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Protein prenylation by farnesyltransferase (FTase) is often described as the targeting of a cysteine-containing motif (CaaX) that is enriched for aliphatic amino acids at the a1 and a2 positions, while quite flexible at the X position. Prenylation prediction methods often rely on these features despite emerging evidence that FTase has broader target specificity than previously considered. Using a machine learning approach and training sets based on canonical (prenylated, proteolyzed, and carboxymethylated) and recently identified shunted motifs (prenylation only), this study aims to improve prenylation predictions with the goal of determining the full scope of prenylation potential among the 8000 possible Cxxx sequence combinations. Further, this study aims to subdivide the prenylated sequences as either shunted (i.e., uncleaved) or cleaved (i.e., canonical). Predictions were determined for Saccharomyces cerevisiae FTase and compared to results derived using currently available prenylation prediction methods. In silico predictions were further evaluated using in vivo methods coupled to two yeast reporters, the yeast mating pheromone a-factor and Hsp40 Ydj1p, that represent proteins with canonical and shunted CaaX motifs, respectively. Our machine learning-based approach expands the repertoire of predicted FTase targets and provides a framework for functional classification.

The spread of COVID-19 and resulting local and national lockdowns have a host of potential consequences for demographic trends. While impacts on mortality and, to some extent, short-term migration flows are beginning to be documented, it is too early to measure actual consequences for family demography. To gain insight into potential future consequences of the lockdown for family demography, we use cross-national Google Trends search data to explore whether trends in searches for words related to fertility, relationship formation, and relationship dissolution changed following lockdowns compared to average, pre-lockdown levels in Europe and the United States. Because lockdowns were not widely anticipated or simultaneous in timing or intensity, we exploit variability over time and between countries (and U.S. states). We use a panel event-study design and difference-in-differences methods, and account for seasonal trends and average country-level (or state-level) differences in searches. We find statistically significant impacts of lockdown timing on changes in searches for terms such as wedding and those related to condom use, emergency contraception, pregnancy tests, and abortion, but little evidence of changes in searches related to fertility. Impacts for union formation and dissolution tended to only be statistically significant at the start of a lockdown with a return to average-levels about 2 to 3 months after lockdown initiation, particularly in Europe. Compared to Europe, returns to average search levels were less evident for the U.S., even 2 to 3 months after lockdowns were introduced. This may be due to the fact, in the U.S., health and social policy responses were less demarcated than in Europe, such that economic uncertainty was likely of larger magnitude. Such pandemic-related economic uncertainty may therefore have the potential to slightly increase already existing polarization in family formation behaviours in the U.S. Alongside contributing to the wider literature on economic uncertainty and family behaviors, this paper also proposes strategies for efficient use of Google Trends data, such as making relative comparisons and testing sensitivity to outliers, and provides a template and cautions for their use in demographic research when actual demographic trends data are not yet available.

Acute myeloid leukemia (AML) is a heterogeneous disease. Prognosis is mainly influenced by patient age at diagnosis and cytogenetic alterations, two of the main factors currently used in AML patient risk stratification. However, additional criteria are required to improve the current risk classification and better adapt patient care. In neoplastic cells, ribosome biogenesis is increased to sustain the high proliferation rate and ribosome composition is altered to modulate specific gene expression driving tumorigenesis. Here, we investigated the usage of ribosome biogenesis factors as clinical markers in adult patients with AML. We showed that nucleoli, the nucleus compartments where ribosome production takes place, are modified in AML by analyzing a panel of AML and healthy donor cells using immunofluorescence staining. Using four AML series, including the TCGA dataset, altogether representing a total of about 270 samples, we showed that not all factors involved in ribosome biogenesis have clinical values although ribosome biogenesis is increased in AML. Interestingly, we identified the regulator of ribosome production nucleolin (NCL) as over-expressed in AML blasts. Moreover, we found in two series that high NCL mRNA expression level was associated with a poor overall survival, particular in elderly patients. Multivariate analyses taking into account age and cytogenetic risk indicated that NCL expression in blast cells is an independent marker of reduced survival. Our study identifies NCL as a potential novel prognostic factor in AML. Altogether, our results suggest that the ribosome biogenesis pathway may be of interest as clinical markers in AML.

The molecular mechanisms that regulate cardiomyocyte proliferation during embryonic heart growth are not completely deciphered yet. In a forward genetic N-ethyl-N-nitrosourea (ENU) mutagenesis screen, we identified the recessive embryonic-lethal zebrafish mutant line weiches herz (whz). Homozygous mutant whz embryos display impaired heart growth due to diminished embryonic cardiomyocyte proliferation resulting in cardiac hypoplasia and weak cardiac contraction. By positional cloning, we found in whz mutant zebrafish a missense mutation within the T-box 20 (Tbx20) transcription factor gene leading to destabilization of Tbx20 protein. Morpholino-mediated knock-down of Tbx20 in wild-type zebrafish embryos phenocopies whz, indicating that the whz phenotype is due to loss of Tbx20 function, thereby leading to significantly reduced cardiomyocyte numbers by impaired proliferation of heart muscle cells. Ectopic overexpression of wild-type Tbx20 in whz mutant embryos restored cardiomyocyte proliferation and heart growth. Interestingly, ectopic overexpression of Tbx20 in wild-type zebrafish embryos resulted, similar to the situation in the embryonic mouse heart, in significantly reduced proliferation rates of ventricular cardiomyocytes, suggesting that Tbx20 activity needs to be tightly fine-tuned to guarantee regular cardiomyocyte proliferation and embryonic heart growth in vivo.

BACKGROUND:Mitochondrial large-conductance Ca2+-sensitive potassium (mBKCa) channels are involved in myocardial ischemic preconditioning. Their role in sildenafil-induced cardioprotection is unknown. We investigated whether sildenafil-induced acute cardioprotection is mediated by activation of mBKCa channels in the rat heart in vitro. METHODS:Male Wistar rats (n = 8 per group) were randomized and anesthetized with pentobarbital (90 mg/kg). Hearts were isolated, mounted on a Langendorff system and perfused with Krebs-Henseleit buffer at a constant pressure of 80 mmHg. Hearts underwent 30 min of global ischemia followed by 60 min of reperfusion. At the end of the experiments infarct size was determined by TTC staining. In the control group rats were not further treated. Sildenafil (3 μM) was administered over 10 min before the beginning of ischemia. The mBKCa channel inhibitor paxilline (1 μM) was administered with and without sildenafil before the onset of ischemia. The pathway underlying sildenafil-induced cardioprotection was further investigated with the protein kinase G blocker KT5823 (1 μM). Myocardial cGMP concentration was measured by ELISA. Data (mean±SD) were analysed with a one and two-way analysis of variance as appropriate. RESULTS:In control animals infarct size was 52±8%. Sildenafil increased cGMP concentration and reduced infarct size to 35±6% (P

Erufosine is a new antineoplastic agent of the group of alkylphosphocholines, which interferes with signal transduction and induces apoptosis in various leukemic and tumor cell lines. The present study was designed to examine for the first time the mechanism of resistance to erufosine in malignant cells with permanently reduced expression of the retinoblastoma (Rb) protein. Bearing in mind the high number of malignancies with reduced level of this tumor-suppressor, this investigation was deemed important for using erufosine, alone or in combination, in patients with compromised RB1 gene expression. For this purpose, clones of the leukemic T-cell line SKW-3 were used, which had been engineered to constantly express differently low Rb levels. The alkylphosphocholine induced apoptosis, stimulated the expression of the cyclin dependent kinase inhibitor p27Kip1 and inhibited the synthesis of cyclin D3, thereby causing a G2 phase cell cycle arrest and death of cells with wild type Rb expression. In contrast, Rb-deficiency impeded the changes induced by erufosine in the expression of these proteins and abrogated the induction of G2 arrest, which was correlated with reduced antiproliferative and anticlonogenic activities of the compound. In conclusion, analysis of our results showed for the first time that the Rb signaling pathway is essential for mediating the antineoplastic activity of erufosine and its efficacy in patients with malignant diseases may be predicted by determining the Rb status.

We previously demonstrated that intratracheally administered S-ketamine inhibits alveolar fluid clearance (AFC), whereas an intravenous (i.v.) bolus injection had no effect. The aim of the present study was to characterize whether continuous i.v. infusion of S-ketamine, yielding clinically relevant plasma concentrations, inhibits AFC and whether its effect is enhanced in acute lung injury (ALI) which might favor the appearance of i.v. S-ketamine at the alveolar surface. AFC was measured in fluid-instilled rat lungs. S-ketamine was administered i.v. over 6 h (loading dose: 20 mg/kg, followed by 20 mg/kg/h), or intratracheally by addition to the instillate (75 µg/ml). ALI was induced by i.v. lipopolysaccharide (LPS; 7 mg/kg). Interleukin (IL)-6 and cytokine-induced neutrophil chemoattractant (CINC)-3 were measured by ELISA in plasma and bronchoalveolar lavage fluid. Isolated rat alveolar type-II cells were exposed to S-ketamine (75 µg/ml) and/or LPS (1 mg/ml) for 6 h, and transepithelial ion transport was measured as short circuit current (ISC). AFC was 27±5% (mean±SD) over 60 min in control rats and was unaffected by i.v. S-ketamine. Tracheal S-ketamine reduced AFC to 18±9%. In LPS-treated rats, AFC decreased to 16±6%. This effect was not enhanced by i.v. S-ketamine. LPS increased IL-6 and CINC-3 in plasma and bronchoalveolar lavage fluid. In alveolar type-II cells, S-ketamine reduced ISC by 37% via a decrease in amiloride-inhibitable sodium transport. Continuous administration of i.v. S-ketamine does not affect rat AFC even in endotoxin-induced ALI. Tracheal application with direct exposure of alveolar epithelial cells to S-ketamine decreases AFC by inhibition of amiloride-inhibitable sodium transport.

Precursors to 1/3 of colorectal cancer (CRC), serrated polyps have been under-detected by screening due to their inconspicuous, non-hemorrhagic, and proximal nature. A new multi-target stool DNA test (multi-target sDNA) shows high sensitivity for both CRC and advanced adenomas. Screen detection of serrated polyps by this approach requires further validation. We sought to assess and compare noninvasive detection of sessile serrated polyps (SSP) ≥ 1 cm by sDNA and an occult blood fecal immunochemical test (FIT).In a blinded prospective study, a single stool sample used for both tests was collected from 456 asymptomatic adults prior to screening or surveillance colonoscopy (criterion standard). All 29 patients with SSP ≥ 1 cm were included as cases and all 232 with no neoplastic findings as controls. Buffered stool samples were processed and frozen on receipt; Exact Sciences performed sDNA in batches using optimized analytical methods. The sDNA multi-marker panel targets methylated BMP3 (mBMP3) and NDRG4, mutant KRAS, β-actin, and hemoglobin. FIT (Polymedco OC-FIT Check) was performed in separate lab ≤ 2 days post defecation and evaluated at cutoffs of 50 (FIT-50) and 100 ng/ml (FIT-100).MEDIAN AGES: cases 61 (range 57-77), controls 62 (52-70), p = NS. Women comprised 59% and 51%, p = NS, respectively. SSP median size was 1.2 cm (1-3 cm), 93% were proximal, and 64% had synchronous diminutive polyps. Among multi-target sDNA markers, mBMP3 proved highly discriminant for detection of SSP ≥ 1 cm (AUC = 0.87, p

Structural biology and structural genomics projects routinely rely on recombinantly expressed proteins, but many proteins and complexes are difficult to obtain by this approach. We investigated native source proteins for high-throughput protein crystallography applications. The Escherichia coli proteome was fractionated, purified, crystallized, and structurally characterized. Macro-scale fermentation and fractionation were used to subdivide the soluble proteome into 408 unique fractions of which 295 fractions yielded crystals in microfluidic crystallization chips. Of the 295 crystals, 152 were selected for optimization, diffraction screening, and data collection. Twenty-three structures were determined, four of which were novel. This study demonstrates the utility of native source proteins for high-throughput crystallography.

Background: COVID-19 infection and its associated consequence, known as long-COVID, lead to a significant burden on the global healthcare system and limitations in people's personal and work lives. This study aims to provide further insight into the impact of acute and ongoing COVID-19 symptoms and investigates the role of patients' gender and vaccination status., Methods: 416 individuals (73.9% female) between the ages of 16 and 80 years (M = 44.18, SD = 12.90) with self-reported symptoms of long-COVID participated in an online survey conducted between March and May 2022., Results: 6.0%, 74.3%, and 19.7% of all respondents reported having had an asymptomatic, mild, or severe acute illness, respectively. Out of all participants, 7.8% required hospitalization. The most prevalent symptoms during the acute infection (Mdn = 23.50 symptoms, IQR = 13-39) included fatigue, exhaustion, cough, brain fog, and memory problems. The median long-COVID disease duration was 12.10 months (IQR = 2.8-17.4). Among 64 inquired long-COVID symptoms (Mdn = 17.00 symptoms, IQR = 9-27), participants reported fatigue, exhaustion, memory problems, brain fog, and dyspnea as the most common ongoing symptoms, which were generally experienced as fluctuating and deteriorating after physical or cognitive activity. Common consequences of long-COVID included financial losses (40.5%), changes in the participants' profession (41.0%), stress resistance (87.5%), sexual life (38.1%), and mood (72.1%), as well as breathing difficulties (41.3%), or an increased drug intake (e.g., medicine, alcohol; 44.6%). In addition, vaccinated individuals exhibited a shorter acute illness duration and an earlier onset of long-COVID symptoms. In general, women reported more long-COVID symptoms than men., Conclusion: Long-COVID represents a heterogeneous disease and impacts multiple life aspects of those affected. Tailored rehabilitation programs targeting the plurality of physical and mental symptoms are needed., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Leitner et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Risk assessment before interventions in elderly patients becomes more and more vital due to an increasing number of elderly patients requiring surgery. Existing risk scores are often not tailored to marginalized groups such as patients aged 80 years or older. We aimed to develop an easy-to-use and readily applicable risk assessment tool that implements pre-interventional predictors of 30-day mortality in elderly patients (≥80 years) undergoing interventions under anesthesia. Using Cox regression analysis, we compared different sets of predictors by taking into account their ease of availability and by evaluating predictive accuracy. Coefficient estimates were utilized to set up a scoring system that was internally validated. Model building and evaluation were based on data from the Peri-Interventional Outcome Study in the Elderly (POSE), which was conducted as a European multicenter, observational prospective cohort study. Our risk assessment tool, named PIRATE, contains three predictors assessable at admission (urgency, severity and living conditions). Discriminatory power, as measured by the concordance index, was 0.75. The estimated prediction error, as measured by the Brier score, was 0.036 (covariate-free reference model: 0.043). PIRATE is an easy-to-use risk assessment tool that helps stratifying elderly patients undergoing interventions with anesthesia at increased risk of mortality. PIRATE is readily available and applies to a wide variety of settings. In particular, it covers patients needing elective or emergency surgery and undergoing in-hospital or day-case surgery. Also, it applies to all types of interventions, from minor to major. It may serve as a basis for multidisciplinary and informed shared decision-making., Competing Interests: AK, MB, MC, MS and RR report grants from ESAIC, during the conduct of the study. AS: No competing interests declared. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Schenk et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Beall's list is widely used to identify potentially predatory journals. With this study, we aim to investigate the impact of Beall's list on the perception of listed journals as well as on the publication and citation behavior of the scientific community. We performed comprehensive bibliometric analyses of data extracted from the ISSN database, PubMed, PubMed Central (PMC), Crossref, Scopus and Web of Science. Citation analysis was performed by data extracted from the Crossref Cited-by database. At the time of analysis, Beall's list consisted of 1,289 standalone journals and 1,162 publishers, which corresponds to 21,735 individual journals. Of these, 3,206 (38.8%) were located in the United States, 2,484 in India (30.0%), and 585 in United Kingdom (7.1%). The majority of journals were listed in the ISSN database (n = 8,266), Crossref (n = 5,155), PubMed (n = 1,139), Scopus (n = 570), DOAJ (n = 224), PMC (n = 135) or Web of Science (n = 50). The number of articles published by journals on Beall's list as well as on the DOAJ continuously increased from 2011 to 2017. In 2018, the number of articles published by journals on Beall's list decreased. Journals on Beall's list were more often cited when listed in Web of Science (CI 95% 5.5 to 21.5; OR = 10.7) and PMC (CI 95% 6.3 to 14.1; OR = 9.4). It seems that the importance of Beall's list for the scientific community is overestimated. In contrast, journals are more likely to be selected for publication or citation when indexed by commonly used and renowned databases. Thus, the providers of these databases must be aware of their impact and verify that good publication practice standards are being applied by the journals listed., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Richtig et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Drosophila melanogaster larvae develop on fermenting fruits with increasing ethanol concentrations. To address the relevance of ethanol in the behavioral response of the larvae, we analyzed the function of ethanol in the context of olfactory associative behavior in Canton S and w1118 larvae. The motivation of larvae to move toward or out of an ethanol-containing substrate depends on the ethanol concentration and the genotype. Ethanol in the substrate reduces the attraction to odorant cues in the environment. Relatively short repetitive exposures to ethanol, which are comparable in their duration to reinforcer representation in olfactory associative learning and memory paradigms, result in positive or negative association with the paired odorant or indifference to it. The outcome depends on the order in which the reinforcer is presented during training, the genotype and the presence of the reinforcer during the test. Independent of the order of odorant presentation during training, Canton S and w1118 larvae do not form a positive or negative association with the odorant when ethanol is not present in the test context. When ethanol is present in the test, w1118 larvae show aversion to an odorant paired with a naturally occurring ethanol concentration of 5%. Our results provide insights into the parameters influencing olfactory associative behaviors using ethanol as a reinforcer in Drosophila larvae and indicate that short exposures to ethanol might not uncover the positive rewarding properties of ethanol for developing larvae., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Berger et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Preliminary empirical data indicates a substantial impact of the COVID-19 pandemic on well-being and mental health. Individuals with minoritized sexual and gender identities are at a higher risk of experiencing such negative changes in their well-being. The objective of this study was to compare levels of well-being among cis-heterosexual individuals and individuals with minoritized sexual and gender identities during the COVID-19 pandemic., Methods: Using data obtained in a cross-sectional online survey between April 20 to July 20, 2020 (N = 2332), we compared levels of well-being (WHO-5) across subgroups (cis-individuals with minoritized sexual identities, individuals with minoritized gender identities and cis-heterosexual individuals) applying univariate (two-sample t-test) and multivariate analysis (multivariate linear regression)., Results: Results indicate overall lower levels of well-being as well as lower levels of well-being in minoritized sexual or gender identities compared to cis-heterosexual individuals. Further, multivariate analyses revealed that living in urban communities as well as being in a relationship were positively associated with higher levels of well-being. Furthermore, a moderation analysis showed that being in a relationship reduces the difference between groups in terms of well-being., Conclusion: Access to mental healthcare for individuals with minoritized sexual and gender identities as well as access to gender-affirming resources should be strengthened during COVID-19 pandemic. Healthcare services with low barriers of access such as telehealth and online peer support groups should be made available, especially for vulnerable groups., Competing Interests: Pichit Buspavanich received a research grant from Gilead. Sven Mahner reports Research support, advisory board, honoraria and travel expenses from AbbVie, AstraZeneca, Clovis, Eisai, GlaxoSmithKline, Medac, MSD, Novartis, Olympus, PharmaMar, Pfizer, Roche, Sensor Kinesis, Teva, and Tesaro. All other authors have no conflict of interest to declare We confirm that our Competing Interest statement does not alter the adherence to PLOS ONE policies on sharing data and materials.

Background: The presentational flow chart "unwell adult" of the Manchester Triage System (MTS) occupies a special role in this triage system, defined as the nonspecific presentation of an emergency patient. Current scientific studies show that a considerable proportion of emergency room patients present with so-called "nonspecific complaints". The aim of the present study is to investigate in detail the initial assessment of emergency patients triaged according to the presentational flow chart "unwell adult"., Methods: Monocentric, retrospective observational study., Results: Data on 14,636 emergency department visits between March 12th and August 12th, 2019 were included. During the observation period, the presentational flow chart "unwell adult" was used 1,143 times and it was the third most frequently used presentational flow chart. Patients triaged with this flow chart often had unspecific complaints upon admission to the emergency department. Patients triaged with the "unwell adult" chart were often classified with a lower triage level. Notably, patients who died in hospital during the observation period frequently received low triage levels. The AUC for the MTS flow chart "unwell adult" and hospitalization in general for older patients (age ≥ 65 years) was 0.639 (95% CI 0.578-0.701), and 0.730 (95% CI 0.714-0.746) in patients triaged with more specific charts. The AUC for the MTS flow chart "unwell adult" and admission to ICU for older patients (age ≥65 years) was 0.631 (95% CI 0.547-0.715) and 0.807 (95% CI 0.790-0.824) for patients triaged with more specific flow charts. Comparison of the predictive ability of the MTS for in-hospital mortality in the group triaged with the presentational flow chart "unwell adult" revealed an AUC of 0.682 (95% CI 0.595-0.769) vs. 0.834 (95% CI 0.799-0.869) in the other presentational flow charts., Conclusion: The presentational flow chart "unwell adult" is frequently used by triage nurses for initial assessment of patients. Patient characteristics assessed with the presentational flow chart "unwell adult" differ significantly from those assessed with MTS presentational flow charts for more specific symptoms. The quality of the initial assessment in terms of a well-functioning triage priority assessment tool is less accurate than the performance of the MTS described in the literature., Competing Interests: The authors have declared that no competing interests exist.

Population size estimation is performed for several reasons including disease surveillance and control, for example to design adequate control strategies such as vaccination programs or to estimate a vaccination campaign coverage. In this study, we aimed at investigating the possibility of using Unmanned Aerial Vehicles (UAV) to estimate the size of free-roaming domestic dog (FRDD) populations and compare the results with two regularly used methods for population estimations: foot-patrol transect survey and the human: dog ratio estimation. Three studies sites of one square kilometer were selected in Petén department, Guatemala. A door-to-door survey was conducted in which all available dogs were marked with a collar and owner were interviewed. The day after, UAV flight were performed twice during two consecutive days per study site. The UAV's camera was set to regularly take pictures and cover the entire surface of the selected areas. Simultaneously to the UAV's flight, a foot-patrol transect survey was performed and the number of collared and non-collared dogs were recorded. Data collected during the interviews and the number of dogs counted during the foot-patrol transects informed a capture-recapture (CR) model fit into a Bayesian inferential framework to estimate the dog population size, which was found to be 78, 259, and 413 in the three study sites. The difference of the CR model estimates compared to previously available dog census count (110 and 289) can be explained by the fact that the study population addressed by the different methods differs. The human: dog ratio covered the same study population as the dog census and tended to underestimate the FRDD population size (97 and 161). Under the conditions within this study, the total number of dogs identified on the UAV pictures was 11, 96, and 71 for the three regions (compared to the total number of dogs counted during the foot-patrol transects of 112, 354 and 211). In addition, the quality of the UAV pictures was not sufficient to assess the presence of a mark on the spotted dogs. Therefore, no CR model could be implemented to estimate the size of the FRDD using UAV. We discussed ways for improving the use of UAV for this purpose, such as flying at a lower altitude in study area wisely chosen. We also suggest to investigate the possibility of using infrared camera and automatic detection of the dogs to increase visibility of the dogs in the pictures and limit workload of finding them. Finally, we discuss the need of using models, such as spatial capture-recapture models to obtain reliable estimates of the FRDD population. This publication may provide helpful directions to design dog population size estimation methods using UAV., Competing Interests: Merlin Benner is an owner and employee of Remote Intelligence, LLC, a private, forprofit company, which provide Unmanned Aerial Drone sales and consulting services. This commercial affiliation does not alter our adherence to all PLOS ONE policies on sharing data and materials

Introduction: For quantification of Optical Coherence Tomography Angiography (OCTA) images, Vessel Density (VD) and Vessel Skeleton Density (VSD) are well established parameters and different algorithms are in use for their calculation. However, comparability, reliability and ability to discriminate healthy and impaired macular perfusion of different algorithms are unclear, yet, of potential high clinical relevance. Hence, we assessed comparability and test-retest reliability of the most common approaches., Materials and Methods: Two consecutive 3×3mm OCTA en face images of the superficial and deep retinal layer were acquired with swept-source OCTA. VD and VSD were calculated with manual thresholding and six automated thresholding algorithms (Huang, Li, Otsu, Moments, Mean, Percentile) using ImageJ and compared in terms of intra-class correlation coefficients, measurement differences and repeatability coefficients. Receiver operating characteristic analyses (healthy vs. macular pathology) were performed and Area Under the Curve (AUC) values were calculated., Results: Twenty-six eyes (8 female, mean age: 47 years) of 15 patients were included (thereof 15 eyes with macular pathology). Binarization thresholds, VD and VSD differed significantly between the algorithms and compared to manual thresholding (p < 0.0001). Inter-measurement differences did not differ significantly between patients with healthy versus pathologic maculae (p ≥ 0.685). Reproducibility was higher for the automated algorithms compared to manual thresholding on all measures of reproducibility assessed. AUC was significantly higher for the Mean algorithm compared to the manual approach with respect to the superficial retinal layer., Conclusions: Automated thresholding algorithms yield a higher reproducibility of OCTA parameters and allow for a more sensitive diagnosis of macular pathology. However, different algorithms are not interchangeable nor results readily comparable. Especially the Mean algorithm should be investigated in further detail. Automated thresholding algorithms are preferable but more standardization is needed for clinical use., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: The Department of Ophthalmology, University of Bonn received imaging devices from Heidelberg Engineering, Optos, Carl Zeiss Meditec, and CenterVue. JHT: No additional conflicts of interest. MWMW: travel grant from DigiSight Technologies, grant from Heine Optotechnik, imaging devices from D-EYE, consultant of Heine Optotechnik GmbH. PF: No additional conflicts of interest. MB: No additional conflicts of interest. FGH: consultant of Acucela, Bayer, Bioeg, Boehringer-Ingelheim, Genentech/Roche, Heidelberg Engineering, Novartis, and Thea, grants from Acucela, Allergan, Bayer, Bioeq, Genentech/Roche, Merz, NightstarX, and Novartis, honoraria from Allergan, Bayer, Carl Zeiss MediTec, Genentech/Roche, Heidelberg Engineering, and Novartis. RPF: consultant of Bayer, Novartis, Santen, Opthea, Novelion, Santhera, Inositec, Alimera and RetinaImplant, honoraria from Bayer, Ellex, Alimera. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Research on sleep after stroke has focused mainly on sleep disordered breathing. However, the extend to which sleep physiology is altered in stroke survivors, how these alterations compare to healthy volunteers, and how sleep changes might affect recovery as well as physical and mental health has yet to be fully researched. Motivated by the view that a deeper understanding of sleep in stroke is needed to account for its role in health and well-being as well as its relevance for recovery and rehabilitation, we conducted a systematic review and meta-analysis of polysomnographic studies comparing stroke to control populations., Method: Medline and PsycInfo databases were searched using "stroke" and words capturing polysomnographic parameters as search terms. This yielded 1692 abstracts for screening, with 15 meeting the criteria for systematic review and 9 for meta-analysis. Prisma best practice guidelines were followed for the systematic review; the Comprehensive Meta-Analysis software was used for random effects modelling., Results: The meta-analysis revealed that patients with stroke have poorer sleep than controls. Patients had lower sleep efficiency (mean 75% vs 84%), shorter total-sleep-time (309.4 vs 340.3 min) and more wake-after-sleep-onset (97.2 vs 53.8 min). Patients also spend more time in stage 1 (13% vs 10%) and less time in stage 2 sleep (36% vs 45%) and slow-wave-sleep (10% vs 12%). No group differences were identified for REM sleep. The systematic review revealed a strong bias towards studies in the early recovery phase of stroke, with no study reporting specifically on patients in the chronic state. Moreover, participants in the control groups included community samples as well as other patients groups., Conclusions: These results indicate poorer sleep in patients with stroke than controls. While strongly suggestive in nature, the evidence base is limited and methodologically diverse, and hands a clear mandate for further research. A particular need regards polysomnographic studies in chronic community-dwelling patients compared to age-matched individuals.

Purpose: Vascular endothelial growth factor receptor (VEGFR2) directed therapies result in a modest survival benefit for patients with advanced esophageal and gastroesophageal (GE) junction cancer. Platelet-derived growth factor receptor (PDGFR) may contribute to escape from VEGFR2 inhibition. We evaluated the efficacy of sorafenib, a broad spectrum tyrosine kinase inhibitor targeting VEGFR2 and PDGFR as well as RET and RAF1, in patients with metastatic chemotherapy refractory esophageal and GE junction cancer., Patients and Methods: This phase II trial of sorafenib 400 mg twice daily enrolled chemotherapy refractory patients with metastatic esophageal and GE junction cancer with primary endpoint of progression-free survival (PFS) rate at two months. Secondary endpoints included overall survival, objective response rate and toxicity., Results: Among 34 patients, 8 week Kaplan-Meier estimated PFS was 61% (90%CI 45 to 73%). Median PFS is 3.6 months (95% CI 1.8 to 3.9 months), with median overall survival OS 9.7 months (95% CI 5.9 to 11.6 months). Grade 3 toxicities were uncommon and included hand foot skin reaction, rash, dehydration and fatigue. One patient (3%) with ongoing complete response and remains on trial for over 5 years. Whole exome sequencing of this tumor revealed mutations in many cancer-associated genes including ARID1A, PIK3CA, and TP53, and focal amplifications of HMGA2 and MET., Conclusion: Sorafenib therapy results in disease stabilization and encouraging PFS in patients with refractory esophageal and GE junction cancer., Trial Registration: ClinicalTrials.gov NCT00917462.

The need for medicinal and aromatic plants for industrial uses creates an opportunity for farmers to produce alternative crops. Stevia rebaudiana Bertoni, a perennial shrub originating from Paraguay, is of increasing interest as a source of zero-calorie natural sweeteners: the steviol glycosides (SVglys). The aim of this study was to investigate the relevance of nitrogen (N) supply for leaf yield and for SVgly concentrations in leaves, which are the two major components of S. rebaudiana productivity. In this regard, the relationship between leaf N concentration, CO2 assimilation, leaf production and SVgly accumulation was investigated. The experiments were conducted consecutively in growth-chamber (CC: controlled conditions), in greenhouse (SCC: semi-controlled conditions) and in field conditions (FC) on two genotypes. In CC and SCC, three levels of N fertilization were applied. Plants were grown on four locations in the FC experiment. Both N supply (CC and SCC) and location (FC) had a significant effect on N content in leaves. When light was not limiting (SCC and FC) N content in leaves was positively correlated with CO2 assimilation rate and biomass accumulation. Irrespective of the growth conditions, N content in leaves was negatively correlated with SVgly content. However, increased SVgly content was correlated with a decreased ratio of rebaudioside A over stevioside. The evidence that the increased SVgly accumulation compensates for the negative effect on biomass production suggests that adequate SVgly productivity per plant may be achieved with relatively low fertilization.

Highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1 not only cause a devastating disease in domestic chickens and turkeys but also pose a continuous threat to public health. In some countries, H5N1 viruses continue to circulate and evolve into new clades and subclades. The rapid evolution of these viruses represents a problem for virus diagnosis and control. In this work, recombinant vesicular stomatitis virus (VSV) vectors expressing HA of subtype H5 were generated. To comply with biosafety issues the G gene was deleted from the VSV genome. The resulting vaccine vector VSV*ΔG(HA) was propagated on helper cells providing the VSV G protein in trans. Vaccination of chickens with a single intramuscular dose of 2×10⁸ infectious replicon particles without adjuvant conferred complete protection from lethal H5N1 infection. Subsequent application of the same vaccine strongly boosted the humoral immune response and completely prevented shedding of challenge virus and transmission to sentinel birds. The vaccine allowed serological differentiation of infected from vaccinated animals (DIVA) by employing a commercially available ELISA. Immunized chickens produced antibodies with neutralizing activity against multiple H5 viruses representing clades 1, 2.2, 2.5, and low-pathogenic avian influenza viruses (classical clade). Studies using chimeric H1/H5 hemagglutinins showed that the neutralizing activity was predominantly directed against the globular head domain. In summary, these results suggest that VSV replicon particles are safe and potent DIVA vaccines that may help to control avian influenza viruses in domestic poultry.

Chromosomal translocations between the EWS gene and members of the ETS gene family are characteristic molecular features of the Ewing sarcoma. The most common translocation t(11;22)(q24;q12) fuses the EWS gene to FLI1, and is present in 85-90% of Ewing sarcomas. In the present study, a specifically designed multiplex long-range PCR assay was applied to amplify genomic EWS-FLI1 fusion sites from as little as 100 ng template DNA. Characterization of the EWS-FLI1 fusion sites of 42 pediatric and young adult Ewing sarcoma patients and seven cell lines revealed a clustering in the 5' region of the EWS-breakpoint cluster region (BCR), in contrast to random distribution of breakpoints in the FLI1-BCR. No association of breakpoints with various recombination-inducing sequence motifs was identified. The occurrence of small deletions and duplications at the genomic junction is characteristic of involvement of the non-homologous end-joining (NHEJ) repair system, similar to findings at chromosomal breakpoints in pediatric leukemia and lymphoma.

The adoptive transfer of antigen-specific effector T cells is being used to treat human infections and malignancy. T cell persistence is a prerequisite for therapeutic efficacy, but reliably establishing a high-level and durable T cell response by transferring cultured CD8(+) T cells remains challenging. Thus, strategies that promote a transferred high-level T cell response may improve the efficacy of T cell therapy. Lymphodepletion enhances persistence of transferred T cells in mice in part by reducing competition for IL-15, a common γ-chain cytokine that promotes T cell memory, but lymphodepleting regimens have toxicity. IL-15 can be safely administered and has minimal effects on CD4(+) regulatory T cells at low doses, making it an attractive adjunct in adoptive T cell therapy. Here, we show in lymphoreplete macaca nemestrina, that proliferation of adoptively transferred central memory-derived CD8(+) effector T (T(CM/E)) cells is enhanced in vivo by administering IL-15. T(CM/E) cells migrated to memory niches, persisted, and acquired both central memory and effector memory phenotypes regardless of the cytokine treatment. Unexpectedly, despite maintaining T cell proliferation, IL-15 did not augment the magnitude of the transferred T cell response in blood, bone marrow, or lymph nodes. T cells induced to proliferate by IL-15 displayed increased apoptosis demonstrating that enhanced cycling was balanced by cell death. These results suggest that homeostatic mechanisms that regulate T cell numbers may interfere with strategies to augment a high-level T cell response by adoptive transfer of CD8(+) T(CM/E) cells in lymphoreplete hosts.

Background: One of the core symptoms of borderline personality disorder (BPD) is the instability in interpersonal relationships. This might be related to existent differences in mindreading between BPD patients and healthy individuals., Methods: We examined the behavioural and neurophysiological (fMRI) responses of BPD patients and healthy controls (HC) during performance of the 'Reading the Mind in the Eyes' test (RMET)., Results: Mental state discrimination was significantly better and faster for affective eye gazes in BPD patients than in HC. At the neurophysiological level, this was manifested in a stronger activation of the amygdala and greater activity of the medial frontal gyrus, the left temporal pole and the middle temporal gyrus during affective eye gazes. In contrast, HC subjects showed a greater activation in the insula and the superior temporal gyri., Conclusion: These findings indicate that BPD patients are highly vigilant to social stimuli, maybe because they resonate intuitively with mental states of others.

Type I interferons (IFN) comprise a family of cytokines that signal through a common cellular receptor to induce a plethora of genes with antiviral and other activities. Recombinant IFNs are used for the treatment of hepatitis C virus infection, multiple sclerosis, and certain malignancies. The capability of type I IFN to suppress virus replication and resultant cytopathic effects is frequently used to measure their bioactivity. However, these assays are time-consuming and require appropriate biosafety containment. In this study, an improved IFN assay is presented which is based on a recombinant vesicular stomatitis virus (VSV) replicon encoding two reporter proteins, firefly luciferase and green fluorescent protein. The vector lacks the essential envelope glycoprotein (G) gene of VSV and is propagated on a G protein-expressing transgenic cell line. Several mammalian and avian cells turned out to be susceptible to infection with the complemented replicon particles. Infected cells readily expressed the reporter proteins at high levels five hours post infection. When human fibroblasts were treated with serial dilutions of human IFN-β prior to infection, reporter expression was accordingly suppressed. This method was more sensitive and faster than a classical IFN bioassay based on VSV cytopathic effects. In addition, the antiviral activity of human IFN-λ (interleukin-29), a type III IFN, was determined on Calu-3 cells. Both IFN-β and IFN-λ were acid-stable, but only IFN-β was resistant to alkaline treatment. The antiviral activities of canine, porcine, and avian type I IFN were analysed with cell lines derived from the corresponding species. This safe bioassay will be useful for the rapid and sensitive quantification of multi-species type I IFN and potentially other antiviral cytokines.

UDP-N-acetylglucosamine 2 epimerase/N-acetylmannosamime kinase (GNE) is a bifunctional enzyme which catalyzes the two key sequential steps in the biosynthetic pathway of sialic acid, the most abundant terminal monosaccharide on glycoconjugates of eukaryotic cells. GNE knock out (GNE KO) mice are embryonically lethal at day E8.5. Although the role of GNE in the sialic pathway has been well established as well as the importance of sialylation in many diverse biological pathways, less is known about the involvement of GNE in muscle development. To address this issue we have studied the role of GNE during in vitro embryogenesis by comparing the developmental profile in culture of embryonic stem cells (ES) from wild type and from GNE KO E3.5 mice embryos, during 45 days. Neuronal cells appeared rarely in GNE KO ES cultures and did not reach an advanced differentiated stage. Although primary cardiac cells appeared at the same time in both normal and GNE KO ES cultures, GNE KO cardiac cells degraded very soon and their beating capacity decayed rapidly. Furthermore very rare skeletal muscle committed cells were detected in the GNE KO ES cultures at any stage of differentiation, as assessed by analysis of the expression of either Pax7, MyoD and MyHC markers. Beyond the supporting evidence that GNE plays an important role in neuronal cell and brain development, these results show that GNE is strongly involved in cardiac tissue and skeletal muscle early survival and organization. These findings could open new avenues in the understanding of muscle function mechanisms in health and in disease.