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Giardia is a prevalent single-celled microaerophilic intestinal parasite causing diarrheal disease and significantly impacting global health. Double diploid (essentially tetraploid) Giardia trophozoites have presented a formidable challenge to the development of molecular genetic tools to interrogate gene function. High sequence divergence and the high percentage of hypothetical proteins lacking homology to proteins in other eukaryotes have limited our understanding of Giardia protein function, slowing drug target validation and development. For more than 25 years, Giardia A and B assemblages have been readily amenable to transfection with plasmids or linear DNA templates. Here, we highlight the utility and power of genetic approaches developed to assess protein function in Giardia, with particular emphasis on the more recent clustered regularly interspaced palindromic repeats/Cas9-based methods for knockdowns and knockouts. Robust and reliable molecular genetic approaches are fundamental toward the interrogation of Giardia protein function and evaluation of druggable targets. New genetic approaches tailored for the double diploid Giardia are imperative for understanding Giardia's unique biology and pathogenesis.

Clonal hematopoiesis (CH) is characterized by the acquisition of a somatic mutation in a hematopoietic stem cell that results in a clonal expansion. These driver mutations can be single nucleotide variants in cancer driver genes or larger structural rearrangements called mosaic chromosomal alterations (mCAs). The factors that influence the variations in mCA fitness and ultimately result in different clonal expansion rates are not well understood. We used the Passenger-Approximated Clonal Expansion Rate (PACER) method to estimate clonal expansion rate as PACER scores for 6,381 individuals in the NHLBI TOPMed cohort with gain, loss, and copy-neutral loss of heterozygosity mCAs. Our mCA fitness estimates, derived by aggregating per-individual PACER scores, were correlated (R2 = 0.49) with an alternative approach that estimated fitness of mCAs in the UK Biobank using population-level distributions of clonal fraction. Among individuals with JAK2 V617F clonal hematopoiesis of indeterminate potential or mCAs affecting the JAK2 gene on chromosome 9, PACER score was strongly correlated with erythrocyte count. In a cross-sectional analysis, genome-wide association study of estimates of mCA expansion rate identified a TCL1A locus variant associated with mCA clonal expansion rate, with suggestive variants in NRIP1 and TERT.

We report a multi-ancestry genome-wide association study on liver cirrhosis and its associated endophenotypes, alanine aminotransferase (ALT) and γ-glutamyl transferase. Using data from 12 cohorts, including 18,265 cases with cirrhosis, 1,782,047 controls, up to 1 million individuals with liver function tests and a validation cohort of 21,689 cases and 617,729 controls, we identify and validate 14 risk associations for cirrhosis. Many variants are located near genes involved in hepatic lipid metabolism. One of these, PNPLA3 p.Ile148Met, interacts with alcohol intake, obesity and diabetes on the risk of cirrhosis and hepatocellular carcinoma (HCC). We develop a polygenic risk score that associates with the progression from cirrhosis to HCC. By focusing on prioritized genes from common variant analyses, we find that rare coding variants in GPAM associate with lower ALT, supporting GPAM as a potential target for therapeutic inhibition. In conclusion, this study provides insights into the genetic underpinnings of cirrhosis.

While studying transgene expression after systemic administration of lentiviral vectors, we found that splenic B cells are robustly transduced, regardless of the types of pseudotyped envelope proteins. However, the administration of two different pseudotypes resulted in transduction of two distinct B cell populations, suggesting that each pseudotype uses unique and specific receptors for its attachment and entry into splenic B cells. Single-cell RNA sequencing analysis of the transduced cells demonstrated that different pseudotypes transduce distinct B cell subpopulations characterized by specific B cell receptor (BCR) genotypes. Functional analysis of the BCRs of the transduced cells demonstrated that BCRs specific to the pseudotyping envelope proteins mediate viral entry, enabling the vectors to selectively transduce the B cell populations that are capable of producing antibodies specific to their envelope proteins. Lentiviral vector entry via the BCR activated the transduced B cells and induced proliferation and differentiation into mature effectors, such as memory B and plasma cells. BCR-mediated viral entry into clonally specific B cell subpopulations raises new concepts for understanding the biodistribution of transgene expression after systemic administration of lentiviral vectors and offers new opportunities for BCR-targeted gene delivery by pseudotyped lentiviral vectors.

Planners and engineers need to know how to assess the impacts of proposed cycling infrastructure projects, so that projects that have the greatest potential impact on the actual and perceived cycling safety are selected over those that would be less effective. Planners also need to be able to communicate these impacts to decision-makers and the public. This research addresses these problems using the BikewaySim cycling shortest path model. BikewaySim uses link impedance functions to account for link attributes (e.g., presence of a bike lane, steep gradients, the number of lanes) and find the least impedance path for any origin-destination pair. In this project, BikewaySim was used to assess the impacts of using time-only and time with attribute impedances, as well as two proposed cycling infrastructure projects, on 28,392 potential trips for a study area in Atlanta, Georgia. These impacts were visualized through bikesheds, individual routing, and betweenness centrality. Two metrics, percent detour and change in impedance, were also calculated. Results demonstrate that BikewaySim can effectively visualize potential improvements of cycling infrastructure and has additional applications for trip planning. An expanded study area was also used to demonstrate bike + transit mode routing for four study area locations. Visualizations examine the accessibility to TAZs, travel time, and the utilized transit modes for each location. Compared to the walk + transit mode, the bike + transit mode provided greater access to other TAZs and reached them in a shorter amount of time. The locations near the center of the transit network where many routes converge offered the greatest accessibility for both the bike + transit and walk + transit modes. The difference in accessibility was greatest for locations near fewer transit routes. This research demonstrated how BikewaySim can be used to both examine the current cycling network and show changes in accessibility likely to result from new infrastructure. Both BikewaySim and TransitSim are open-source Python based tools that will be made available for practitioners to use in bicycle network planning. Future research will focus on calibrating link impedance functions with revealed preference data (cycling GPS traces) and survey response data (surveys on user preference for cycling infrastructure). View the NCST Project Webpage

Small animal models are of crucial importance for assessing COVID-19 countermeasures. Common laboratory mice would be well-suited for this purpose but are not susceptible to infection with wild-type SARS-CoV-2. However, the development of mouse-adapted virus strains has revealed key mutations in the SARS-CoV-2 spike protein that increase infectivity, and interestingly, many of these mutations are also present in naturally occurring SARS-CoV-2 variants of concern. This suggests that these variants might have the ability to infect common laboratory mice. Herein we show that the SARS-CoV-2 beta variant attains infectibility to BALB/c mice and causes pulmonary changes within 2–3 days post infection, consistent with results seen in other murine models of COVID-19, at a reasonable virus dose (2 × 105 PFU). The findings suggest that common laboratory mice can serve as the animal model of choice for testing the effectiveness of antiviral drugs and vaccines against SARS-CoV-2.

Racism is pervasive in education in Aotearoa New Zealand, including in early childhood education. The preparedness of early childhood teachers to respond to the Ministry of Education's current anti-racism policy direction is a pressing concern. This is particularly the case, given the early childhood curriculum "Te Whariki: He whariki matauranga mo nga mokopuna o Aotearoa" offers little guidance to support early childhood teachers to develop anti-racism pedagogies. This primarily theoretical article seeks to contribute to dialogue with early childhood teachers about both racism and anti-racism pedagogies. The theoretical arguments advanced in the article focus on document analysis of "Te Whariki." Analysis includes consideration of the themes of inclusion, equity and social justice. It also includes consideration of what these themes might imply about expectations for early childhood teachers' uptake of anti-racism approaches in their practice. Document analysis is supplemented by limited preliminary survey data drawn from the initial findings of the "Anti-racism Commitment in Early Childhood Education: Pathways to Inclusion, Equity and Social Justice" (ARC-ECE) study. Drawing from race-critical scholarship to further advance the theoretical arguments, the article highlights tensions in early childhood teachers' understandings about racism. The limits of narrow definitions of racism that explain it as the result of 'cultural difference' are explored. In making a case for thinking beyond cultural competence and culturally responsive practice, the article calls for an immediate rethinking of racism in (and beyond) the sector.

Deploying systems-theoretical conceptuality, this paper improves understanding of the organisational consequences of the intensified societal engagement of a research university. Aligning its work with Luhmannian organisational analysis, it addresses the dynamic interplay between two modes of administrative decision-making communication, namely, the traditional professional administration and the New-Public-Management-oriented (NPM) managerial techniques. Our research observes how the politico-economic conditions of the society translate into the university's decisions concerning an initiative to engage in start-up entrepreneurship. The article contributes to higher education literature by showing that the university's professional administration is a discrete organisational function internally differentiated into specialised administrative branches, each of which operates according to a sense-making regime associated with its primary societal system reference, such as education, science and the economy. The article also demonstrates the structurally conditioned differences in branch-specific temporalisations of the entrepreneurial initiative during decision-making. Inspired by the Luhmannian view on temporality, we demonstrate how administrative decisions synchronise the varied structural time horizons within the university's professional administration. Focus on temporality in decision-making thus allows us to see how the NPM-inspired managerial techniques are operationalised in administrative communication at universities. Consequently, the paper argues that university administration is a complex dynamic entity, which varyingly aligns itself to national policy scripts, and only selectively enacts features of a global trend known as NPM.

Groundwater-filled boreholes are a common solution in Scandinavian installations of ground source heat pumps (GSHP) due to the particular hydro-geological conditions with existing bedrock, and groundwater levels close to the surface. Different studies have highlighted the advantage of water-filled boreholes compared with their grouted counterparts since the natural convection of water within the borehole tends to decrease the effective thermal resistance Rb*. In this study, several methods are proposed for the evaluation and modeling of the effective thermal resistance of groundwater-filled boreholes. They are based on distributed temperature sensing (DTS) measurements of six representative boreholes within the irregular 74-single-U 300 m-deep borehole field of Aalto New Campus Complex (ANCC). These methods are compared with the recently developed correlations for groundwater-filled boreholes, which are implemented within the python-based simulation toolbox Pygfunction. The results from the enhanced Pygfunction simulation with daily update of Rb* show very good agreement with the measured mean fluid temperature of the first 39 months of system operation (March 2018–May 2021). It is observed that in real operation the effective thermal resistance Rb* can vary significantly, and therefore it is concluded that the update of Rb* is crucial for a reliable long-term simulation of groundwater-filled boreholes.

Purpose: The purpose of this study was to identify commonalities and differences between content components in stuttering treatment programs for preschool-age children. Method: In this document analysis, a thematic analysis of the content was conducted of handbooks and manuals describing Early Childhood Stuttering Therapy, the Lidcombe Program, Mini-KIDS, Palin Parent--Child Interaction Therapy, RESTART Demands and Capacities Model Method, and the Westmead Program. First, a theoretical framework defining a content component in treatment was developed. Second, we coded and categorized the data following the procedure of reflexive thematic analysis. In addition, the first authors of the treatment documents have reviewed the findings in this study, and their feedback has been analyzed and taken into consideration. Results: Sixty-one content components within the seven themes--"interaction, coping, reactions, everyday life, information, language, and speech"--were identified across the treatment programs. The content component "SLP providing information about the child's stuttering" was identified across all treatment programs. All programs are multithematic, and no treatment program has a single focus on speech, language, or parent-child interaction. A comparison of the programs with equal treatment goals highlighted more commonalities in content components across the programs. The differences between the treatment programs were evident in both the number of content components that varied from seven to 39 and the content included in each treatment program. Conclusions: Only one common content component was identified across programs, and the number and types of components vary widely. The role that the common content component plays in treatment effects is discussed, alongside implications for research and clinical practice.

An amendment to this paper has been published and can be accessed via the original article.

Abstract Background Interventions in infectious diseases can have both direct effects on individuals who receive the intervention as well as indirect effects in the population. In addition, intervention combinations can have complex interactions at the population level, which are often difficult to adequately assess with standard study designs and analytical methods. Discussion Herein, we urge the adoption of a new paradigm for the design and interpretation of intervention trials in infectious diseases, particularly with regard to emerging infectious diseases, one that more accurately reflects the dynamics of the transmission process. In an increasingly complex world, simulations can explicitly represent transmission dynamics, which are critical for proper trial design and interpretation. Certain ethical aspects of a trial can also be quantified using simulations. Further, after a trial has been conducted, simulations can be used to explore the possible explanations for the observed effects. Conclusion Much is to be gained through a multidisciplinary approach that builds collaborations among experts in infectious disease dynamics, epidemiology, statistical science, economics, simulation methods, and the conduct of clinical trials.

During a large outbreak of Shiga toxin−producing Escherichia coli illness associated with an agricultural show in Australia, we used whole-genome sequencing to detect an IS1203v insertion in the Shiga toxin 2c subunit A gene of Shiga toxin−producing E. coli. Our study showed that clinical illness was mild, and hemolytic uremic syndrome was not detected.

Though coronary arteries are crucial for heart function, the mechanisms guiding their formation are largely unknown. Here, Wang et al. identify a unique, endocardially-derived angiogenic precursor cell population for coronary artery formation in mice and show that a DLL4/NOTCH1/VEGFA/VEGFR2 signaling axis is key for coronary artery development.

Although many studies have reported on physical activity (PA) levels using accelerometers, a thorough description of the PA pattern in preschool children during their stay in the preschool, is lacking in the current literature. Furthermore, there remains a lack of understanding of the PA level and pattern in children in the lower end of the PA continuum. The first aim of this study was therefore to describe the PA pattern during a week-long stay in a preschool in all children born in 2011 (either three or four-year-olds) attending public preschools in a municipality in Norway. The second aim was to describe the PA level and pattern of the children who are the least physically active. This cross-sectional study included baseline data from 95% of children (N = 111) participating in a randomized controlled physical activity intervention (Active Kindergarten – Active Children study). The participants wore an Actigraph accelerometer, in week 39 in 2015, from when they arrived at the preschool on Monday morning and throughout their stay of five consecutive days. The amount of moderate- to vigorous-intensity PA (MVPA) per day ranged from 16 min to 116 min, and sedentary time ranged from 2.7 h to 6.5 h per day in the least and most sedentary child, respectively. The least physically active 25% of children were less active throughout the entire day, and only a few of them managed to achieve the recommended level of MVPA on any weekday. The physical activity levels and patterns among the least active children described in this study may help to inform interventions targeting this group.

Abstract Obesity and dyslipidemia are hallmarks of metabolic and cardiovascular diseases. Polydextrose (PDX), a soluble fiber has lipid lowering effects. We hypothesize that PDX reduces triglycerides and cholesterol by influencing gut microbiota, which in turn modulate intestinal gene expression. C57BL/6 male mice were fed a Western diet (WD) ±75 mg PDX twice daily by oral gavage for 14 days. Body weight and food intake were monitored daily. Fasting plasma lipids, caecal microbiota and gene expression in intestine and liver were measured after 14 days of feeding. PDX supplementation to WD significantly reduced food intake (p

Abstract The collagen- and calcium-binding EGF domains 1 (CCBE1) protein is necessary for lymphangiogenesis. Its C-terminal collagen-like domain was shown to be required for the activation of the major lymphangiogenic growth factor VEGF-C (Vascular Endothelial Growth Factor-C) along with the ADAMTS3 (A Disintegrin And Metalloproteinase with Thrombospondin Motifs-3) protease. However, it remained unclear how the N-terminal domain of CCBE1 contributed to lymphangiogenic signaling. Here, we show that efficient activation of VEGF-C requires its C-terminal domain both in vitro and in a transgenic mouse model. The N-terminal EGF-like domain of CCBE1 increased VEGFR-3 signaling by colocalizing pro-VEGF-C with its activating protease to the lymphatic endothelial cell surface. When the ADAMTS3 amounts were limited, proteolytic activation of pro-VEGF-C was supported by the N-terminal domain of CCBE1, but not by its C-terminal domain. A single amino acid substitution in ADAMTS3, identified from a lymphedema patient, was associated with abnormal CCBE1 localization. These results show that CCBE1 promotes VEGFR-3 signaling and lymphangiogenesis by different mechanisms, which are mediated independently by the two domains of CCBE1: by enhancing the cleavage activity of ADAMTS3 and by facilitating the colocalization of VEGF-C and ADAMTS3. These new insights should be valuable in developing new strategies to therapeutically target VEGF-C/VEGFR-3-induced lymphangiogenesis.

Abstract Thyroid gland vasculature has a distinguishable characteristic of endothelial fenestrae, a critical component for proper molecular transport. However, the signaling pathway that critically governs the maintenance of thyroid vascular integrity, including endothelial fenestrae, is poorly understood. Here, we found profound and distinct expression of follicular epithelial VEGF‐A and vascular VEGFR2 that were precisely regulated by circulating thyrotropin, while there were no meaningful expression of angiopoietin–Tie2 system in the thyroid gland. Our genetic depletion experiments revealed that VEGFR2, but not VEGFR3, is indispensable for maintenance of thyroid vascular integrity. Notably, blockade of VEGF‐A or VEGFR2 not only abrogated vascular remodeling but also inhibited follicular hypertrophy, which led to the reduction of thyroid weights during goitrogenesis. Importantly, VEGFR2 blockade alone was sufficient to cause a reduction of endothelial fenestrae with decreases in thyrotropin‐responsive genes in goitrogen‐fed thyroids. Collectively, these findings establish follicular VEGF‐A–vascular VEGFR2 axis as a main regulator for thyrotropin‐dependent thyroid angiofollicular remodeling and goitrogenesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Background: Research demonstrates that the placebo effect can influence the effectiveness of medical treatments and accounts for a significant proportion of healing in many conditions. However, providers may differ in the degree to which they consciously or unconsciously leverage the forces that produce placebo effects in clinical practice. Some studies suggest that the manner in which providers interact with patients shapes the magnitude of placebo effects, but this research has yet to distill the specific dimensions of patient–provider interactions that are most likely to influence placebo response and the mechanisms through which aspects of patient–provider interactions impact placebo response.Methods: We offer a simplifying and unifying framework in which interactions that boost placebo response can be dissected into two key dimensions: patients’ perceptions of competence, or whether a doctor “gets it” (i.e., displays of efficiency, knowledge, and skill), and patients’ perceptions of warmth, or whether a doctor “gets me” (i.e., displays of personal engagement, connection, and care for the patient).Results: First, we discuss how this framework builds on past research in psychology on social perception of competence and warmth and in medical literature on models of effective medical care, patient satisfaction, and patient–provider interactions. Then we consider possible mechanisms through which competence and warmth may affect the placebo response in healthcare. Finally, we share original data from patients and providers highlighting how this framework applies to healthcare. Both patient and provider data illustrate actionable ways providers can demonstrate competence and warmth to patients.Discussion: We conclude with recommendations for how researchers and practitioners alike can more systematically consider the role of provider competence and warmth in patient–provider interactions to deepen our understanding of placebo effects and, ultimately, enable providers to boost placebo effects alongside active medications (i.e., with known medical ingredients) and treatment in clinical care.

ObjectiveTo assess which mental health-related states of being are perceived as diseases by psychiatrists, non-psychiatric physicians, nurses, parliament members and laypeople.Design and settingA population-based, mailed survey in Finland.ParticipantsRespondents from a random sample of 3000 laypeople, 1500 physicians, 1500 nurses and all 200 members of the parliament (MPs) of Finland.Primary outcome measuresRespondents’ perspectives on 20 mental health-related states of being as diseases, measuring the extent of agreement with the claim: ‘[This state of being] is a disease’.ResultsOf the 6200 people approached, we received 3259 eligible responses (53%). Two conditions (schizophrenia and autism) were considered to be diseases by at least 75% and two states (grief and homosexuality) were considered not to be diseases by at least 75% in each group. A majority (at least 50% in each group) considered seven states as diseases (anorexia, attention deficit hyperactivity disorder, bulimia, depression, generalised anxiety disorder, panic disorder and personality disorder) and three not to be diseases (absence of sexual desire, premature ejaculation and transsexualism). In six states, there was a wide divergence of opinion (alcoholism, drug addiction, gambling addiction, insomnia, social anxiety disorder and work exhaustion). Psychiatrists were significantly more inclined to considering states of being as diseases relative to other groups, followed by non-psychiatric physicians, nurses, MPs and laypeople.ConclusionsRespondents agreed that some conditions, such as schizophrenia and autism, are diseases and other states, such as grief and homosexuality, are not; for others, there was considerable disagreement. Psychiatrists are more inclined to consider mental health-related states of being as diseases compared with other physicians, who, in turn, are more inclined than other constituencies. Understanding notions of disease may underlie important debates in public policy and practice in areas of mental health and behaviour, and have implications for resource allocation and stigma.

ABSTRACT We previously reported that the Toll-like receptor 4 (TLR4) antagonist Eritoran blocks acute lung injury (ALI) therapeutically in mouse and cotton rat models of influenza. However, secondary (2°) bacterial infection following influenza virus infection is associated with excess morbidity and mortality. Wild-type (WT) mice infected with mouse-adapted influenza A/Puerto Rico/8/34 virus (PR8) and, 7 days later, with Streptococcus pneumoniae serotype 3 (Sp3) exhibited significantly enhanced lung pathology and lethality that was reversed by Eritoran therapy after PR8 infection but before Sp3 infection. Cotton rats infected with nonadapted pH1N1 influenza virus and then superinfected with methicillin-resistant Staphylococcus aureus also exhibited increased lung pathology and serum high-mobility-group box 1 (HMGB1) levels, both of which were blunted by Eritoran therapy. In mice, PR8 infection suppressed Sp3-induced CXCL1 and CXCL2 mRNA, reducing neutrophil infiltration and increasing the bacterial burden, all of which were reversed by Eritoran treatment. While beta interferon (IFN-β)-deficient (IFN-β−/−) mice are highly susceptible to PR8, they exhibited delayed death upon Sp3 superinfection, indicating that while IFN-β was protective against influenza, it negatively impacted the host response to Sp3. IFN-β-treated WT macrophages selectively suppressed Sp3-induced CXCL1/CXCL2 transcriptionally, as evidenced by reduced recruitment of RNA polymerase II to the CXCL1 promoter. Thus, influenza establishes a “trained” state of immunosuppression toward 2° bacterial infection, in part through the potent induction of IFN-β and its downstream transcriptional regulation of chemokines, an effect reversed by Eritoran. IMPORTANCE Enhanced susceptibility to 2° bacterial infections following infection with influenza virus is a global health concern that accounts for many hospitalizations and deaths, particularly during pandemics. The complexity of the impaired host immune response during 2° bacterial infection has been widely studied. Both type I IFN and neutrophil dysfunction through decreased chemokine production have been implicated as mechanisms underlying enhanced susceptibility to 2° bacterial infections. Our findings support the conclusion that selective suppression of CXCL1/CXCL2 represents an IFN-β-mediated “training” of the macrophage transcriptional response to TLR2 agonists and that blocking of TLR4 therapeutically with Eritoran after influenza virus infection reverses this suppression by blunting influenza-induced IFN-β.

Climate change is modifying temperature and precipitation regimes across all seasons in northern ecosystems. Summer temperatures are higher, growing seasons extend into spring and fall and snow cover conditions are more variable during winter. The resistance of dominant tundra species to these season-specific changes, with each season potentially having contrasting effects on their growth and survival, can determine the future of tundra plant communities under climate change. In our study, we evaluated the effects of several spring/summer and winter climatic variables (i.e., summer temperature, growing season length, growing degree days, and number of winter freezing days) on the resistance of the dwarf shrub Empetrum nigrum. We measured over six years the ability of E. nigrum to keep a stable shoot growth, berry production, and vegetative cover in five E. nigrum dominated tundra heathlands, in a total of 144 plots covering a 200-km gradient from oceanic to continental climate. Overall, E. nigrum displayed high resistance to climatic variation along the gradient, with positive growth and reproductive output during all years and sites. Climatic conditions varied sharply among sites, especially during the winter months, finding that exposure to freezing temperatures during winter was correlated with reduced shoot length and berry production. These negative effects however, could be compensated if the following growing season was warm and long. Our study demonstrates that E. nigrum is a species resistant to fluctuating climatic conditions during the growing season and winter months in both oceanic and continental areas. Overall, E. nigrum appeared frost hardy and its resistance was determined by interactions among different season-specific climatic conditions with contrasting effects.

Background Reduced left ventricular ejection fraction (LVEF) is a risk marker for mortality after an acute coronary syndrome (ACS). Global Registry of Acute Coronary Events (GRACE) risk score, developed almost two decades ago, is the preferred scoring system for risk stratification in ACS. The aim of this study was to validate the GRACE score and evaluate whether LVEF has incremental predictive value over the GRACE in predicting 6-month mortality after ACS in a contemporary setting.Methods A retrospective analysis of all 1576 consecutive patients who were admitted to Tays Heart Hospital and underwent coronary angiography for a first episode of ACS (2015–2016). Clinical risk factors were extensively recorded. Adjusted Cox regression analysis was used to analyse the associations between LVEF and the GRACE score with 6-month all-cause mortality. The incremental predictive value was assessed by the change in C-statistic by Delong’s method for paired samples and by index of discrimination improvement (IDI).Results In univariable analysis, both LVEF and the GRACE were associated with 6-month mortality, and after applying both variables into the same model, the results remained significant (GRACE score: HR: 1.036, 95% CI 1.030 to 1.042; LVEF: HR: 0.965, 95% CI 0.948 to 0.982, both HRs corresponding to a one unit change in the exposure variable). The GRACE score demonstrated good discrimination for mortality (C-statistic: 0.833, 95% CI 0.795 to 0.871). Adding LVEF to the model with the GRACE score improved model performance significantly (C-statistic: 0.848, 95% CI 0.813 to 0.883, p=0.029 for the improvement and IDI 0.0171, 95% CI 0.0016 to 0.0327, p=0.031).Conclusions Adding LVEF to the GRACE score significantly improves risk prediction of 6-month mortality after ACS.

The human cell lines HepG2, HuH-7, and Jurkat are commonly used for amplification of the RNA viruses present in environmental samples. To assist with assays by RNAseq, we sequenced these cell lines and developed a subtraction database that contains sequences expected in sequence data from uninfected cells. RNAseq data from cell lines infected with Sendai virus were analyzed to test host subtraction. The process of mapping RNAseq reads to our subtraction database vastly reduced the number non-viral reads in the dataset to allow for efficient secondary analyses.

Vascular endothelial growth factor-C (VEGF-C) acts primarily on endothelial cells, but also on non-vascular targets, for example in the CNS and immune system. Here we describe a novel, unique VEGF-C form in the human reproductive system produced via cleavage by kallikrein-related peptidase 3 (KLK3), aka prostate-specific antigen (PSA). KLK3 activated VEGF-C specifically and efficiently through cleavage at a novel N-terminal site. We detected VEGF-C in seminal plasma, and sperm liquefaction occurred concurrently with VEGF-C activation, which was enhanced by collagen and calcium binding EGF domains 1 (CCBE1). After plasmin and ADAMTS3, KLK3 is the third protease shown to activate VEGF-C. Since differently activated VEGF-Cs are characterized by successively shorter N-terminal helices, we created an even shorter hypothetical form, which showed preferential binding to VEGFR-3. Using mass spectrometric analysis of the isolated VEGF-C-cleaving activity from human saliva, we identified cathepsin D as a protease that can activate VEGF-C as well as VEGF-D.

Introduction: Physical activity (PA) has been associated with telomere shortening. The association of PA intensity or volume with telomere length (TL) is nonetheless unclear. The aim of our study was to investigate the associations of exercise intensity and volume with TL in elderly adults from Northern Finland (65° latitude North).Methods: Seven hundred elderly subjects born in 1945 in the Oulu region were investigated. PA was measured during a 2-week period with a wrist-worn accelerometer. In addition, a questionnaire was used to assess sedentary time and to achieve a longitudinal PA history and intensity. Relative telomere lengths (RTL) were determined from frozen whole blood samples using a qPCR-based method.Results: Relative telomere lengths were significantly longer in women than men and negatively correlated with age in both genders (men r = -0.210, p = 0.000, women r = -0.174, and p = 0.000). During the 2-week study period, women took more steps than men (p = 0.001), but the association between steps and RTL was only seen in men (p = 0.05). Total steps taken (r = 0.202 and p = 0.04) and sedentary time (r = -0.247 and p = 0.007) significantly correlated with RTLs in 70-year old subjects. Moderate PA was associated with RTL in subjects with the highest quartile of moderate PA compared to the three lower quartiles (p-values: 0.023 between 4th and 1st, 0.04 between 4th and 2nd, and 0.027 between 4th and 3rd) in the 70-year old subjects.Conclusion: Women had longer RTL and a higher step count compared to men. However, exercise volume and RTL correlated positively only in men. Surprisingly, age correlated negatively with RTL already within an age difference of 2 years. This suggests that telomere attrition rate may accelerate in older age. Moderate physical activity at the time of study was associated with RTL.

BackgroundProprotein convertase subtilisin/kexin type 9 (PCSK9) is a liver serine protease regulating LDL cholesterol metabolism. PCSK9 binds to LDL receptors and guides them to lysosomes for degradation, thus increasing the amount of circulating LDL cholesterol. The aim of the study was to investigate associations between physical activity and plasma PCSK9 in subjects with high risk for type 2 diabetes (T2D).MethodsSixty-eight subjects from both genders with a high risk for T2D were included to a randomized controlled trial with a 3-month physical activity intervention. Physical activity intensities and frequencies were monitored throughout the intervention using a hip worn portable accelerometer. The plasma was collected before and after intervention for analysis of PCSK9 and cardiovascular biomarkers.ResultsPlasma PCSK9 did not relate to physical activity although number of steps were 46% higher in the intervention group than in the control group (p < 0.029). Total cholesterol was positively correlated with plasma PCSK9 (R = 0.320, p = 0.008), while maximal oxygen uptake was negatively associated (R = -0.252, p = 0.044). After the physical activity intervention PCSK9 levels were even stronger inversely associated with maximal oxygen uptake (R = -0.410, p = 0.0008) and positively correlated with HDL cholesterol (R = 0.264, p = 0.030). Interestingly, plasma PCSK9 levels were higher in the beginning than at the end of the study.ConclusionThe low physical activity that our subjects with high risk for T2D could perform did not influence plasma PCSK9 levels. Intervention with higher physical activities might be more effective in influencing PCSK9 levels.

BACKGROUND:Vitamin A and D deficiency is prevalent in pregnant women worldwide. Both vitamins are involved in fetal skeletal development. A positive association between maternal vitamin D levels and offspring bone mineral density (BMD) at adulthood has been observed. The impact of maternal vitamin A status in pregnancy on offspring peak bone mass remains unclear. METHOD AND FINDINGS:Forty-one mother-child pairs were recruited from a population-based prospective cohort study in Trondheim, Norway, where pregnant women were followed from gestational week 17. Their term-born infants were followed from birth (1986-88). Regression analyses were performed for vitamin A (retinol), 25-hydroxyvitamin D [25(OH)D] and 1,25-dihydroxyvitamin D [1,25(OH)2D] in maternal serum (gestational weeks 17, 33, 37) and cord blood. Offspring BMD and spine trabecular bone score (TBS), a measure of bone quality, were analyzed by dual x-ray absorptiometry at 26 years. Average levels during pregnancy of retinol, 25(OH)D and 1,25(OH)2D were 1.66 (0.32) μmol/L, 59.0 (20.6) nmol/L, and 251.3 (62.4) pmol/L, respectively. 1,25(OH)2D levels were similar in those with 25(OH)D levels 75 nmol/L. After adjustment for maternal age, BMI, smoking, and education, and offspring birth weight, maternal serum retinol was positively associated with offspring spine BMD [mean change 30.8 (CI 7.6, 54.0) mg/cm2 per 0.2 μmol/L retinol], and with offspring TBS, although non-significant (p = 0.08). No associations were found between maternal 25(OH)D and 1,25(OH)2D levels and offspring bone parameters. Vitamin levels in cord blood were not associated with offspring BMD or TBS. CONCLUSIONS:This is the first study to show an association between maternal vitamin A status and offspring peak bone mass. Our findings may imply increase future risk for osteoporotic fracture in offspring of mothers with suboptimal vitamin A level. No associations were observed between 25(OH)D and 1,25(OH)2D and offspring BMD.

Blue mold is a postharvest rot of pomaceous fruits caused by Penicillium expansum and a number of other Penicillium species. The genome of the highly aggressive P. expansum strain R19 was re-sequenced and analyzed together with the genome of the less aggressive P. solitum strain RS1. Whole genome scale similarities and differences were examined. A phylogenetic analysis of P. expansum, P. solitum, and several closely related Penicillium species revealed that the two pathogens isolated from decayed apple with blue mold symptoms are not each other’s closest relatives. Among a total of 10,560 and 10,672 protein coding sequences respectively, a comparative genomics analysis revealed 41 genes in P. expansum R19 and 43 genes in P. solitum RS1 that are unique to these two species. These genes may be associated with pome fruit–fungal interactions, subsequent decay processes, and mycotoxin accumulation. An intact patulin gene cluster consisting of 15 biosynthetic genes was identified in the patulin producing P. expansum strain R19, while only a remnant, seven-gene cluster was identified in the patulin-deficient P. solitum strain. However, P. solitum contained a large number of additional secondary metabolite gene clusters, indicating that this species has the potential capacity to produce an array of known as well as not-yet-identified products of possible toxicological or biotechnological interest.

Orthotopic liver transplantation remains the only curative therapy for inborn errors of metabolism. Given the tremendous success for primary immunodeficiencies using ex-vivo gene therapy with lentiviral vectors, there is great interest in developing similar curative therapies for metabolic liver diseases. We have previously generated a pig model of hereditary tyrosinemia type 1 (HT1), an autosomal recessive disorder caused by deficiency of fumarylacetoacetate hydrolase (FAH). Using this model, we have demonstrated curative ex-vivo gene and cell therapy using a lentiviral vector to express FAH in autologous hepatocytes. To further evaluate the long-term clinical outcomes of this therapeutic approach, we continued to monitor one of these pigs over the course of three years. The animal continued to thrive off the protective drug NTBC, gaining weight appropriately, and maintaining sexual fecundity for the course of his life. The animal was euthanized 31 months after transplantation to perform a thorough biochemical and histological analysis. Biochemically, liver enzymes and alpha-fetoprotein levels remained normal and abhorrent metabolites specific to HT1 remained corrected. Liver histology showed no evidence of tumorigenicity and Masson’s trichrome staining revealed minimal fibrosis and no evidence of cirrhosis. FAH-immunohistochemistry revealed complete repopulation of the liver by transplanted FAH-positive cells. A complete histopathological report on other organs, including kidney, revealed no abnormalities. This study is the first to demonstrate long-term safety and efficacy of hepatocyte-directed gene therapy in a large animal model. We conclude that hepatocyte-directed ex-vivo gene therapy is a rational choice for further exploration as an alternative therapeutic approach to whole organ transplantation for metabolic liver disease, including HT1.

The value of high-impact practices in undergraduate higher education is well documented. However, very little is known about high-impact practices in Human Development and Family Science (HDFS) programs. The goal of this study was to explore one specific high-impact practice: undergraduate research. Specifically, our study explored the benefits and barriers of undergraduate research for HDFS students as well as the pathways HDFS students use to become involved in undergraduate research. College student identity theory served as a guide for our inquiry. Semi-structured interviews were conducted with 22 undergraduate HDFS students, 13 of whom had participated in undergraduate research and nine who had not. Results revealed three pathways students took to participate in undergraduate research: interest in a specific research topic, connection with the researcher, and preparation for graduate school. Enhanced learning, professional skills, personal growth, and relationships with others were identified as benefits of participation in undergraduate research. Barriers to participation in research included time, lack of awareness, and feelings of fear or intimidation. Specific processes for students and faculty to overcome these barriers are suggested. It is recommended that HDFS faculty and administrators provide multiple pathways and supports for undergraduate students to become involved in research.

Educators in rural settings experience many unique professional circumstances due to the rural context of their schools; one such unique circumstance is professional development opportunities that are unavailable locally. Rural reading specialists have limited opportunities for local professional development as well as limited opportunities for collaboration with other reading specialists. This qualitative case study explored the characteristics of effective professional development for rural reading specialists. Reading specialists located in rural, Eastern Montana were surveyed about their professional development opportunities. From the survey results, focus group participants were identified to further elaborate on professional development considerations and opportunities for rural reading specialists. A key finding was rural reading specialists relied on the More Knowledgeable Other.

Teacher education faculty are often expected to engage in accreditation work. Our purpose was to document the collective driving mechanisms of junior faculty who volunteered to redesign key accreditation assessments. Specifically, we explored the values and expectations for success that led junior faculty to engage in and persist through key accreditation reform. Findings include eleven themes organized into three categories: drivers to join, hurdles, and drivers to continue. We interpret the results through Eccles and Wigfield's (2020) Situated Expectancy Value Theory and highlight the critical role that other colleagues and internal and external pressures played in undertaking the accreditation process.

This study examines how nursing students in their final year use skills in information literacy (IL) in clinical placements when working on an assignment in evidence-based practice. The IL instruction and the assignment were designed to give the students room for reflective thinking. Reflective thinking is crucial for developing good practice and adequate care in nursing. The study is qualitatively oriented and sets out to examine what kind of skills and knowledge the students demonstrate when doing an assignment that is practically oriented, and where IL is integrated in other tasks. The empirical material consists of written reflection notes from clinical placement. These notes have been analysed using a version of Bloom's taxonomy. For educators it is important to focus on how to bridge the theory-practice gap and combine IL with practical work. In this study we have identified some factors that may need more attention when designing similar assignments for students. The conclusion drawn from the reflection notes is that the students are on different levels of maturity and that in designing assignments related to real-life contexts, this should be given more attention.

High-Leverage Practices (HLPs) are a fundamental set of skills teachers should use consistently in the classroom to positively impact both their own capabilities and their students' learning. In our state, the five most important HLPs have been embedded into multiple courses across several educator preparation programs (EPPs). We asked preservice teachers to share their perceptions of their knowledge of and confidence in implementing HLPs prior to being introduced to them in their courses. Preservice teachers reported overwhelmingly strong knowledge and confidence, despite having never learned about the HLPs. In addition to our results, we share our thoughts about the illusion of fluency, the Dunning-Kruger Effect, and implications for EPPs with regard to field experiences and implementing HLPs.