, 0.87 and 0.53 (C-GHQ), respectively. Discrimination (Delta) also varied according to scoring method: 0.94 (Likert method), 0.63 (GHQ method) and 0.97 (C-GHQ method). Conventional psychometric assessments using factor analysis and reliability estimates have obscured substantial measurement error in the GHQ-12 due to response bias on the negative items, which limits its utility as a screening instrument for psychiatric morbidity.


Role of in silico structural modeling in predicting immunogenic neoepitopes for cancer vaccine development.
In prior studies, we delineated the landscape of neoantigens arising from nonsynonymous point mutations in a murine pancreatic cancer model, Panc02. We developed a peptide vaccine by targeting neoantigens predicted using a pipeline that incorporates the MHC binding algorithm NetMHC. The vaccine, when combined with immune checkpoint modulators, elicited a robust neoepitope-specific antitumor immune response and led to tumor clearance. However, only a small fraction of the predicted neoepitopes induced T cell immunity, similarly to that reported for neoantigen vaccines tested in clinical studies. While these studies have used binding affinities to MHC I as surrogates for T cell immunity, this approach does not include spatial information on the mutated residue that is crucial for TCR activation. Here, we investigate conformational alterations in and around the MHC binding groove induced by selected minimal neoepitopes, and we examine the influence of a given mutated residue as a function of its spatial position. We found that structural parameters, including the solvent-accessible surface area (SASA) of the neoepitope and the position and spatial configuration of the mutated residue within the sequence, can be used to improve the prediction of immunogenic neoepitopes for inclusion in cancer vaccines.


Reproducibility of repeated measures of carbon-11-raclopride binding in the human brain.
Carbon-11-raclopride has been successfully utilized with PET to assess changes in endogenous dopamine concentration after pharmacological intervention in the living baboon brain. For similar studies to be done in humans, measurements of 11C-raclopride with no intervention need to be reproducible. In order to test the reproducibility (test-retest) of 11C-raclopride binding in the human brain, we performed repeated studies on two different days. Studies were done in five normal controls with no pharmacological intervention. Time-activity (%dose/cc) curves for 11C-raclopride in the basal ganglia (BG) and cerebellum (CBL) were highly reproducible with an average difference in peak uptake for repeated studies in the same individual of 4%. The BG to CBL ratio for the average activity concentration between 30 and 60 min showed differences that ranged from -7% to 8% between the repeated studies. Graphical analysis to obtain the distribution volume revealed intrasubject values that ranged from -9% to 7% for the ratio of the distribution volume in BG to that in CBL. These studies demonstrate that in order to use 11C-raclopride to measure an individual's change in relative dopamine concentration secondary to pharmacological or behavioral intervention, a change in striatal 11C-raclopride binding in excess of 10% is required.


The Biological Basis for Cardiac Repair After Myocardial Infarction: From Inflammation to Fibrosis.
In adult mammals, massive sudden loss of cardiomyocytes after infarction overwhelms the limited regenerative capacity of the myocardium, resulting in the formation of a collagen-based scar. Necrotic cells release danger signals, activating innate immune pathways and triggering an intense inflammatory response. Stimulation of toll-like receptor signaling and complement activation induces expression of proinflammatory cytokines (such as interleukin-1 and tumor necrosis factor-α) and chemokines (such as monocyte chemoattractant protein-1/ chemokine (C-C motif) ligand 2 [CCL2]). Inflammatory signals promote adhesive interactions between leukocytes and endothelial cells, leading to extravasation of neutrophils and monocytes. As infiltrating leukocytes clear the infarct from dead cells, mediators repressing inflammation are released, and anti-inflammatory mononuclear cell subsets predominate. Suppression of the inflammatory response is associated with activation of reparative cells. Fibroblasts proliferate, undergo myofibroblast transdifferentiation, and deposit large amounts of extracellular matrix proteins maintaining the structural integrity of the infarcted ventricle. The renin-angiotensin-aldosterone system and members of the transforming growth factor-β family play an important role in activation of infarct myofibroblasts. Maturation of the scar follows, as a network of cross-linked collagenous matrix is formed and granulation tissue cells become apoptotic. This review discusses the cellular effectors and molecular signals regulating the inflammatory and reparative response after myocardial infarction. Dysregulation of immune pathways, impaired suppression of postinfarction inflammation, perturbed spatial containment of the inflammatory response, and overactive fibrosis may cause adverse remodeling in patients with infarction contributing to the pathogenesis of heart failure. Therapeutic modulation of the inflammatory and reparative response may hold promise for the prevention of postinfarction heart failure.


Pyridine-Based Electron-Transport Materials with High Solubility, Excellent Film-Forming Ability, and Wettability for Inkjet-Printed OLEDs.
Film morphology has predominant influence on the performance of multilayered organic light-emitting diodes (OLEDs), whereas there is little reported literature from the angle of the molecular level to investigate the impact on film-forming ability and device performance. In this work, four isomeric cross-linkable electron-transport materials constructed with pyridine, 1,2,4-triazole, and vinylbenzyl ether groups were developed for inkjet-printed OLEDs. Their lowest unoccupied molecular orbital (∼3.20 eV) and highest occupied molecular orbital (∼6.50 eV) levels are similar, which are mainly determined by the 1,2,4-triazole groups. The triplet energies of these compounds can be tuned from 2.51 to 2.82 eV by different coupling modes with the core of pyridine, where the 2,6-pyridine-based compound has the highest value of 2.82 eV. Film formation and solubility of the compounds were investigated. It was found that the 2,6-pyridine-based compound outperformed the 2,4-pyridine, 2,5-pyridine, and 3,5-pyridine-based compounds. The spin-coated blue OLEDs based on the four compounds have achieved over 14.0% external quantum efficiencies (EQEs) at the luminance of 100 cd m-2, and a maximum EQE of 12.1% was obtained for the inkjet-printed device with 2,6-pyridine-based compound.


VERO cells harbor a poly-ADP-ribose belt partnering their epithelial adhesion belt.
Poly-ADP-ribose (PAR) is a polymer of up to 400 ADP-ribose units synthesized by poly-ADP-ribose-polymerases (PARPs) and degraded by poly-ADP-ribose-glycohydrolase (PARG). Nuclear PAR modulates chromatin compaction, affecting nuclear functions (gene expression, DNA repair). Diverse defined PARP cytoplasmic allocation patterns contrast with the yet still imprecise PAR distribution and still unclear functions. Based on previous evidence from other models, we hypothesized that PAR could be present in epithelial cells where cadherin-based adherens junctions are linked with the actin cytoskeleton (constituting the adhesion belt). In the present work, we have examined through immunofluorescence and confocal microscopy, the subcellular localization of PAR in an epithelial monkey kidney cell line (VERO). PAR was distinguished colocalizing with actin and vinculin in the epithelial belt, a location that has not been previously reported. Actin filaments disruption with cytochalasin D was paralleled by PAR belt disruption. Conversely, PARP inhibitors 3-aminobenzamide, PJ34 or XAV 939, affected PAR belt synthesis, actin distribution, cell shape and adhesion. Extracellular calcium chelation displayed similar effects. Our results demonstrate the existence of PAR in a novel subcellular localization. An initial interpretation of all the available evidence points towards TNKS-1 as the most probable PAR belt architect, although TNKS-2 involvement cannot be discarded. Forthcoming research will test this hypothesis as well as explore the existence of the PAR belt in other epithelial cells and deepen into its functional implications.


Interplay of Direct and Indirect Charge-Transfer Pathways in Donor-Bridge-Acceptor Systems.
Charge transfer in donor-bridge-acceptor (DBA) structures typically takes place through the combination of donor-bridge and bridge-acceptor overlap integrals forming an effective, indirect electronic coupling between the donor (D) and acceptor (A) moieties. Here we examine the effects of an additional direct DA electronic coupling of charge-transfer processes in DBA systems with local interaction with thermal baths. First, using the exact Nakajima-Zwanzig master equation (NZME) for the reduced density matrix, we rigorously define probability currents as the coherent part of the NZME, thereby allowing us to quantify the contribution of the different electronic pathways (direct and indirect) to the charge-transfer dynamics. Focusing on two minimal DBA systems of three sites (V and Λ models) and adopting well-developed methods, we find that the interplay between different transfer pathways can be assessed by the McConnell formula in the weak system-bath coupling regime. We then demonstrate that the combination of indirect and direct donor-acceptor coupling either enhances or leads to a destructive quantum interference effect on charge-transport processes, depending on the energy landscape of the DBA system.


The role of the substantia nigra pars reticulata in kindling resistance in rats with genetic absence epilepsy.
Genetic Absence Epilepsy Rats from Strasbourg (GAERS) show a resistance to secondary generalization of focal limbic seizures evoked by kindling. The substantia nigra pars reticulata (SNR) is involved in the propagation and modulation of seizures in kindling. We first examined the role of the SNRanterior and SNRposterior subregions in the resistance to the development of kindling in GAERS. Subsequently, to determine whether kindling resistance relates to differential sensitivity of γ-aminobutyric acid γ-aminobutyric acid (GABA)ergic or dopaminergic SNR neurons to kindling, we studied the effects of kindling-inducing stimulations on parvalbumin (PRV; GABAergic neuron marker) or tyrosine hydroxylase (TH; dopaminergic neuron marker) immunoreactivity (ir), respectively, in GAERS and in nonepileptic control (NEC) Wistar rats that lack kindling resistance. Adult male GAERS were implanted with a stimulation electrode in the amygdala, and bilateral injection cannulas for lidocaine or saline injection (30 min before each kindling stimulation until the animals reached three stage 5 seizures or the 22 stimulations) into the SNRanterior or SNRposterior . In another experiment, PRV-ir in SNRanterior and SNRposterior and TH-ir in SNRposterior only were densitometrically compared in GAERS-SHAM, NEC-SHAM GAERS-STIM, and NEC-STIM animals (6 kindling stimulations). Bilateral SNRposterior infusions of lidocaine eliminated the kindling resistance and resulted in stage 5 generalized motor seizures in all kindled rats. Bilateral lidocaine infusions in the SNRanterior failed to alter the kindling resistance in GAERS. PRV-ir in the SNRposterior was unaltered in GAERS-STIM but increased in NEC-STIM group. Cellular TH-ir in the SNRposterior significantly increased by kindling stimulations in both NEC-STIM and GAERS-STIM groups. The kindling resistance in GAERS is mediated by the SNRposterior in a lidocaine-sensitive manner. The insensitivity to kindling stimulation of PRV-ir in SNRposterior of GAERS but not NEC rats, implicate GABAergic SNRposterior neurons in kindling resistance. In contrast, the observed stimulation-specific increase in TH-ir in the SNRposterior is unrelated to kindling resistance.


Regulation of in vivo behavior of TAT-modified liposome by associated protein corona and avidity to tumor cells.
PEGylated liposomes are widely used and studied as carriers for chemotherapeutics. While pharmacokinetics of the encapsulated drug is drastically altered resulting in favorable circulation time, improved tumor accumulation, and better manageable or reduced side effects, therapeutic efficacy has been disappointing. Major drawbacks are a failure to reach the tumor cell, limited penetration depth, and impaired uptake by tumor cells. Here, we study the implication of HIV-1 transactivator of transcription (TAT)-derived peptides inserted on PEGylated liposomal doxorubicin (PLD) and followed in vitro and in vivo fate. PLDs were installed with 25-400 TAT peptides per liposome without an effect on PLD stability. While TAT peptides facilitate active endocytosis of the carriers, we observed that these peptides did not promote endosomal escape or enhanced intracellular availability of doxorubicin. Interestingly, incorporation of TAT peptides did not change pharmacokinetics or biodistribution, which we found to result from a dysopsonization of the TAT-modified liposomes by serum proteins. A protein corona (PC) on TAT peptide-modified PLDs shields the active moieties and effectively reduces clearance of the TAT peptide containing nanoparticles. However, intratumoral activity was influenced by the number of TAT peptides present. The best antitumor efficacy was observed with a TAT peptide density of 100, while lower amounts showed results comparable to unmodified PLDs. At 200 TAT peptides, the preparation appeared to be least effective, which likely results from augmented interaction with tumor cells directly upon extravasation. We conclude that by optimizing TAT-modified PLDs, the occurring PC balances pharmacokinetics and tumor penetration through interference with avidity.


Punishing second-order free riders before first-order free riders: The effect of pool punishment priority on cooperation.
Second-order free riders, who do not owe punishment cost to first-order free riders in public goods games, lead to low cooperation. Previous studies suggest that for stable cooperation, it is critical to have a pool punishment system with second-order punishment, which gathers resources from group members and punishes second-order free riders as well as first-order free riders. In this study, we focus on the priority of punishment. We hypothesize that the pool punishment system that prioritizes second-order punishment is more likely to achieve cooperation than the system that prioritizes first-order punishment, because the former is more likely to obtain sufficient punishment resources. In the experiments, we compare four pool punishment systems: 1To2 (first-order punishment to second-order punishment), 2To1 (second-order punishment to first-order punishment), 1ONLY (first-order punishment only), and 2ONLY (second-order punishment only). We find that the 2To1 and 2ONLY systems can receive more support than the 1To2 and 1ONLY systems and only the 2To1 system can achieve high cooperation. However, the effect of priority of second-order punishment is observed only when the punishment ratio (PR) is low (Experiment 1), not high (Experiment 2), in which the punishment resource is relatively abundant.


Small Molecule Modulators of the Circadian Molecular Clock With Implications for Neuropsychiatric Diseases.
Circadian rhythms regulate many biological processes and play fundamental roles in behavior, physiology, and metabolism. Such periodicity is critical for homeostasis because disruption or misalignment of the intrinsic rhythms is associated with the onset and progression of various human diseases and often directly leads to pathological states. Since the first identification of mammalian circadian clock genes, numerous genetic and biochemical studies have revealed the molecular basis of these cell-autonomous and self-sustainable rhythms. Specifically, these rhythms are generated by two interlocking transcription/translation feedback loops of clock proteins. As our understanding of these underlying mechanisms and their functional outputs has expanded, strategies have emerged to pharmacologically control the circadian molecular clock. Small molecules that target the molecular clock may present novel therapeutic strategies to treat chronic circadian rhythm-related diseases. These pharmaceutical approaches may include the development of new drugs to treat circadian clock-related disorders or combinational use with existing therapeutic strategies to improve efficacy via intrinsic clock-dependent mechanisms. Importantly, circadian rhythm disruptions correlate with, and often precede, many symptoms of various neuropsychiatric disorders such as sleep disorders, affective disorders, addiction-related disorders, and neurodegeneration. In this mini-review, we focus on recent discoveries of small molecules that pharmacologically modulate the core components of the circadian clock and their potential as preventive and/or therapeutic strategies for circadian clock-related neuropsychiatric diseases.


STFM Behavioral Science/Family Systems Educator Fellowship: Evaluation of the First 4 Years.
The discipline of family medicine has long valued the behavioral sciences. Most residency training programs employ a clinical psychologist, social worker, or family therapist to deliver behavioral science curriculum to their residents. However, the cultures and content of training for behavioral sciences and medical professions are quite different, leaving the lone behavioral scientist feeling professionally isolated and unprepared to translate knowledge and skills into tools for the family physician. In response to this need, a group of family medicine educators developed an STFM-sponsored fellowship for behavioral science faculty. The goals of the program were to improve fellows' understanding of the culture of family medicine, provide a curricular toolbox for the behavioral sciences, promote scholarship, and develop a supportive professional network. Senior behavioral science faculty at STFM developed a 1-year fellowship program, featuring "classroom learning" at relevant conferences, mentored small-group interactions, and scholarly project requirements. Achievement of program goals was evaluated annually with pre- and post-fellowship surveys. From 2010 to 2014, 59 fellows completed the program; most were psychologists or social workers; two thirds were women. One month after graduation, fellows reported significant increases in understanding the culture of medicine, improved confidence in their curricula and scholarship, and expanded professional networks, compared to pre-fellowship levels. The program required many hours of volunteer time by leaders, faculty, and mentors plus modest support from STFM staff. Leaders in family medicine education, confronted by the need for inter-professional development, designed and implemented a successful training program for behavioral science faculty.


Clinicians' attitudes toward standardized assessment and diagnosis within child and adolescent psychiatry.
There is a strong call for clinically useful standardized assessment tools in everyday child and adolescent psychiatric practice. The attitudes of clinicians have been raised as a key-facilitating factor when implementing new methods. An explorative study was conducted aimed to investigate the clinicians' attitudes regarding standardized assessments and usefulness of diagnoses in treatment planning. 411 mental health service personnel working with outpatient and inpatient assessment and treatment within the specialist child and adolescent mental health services, Stockholm County Council were asked to participate in the study, of which 345 (84%) agreed answer a questionnaire. The questionnaire included questions regarding Attitudes toward Standardized Assessment and Utility of Diagnosis. Descriptive analyses were performed and four subscales were compared with information from a similar study in US using the same instruments. The demographic and professional characteristics (age, working years, gender, education, profession, management position, involvement in assessment, level of service) in terms of prediction of attitudes were studied by univariate and multivariate linear regressions. Overall, the clinicians had quite positive attitudes and were more positive compared to a similar study conducted in the US earlier. There were differences in attitudes due to several characteristics but the only characteristic predicting all subscales was type of profession (counselor, nurse, psychiatrist, psychologist, other), with counselors being less positive than other groups. The overall positive attitudes toward standard assessment are of importance in the development of evidence-based practice and our study implies that clinicians in general value and are willing to use standardized assessment. Nevertheless, there are specific issues such as adequate training and available translated assessment instrument that need to be addressed. When implementing new methods in practice, there are general as well as specific resistances that need to be overcome. Studies in different cultural settings are of importance to further extend the knowledge of what is general and what is specific barriers.


The metabolic disposition of laudanosine in dog, rabbit, and man.
This paper summarizes the results of studies of the metabolic fate of laudanosine, a major degradation product of atracurium. Intravenous bolus doses of laudanosine (1-3 mg/kg) were administered to eight dogs and two rabbits anesthetized with halothane, and urine and bile samples were collected for up to 6 hr. Urine samples also were collected from two surgical patients given repetitive doses of atracurium. Metabolites were isolated from all samples using C18-Sep Paks. Treatment of the isolates with beta-glucuronidase followed by purification of the hydrolysate by preparative liquid chromatography provided metabolite fractions which were characterized by analytical liquid chromatography and capillary gas chromatography combined with nitrogen-phosphorus and/or electron ionization-mass spectrometric detection. Reference compounds were employed as chromatographic retention time markers. O-Trimethylsilyl, O-tert-butyldimethylsilyl, and N-trifluoroacetyl derivatives of the metabolites and reference compounds were used for gas chromatographic and mass spectrometric analysis. In all three species, the following metabolites of laudanosine were identified: pseudocodamine (4'-desmethyllaudanosine), pseudolaudanine (6-desmethyllaudanosine), laudanine (3'-desmethyllaudanosine), codamine (7-desmethyllaudanosine), N-norlaudanosine, N-norpseudocodamine, and N-norpseudolaudanine.


Efficacy and Safety of Direct-Acting Antivirals in Elderly Patients with Chronic Hepatitis C: A Nationwide Real-Life, Observational, Multicenter Study from Turkey.
The number and proportion of elderly patients living with chronic hepatitis C are expected to increase in the coming years. We aimed to compare the real-world efficacy and safety of direct-acting antiviral treatment in elderly and younger Turkish adults infected with chronic hepatitis C. In this multicenter prospective study, 2629 eligible chronic hepatitis C patients treated with direct-acting antivirals between April 2017 and December 2019 from 37 Turkish referral centers were divided into 2 age groups: elderly (≥65 years) and younger adults (<65 years) and their safety was compared between 2 groups in evaluable population. Then, by matching the 2 age groups for demographics and pretreatment risk factors for a non-sustained virological response, a total of 1516 patients (758 in each group) and 1244 patients (622 in each group) from the modified evaluable population and per-protocol population were included in the efficacy analysis and the efficacy was compared between age groups. The sustained virological response in the chronic hepatitis C patients was not affected by the age and the presence of cirrhosis both in the modified evaluable population and per-protocol population (P = .879, P = .508 for modified evaluable population and P = .058, P = .788 for per-protocol population, respectively). The results of the per-protocol analysis revealed that male gender, patients who had a prior history of hepatocellular carcinoma, patients infected with non-genotype 1 hepatitis C virus, and patients treated with sofosbuvir+ribavirin had a significantly lower sustained virological response 12 rates (P < .001, P = .047, P = .013, and P = .025, respectively). Direct-acting antivirals can be safely used to treat Turkish elderly chronic hepatitis C patients with similar favorable efficacy and safety as that in younger adults.


Challenges to liver transplantation and strategies to improve outcomes.
Liver transplantation (LT) is a highly successful treatment for many patients with nonmalignant and malignant liver diseases. However, there is a worldwide shortage of available organs; many patients deteriorate or die while on waiting lists. We review the important clinical challenges to LT and the best use of the scarce organs. We focus on changes in indications for LT and discuss scoring systems to best match donors with recipients and optimize outcomes, particularly for the sickest patients. We also cover controversial guidelines for the use of LT in patients with hepatocellular carcinoma and cholangiocarcinoma. Strategies to increase the number of functional donor organs involve techniques to perfuse the organs before implantation. Partial LT (living donor and split liver transplantation) techniques might help to overcome organ shortages, and we discuss small-for-size syndrome. Many new developments could increase the success of this procedure, which is already one of the major achievements in medicine during the second part of the 20th century.


Extending thrombolysis to 4·5-9 h and wake-up stroke using perfusion imaging: a systematic review and meta-analysis of individual patient data.
Stroke thrombolysis with alteplase is currently recommended 0-4·5 h after stroke onset. We aimed to determine whether perfusion imaging can identify patients with salvageable brain tissue with symptoms 4·5 h or more from stroke onset or with symptoms on waking who might benefit from thrombolysis. In this systematic review and meta-analysis of individual patient data, we searched PubMed for randomised trials published in English between Jan 1, 2006, and March 1, 2019. We also reviewed the reference list of a previous systematic review of thrombolysis and searched ClinicalTrials.gov for interventional studies of ischaemic stroke. Studies of alteplase versus placebo in patients (aged ≥18 years) with ischaemic stroke treated more than 4·5 h after onset, or with wake-up stroke, who were imaged with perfusion-diffusion MRI or CT perfusion were eligible for inclusion. The primary outcome was excellent functional outcome (modified Rankin Scale [mRS] score 0-1) at 3 months, adjusted for baseline age and clinical severity. Safety outcomes were death and symptomatic intracerebral haemorrhage. We calculated odds ratios, adjusted for baseline age and National Institutes of Health Stroke Scale score, using mixed-effects logistic regression models. This study is registered with PROSPERO, number CRD42019128036. We identified three trials that met eligibility criteria: EXTEND, ECASS4-EXTEND, and EPITHET. Of the 414 patients included in the three trials, 213 (51%) were assigned to receive alteplase and 201 (49%) were assigned to receive placebo. Overall, 211 patients in the alteplase group and 199 patients in the placebo group had mRS assessment data at 3 months and thus were included in the analysis of the primary outcome. 76 (36%) of 211 patients in the alteplase group and 58 (29%) of 199 patients in the placebo group had achieved excellent functional outcome at 3 months (adjusted odds ratio [OR] 1·86, 95% CI 1·15-2·99, p=0·011). Symptomatic intracerebral haemorrhage was more common in the alteplase group than the placebo group (ten [5%] of 213 patients vs one [<1%] of 201 patients in the placebo group; adjusted OR 9·7, 95% CI 1·23-76·55, p=0·031). 29 (14%) of 213 patients in the alteplase group and 18 (9%) of 201 patients in the placebo group died (adjusted OR 1·55, 0·81-2·96, p=0·66). Patients with ischaemic stroke 4·5-9 h from stroke onset or wake-up stroke with salvageable brain tissue who were treated with alteplase achieved better functional outcomes than did patients given placebo. The rate of symptomatic intracerebral haemorrhage was higher with alteplase, but this increase did not negate the overall net benefit of thrombolysis. None.


Fetal cell-free DNA fraction in maternal plasma for the prediction of hypertensive disorders of pregnancy.
The purpose of this study is to compare the fetal fractions during non-invasive prenatal testing (NIPT) in singleton pregnancies according to gestational age and maternal characteristics to evaluate the utility of this parameter for the prediction of pregnancy complications including gestational diabetes mellitus (GDM) and hypertensive disorders of pregnancy (HDP). This study was a multicenter prospective cohort study. The present data were collected from women whose NIPT results were negative. The relationships between the fetal fractions and the gestational age, maternal weight and height, and incidences of miscarriage, preterm delivery, and pregnancy complications including GDM, HDP and placental abruption were assessed. A total of 5582 pregnant women with verified NIPT negative results were registered in the study. The demographic characteristics of the study populations were statistically analyzed, and the women with HDP tended to have a low fetal fraction in samples taken during early gestation. The area under the curve (AUC) in a receiver operating characteristic curve (ROC) analysis was 0.608 for women with HDP. A low fetal fraction on NIPT might be correlated with future HDP. However, predicting HDP during early pregnancy in women with a low fetal fraction might be difficult.


Cardiotoxicity Hazard and Risk Characterization of ToxCast Chemicals Using Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes from Multiple Donors.
Heart disease remains a significant human health burden worldwide with a significant fraction of morbidity attributable to environmental exposures. However, the extent to which the thousands of chemicals in commerce and the environment may contribute to heart disease morbidity is largely unknown, because in contrast to pharmaceuticals, environmental chemicals are seldom tested for potential cardiotoxicity. Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes have become an informative in vitro model for cardiotoxicity testing of drugs with the availability of cells from multiple individuals allowing in vitro testing of population variability. In this study, we hypothesized that a panel of iPSC-derived cardiomyocytes from healthy human donors can be used to screen for the potential cardiotoxicity hazard and risk of environmental chemicals. We conducted concentration-response testing of 1029 chemicals (drugs, pesticides, flame retardants, polycyclic aromatic hydrocarbons (PAHs), plasticizers, industrial chemicals, food/flavor/fragrance agents, etc.) in iPSC-derived cardiomyocytes from 5 donors. We used kinetic calcium flux and high-content imaging to derive quantitative measures as inputs into Bayesian population concentration-response modeling of the effects of each chemical. We found that many environmental chemicals pose a hazard to human cardiomyocytes in vitro with more than half of all chemicals eliciting positive or negative chronotropic or arrhythmogenic effects. However, most of the tested environmental chemicals for which human exposure and high-throughput toxicokinetics data were available had wide margins of exposure and, thus, do not appear to pose a significant human health risk in a general population. Still, relatively narrow margins of exposure (<100) were estimated for some perfuoroalkyl substances and phthalates, raising concerns that cumulative exposures may pose a cardiotoxicity risk. Collectively, this study demonstrated the value of using a population-based human in vitro model for rapid, high-throughput hazard and risk characterization of chemicals for which little to no cardiotoxicity data are available from guideline studies in animals.


Transport Kinetics, Selective Inhibition, and Successful Prediction of In Vivo Inhibition of Rat Hepatic Organic Anion Transporting Polypeptides.
For successful in vitro-to-in vivo extrapolation of hepatic drug uptake and drug-drug interactions (DDI), it is important to characterize the kinetic properties of the individual transporters involved, their fraction (ft) contribution to hepatic uptake, and their selective inhibitors. Here, we characterized the in vitro transport kinetics of two model drugs, rosuvastatin (RSV) and olmesartan acid (OLM), by rat hepatic organic anion transporting polypeptides (Oatp1a1, 1a4, and 1b2) and identified selective inhibitors of these transporters. [3H]-RSV was transported by Oatp1a1, 1a4, and 1b2, and their Michaelis-Menten constant (Km) values were estimated to be 9.61, 67.2, and 28.1 µM, respectively. In contrast, [3H]-OLM was transported by only Oatp1b2 (Km: 72.8 µM). Digoxin (IC50: 0.107 µM) and rifamycin SV (IC50: 0.140 and 0.088 µM for RSV and OLM, respectively) were potent and selective inhibitors of Oatp1a4 and 1b2, respectively, and glyburide (100 µM) completely inhibited all three rat hepatic Oatps. These inhibitors can therefore be used alone and in combination to determine the contribution of each Oatp to hepatic influx. In addition, the magnitude of in vivo inhibition of sinusoidal uptake clearance of RSV by rifampin was well predicted using rifampin IC50 profiles for each Oatps and RSV ft by each Oatp. This is the first report to 1) detail the transport kinetics of RSV and OLM by rat hepatic Oatps, 2) identify selective inhibitor concentrations of rat Oatps, and 3) demonstrate successful prediction of the magnitude of transporter-mediated in vivo DDI from IC50 profiles of an inhibitor and ft of a drug by each transporter.


Synthetic microbial consortia: from systematic analysis to construction and applications.
Synthetic biology is an emerging research field that focuses on using rational engineering strategies to program biological systems, conferring on them new functions and behaviours. By developing genetic parts and devices based on transcriptional, translational, post-translational modules, many genetic circuits and metabolic pathways had been programmed in single cells. Extending engineering capabilities from single-cell behaviours to multicellular microbial consortia represents a new frontier of synthetic biology. Herein, we first reviewed binary interaction modes of microorganisms in microbial consortia and their underlying molecular mechanisms, which lay the foundation of programming cell-cell interactions in synthetic microbial consortia. Systems biology studies on cellular systems enable systematic understanding of diverse physiological processes of cells and their interactions, which in turn offer insights into the optimal design of synthetic consortia. Based on such fundamental understanding, a comprehensive array of synthetic microbial consortia constructed in the last decade were reviewed, including isogenic microbial communities programmed by quorum sensing-based cell-cell communications, sender-receiver microbial communities with one-way communications, and microbial ecosystems wired by two-way (bi-directional) communications. Furthermore, many applications including using synthetic microbial consortia for distributed bio-computations, chemicals and bioenergy production, medicine and human health, and environments were reviewed. Synergistic development of systems and synthetic biology will provide both a thorough understanding of naturally occurring microbial consortia and rational engineering of these complicated consortia for novel applications.


Section-level relationships of North American Agalinis (Orobanchaceae) based on DNA sequence analysis of three chloroplast gene regions.
The North American Agalinis are representatives of a taxonomically difficult group that has been subject to extensive taxonomic revision from species level through higher sub-generic designations (e.g., subsections and sections). Previous presentations of relationships have been ambiguous and have not conformed to modern phylogenetic standards (e.g., were not presented as phylogenetic trees). Agalinis contains a large number of putatively rare taxa that have some degree of taxonomic uncertainty. We used DNA sequence data from three chloroplast genes to examine phylogenetic relationships among sections within the genus Agalinis Raf. (=Gerardia), and between Agalinis and closely related genera within Orobanchaceae. Maximum likelihood analysis of sequences data from rbcL, ndhF, and matK gene regions (total aligned length 7323 bp) yielded a phylogenetic tree with high bootstrap values for most branches. Likelihood ratio tests showed that all but a few branch lengths were significantly greater than zero, and an additional likelihood ratio test rejected the molecular clock hypothesis. Comparisons of substitution rates between gene regions based on linear models of pairwise distance estimates between taxa show both ndhF and matK evolve more rapidly than rbcL, although the there is substantial rate heterogeneity within gene regions due in part to rate differences among codon positions. Phylogenetic analysis supports the monophyly of Agalinis, including species formerly in Tomanthera, and this group is sister to a group formed by the genera Aureolaria, Brachystigma, Dasistoma, and Seymeria. Many of the previously described sections within Agalinis are polyphyletic, although many of the subsections appear to form natural groups. The analysis reveals a single evolutionary event leading to a reduction in chromosome number from n = 14 to n = 13 based on the sister group relationship of section Erectae and section Purpureae subsection Pedunculares. Our results establish the evolutionary distinctiveness of A. tenella from the more widespread and common A. obtusifolia. However, further data are required to clearly resolve the relationship between A. acuta and A. tenella.


Possible relevance of phospholipid abnormalities and genetic interactions in psychiatric disorders: the relationship between dyslexia and schizophrenia.
The fatty acids of cell membrane phospholipids are essential for normal membrane structures, for the functioning of membrane-bound and membrane-associated proteins and for normal cell-signalling responses. In dyslexia, there is evidence for reduced incorporation of docosahexaenoic acid and arachidonic acid into cell membranes, while in schizophrenia, there is evidence for an increased rate of docosahexaenoic acid and arachidonic acid loss from membranes because of enhanced phospholipase A2 activity. The presence of both defects will cause a much greater degree of abnormality than either one alone. It is hypothesized that unequivocal clinical schizophrenia may occur when both genes are present in the same individual. The dyslexia gene along will produce dyslexia while the schizophrenia gene alone may produce bipolar or schizoaffective disorders. These proposals could explain: 1. The reduced asymmetry of the brain, especially of the planum temporale in both schizophrenia and dyslexia; 2. The schizotypal personality characteristics of dyslexics; 3. The increased risks of dyslexia in families with a schizophrenic proband; 4. The increased risks of bipolar and schizoaffective disorders in families with a schizophrenic proband; 5. The earlier onset and possibly increased severity of both disorders in males since females have a lower requirement for arachidonic acid and docosahexaenoic acid; 6. The absence of selective pressure against schizophrenia since reproduction would be impaired only when the schizophrenic gene coexisted with a dyslexic gene. The schizophrenic gene alone might even lead to improved reproductive performance.


Smoking, smoking cessation, and risk for type 2 diabetes mellitus: a cohort study.
Cigarette smoking is an established predictor of incident type 2 diabetes mellitus, but the effects of smoking cessation on diabetes risk are unknown. To test the hypothesis that smoking cessation increases diabetes risk in the short term, possibly owing to cessation-related weight gain. Prospective cohort study. The ARIC (Atherosclerosis Risk in Communities) Study. 10,892 middle-aged adults who initially did not have diabetes in 1987 to 1989. Smoking was assessed by interview at baseline and at subsequent follow-up. Incident diabetes was ascertained by fasting glucose assays through 1998 and self-report of physician diagnosis or use of diabetes medications through 2004. During 9 years of follow-up, 1254 adults developed type 2 diabetes. Compared with adults who never smoked, the adjusted hazard ratio of incident diabetes in the highest tertile of pack-years was 1.42 (95% CI, 1.20 to 1.67). In the first 3 years of follow-up, 380 adults quit smoking. After adjustment for age, race, sex, education, adiposity, physical activity, lipid levels, blood pressure, and ARIC Study center, compared with adults who never smoked, the hazard ratios of diabetes among former smokers, new quitters, and continuing smokers were 1.22 (CI, 0.99 to 1.50), 1.73 (CI, 1.19 to 2.53), and 1.31 (CI, 1.04 to 1.65), respectively. Further adjustment for weight change and leukocyte count attenuated these risks substantially. In an analysis of long-term risk after quitting, the highest risk occurred in the first 3 years (hazard ratio, 1.91 [CI, 1.19 to 3.05]), then gradually decreased to 0 at 12 years. Residual confounding is possible even with meticulous adjustment for established diabetes risk factors. Cigarette smoking predicts incident type 2 diabetes, but smoking cessation leads to higher short-term risk. For smokers at risk for diabetes, smoking cessation should be coupled with strategies for diabetes prevention and early detection.


Microarray test results should not be compensated for multiplicity of gene contents.
Microarray technology has enabled the measurement of comprehensive transcriptomic information. However, each data entry may reflect trivial individual differences among samples and also contain technical noise. Therefore, the certainty of each observed difference should be confirmed at earlier steps of the analyses, and statistical tests are frequently used for this purpose. Since microarrays analyze a huge number of genes simultaneously, concerns of multiplicity, i.e. the family wise error rate (FWER) and false discovery rate (FDR), have been raised in testing the data. To deal with these concerns, several compensation methodologies have been proposed, making the tests very conservative to the extent that arbitrary tuning of the threshold has been introduced to relax the conditions. Unexpectedly, however, the appropriateness of the test methodologies, the concerns of multiplicity, and the compensation methodologies have not been sufficiently confirmed. The appropriateness was checked by means of coincidence between the methodologies' premises and the statistical characteristics of data found in two typical microarray platforms. As expected, normality was observed in within-group data differences, supporting application of t-test and F-test statistics. However, genes displayed their own tendencies in the magnitude of variations, and the distributions of p-values were rather complex. These characteristics are inconsistent with premises underlying the compensation methodologies, which assume that most of the null hypotheses are true. The evidence also raised concerns about multiplicity. In transcriptomic studies, FWER should not be critical, as analyses at higher levels would not be influenced by a few false positives. Additionally, the concerns for FDR are not suitable for the sharp null hypotheses on expression levels. Therefore, although compensation methods have been recommended to deal with the problem of multiplicity, the compensations are actually inappropriate for transcriptome analyses. Compensations are not only unnecessary, but will increase the occurrence of false negative errors, and arbitrary adjustment of the threshold damages the objectivity of the tests. Rather, the results of parametric tests should be evaluated directly.


How well tolerated are lipid-lowering drugs?
It has been clearly established that lipid-lowering treatments [such as 3-hydroxyl-3-methylglutamyl coenzyme A reductase inhibitors ('statins') or fibrates] can reduce cardiovascular events, and with one of the statins even total mortality, in high-risk populations. Intervention studies have not included the very old, but it is generally assumed that this patient group would benefit from these treatments to an extent similar to younger patients. Worries about the associations seen in observational studies between low cholesterol levels and cancer, cerebral haemorrhage or mood and behaviour change have been largely overcome by findings from the latest large drug intervention trials, which do not show any increase in these conditions with statin or fibrate treatments. The common adverse effects associated with these drugs are relatively mild and often transient in nature. Potentially more serious adverse effects, which are more clearly related to drug treatment and are probably dose-dependent, include elevations in hepatic transaminase levels and myopathy; however, these effects are uncommon and generally resolve rapidly when treatment is stopped. The risk of myopathy with fibrate treatment is increased in patients with renal impairment, and the risk of myopathy with statin treatment increases with co-administration of drugs that inhibit statin metabolism or transport. Other adverse effects are related to specific drugs, for example, clofibrate is associated with an increased risk of gallstones. Studies in elderly patients have not shown an increased risk of adverse effects with lipid-lowering drugs compared with younger patients, but in clinical practice there may be some increased risk, particularly with regards to drug interactions. Therefore, lipid-lowering drugs should be administered with extra caution to elderly patients.


Type of β-blocker use among patients with versus without diabetes after myocardial infarction.
Discharge β-blocker prescription after myocardial infarction (MI) is recommended for all eligible patients. Numerous β-blocker choices are presently available with variable glycometabolic effects, which could be an important consideration in patients with diabetes mellitus (DM). Whether patients with DM preferentially receive β-blockers with favorable metabolic effects after MI and if this choice is associated with better glycemic control postdischarge is unknown. Among patients from 24 US hospitals enrolled in an MI registry (2005-2008), we investigated the frequency of "DM-friendly" β-blocker prescription at discharge by DM status. β-Blockers were classified as DM-friendly (eg, carvedilol and labetalol) or non-DM-friendly (eg, metoprolol and atenolol), based on their effects on glycemic control. Hierarchical, multivariable logistic regression examined the association of DM with DM-friendly β-blocker use. Among DM patients, we examined the association of DM-friendly β-blockers with worsened glycemic control at 6 months after MI. Of 4,031 MI patients, 1,382 (34%) had DM. β-Blockers were prescribed at discharge in 93% of patients. Diabetes mellitus-friendly β-blocker use was low regardless of DM status, although patients with DM were more likely to be discharged on a DM-friendly β-blocker compared with patients without DM (13.5% vs 10.3%, P = .003), an association that remained after multivariable adjustment (odds ratio 1.41, 95% CI 1.13-1.77). There was a trend toward a lower risk of worsened glucose control at 6 months in DM patients prescribed DM-friendly versus non-DM-friendly β-blockers (Relative Risk 0.80, 95% CI 0.60-1.08). Most DM patients were prescribed non-DM-friendly β-blockers-a practice that was associated with a trend toward worse glycemic control postdischarge. Although in need of further confirmation in larger studies, our findings highlight an opportunity to improve current practices of β-blockers use in patients with DM.


Facilitating pharmacogenetic studies using electronic health records and natural-language processing: a case study of warfarin.
DNA biobanks linked to comprehensive electronic health records systems are potentially powerful resources for pharmacogenetic studies. This study sought to develop natural-language-processing algorithms to extract drug-dose information from clinical text, and to assess the capabilities of such tools to automate the data-extraction process for pharmacogenetic studies. A manually validated warfarin pharmacogenetic study identified a cohort of 1125 patients with a stable warfarin dose, in which 776 patients were managed by Coumadin Clinic physicians, and the remaining 349 patients were managed by their providers. The authors developed two algorithms to extract weekly warfarin doses from both data sets: a regular expression-based program for semistructured Coumadin Clinic notes; and an advanced weekly dose calculator based on an existing medication information extraction system (MedEx) for narrative providers' notes. The authors then conducted an association analysis between an automatically extracted stable weekly dose of warfarin and four genetic variants of VKORC1 and CYP2C9 genes. The performance of the weekly dose-extraction program was evaluated by comparing it with a gold standard containing manually curated weekly doses. Precision, recall, F-measure, and overall accuracy were reported. Associations between known variants in VKORC1 and CYP2C9 and warfarin stable weekly dose were performed with linear regression adjusted for age, gender, and body mass index. The authors' evaluation showed that the MedEx-based system could determine patients' warfarin weekly doses with 99.7% recall, 90.8% precision, and 93.8% accuracy. Using the automatically extracted weekly doses of warfarin, the authors successfully replicated the previous known associations between warfarin stable dose and genetic variants in VKORC1 and CYP2C9.


Cell Adhesion on Dynamic Supramolecular Surfaces Probed by Fluid Force Microscopy-Based Single-Cell Force Spectroscopy.
Biomimetic and stimuli-responsive cell-material interfaces are actively being developed to study and control various cell-dynamics phenomena. Since cells naturally reside in the highly dynamic and complex environment of the extracellular matrix, attempts are being made to replicate these conditions in synthetic biomaterials. Supramolecular chemistry, dealing with noncovalent interactions, has recently provided possibilities to incorporate such dynamicity and responsiveness in various types of architectures. Using a cucurbit[8]uril-based host-guest system, we have successfully established a dynamic and electrochemically responsive interface for the display of the integrin-specific ligand, Arg-Gly-Asp (RGD), to promote cell adhesion. Due to the weak nature of the noncovalent forces by which the components at the interface are held together, we expected that cell adhesion would also be weaker in comparison to traditional interfaces where ligands are usually immobilized by covalent linkages. To assess the stability and limitations of our noncovalent interfaces, we performed single-cell force spectroscopy studies using fluid force microscopy. This technique enabled us to measure rupture forces of multiple cells that were allowed to adhere for several hours on individual substrates. We found that the rupture forces of cells adhered to both the noncovalent and covalent interfaces were nearly identical for up to several hours. We have analyzed and elucidated the reasons behind this result as a combination of factors including the weak rupture force between linear Arg-Gly-Asp and integrin, high surface density of the ligand, and increase in effective concentration of the supramolecular components under spread cells. These characteristics enable the construction of highly dynamic biointerfaces without compromising cell-adhesive properties.


Monitoring the aquatic toxicity of mosquito vector control spray pesticides to freshwater receiving waters.
Pesticides are applied to state and local waterways in California to control insects such as mosquitoes, which are known to serve as a vector for West Nile Virus infection of humans. The California State Water Resources Control Board adopted a National Pollutant Discharge Elimination System General Permit to address the discharge to waters of the United States of pesticides resulting from adult and larval mosquito control. Because pesticides used in spray activities have the potential to cause toxicity to nontarget organisms in receiving waters, the current study was designed to determine whether toxicity testing provides additional, useful environmental risk information beyond chemical analysis in monitoring spray pesticide applications. Monitoring included a combination of aquatic toxicity tests and chemical analyses of receiving waters from agricultural, urban, and wetland habitats. The active ingredients monitored included the organophosphate pesticides malathion and naled, the pyrethroid pesticides etofenprox, permethrin, and sumithrin, pyrethrins, and piperonyl butoxide (PBO). Approximately 15% of the postapplication water samples were significantly toxic. Toxicity of half of these samples was attributed to the naled breakdown product dichlorvos. Toxicity of 2 other water samples likely occurred when PBO synergized the effects of pyrethroid pesticides that were likely present in the receiving system. Four of 43 postapplication sediment samples were significantly more toxic than their corresponding pre-application samples, but none of the observed toxicity was attributed to the application events. These results indicate that many of the spray pesticides used for adult mosquito control do not pose significant acute toxicity risk to invertebrates in receiving systems. In the case of naled in water, analysis of only the active ingredient underestimated potential impacts to the receiving system, because toxicity was attributed to the breakdown product, dichlorvos. Toxicity testing can provide useful risk information about unidentified, unmeasured toxicants or mixtures of toxicants. In this case, toxicity testing provided information that could lead to the inclusion of dichlorvos monitoring as a permit requirement.


Combining Multiple Hypothesis Testing with Machine Learning Increases the Statistical Power of Genome-wide Association Studies.
The standard approach to the analysis of genome-wide association studies (GWAS) is based on testing each position in the genome individually for statistical significance of its association with the phenotype under investigation. To improve the analysis of GWAS, we propose a combination of machine learning and statistical testing that takes correlation structures within the set of SNPs under investigation in a mathematically well-controlled manner into account. The novel two-step algorithm, COMBI, first trains a support vector machine to determine a subset of candidate SNPs and then performs hypothesis tests for these SNPs together with an adequate threshold correction. Applying COMBI to data from a WTCCC study (2007) and measuring performance as replication by independent GWAS published within the 2008-2015 period, we show that our method outperforms ordinary raw p-value thresholding as well as other state-of-the-art methods. COMBI presents higher power and precision than the examined alternatives while yielding fewer false (i.e. non-replicated) and more true (i.e. replicated) discoveries when its results are validated on later GWAS studies. More than 80% of the discoveries made by COMBI upon WTCCC data have been validated by independent studies. Implementations of the COMBI method are available as a part of the GWASpi toolbox 2.0.


The structure, distribution and evolution of the Ta1 retrotransposable element family of Arabidopsis thaliana.
The Ta1 elements are a low copy number, copia-like retrotransposable element family of Arabidopsis thaliana. Six Ta1 insertions comprise all of the Ta1 element copies found in three geographically diverse A. thaliana races. These six elements occupy three distinct target sites: Ta1-1 is located on chromosome 5 and is common to all three races (Col-0, Kas-1 and La-0). Ta1-2 is present in two races on chromosome 4 (Kas-1 and La-0), and Ta1-3, also located on chromosome 4, is present only in one race (La-0). The six Ta1 insertions share greater than 96% nucleotide identity, yet are likely to be incapable of further transposition due to deletions or nucleotide changes that alter either the coding capacity of the elements or conserved protein domains required for retrotransposition. Nucleotide sequence comparisons of these elements and the distribution of Ta1 among 12 additional A. thaliana geographical races suggest that Ta1-1 predated the global dispersal of A. thaliana. As the species spread throughout the world, two additional transposition events occurred which gave rise first to Ta1-2 and finally to Ta1-3.


[Hepatitis C virus: 25 years-old, the end?].
The treatment of hepatitis C virus (HCV) infection markedly progressed these two last decades. Since 15 years, the combination of pegylated interferon α and ribavirin led to a sustained virologic response (SVR) which corresponds to a complete recovery in around 45 % of patients with HCV genotype 1, 65 % with HCV genotype 4, 70 % with HCV genotype 3 and around 85 % with HCV genotype 2. A better understanding of the HCV life-cycle recently resulted in the development of several potential direct-acting antiviral drugs (DAA) targeting viral proteins (NS3/4A protease inhibitors, NS5B nucleosidic and non nucleosidic polymerase inhibitors, NS5A replication complex inhibitors). A lot of data has been reported with the combinations of pegylated interferon α/ribavirin and the first generation oral DAA, Telaprevir and Boceprevir. These regimens have demonstrated a high level of antiviral efficacy (75 % of SVR) and an acceptable safety profile. After this first major step, the combination of the second generation DAA with pegylated interferon α/ribavirin will impact antiviral potency (75 to 90 % of SVR) and tolerance and will reduce the duration of therapies and the pill burden. The next step, which is an actual revolution, will be the oral combination of new DAA which is likely to become the standard of care for chronic HCV after 2015. Most studies have been conducted in small numbers of "easy-to-treat" patients with short post-treatment period with outstanding results but we are now waiting for confirming these results in more difficult-to-treat patients (experienced genotype 3-infected or genotype 1-infected patients who failed to first generation protease inhibitors, cirrhotic, HIV co-infected patients, allograft recipients or candidates to transplantation).


Controlled and localized genetic manipulation in the brain.
Brain structure and function are determined in part through experience and in part through our inherited genes. A powerful approach for unravelling the balance between activity-dependent neuronal plasticity and genetic programs is to directly manipulate the genome. Such molecular genetic studies have been greatly aided by the remarkable progress of large-scale genome sequencing efforts. Sophisticated mouse genetic manipulations allow targeted point-mutations, deletions and additions to the mouse genome. These can be regulated through inducible promoters expressing in genetically specified neuronal cell types. However, despite significant progress it remains difficult to target specific brain regions through transgenesis alone. Recent work suggests that transduction vectors, like lentiviruses and adeno-associated viruses, may provide suitable additional tools for localized and controlled genetic manipulation. Furthermore, studies with such vectors may aid the development of human genetic therapies for brain diseases.