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How do parenthood and publishing contribute to gender gaps in academic career advancement? While extensive research examines the causes of gender disparities in science, technology, engineering, and mathematics (STEM) careers, we know much less about the factors that constrain women's advancement in the social sciences. Combining detailed career- and administrative register data on 976 Danish social scientists in Business and Management, Economics, Political Science, Psychology, and Sociology (5703 person-years) that obtained a PhD degree between 2000 and 2015, we estimate gender differences in attainment of senior research positions and parse out how publication outputs, parenthood and parental leave contribute to these differences. Our approach is advantageous over previous longitudinal studies in that we track the careers and publication outputs of graduates from the outset of their PhD education and match this data with time-sensitive information on each individual's publication activities and family situation. In discrete time-event history models, we observe a ∼24 per cent female disadvantage in advancement likelihoods within the first 7 years after PhD graduation, with gender differences increasing over the observation period. A decomposition indicates that variations in publishing, parenthood and parental leave account for ∼ 40 per cent of the gender gap in career advancement, suggesting that other factors, including recruitment disparities, asymmetries in social capital and experiences of unequal treatment at work, may also constrain women's careers., (© 2024 The Authors. The British Journal of Sociology published by John Wiley & Sons Ltd on behalf of London School of Economics and Political Science.)

Endogenous and bacterial proteases play important roles in wound healing and infection. Analysis of alterations in the low-molecular-weight peptidome by individual enzymes could therefore provide insight into proteolytic events occurring in wounds and may aid in the discovery of biomarkers. Using liquid chromatography with tandem mass spectrometry, we characterized the peptidome of plasma and acute wound fluids digested ex vivo with human (neutrophil elastase and cathepsin G) and bacterial proteases ( Pseudomonas aeruginosa LasB and Staphyloccocus aureus V8). We identified over 100 protein targets for each enzyme and characterized enzyme specific peptides and cleavage patterns. Moreover, we found unique peptide regions in V8 digested samples that were also present in dressing extracts from S. aureus infected wounds. Finally, the work indicates that peptidomic analysis of qualitative differences of proteolytic activity of individual enzymes may aid in the discovery of potential diagnostic biomarkers for wound healing status., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Background and Aim: One of the greatest barriers to the treatment of T2DM is nonadherence which particularly applies to insulin therapy. There is a need for a comprehensive overview of all factors associated with nonadherence to insulin therapy. The aim of this study was to identify factors associated with adherence or nonadherence to insulin therapy among adults with T2DM., Methods: A scoping review was conducted in accordance with the PRISMA 2020 statement. A systematic search was performed in PubMed, Cinahl, and Web of Science (January 2013 to March 2023)., Results: A final sample of 48 studies was included in the scoping review. The synthesis revealed 30 factors associated with adherence or nonadherence. The factors were grouped into 6 themes: demographics, attitude and perceptions, management of diabetes, impact on daily living, disease and medication, and healthcare system., Conclusion: The most prominent factors identified were age, cost of healthcare, personal beliefs towards insulin therapy, social stigma, patient education, complexity of diabetes treatment, impact of insulin therapy on daily life, and fear of side effects. The results indicate a need for further research to determine threshold values for the factors associated with adherence or nonadherence., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Open data sharing is critical for scientific progress. Yet, many authors refrain from sharing scientific data, even when they have promised to do so. Through a preregistered, randomized audit experiment (N = 1,634), we tested possible ethnic, gender and status-related bias in scientists' data-sharing willingness. 814 (54%) authors of papers where data were indicated to be 'available upon request' responded to our data requests, and 226 (14%) either shared or indicated willingness to share all or some data. While our preregistered hypotheses regarding bias in data-sharing willingness were not confirmed, we observed systematically lower response rates for data requests made by putatively Chinese treatments compared to putatively Anglo-Saxon treatments. Further analysis indicated a theoretically plausible heterogeneity in the causal effect of ethnicity on data-sharing. In interaction analyses, we found indications of lower responsiveness and data-sharing willingness towards male but not female data requestors with Chinese names. These disparities, which likely arise from stereotypic beliefs about male Chinese requestors' trustworthiness and deservingness, impede scientific progress by preventing the free circulation of knowledge., (© 2023. The Author(s).)

In the marine environment, surface-associated bacteria often produce an array of antimicrobial secondary metabolites, which have predominantly been perceived as competition molecules. However, they may also affect other hallmarks of surface-associated living, such as motility and biofilm formation. Here, we investigate the ecological significance of an antibiotic secondary metabolite, tropodithietic acid (TDA), in the producing bacterium, Phaeobacter piscinae S26. We constructed a markerless in-frame deletion mutant deficient in TDA biosynthesis, S26Δ tdaB . Molecular networking demonstrated that other chemical sulfur-containing features, likely related to TDA, were also altered in the secondary metabolome. We found several changes in the physiology of the TDA-deficient mutant, Δ tdaB , compared to the wild type. Growth of the two strains was similar; however, Δ tdaB cells were shorter and more motile. Transcriptome and proteome profiling revealed an increase in gene expression and protein abundance related to a type IV secretion system, and to a prophage, and a gene transfer agent in Δ tdaB . All these systems may contribute to horizontal gene transfer (HGT), which may facilitate adaptation to novel niches. We speculate that once a TDA-producing population has been established in a new niche, the accumulation of TDA acts as a signal of successful colonization, prompting a switch to a sessile lifestyle. This would lead to a decrease in motility and the rate of HGT, while filamentous cells could form the base of a biofilm. In addition, the antibiotic properties of TDA may inhibit invading competing microorganisms. This points to a role of TDA in coordinating colonization and adaptation. IMPORTANCE Despite the broad clinical usage of microbial secondary metabolites with antibiotic activity, little is known about their role in natural microbiomes. Here, we studied the effect of production of the antibiotic tropodithietic acid (TDA) on the producing strain, Phaeobacter piscinae S26, a member of the Roseobacter group. We show that TDA affects several phenotypes of the producing strain, including motility, cell morphology, metal metabolism, and three horizontal gene transfer systems: a prophage, a type IV secretion system, and a gene transfer agent. Together, this indicates that TDA participates in coordinating the colonization process of the producer. TDA is thus an example of a multifunctional secondary metabolite that can mediate complex interactions in microbial communities. This work broadens our understanding of the ecological role that secondary metabolites have in microbial community dynamics.

We present the influence of treating progeroid fibroblasts with two modified antisense oligonucleotides (ONs) on the nuclear envelope. Two modified ONs were designed to block ribosome binding during translation and spliceosome binding at the cryptic splice site. We analysed the changes in the nuclear morphology of progeria cell nuclei after repetitive transfection with modified ONs as a physical analysis tool for estimating alteration of the gene expression at the protein level. Confocal microscopy was used to image the nuclei, and the nuclear lobulations were quantified to study the changes in the morphology of the nuclear envelope upon treatment. PCR was used to identify the changes in the expression of lamin A and progerin after antisense treatment at the RNA level. We found a significant decrease in the number of nuclear envelope lobulations and a lower progerin expression in progeria cells after transfection with modified ONs., Competing Interests: “The authors declare no conflict of interest. Also, the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results”., (© 2022 The Authors.)

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The marine bacterium Photobacterium galatheae S2753 produces a group of cyclodepsipeptides, called solonamides, which impede the virulence but not the survival of Staphylococcus aureus. In addition to their invaluable antivirulence activity, little is known about the biosynthesis and physiological function of solonamides in the native producer. This study generated a solonamide-deficient (Δ sol ) mutant by in-frame deletion of the sol gene, thereby identifying the core gene for solonamide biosynthesis. By annotation from antiSMASH, the biosynthetic pathway of solonamides in S2753 was also proposed. Mass spectrometry analysis of cell extracts found that deficiency of solonamide production influenced the production of a group of unknown compounds but otherwise did not alter the overall secondary metabolite profile. Physiological comparison between Δ sol and wild-type S2753 demonstrated that growth dynamics and biofilm formation of both strains were similar; however, the Δ sol mutant displayed reduced motility rings compared to the wild type. Reintroduction of sol restored solonamide production and motility to the mutant, indicating that solonamides influence the motility behavior of P. galatheae S2753. Proteomic analysis of the Δ sol and wild-type strains found that eliminating solonamides influenced many cellular processes, including swimming-related proteins and proteins adjusting the cellular cyclic di-GMP concentration. In conclusion, our results revealed the biosynthetic pathway of solonamides and their ecological benefits to P. galatheae S2753 by enhancing motility, likely by altering the motile physiology. IMPORTANCE The broad range of bioactive potentials of cyclodepsipeptides makes these compounds invaluable in the pharmaceutical industry. Recently, a few novel cyclodepsipeptides have been discovered in marine Proteobacteria ; however, their biosynthetic pathways remain to be revealed. Here, we demonstrated the biosynthetic genetic basis and pathway of the antivirulence compounds known as solonamides in P. galatheae S2753. This can pave the way for the biological overproduction of solonamides on an industrial scale. Moreover, the comparison of a solonamide-deficient mutant and wild-type S2753 demonstrated that solonamides stimulate the swimming behavior of S2753 and also influence a few key physiological processes of the native producers. These results evidenced that, in addition to their importance as novel drug candidates, these compounds play a pivotal role in the physiology of the producing microorganisms and potentially provide the native producer competitive benefits for their survival in nature.

Publications are essential for a successful academic career, and there is evidence that the COVID-19 pandemic has amplified existing gender disparities in the publishing process. We used longitudinal publication data on 431,207 authors in four disciplines - basic medicine, biology, chemistry and clinical medicine - to quantify the differential impact of COVID-19 on the annual publishing rates of men and women. In a difference-in-differences analysis, we estimated that the average gender difference in publication productivity increased from -0.26 in 2019 to -0.35 in 2020; this corresponds to the output of women being 17% lower than the output of men in 2109, and 24% lower in 2020. An age-group comparison showed a widening gender gap for both early-career and mid-career scientists. The increasing gender gap was most pronounced among highly productive authors and in biology and clinical medicine. Our study demonstrates the importance of reinforcing institutional commitments to diversity through policies that support the inclusion and retention of women in research., Competing Interests: EM, MN, JB, RJ, JA No competing interests declared, (© 2022, Madsen et al.)

In this paper, we introduce the Societal Readiness (SR) Thinking Tool to aid researchers and innovators in developing research projects with greater responsiveness to societal values, needs, and expectations. The need for societally-focused approaches to research and innovation-complementary to Technology Readiness (TR) frameworks-is presented. Insights from responsible research and innovation (RRI) concepts and practice, organized across critical stages of project-life cycles are discussed with reference to the development of the SR Thinking Tool. The tool is designed to complement not only shortfalls in TR approaches, but also improve upon other efforts to integrate RRI, sustainability, and design thinking in research and innovation cycles. Operationalization and early-stage user tests of the Tool are reported, along with discussion of potential future iterations and applications., (© 2022. The Author(s).)

Background: In recent years, interest has grown in whether and to what extent demographic diversity sparks discovery and innovation in research. At the same time, topic modeling has been employed to discover differences in what women and men write about. This study engages these two strands of scholarship to explore associations between changing researcher demographics and research questions asked in the discipline of history. Specifically, we analyze developments in history as women entered the field., Methods: We focus on author gender in diachronic analysis of history dissertations from 1980 (when online data is first available) to 2015 and a select set of general history journals from 1950 to 2015. We use correlated topic modeling and network visualizations to map developments in research agendas over time and to examine how women and men have contributed to these developments., Results: Our summary snapshot of aggregate interests of women and men for the period 1950 to 2015 identifies new topics associated with women authors: gender and women's history, body history, family and households, consumption and consumerism, and sexuality. Diachronic analysis demonstrates that while women pioneered topics such as gender and women's history or the history of sexuality, these topics broaden over time to become methodological frameworks that historians widely embraced and that changed in interesting ways as men engaged with them. Our analysis of history dissertations surface correlations between advisor/advisee gender pairings and choice of dissertation topic., Conclusions: Overall, this quantitative longitudinal study suggests that the growth in women historians has coincided with the broadening of research agendas and an increased sensitivity to new topics and methodologies in the field., Competing Interests: The authors have declared that no competing interests exist.

Staphylococcus aureus causes various infections in humans and animals, the skin being the principal reservoir of this pathogen. The widespread occurrence of methicillin-resistant S. aureus (MRSA) limits the elimination and treatment of this pathogen. Phage lytic proteins have been proven as efficient antimicrobials against S. aureus . Here, a set of 12 engineered proteins based on endolysins were conceptualized to select the most optimal following a stepwise funnel approach assessing parameters including turbidity reduction, minimum inhibitory concentration (MIC), time-kill curves, and antibiofilm assays, as well as testing their stability in a broad range of storage conditions (pH, temperature, and ionic strength). The engineered phage lysins LysRODIΔAmi and ClyRODI-H5 showed the highest specific lytic activity (5 to 50 times higher than the rest), exhibited a shelf-life up to 6 months and remained stable at temperatures up to 50°C and in a pH range from 3 to 9. LysRODIΔAmi showed the lower MIC values against all staphylococcal strains tested. Both proteins were able to kill 6 log units of the strain S. aureus Sa9 within 5 min and could remove preformed biofilms (76 and 65%, respectively). Moreover, LysRODIΔAmi could prevent biofilm formation at low protein concentrations (0.15-0.6 μM). Due to its enhanced antibiofilm properties, LysRODIΔAmi was selected to effectively remove S. aureus contamination in both intact and disrupted keratinocyte monolayers. Notably, this protein did not demonstrate any toxicity toward human keratinocytes, even at high concentrations (22.1 μM). Finally, a pig skin ex vivo model was used to evaluate treatment of artificially contaminated pig skin using LysRODIΔAmi (16.5 μg/cm 2 ). Following an early reduction of S. aureus , a second dose of protein completely eradicated S. aureus . Overall, our results suggest that LysRODIΔAmi is a suitable candidate as antimicrobial agent to prevent and treat staphylococcal skin infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gutiérrez, Rodríguez-Rubio, Ruas-Madiedo, Fernández, Campelo, Briers, Nielsen, Pedersen, Lavigne, García and Rodríguez.)

Sex and gender differences impact the incidence of SARS-CoV-2 infection and COVID-19 mortality. Furthermore, sex differences influence the frequency and severity of pharmacological side effects. A large number of clinical trials to develop new therapeutic approaches and vaccines for COVID-19 are ongoing. We investigated the inclusion of sex and/or gender in COVID-19 studies on ClinicalTrials.gov, collecting data for the period January 1, 2020 to January 26, 2021. Here, we show that of the 4,420 registered SARS-CoV-2/COVID-19 studies, 935 (21.2%) address sex/gender solely in the context of recruitment, 237 (5.4%) plan sex-matched or representative samples or emphasized sex/gender reporting, and only 178 (4%) explicitly report a plan to include sex/gender as an analytical variable. Just eight (17.8%) of the 45 COVID-19 related clinical trials published in scientific journals until December 15, 2020 report sex-disaggregated results or subgroup analyses.

Spread of livestock-associated methicillin resistant Staphylococcus aureus (LA-MRSA) to farmworkers has been recognized as a risk when working in LA-MRSA positive stables, due to LA-MRSA being present on airborne dust particles. Based on this, airborne spread of LA-MRSA through stable vents is a concern that is addressed in this study. The aim of the investigation was to quantify the airborne spread of LA-MRSA from a MRSA positive swine farm. In order to achieve this, a method for sampling large volumes of air was applied. The results were compared to meteorological data and bacteriological investigation of samples from the air inside the swine barn, soil outside the farm, and nasal samples from the individuals participating in the sampling process. MRSA was detected up to 300 m (the maximal measuring distance) from the swine farm in the air but only at low levels at distances above 50 meters (0.085 CFU/m 3 at a distance of 50 m in the wind plume). MRSA was detected in sock samples obtained at the soil surfaces up to 400 m (the maximal measuring distance) from the farm building. The proportion of MRSA positive soil samples decreased from ~80 to 30% with increasing distance from the farm. A total of 25 human nasal samples were sampled after the farm visits after the participants had stayed in the surroundings of the farm for an average of 10.5 h. When leaving the farm, only two of the samples (8%) were LA-MRSA-positive both obtained from one individual who was the one who had sampled the ventilation shafts. In conclusion, airborne spread of MRSA from swine farms does not seem to be an important route for human contamination for individuals staying a whole working day outside a swine farm., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Angen, Nielsen, Løfstrøm, Larsen and Hendriksen.)

Research suggests that scientists based at prestigious institutions receive more credit for their work than scientists based at less prestigious institutions, as do scientists working in certain countries. We examined the extent to which country- and institution-related status signals drive such differences in scientific recognition. In a preregistered survey experiment, we asked 4,147 scientists from six disciplines (astronomy, cardiology, materials science, political science, psychology and public health) to rate abstracts that varied on two factors: (i) author country (high status vs lower status in science); (ii) author institution (high status vs lower status university). We found only weak evidence of country- or institution-related status bias, and mixed regression models with discipline as random-effect parameter indicated that any plausible bias not detected by our study must be small in size., Competing Interests: MN, CB, EB, MP, JA No competing interests declared, (© 2021, Nielsen et al.)

Background: In this paper, we argue for Gender as a Sociocultural Variable (GASV) as a complement to Sex as a Biological Variable (SABV). Sex (biology) and gender (sociocultural behaviors and attitudes) interact to influence health and disease processes across the lifespan-which is currently playing out in the COVID-19 pandemic. This study develops a gender assessment tool-the Stanford Gender-Related Variables for Health Research-for use in clinical and population research, including large-scale health surveys involving diverse Western populations. While analyzing sex as a biological variable is widely mandated, gender as a sociocultural variable is not, largely because the field lacks quantitative tools for analyzing the influence of gender on health outcomes., Methods: We conducted a comprehensive review of English-language measures of gender from 1975 to 2015 to identify variables across three domains: gender norms, gender-related traits, and gender relations. This yielded 11 variables tested with 44 items in three US cross-sectional survey populations: two internet-based (N = 2051; N = 2135) and a patient-research registry (N = 489), conducted between May 2017 and January 2018., Results: Exploratory and confirmatory factor analyses reduced 11 constructs to 7 gender-related variables: caregiver strain, work strain, independence, risk-taking, emotional intelligence, social support, and discrimination. Regression analyses, adjusted for age, ethnicity, income, education, sex assigned at birth, and self-reported gender identity, identified associations between these gender-related variables and self-rated general health, physical and mental health, and health-risk behaviors., Conclusion: Our new instrument represents an important step toward developing more comprehensive and precise survey-based measures of gender in relation to health. Our questionnaire is designed to shed light on how specific gender-related behaviors and attitudes contribute to health and disease processes, irrespective of-or in addition to-biological sex and self-reported gender identity. Use of these gender-related variables in experimental studies, such as clinical trials, may also help us understand if gender factors play an important role as treatment-effect modifiers and would thus need to be further considered in treatment decision-making.

Citations are important building blocks for status and success in science. We used a linked dataset of more than 4 million authors and 26 million scientific papers to quantify trends in cumulative citation inequality and concentration at the author level. Our analysis, which spans 15 y and 118 scientific disciplines, suggests that a small stratum of elite scientists accrues increasing citation shares and that citation inequality is on the rise across the natural sciences, medical sciences, and agricultural sciences. The rise in citation concentration has coincided with a general inclination toward more collaboration. While increasing collaboration and full-count publication rates go hand in hand for the top 1% most cited, ordinary scientists are engaging in more and larger collaborations over time, but publishing slightly less. Moreover, fractionalized publication rates are generally on the decline, but the top 1% most cited have seen larger increases in coauthored papers and smaller relative decreases in fractional-count publication rates than scientists in the lower percentiles of the citation distribution. Taken together, these trends have enabled the top 1% to extend its share of fractional- and full-count publications and citations. Further analysis shows that top-cited scientists increasingly reside in high-ranking universities in western Europe and Australasia, while the United States has seen a slight decline in elite concentration. Our findings align with recent evidence suggesting intensified international competition and widening author-level disparities in science., Competing Interests: The authors declare no competing interest.

Genome mining of pigmented Pseudoalteromonas has revealed a large potential for the production of bioactive compounds and hydrolytic enzymes. The purpose of the present study was to explore this bioactivity potential in a potent antibiotic and enzyme producer, Pseudoalteromonas rubra strain S4059. Proteomic analyses (data are available via ProteomeXchange with identifier PXD023249) indicated that a highly efficient chitin degradation machinery was present in the red-pigmented P. rubra S4059 when grown on chitin. Four GH18 chitinases and two GH20 hexosaminidases were significantly upregulated under these conditions. GH19 chitinases, which are not common in bacteria, are consistently found in pigmented Pseudoalteromonas, and in S4059, GH19 was only detected when the bacterium was grown on chitin. To explore the possible role of GH19 in pigmented Pseudoalteromonas , we developed a protocol for genetic manipulation of S4059 and deleted the GH19 chitinase, and compared phenotypes of the mutant and wild type. However, none of the chitin degrading ability, secondary metabolite profile, or biofilm-forming capacity was affected by GH19 deletion. In conclusion, we developed a genetic manipulation protocol that can be used to unravel the bioactive potential of pigmented pseudoalteromonads. An efficient chitinolytic enzyme cocktail was identified in S4059, suggesting that this strain could be a candidate with industrial potential.

Transition milk (TM, defined here as the second through fourth milkings after calving) supplies additional fat, protein, and immunoglobulins to the calf compared with milk replacer at industry-suggested feeding rates (∼14% solids). Our objective was to determine whether 9 feedings of TM on d 2 through 4 of life increase the growth rate and overall health of calves. Holstein heifer calves on a commercial farm were randomly assigned to 1 of 3 diets (n = 35/diet): milk replacer (MR; Purina Warm Front BOV MOS Medicated Milk Replacer, St. Louis, MO), TM, or a 50:50 blend of MR and colostrum replacer (MCR; Alta HiCal Colostrum Powder Replacer, the Saskatoon Colostrum Company Ltd., Saskatoon, SK, Canada). The TM was harvested from Holstein cows on the farm, pooled, and pasteurized at 71.7°C for 15 s. Nutrient composition on a dry matter basis of TM was 25.9% fat, 41.8% protein, and 14% solids; MR was 10.3% fat, 27.8% protein, and 14% solids; and MCR was 14.6% fat, 38.6% protein, and 15% solids. All calves received IgG-enriched colostrum replacer for the first 2 feedings after birth. Subsequently, calves were fed 1.9 L of MR, TM, or MCR 3 times per day for 3 d (starting on d 2). After initial diets ended, calves were fed and managed similarly. Body weights (d 1, 7, 14, 21, and 56), blood samples (d 1, 7, 14, and 21), and daily health scores (scale of 0 to 3, with 0 representing normal or healthy and 3 representing severe symptoms or ill) were collected through weaning at 56 d. All except 1 calf achieved successful transfer of passive immunity, with serum IgG values greater than 10.0 mg/mL. From birth through weaning, calves fed TM and MCR gained 3 kg more total body weight than those fed MR (34.3, 34.3, and 31.3 kg, respectively). Increased metabolizable energy (using NRC 2001 recommendations) in TM accounts for 0.68 kg of the increased gain compared with MR. Treatment did not alter health scores for ears, eyes, or feces. Haptoglobin concentrations were lower in TM and MCR than in MR calves (4.63, 3.62, and 7.54 µg/mL, respectively), whereas lipopolysaccharide binding protein concentrations were not different. In conclusion, compared with MR alone, feeding TM or MR with colostrum replacer for 3 d increased growth rate of calves throughout the preweaning period., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Background: Lytic polysaccharide monooxygenases (LPMOs) are often studied in simple models involving activity measurements of a single LPMO or a blend thereof with hydrolytic enzymes towards an insoluble substrate. However, the contribution of LPMOs to polysaccharide breakdown in complex cocktails of hydrolytic and oxidative enzymes, similar to fungal secretomes, remains elusive. Typically, two starch-specific AA13 LPMOs are encoded by mainly Ascomycota genomes. Here, we investigate the impact of LPMO loss on the growth and degradation of starches of varying resistance to amylolytic hydrolases by Aspergillus nidulans ., Results: Deletion of the genes encoding An AA13A that possesses a CBM20 starch-binding module, An AA13B (lacking a CBM20) or both AA13 genes resulted in reduced growth on solid media with resistant, but not soluble processed potato starch. Larger size and amount of residual starch granules were observed for the AA13-deficient strains as compared to the reference and the impairment of starch degradation was more severe for the strain lacking An AA13A based on a microscopic analysis. After 5 days of growth on raw potato starch in liquid media, the mount of residual starch was about fivefold higher for the AA13 gene deletion strains compared to the reference, which underscores the importance of LPMOs for degradation of especially resistant starches. Proteomic analyses revealed substantial changes in the secretomes of the double AA13 gene deletion, followed by the An AA13A-deficient strain, whereas only a single protein was significantly different in the proteome of the An AA13B-deficient strain as compared to the reference., Conclusions: This study shows that the loss of AA13, especially the starch-binding An AA13A, impairs degradation of resistant potato starch, but has limited impact on less-resistant wheat starch and no impact on processed solubilized starch. The effects of LPMO loss are more pronounced at the later stages of fungal growth, likely due to the accumulation of the less-accessible regions of the substrate. The striking impairment in granular starch degradation due to the loss of a single LPMO from the secretome offers insight into the crucial role played by AA13 in the breakdown of resistant starch and presents a methodological framework to analyse the contribution of distinct LPMOs towards semi-crystalline polysaccharides under in vivo conditions., Competing Interests: Competing interestsThe authors declare no competing interest., (© The Author(s) 2020.)

The COVID-19 pandemic has resulted in school closures and distancing requirements that have disrupted both work and family life for many. Concerns exist that these disruptions caused by the pandemic may not have influenced men and women researchers equally. Many medical journals have published papers on the pandemic, which were generated by researchers facing the challenges of these disruptions. Here we report the results of an analysis that compared the gender distribution of authors on 1893 medical papers related to the pandemic with that on papers published in the same journals in 2019, for papers with first authors and last authors from the United States. Using mixed-effects regression models, we estimated that the proportion of COVID-19 papers with a woman first author was 19% lower than that for papers published in the same journals in 2019, while our comparisons for last authors and overall proportion of women authors per paper were inconclusive. A closer examination suggested that women's representation as first authors of COVID-19 research was particularly low for papers published in March and April 2020. Our findings are consistent with the idea that the research productivity of women, especially early-career women, has been affected more than the research productivity of men., Competing Interests: JA, MN, NS No competing interests declared, RL Founder of TIME'S UP Healthcare, a non-profit initiative that advocates for safety and equity in healthcare; advisor for FeminEM.org, a website that supports the careers of women in medicine. RJ Has stock options as compensation for her advisory board role in Equity Quotient, a company that evaluates culture in health care companies; has received personal fees from Amgen and Vizient and grants for unrelated work from the National Institutes of Health, the Doris Duke Foundation, the Greenwall Foundation, the Komen Foundation, and Blue Cross Blue Shield of Michigan for the Michigan Radiation Oncology Quality Consortium; has a contract to conduct an investigator-initiated study with Genentech; has served as an expert witness for Sherinian and Hasso and Dressman Benzinger LaVelle; uncompensated founding member of TIME'S UP Healthcare; member of the Board of Directors of ASCO., (© 2020, Andersen et al.)

A number of studies suggest that scientific papers with women in leading-author positions attract fewer citations than those with men in leading-author positions. We report the results of a matched case-control study of 1,269,542 papers in selected areas of medicine published between 2008 and 2014. We find that papers with female authors are, on average, cited between 6.5 and 12.6% less than papers with male authors. However, the standardized mean differences are very small, and the percentage overlaps between the distributions for male and female authors are extensive. Adjusting for self-citations, number of authors, international collaboration and journal prestige, we find near-identical per-paper citation impact for women and men in first and last author positions, with self-citations and journal prestige accounting for most of the small average differences. Our study demonstrates the importance of focusing greater attention on within-group variability and between-group overlap of distributions when interpreting and reporting results of gender-based comparisons of citation impact., Competing Interests: JA, JS, MN No competing interests declared, RJ RJ: Stock options in Equity Quotient; advisory role and personal fees from Amgen; and consulting for Vizient., (© 2019, Andersen et al.)

Gender diversity has the potential to drive scientific discovery and innovation. Here, we distinguish three approaches to gender diversity: diversity in research teams, diversity in research methods and diversity in research questions. While gender diversity is commonly understood to refer only to the gender composition of research teams, fully realizing the potential of diversity for science and innovation also requires attention to the methods employed and questions raised in scientific knowledge-making. We provide a framework for understanding the best ways to support the three approaches to gender diversity across four interdependent domains - from research teams to the broader disciplines in which they are embedded to research organizations and ultimately to the different societies that shape them through specific gender norms and policies. Our analysis demonstrates that realizing the benefits of diversity for science requires careful management of these four interdependent domains.

Digital dermatitis (DD) is one of the main causes of lameness in dairy cattle worldwide, and it is frequently reported in high-yielding, free stall dairy herds from regions with a temperate climate. However, DD is also observed with high prevalence in grazing cattle with a low milk yield in tropical regions. To clarify whether these differences have an impact on the etiology of the disease, we studied DD lesions from all year round grazing cattle of mixed breed in Brazil using high-throughput 16S rRNA gene sequencing and fluorescent in situ hybridization. The study included samples from 66 skin lesions and 5 healthy skins collected from five farms. Both techniques showed Treponema spp. to be the most abundant bacteria, present in all but one of the samples with minimal epidermal alterations. We identified eleven different Treponema strains belonging to the six major phylotypes of Treponema which have all previously been identified in DD lesions. Furthermore, we identify Dichelobacter nodosus in DD lesions by gene sequencing and also by fluorescent in situ hybridization in almost half of biopsy specimens in areas with mild epithelial damage and together with Treponema. The present data support the hypothesis that Treponema constitutes the main pathogen responsible for DD, independent of the environment and region where cows are kept, and it further suggests D. nodosus as another potentially important pathogen.

Gender and sex analysis is increasingly recognized as a key factor in creating better medical research and health care 1-7 . Using a sample of more than 1.5 million medical research papers, our study examined the potential link between women's participation in medical science and attention to gender-related and sex-related factors in disease-specific research. Adjusting for variations across countries, disease topics and medical research areas, we compared the participation of women authors in studies that do and do not involve gender and sex analysis. Overall, our results show a robust positive correlation between women's authorship and the likelihood of a study including gender and sex analysis. These findings corroborate discussions of how women's participation in medical science links to research outcomes, and show the mutual benefits of promoting both the scientific advancement of women and the integration of gender and sex analysis into medical research.

At present, very little information exists regarding what role the environmental slurry may play as an infection reservoir and/or route of transmission for bovine digital dermatitis (DD), a disease which is a global problem in dairy herds. To investigate whether DD-related bacteria belong to the indigenous microbiota of the dairy herd environment, we used deep amplicon sequencing of the 16S rRNA gene in 135 slurry samples collected from different sites in 22 dairy farms, with and without DD-infected cows. Both the general bacterial populations and digital dermatitis-associated Treponema were targeted in this study. The results revealed significant differences in the bacterial communities between the herds, with only 12 bacterial taxa shared across at least 80% of all the individual samples. These differences in the herd microbiota appeared to reflect mainly between-herd variation. Not surprisingly, the slurry was dominated by ubiquitous gastrointestinal bacteria, such as Ruminococcaceae and Lachnospiraceae Despite the low relative abundance of spirochetes, which ranged from 0 to 0.6%, we were able to detect small amounts of bacterial DNA from DD-associated treponemes in the slurry. However, the DD-associated Treponema spp. were detected only in samples from herds with reported DD problems. These data indicate that treponemes involved in the pathogenesis of DD are not part of the normal environmental microflora in dairy herds without clinical DD and, consequently, that slurry is not a primary reservoir of infection. IMPORTANCE Bovine digital dermatitis (DD), a dermal disease which causes lameness in dairy cattle, is a serious problem worldwide. To control this disease, the infection reservoirs and transmission routes of DD pathogens need to be clarified. The dairy herd slurry may be a pathogen reservoir of DD-associated bacteria. The rationale for the present study was, therefore, to examine whether DD-associated bacteria are always present in slurry or if they are found only in DD-afflicted herds. The results strongly indicated that DD Treponema spp. are not part of the indigenous slurry and, therefore, do not comprise an infection reservoir in healthy herds. This study applied next-generation sequencing technology to decipher the microbial compositions of environmental slurry of dairy herds with and without digital dermatitis., (Copyright © 2017 American Society for Microbiology.)

In Lotus japonicus, a LysM receptor kinase, EPR3, distinguishes compatible and incompatible rhizobial exopolysaccharides at the epidermis. However, the role of this recognition system in bacterial colonization of the root interior is unknown. Here we show that EPR3 advances the intracellular infection mechanism that mediates infection thread invasion of the root cortex and nodule primordia. At the cellular level, Epr3 expression delineates progression of infection threads into nodule primordia and cortical infection thread formation is impaired in epr3 mutants. Genetic dissection of this developmental coordination showed that Epr3 is integrated into the symbiosis signal transduction pathways. Further analysis showed differential expression of Epr3 in the epidermis and cortical primordia and identified key transcription factors controlling this tissue specificity. These results suggest that exopolysaccharide recognition is reiterated during the progressing infection and that EPR3 perception of compatible exopolysaccharide promotes an intracellular cortical infection mechanism maintaining bacteria enclosed in plant membranes.

Background: Polymicrobial infections represent a great challenge for the clarification of disease etiology and the development of comprehensive diagnostic or therapeutic tools, particularly for fastidious and difficult-to-cultivate bacteria. Using bovine digital dermatitis (DD) as a disease model, we introduce a novel strategy to study the pathogenesis of complex infections., Results: The strategy combines meta-transcriptomics with high-density peptide-microarray technology to screen for in vivo-expressed microbial genes and the host antibody response at the site of infection. Bacterial expression patterns supported the assumption that treponemes were the major DD pathogens but also indicated the active involvement of other phyla (primarily Bacteroidetes). Bacterial genes involved in chemotaxis, flagellar synthesis and protection against oxidative and acidic stress were among the major factors defining the disease., Conclusions: The extraordinary diversity observed in bacterial expression, antigens and host antibody responses between individual cows pointed toward microbial variability as a hallmark of DD. Persistence of infection and DD reinfection in the same individual is common; thus, high microbial diversity may undermine the host's capacity to mount an efficient immune response and maintain immunological memory towards DD. The common antigenic markers identified here using a high-density peptide microarray address this issue and may be useful for future preventive measures against DD.

Global investment in biomedical research has grown significantly over the last decades, reaching approximately a quarter of a trillion US dollars in 2010. However, not all of this investment is distributed evenly by gender. It follows, arguably, that scarce research resources may not be optimally invested (by either not supporting the best science or by failing to investigate topics that benefit women and men equitably). Women across the world tend to be significantly underrepresented in research both as researchers and research participants, receive less research funding, and appear less frequently than men as authors on research publications. There is also some evidence that women are relatively disadvantaged as the beneficiaries of research, in terms of its health, societal and economic impacts. Historical gender biases may have created a path dependency that means that the research system and the impacts of research are biased towards male researchers and male beneficiaries, making it inherently difficult (though not impossible) to eliminate gender bias. In this commentary, we - a group of scholars and practitioners from Africa, America, Asia and Europe - argue that gender-sensitive research impact assessment could become a force for good in moving science policy and practice towards gender equity. Research impact assessment is the multidisciplinary field of scientific inquiry that examines the research process to maximise scientific, societal and economic returns on investment in research. It encompasses many theoretical and methodological approaches that can be used to investigate gender bias and recommend actions for change to maximise research impact. We offer a set of recommendations to research funders, research institutions and research evaluators who conduct impact assessment on how to include and strengthen analysis of gender equity in research impact assessment and issue a global call for action.

Although treponemes are consistently identified in tissue from bovine digital dermatitis (DD) lesions, the definitive etiology of this debilitating polymicrobial disease is still unresolved. To study the microbiomes of 27 DD-infected and 10 healthy interdigital skin samples, we used a combination of different molecular methods. Deep sequencing of the 16S rRNA gene variable regions V1-V2 showed that Treponema, Mycoplasma, Fusobacterium and Porphyromonas were the genera best differentiating the DD samples from the controls. Additional deep sequencing analysis of the most abundant genus, Treponema, targeting another variable region of the 16S rRNA gene, V3-V4, identified 15 different phylotypes, among which Treponema phagedenis-like and Treponema refringens-like species were the most abundant. Although the presence of Treponema spp., Fusobacterium necrophorum and Porphyromonas levii was confirmed by fluorescence in situ hybridization (FISH), the results for Mycoplasma spp. were inconclusive. Extensive treponemal epidermal infiltration, constituting more than 90% of the total bacterial population, was observed in 24 of the 27 DD samples. F. necrophorum and P. levii were superficially located in the epidermal lesions and were present in only a subset of samples. RT-qPCR analysis showed that treponemes were also actively expressing a panel of virulence factors at the site of infection. Our results further support the hypothesis that species belonging to the genus Treponema are major pathogens of DD and also provide sufficient clues to motivate additional research into the role of M. fermentans, F. necrophorum and P. levii in the etiology of DD., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Surface polysaccharides are important for bacterial interactions with multicellular organisms, and some are virulence factors in pathogens. In the legume-rhizobium symbiosis, bacterial exopolysaccharides (EPS) are essential for the development of infected root nodules. We have identified a gene in Lotus japonicus, Epr3, encoding a receptor-like kinase that controls this infection. We show that epr3 mutants are defective in perception of purified EPS, and that EPR3 binds EPS directly and distinguishes compatible and incompatible EPS in bacterial competition studies. Expression of Epr3 in epidermal cells within the susceptible root zone shows that the protein is involved in bacterial entry, while rhizobial and plant mutant studies suggest that Epr3 regulates bacterial passage through the plant's epidermal cell layer. Finally, we show that Epr3 expression is inducible and dependent on host perception of bacterial nodulation (Nod) factors. Plant-bacterial compatibility and bacterial access to legume roots is thus regulated by a two-stage mechanism involving sequential receptor-mediated recognition of Nod factor and EPS signals.

It is important to investigate the molecular causes of the variation in ecologically important traits to fully understand phenotypic responses to climate change. In the Mississippi River Delta, two distinct, sympatric invasive lineages of common reed (Phragmites australis) are known to differ in several ecophysiological characteristics and are expected to become more salt resistant due to increasing atmospheric CO2 and temperature. We investigated whether different patterns of gene expression can explain their ecophysiological differences and increased vigor under future climatic conditions. We compared the transcript abundance of photosynthetic genes of the Calvin cycle (Rubisco small subunit, RbcS; Phosphoglycerate kinase, PGK; Phosphoribulokinase, PRK), genes related with salt transport (Na(+)/H(+) antiporter, PhaNHA) and oxidative stress response genes (Manganese Superoxide dismutase, MnSOD; Glutathione peroxidase, GPX), and the total aboveground biomass production between two genotypes representing the two lineages. The two genotypes (Delta-type, Mediterranean lineage, and EU-type, Eurasian lineage) were grown under an ambient and a future climate scenario with simultaneously elevated CO2 and temperature, and under two different soil salinities (0‰ or 20‰). We found neither differences in the aboveground biomass production nor the transcript abundances of the two genotypes, but soil salinity significantly affected all the investigated parameters, often interacting with the climatic conditions. At 20‰ salinity, most genes were higher expressed in the future than in the ambient climatic conditions. Higher transcription of the genes suggests higher abundance of the protein they code for, and consequently increased photosynthate production, improved stress responses, and salt exclusion. Therefore, the higher expression of these genes most likely contributed to the significantly ameliorated salinity impact on the aboveground biomass production of both P. australis genotypes under elevated temperature and CO2. Although transcript abundances did not explain differences between the lineages, they correlated with the increased vigor of both lineages under anticipated future climatic conditions.

Osseointegration of titanium implants can be improved by organic and inorganic nanocoating of the surface. The aim of our study was to evaluate the effect of organic nanocoating of titanium surface with unmodified and modified pectin Rhamnogalacturonan-Is (RG-Is) isolated from potato and apple with respect to surface properties and osteogenic response in osteoblastic cells. Nanocoatings on titanium surfaces were evaluated by scanning electron microscopy, contact angle measurements, atomic force microscopy, and X-ray photoelectron spectroscopy. The effect of coated RG-Is on cell adhesion, cell viability, bone matrix formation and mineralization was tested using SaOS-2 cells. Nanocoating with pectin RG-Is affected surface properties and in consequence changed the environment for cellular response. The cells cultured on surfaces coated with RG-Is from potato with high content of linear 1.4-linked galactose produced higher level of mineralized matrix compared with control surfaces and surfaces coated with RG-I with low content of linear 1.4-linked galactose. The study showed that the pectin RG-Is nanocoating not only changed chemical and physical titanium surface properties, but also specific coating with RG-Is containing high amount of galactan increased mineralized matrix formation of osteoblastic cells in vitro., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The bacteria associated with the infectious claw disease bovine digital dermatitis (DD) are spirochetes of the genus Treponema; however, their environmental reservoir remains unknown. To our knowledge, the current study is the first report of the discovery and phylogenetic characterization of rRNA gene sequences from DD-associated treponemes in the dairy herd environment. Although the spread of DD appears to be facilitated by wet floors covered with slurry, no DD-associated treponemes have been isolated from this environment previously. Consequently, there is a lack of knowledge about the spread of this disease among cows within a herd as well as between herds. To address the issue of DD infection reservoirs, we searched for evidence of DD-associated treponemes in fresh feces, in slurry, and in hoof lesions by deep sequencing of the V3 and V4 hypervariable regions of the 16S rRNA gene coupled with identification at the operational-taxonomic-unit level. Using treponeme-specific primers in this high-throughput approach, we identified small amounts of DNA (on average 0.6% of the total amount of sequence reads) from DD-associated treponemes in 43 of 64 samples from slurry and cow feces collected from six geographically dispersed dairy herds. Species belonging to the Treponema denticola/Treponema pedis-like and Treponema phagedenis-like phylogenetic clusters were among the most prevalent treponemes in both the dairy herd environment and the DD lesions. By the high-throughput approach presented here, we have demonstrated that cow feces and environmental slurry are possible reservoirs of DD-associated treponemes. This method should enable further clarification of the etiopathogenesis of DD., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Atrial fibrillation (AF) is the most common cardiac arrhythmia affecting 1-2% of the general population. A number of studies have demonstrated that AF, and in particular lone AF, has a substantial genetic component. Monogenic mutations in lone and familial AF, although rare, have been recognized for many years. Presently, mutations in 25 genes have been associated with AF. However, the complexity of monogenic AF is illustrated by the recent finding that both gain- and loss-of-function mutations in the same gene can cause AF. Genome-wide association studies (GWAS) have indicated that common single-nucleotide polymorphisms (SNPs) have a role in the development of AF. Following the first GWAS discovering the association between PITX2 and AF, several new GWAS reports have identified SNPs associated with susceptibility of AF. To date, nine SNPs have been associated with AF. The exact biological pathways involving these SNPs and the development of AF are now starting to be elucidated. Since the first GWAS, the number of papers concerning the genetic basis of AF has increased drastically and the majority of these papers are for the first time included in a review. In this review, we discuss the genetic basis of AF and the role of both common and rare genetic variants in the susceptibility of developing AF. Furthermore, all rare variants reported to be associated with AF were systematically searched for in the Exome Sequencing Project Exome Variant Server.

Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a lethal, rare hereditary disease with an estimated prevalence of 1:10 000. The genetic variants that cause CPVT are usually highly penetrant. To date, about 189 variants in 5 genes (RYR2, CASQ2, CALM1, TRND, and KCNJ2) have been associated with CPVT pathogenesis., Methods and Results: The Exome Sequencing Project database (ESP; n=6503) was systematically searched for previously published missense and nonsense CPVT-associated variants reported in several comprehensive reviews and in 2 databases: The Human Gene Mutation Database and The Inherited Arrhythmias Database. We used 4 different prediction tools to assess all missense variants previously associated with CPVT and compared the prediction of protein damage between CPVT-associated variants identified in the ESP and those variants not identified in the ESP. We identified 11% of the variants previously associated with CPVT in the ESP population. In the literature, 57% of these variants were reported as novel disease-causing variants absent in the healthy control subjects. These putative CPVT variants were identified in 41 out of 6131 subjects in the ESP population, corresponding to a prevalence of CPVT of up to 1:150. Using an agreement of ≥3, in silico prediction tools showed a significantly higher frequency of damaging variants among the CPVT-associated variants not identified in the ESP database (83%) compared with those variants identified in the ESP (50%; P=0.021)., Conclusions: We identified a substantial overrepresentation of CPVT-associated variants in a large exome database, suggesting that these variants are not necessarily the monogenic cause of CPVT.

Brugada syndrome (BrS) is a clinical entity first described in 1992. BrS is characterized by ST-segment elevations in the right precordial leads and susceptibility to ventricular arrhythmias and sudden cardiac death. It affects young subjects, predominantly males, with structurally normal hearts. The prevalence varies with ethnicity ranging from 1:2,000 to 1:100,000 in different parts of the world. Today, hundreds of variants in 17 genes have been associated with BrS of which mutations in SCN5A, coding for the cardiac voltage-gated sodium channel, accounts for the vast majority. Despite this, approximately 70% of BrS cases cannot be explained genetically with the current knowledge. Moreover, the monogenic role of some of the variants previously described as being associated with BrS has been questioned by their occurrence in about 4% (1:23) of the general population as found in NHLBI GO Exome Sequencing Project (ESP) currently including approximately 6500 individuals. If we add the variants described in the five newest identified genes associated with BrS, they appear at an even higher prevalence in the ESP (1:21). The current standard treatment of BrS is an implantable cardioverter-defibrillator (ICD). The risk stratification and indications for ICD treatment are based on the ECG and on the clinical and family history. In this review we discuss the genetic basis of BrS.

Several studies have associated mutations in genes encoding potassium channels and accessory subunits involved in cardiac repolarization with increased susceptibility of atrial fibrillation (AF). Recently, the Krüppel-like factor 15 (Klf15) was found to transcriptionally control rhythmic expression of KChIP2, a critical subunit required for generating the transient outward potassium current (Ito), and that deficiency or excess of Klf15 increased the susceptibility of arrhythmias. On this basis we hypothesized that mutations in Klf15 could be associated with AF. A total of 209 unrelated Caucasian lone AF patients were screened for mutations in Klf15 by direct sequencing. No mutations in the lone AF cohort were found. In one patient we found a synonymous variant (c.36C > T). In NHLBI GO Exome Sequencing Project (ESP) the variant was present in 31 of 4269 Caucasian individuals and in 3 of 2200 African Americans. In our cohort Klf15 was not associated with lone AF.

One of the non-classical human leukocyte antigen (HLA) class Ib proteins, HLA-G, is believed to exert important immunoregulatory functions, especially during pregnancy. The presence of HLA protein in paternal seminal fluid has been suggested to have an influence on the risk of developing pre-eclampsia. We have investigated whether HLA-G protein is present in human seminal plasma and in different tissue samples of the male reproductive system. Western blot technique and a soluble HLA-G (sHLA-G) assay were used to detect sHLA-G in human seminal plasma samples. Immunohistochemical staining was performed on paraffin-embedded tissue samples. We detected sHLA-G protein in seminal plasma, and HLA-G expression in normal testis and in epididymal tissue of the male reproductive system but not in the seminal vesicle. Furthermore, the results indicated a weak expression of HLA-G in hyperplastic prostatic tissue. In summary, several of the findings reported in this study suggest an immunoregulatory role of HLA-G in the male reproductive system and in seminal plasma.

Cytokinin plays a central role in the formation of nitrogen-fixing root nodules following inoculation with rhizobia. We show that exogenous cytokinin induces formation of discrete and easily visible nodule primordia in Lotus japonicus roots. The expression of nodulin genes was up-regulated upon cytokinin treatment, suggesting that the genuine nodulation program was indeed activated. This offers a simple approach for dissecting the underlying mechanism. Cytokinin-induced nodule primordia formation was unperturbed in several loss-of-function mutants impaired in epidermal responses to either rhizobial infection, Nod factor application, or both. However, absence of primordia in nsp1, nsp2, and nin mutants showed the requirement for these transcriptional regulators in the cytokinin-mediated activation of the root cortex. Distinguishing the epidermal and cortical responses further, we found that external cytokinin application induced expression of the Nin::GUS reporter gene within the root cortex but not in the root epidermis. Using L. japonicus lhk1-1 and har1 mutants, we demonstrate that discrete activation of root cortical cells by cytokinin depends on the LHK1 cytokinin receptor and is subjected to HAR1-mediated autoregulation.

T cell receptor (TCR) structure and function have been thoroughly studied for decades. Production and analyses of knock-out and knock-in mice with mutations in the CD3 chains have contributed significantly to these studies. The generation of such gene-modified mice relies on the availability of suitable embryonic stem (ES) cell lines. Traditionally, ES cell lines from the 129 mouse strains have been used followed by backcrossing to the C57BL/6 strain. In the present study, we demonstrate the existence of polymorphisms in the CD3 genes from mice of the 129 and C57BL/6 strains. These polymorphisms result in amino acid substitutions in the ectodomains of both the CD3delta and CD3epsilon chains in 129 mice compared to C57BL/6 mice. The amino acid substitutions do not change the stoichiometry or surface expression level of the TCR complex in 129 T cells but cause reduced anti-CD3 antibody binding to 129 T cells. Further, when stimulated with mitogenic anti-CD3 antibodies, T cells from the 129 strains show reduced expression of the activation marker CD69, Ca(2+) flux, IL-2 production and proliferative responses compared to C57BL/6 T cells. These findings demonstrate that polymorphisms of the CD3delta and epsilon ectodomains exist in mice, and that some of these polymorphisms lead to amino acid substitutions which cause structural changes and affect anti-CD3 antibody binding. Thus, functional T cell studies should be interpreted with caution when anti-CD3 antibodies are used for stimulation of T cells derived from gene-modified mice originating from 129 ES cell lines., (Copyright 2010 Elsevier Ltd. All rights reserved.)

A microfluidic chip for generation of gradients of dissolved oxygen was designed, fabricated and tested. The novel way of active oxygen depletion through a gas permeable membrane was applied. Numerical simulations for generation of O(2) gradients were correlated with measured oxygen concentrations. The developed microsystem was used to study growth patterns of the bacterium Pseudomonas aeruginosa in medium with different oxygen concentrations. The results showed that attachment of Pseudomonas aeruginosa to the substrate changed with oxygen concentration. This demonstrates that the device can be used for studies requiring controlled oxygen levels and for future studies of microaerobic and anaerobic conditions.

LysM receptor kinases were identified as receptors of acylated chitin (Nod factors) or chitin produced by plant-interacting microbes. Here, we present the identification and characterization of the LysM receptor kinase gene (Lys) family (17 members) in Lotus japonicus. Comprehensive phylogenetic analysis revealed a correlation between Lys gene structure and phylogeny. Further mapping coupled with sequence-based anchoring on the genome showed that the family has probably expanded by a combination of tandem and segmental duplication events. Using a sliding-window approach, we identified distinct regions in the LysM and kinase domains of recently diverged Lys genes where positive selection may have shaped ligand interaction. Interestingly, in the case of NFR5 and its closest paralog, LYS11, one of these regions coincides with the predicted Nod-factor binding groove and the suggested specificity determining area of the second LysM domain. One hypothesis for the evolutionary diversification of this receptor family in legumes is their unique capacity to decipher various structures of chitin-derived molecules produced by an extended spectrum of interacting organisms: symbiotic, associative, endophytic, and parasitic. In a detailed expression analysis, we found several Lotus Lys genes regulated not only during the symbiotic association with Mesorhizobium loti but also in response to chitin treatment.

Infection thread-dependent invasion of legume roots by rhizobia leads to internalization of bacteria into the plant cells, which is one of the salient features of root nodule symbiosis. We found that two genes, Nap1 (for Nck-associated protein 1) and Pir1 (for 121F-specific p53 inducible RNA), involved in actin rearrangements were essential for infection thread formation and colonization of Lotus japonicus roots by its natural microsymbiont, Mesorhizobium loti. nap1 and pir1 mutants developed an excess of uncolonized nodule primordia, indicating that these two genes were not essential for the initiation of nodule organogenesis per se. However, both the formation and subsequent progression of infection threads into the root cortex were significantly impaired in these mutants. We demonstrate that these infection defects were due to disturbed actin cytoskeleton organization. Short root hairs of the mutants had mostly transverse or web-like actin filaments, while bundles of actin filaments in wild-type root hairs were predominantly longitudinal. Corroborating these observations, temporal and spatial differences in actin filament organization between wild-type and mutant root hairs were also observed after Nod factor treatment, while calcium influx and spiking appeared unperturbed. Together with various effects on plant growth and seed formation, the nap1 and pir1 alleles also conferred a characteristic distorted trichome phenotype, suggesting a more general role for Nap1 and Pir1 in processes establishing cell polarity or polar growth in L. japonicus.

Objective: This study compared national self-reported data on medicine use and national prescription records at the individual level., Study Design and Setting: Data from the nationally representative Danish health survey conducted in 2000 (n=16,688) were linked at the individual level to national prescription records covering 1999-2000. Kappa statistics and 95% confidence intervals were calculated., Results: Applying the legend time method to medicine groups used mainly on a chronic basis revealed good to very good agreement between the two data sources, whereas medicines used as needed showed fair to moderate agreement. When a fixed-time window was applied for analysis, agreement was unchanged for medicines used mainly on a chronic basis, whereas agreement increased somewhat compared to the legend time method when analyzing medicines used as needed., Conclusion: Agreement between national self-reported data and national prescription records differed according to method of analysis and therapeutic group. A fixed-time window is an appropriate method of analysis for most therapeutic groups.

Background: The different role of various immunological effector cells in contact hypersensitivity (CHS) is receiving increased attention. During the past decade, the involvement of different cell types in CHS has been investigated by the use of antibody-induced depletion of specific subtypes of immunological cells and by studying knockout mice lacking one or more of these immunological cell populations., Objectives: To develop a method for studying the collective cellular dynamics of immune cells in the draining lymph nodes during CHS in intact animals., Patients/methods: Mice were sensitized and/or challenged with 2,4-dinitrofluorobenzene or oxazolone. Using multi-parameter flow cytometry we determined the proliferation, activation state, and absolute number of helper T cells, cytotoxic T cells, B cells, and natural killer cells in the draining lymph nodes., Results: The presented method can be applied to evaluate the effect of different contact allergens on various cell populations of the immune system., Conclusions: Our study support recent findings that several cell types seem to be involved in CHS.

It is well known that protein kinase C (PKC) plays an important role in regulation of TCR cell surface expression levels. However, eight different PKC isotypes are present in T cells, and to date the particular isotype(s) involved in TCR down-regulation remains to be identified. The aim of this study was to identify the PKC isotype(s) involved in TCR down-regulation and to elucidate the mechanism by which they induce TCR down-regulation. To accomplish this, we studied TCR down-regulation in the human T cell line Jurkat, in primary human T cells, or in the mouse T cell line DO11.10 in which we either overexpressed constitutive active or dominant-negative forms of various PKC isotypes. In addition, we studied TCR down-regulation in PKC knockout mice and by using small interfering RNA-mediated knockdown of specific PKC isotypes. We found that PKCalpha and PKCtheta were the only PKC isotypes able to induce significant TCR down-regulation. Both isotypes mediated TCR down-regulation via the TCR recycling pathway that strictly depends on Ser(126) and the di-leucine-based receptor-sorting motif of the CD3gamma chain. Finally, we found that PKCtheta was mainly implicated in down-regulation of directly engaged TCR, whereas PKCalpha was involved in down-regulation of nonengaged TCR.