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BackgroundEarly antenatal care (ANC) reduces maternal and neonatal morbidity and mortality through identification of pregnancy-related complications, yet 44% of Rwandan women present to ANC after 16 weeks gestational age (GA). The objective of this study was to identify factors associated with delayed initiation of ANC and describe differences in the obstetric risks identified at the first ANC visit (ANC-1) between women presenting early and late to care.MethodsThis secondary data analysis included 10,231 women presenting for ANC-1 across 18 health centers in Rwanda (May 2017-December 2018). Multivariable logistic regression models were constructed using backwards elimination to identify predictors of presentation to ANC at ≥16 and ≥24 weeks GA. Logistic regression was used to examine differences in obstetric risk factors identified at ANC-1 between women presenting before and after 16- and 24-weeks GA.ResultsSixty-one percent of women presented to ANC at ≥16 weeks and 24.7% at ≥24 weeks GA, with a mean (SD) GA at presentation of 18.9 (6.9) weeks. Younger age (16 weeks: OR = 1.36, 95% CI: 1.06, 1.75; 24 weeks: OR = 1.33, 95% CI: 0.95, 1.85), higher parity (16 weeks: 1-4 births, OR = 1.55, 95% CI: 1.39, 1.72; five or more births, OR = 2.57, 95% CI: 2.17, 3.04; 24 weeks: 1-4 births, OR = 1.93, 95% CI: 1.78, 2.09; five or more births, OR = 3.20, 95% CI: 2.66, 3.85), lower educational attainment (16 weeks: primary, OR = 0.75, 95% CI: 0.65, 0.86; secondary, OR = 0.60, 95% CI: 0.47,0.76; university, OR = 0.48, 95% CI: 0.33, 0.70; 24 weeks: primary, OR = 0.64, 95% CI: 0.53, 0.77; secondary, OR = 0.43, 95% CI: 0.29, 0.63; university, OR = 0.12, 95% CI: 0.04, 0.32) and contributing to household income (16 weeks: OR = 1.78, 95% CI: 1.40, 2.25; 24 weeks: OR = 1.91, 95% CI: 1.42, 2.55) were associated with delayed ANC-1 (≥16 and ≥24 weeks GA). History of a spontaneous abortion (16 weeks: OR = 0.74, 95% CI: 0.66, 0.84; 24 weeks: OR = 0.70, 95% CI: 0.58, 0.84), pregnancy testing (16 weeks: OR = 0.48, 95% CI: 0.33, 0.71; 24 weeks: OR = 0.41, 95% CI: 0.27, 0.61; 24 weeks) and residing in the same district (16 weeks: OR = 1.55, 95% CI: 1.08, 2.22; 24 weeks: OR = 1.73, 95% CI: 1.04, 2.87) or catchment area (16 weeks: OR = 1.53, 95% CI: 1.05, 2.23; 24 weeks: OR = 1.84, 95% CI: 1.28, 2.66; 24 weeks) as the health facility were protective against delayed ANC-1. Women with a prior preterm (OR, 0.71, 95% CI, 0.53, 0.95) or low birthweight delivery (OR, 0.72, 95% CI, 0.55, 0.95) were less likely to initiate ANC after 16 weeks. Women with no obstetric history were more likely to present after 16 weeks GA (OR, 1.18, 95% CI, 1.06, 1.32).ConclusionThis study identified multiple predictors of delayed ANC-1. Focusing existing Community Health Worker outreach efforts on the populations at greatest risk of delaying care and expanding access to home pregnancy testing may improve early care attendance. While women presenting late to care were less likely to present without an identified obstetric risk factor, lower than expected rates were identified in the study population overall. Health centers may benefit from provider training and standardized screening protocols to improve identification of obstetric risk factors at ANC-1.

INTRODUCTION:Telomerase repairs the telomeric ends of chromosomes and is active in nearly all malignant cells. Hepatitis C virus (HCV) is known to be oncogenic and potential interactions with the telomerase system require further study. We determined the effects of HCV infection on human telomerase reverse transcriptase (TERT) expression and enzyme activity in primary human hepatocytes and continuous cell lines. RESULTS:Primary human hepatocytes and Huh-7.5 hepatoma cells showed early de novo TERT protein expression 2-4 days after infection and these events coincided with increased TERT promoter activation, TERT mRNA, and telomerase activity. Immunoprecipitation studies demonstrated that NS3-4A protease-helicase, in contrast to core or NS5A, specifically bound to the C-terminal region of TERT through interactions between helicase domain 2 and protease sequences. Increased telomerase activity was noted when NS3-4A was transfected into cells, when added to reconstituted mixtures of TERT and telomerase RNA, and when incubated with high molecular weight telomerase 'holoenzyme' complexes. The NS3-4A catalytic effect on telomerase was inhibited with primuline or danoprevir, agents that are known to inhibit NS3 helicase and protease activities respectively. In HCV infected cells, NS3-4A could be specifically recovered with telomerase holoenzyme complexes in contrast to NS5A or core protein. HCV infection also activated the effector caspase 7 which is known to target TERT. Activation coincided with the appearance of lower molecular weight carboxy-terminal fragment(s) of TERT, chiefly sized at 45 kD, which could be inhibited with pancaspase or caspase 7 inhibitors. CONCLUSIONS:HCV infection induces TERT expression and stimulates telomerase activity in addition to triggering Caspase activity that leads to increased TERT degradation. These activities suggest multiple points whereby the virus can influence neoplasia. The NS3-4A protease-helicase can directly bind to TERT, increase telomerase activity, and thus potentially influence telomere repair and host cell neoplastic behavior.

Peptides are important affinity ligands for microscopy, biosensing, and targeted delivery. However, because they can have low affinity for their targets, their selection from large naïve libraries can be challenging. When selecting peptidic ligands from display libraries, it is important to: 1) ensure efficient display; 2) maximize the ability to select high affinity ligands; and 3) minimize the effect of the display context on binding. The "helper cell" packaging system has been described as a tool to produce filamentous phage particles based on phagemid constructs with varying display levels, while remaining free of helper phage contamination. Here we report on the first use of this system for peptide display, including the systematic characterization and optimization of helper cells, their inefficient use in antibody display and their use in creating and selecting from a set of phage display peptide libraries. Our libraries were analyzed with unprecedented precision by standard or deep sequencing, and shown to be superior in quality than commercial gold standards. Using our helper cell libraries, we have obtained ligands recognizing Yersinia pestis surface antigen F1V and L-glutamine-binding periplasmic protein QBP. In the latter case, unlike any of the peptide library selections described so far, we used a combination of phage and yeast display to select intriguing peptide ligands. Based on the success of our selections we believe that peptide libraries obtained with helper cells are not only suitable, but preferable to traditional phage display libraries for selection of peptidic ligands.

Fosmidomycin is a time-dependent nanomolar inhibitor of methylerythritol phosphate (MEP) synthase, which is the enzyme that catalyzes the first committed step in the MEP pathway to isoprenoids. Importantly, fosmidomycin is one of only a few MEP pathway-specific inhibitors that exhibits antimicrobial activity. Most inhibitors identified to date only exhibit activity against isolated pathway enzymes. The MEP pathway is the sole route to isoprenoids in many bacteria, yet has no human homologs. The development of inhibitors of this pathway holds promise as novel antimicrobial agents. Similarly, analyses of the bacterial response toward MEP pathway inhibitors provides valuable information toward the understanding of how emergent resistance may ultimately develop to this class of antibiotics. We have examined the transcriptional response of Salmonella enterica serovar typhimurium LT2 to sub-inhibitory concentrations of fosmidomycin via cDNA microarray and RT-PCR. Within the regulated genes identified by microarray were a number of genes encoding enzymes associated with the mediation of reactive oxygen species (ROS). Regulation of a panel of genes implicated in the response of cells to oxidative stress (including genes for catalases, superoxide dismutases, and alkylhydrogen peroxide reductases) was investigated and mild upregulation in some members was observed as a function of fosmidomycin exposure over time. The extent of regulation of these genes was similar to that observed for comparable exposures to kanamycin, but differed significantly from tetracycline. Furthermore, S. typhimurium exposed to sub-inhibitory concentrations of fosmidomycin displayed an increased sensitivity to exogenous H2O2 relative to either untreated controls or kanamycin-treated cells. Our results suggest that endogenous oxidative stress is one consequence of exposures to fosmidomycin, likely through the temporal depletion of intracellular isoprenoids themselves, rather than other mechanisms that have been proposed to facilitate ROS accumulation in bacteria (e.g. cell death processes or the ability of the antibiotic to redox cycle).

The largest digitized dataset of land plant distributions in Australia assembled to date (750,741 georeferenced herbarium records; 6,043 species) was used to partition the Australian continent into phytogeographical regions. We used a set of six widely distributed vascular plant groups and three non-vascular plant groups which together occur in a variety of landscapes/habitats across Australia. Phytogeographical regions were identified using quantitative analyses of species turnover, the rate of change in species composition between sites, calculated as Simpson's beta. We propose six major phytogeographical regions for Australia: Northern, Northern Desert, Eremaean, Eastern Queensland, Euronotian and South-Western. Our new phytogeographical regions show a spatial agreement of 65% with respect to previously defined phytogeographical regions of Australia. We also confirm that these new regions are in general agreement with the biomes of Australia and other contemporary biogeographical classifications. To assess the meaningfulness of the proposed phytogeographical regions, we evaluated how they relate to broad scale environmental gradients. Physiographic factors such as geology do not have a strong correspondence with our proposed regions. Instead, we identified climate as the main environmental driver. The use of an unprecedentedly large dataset of multiple plant groups, coupled with an explicit quantitative analysis, makes this study novel and allows an improved historical bioregionalization scheme for Australian plants. Our analyses show that: (1) there is considerable overlap between our results and older biogeographic classifications; (2) phytogeographical regions based on species turnover can be a powerful tool to further partition the landscape into meaningful units; (3) further studies using phylogenetic turnover metrics are needed to test the taxonomic areas.

The ability to remember future intentions (i.e., prospective memory) is influenced by attentional control. At the neuronal level, frontal and parietal brain regions have been related to attentional control and prospective memory. It is debated, however, whether more or less activity in these regions is beneficial for older adults' performance. We will test that by systematically enhancing or inhibiting activity in these regions with anodal or cathodal high-definition transcranial direct current stimulation in older adults. We will include n = 105 healthy older volunteers (60-75 years of age) in a randomized, double-blind, sham-controlled, and parallel-group design. The participants will receive either cathodal, anodal, or sham high-definition transcranial direct current stimulation of the left or right inferior frontal gyrus, or the right superior parietal gyrus (1mA for 20 min). During and after stimulation, the participants will complete tasks of attentional control and prospective memory. The results of this study will clarify how frontal and parietal brain regions contribute to attentional control and prospective memory in older healthy adults. In addition, we will elucidate the relationship between attentional control and prospective memory in that age group. The study has been registered with ClinicalTrials.gov on the 12th of May 2021 (trial identifier: NCT04882527)., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Schmidt et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objectives: Few is known on pregnant women with mild COVID-19 managed in a community setting with a telemedicine solution, including their outcomes. The objective of this study is to evaluate the adverse fetal outcomes and hospitalization rates of pregnant COVID-19 outpatients who were monitored with the Covidom© telemedicine solution., Methods: A nested study was conducted on pregnant outpatients with confirmed COVID-19, who were managed with Covidom© between March and November 2020. The patients were required to complete a standard medical questionnaire on co-morbidities and symptoms at inclusion, and were then monitored daily for 30 days after symptom onset. Adverse fetal outcome was defined as a composite of preterm birth, low birthweight, or stillbirth, and was collected retrospectively through phone contact with a standardized questionnaire., Results: The study included 714 pregnant women, with a median age of 32.0 [29.0-35.0] and a median BMI of 23.8 [21.3-27.0]. The main comorbidities observed were smoking (53%), hypertension (19%). The most common symptoms were asthenia (45.6%), cough (40.3%) and headache (25.7%), as well as anosmia (28.4%) and agueusia (32.3%). Adverse fetal outcomes occurred in 64 (9%) cases, including 38 (5%) preterm births, 33 (5%) low birthweights, and 6 (1%) stillbirths. Hospitalization occurred in 102 (14%) cases and was associated with adverse fetal outcomes (OR 2.4, 95% CI 1.3-4.4)., Conclusions: Our study suggests that adverse fetal outcomes are rare in pregnant women with mild COVID-19 who are monitored at home with telemedicine. However, hospitalization for COVID-19 and pregnancy-induced hypertension are associated with a higher risk of adverse fetal outcome., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Dinh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The aim of the research was to analyse the process of evacuation from the point of view of the individual's perception, behaviour and decision making. The study used a survey method that was conducted during two real-scale evacuation experiments in real road tunnels under smoky conditions. All experiments, with fire scenarios and procedures were very similar to real accident. Respondents' observations and important aspects affecting the evacuation process were verified, including decision-making during evacuation, loss of bearing in smoky conditions and group evacuation. The results indicate that participants in the experiments had started the evacuation due to smoke in the tunnel and fire drill. The evacuees observed decreased visibility on the escape route as well as a loss of bearing in the tunnel when smoke levels were high (extinction coefficient Cs > 0,7 m-1). The participants in the experiment evacuated in a group (when the tunnel infrastructure was unknown and there was no instruction as to what to do) and in twos under the smokiest conditions (extinction coefficient Cs~1.0-1.1m-1). During the experiments, the large impact of herding behaviour and following the group was noticed. The results of such studies based on real-scale evacuation experiments in road tunnels are essential to improve the level of safety in the road tunnel. In the surveys, the participants pointed to important issues related to evacuation, which require particular attention during the design, implementation and acceptance of this type of construction. The results of the study provide a better understanding of the behaviour of evacuees and indicate areas where tunnel infrastructure needs to be improved., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Natalia Schmidt-Polończyk. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: The aim of this study is to explore factors influencing the study engagement of health and social professions students during the COVID-19 pandemic. While antecedents of study engagement have been studied previously, the factors influencing engagement under pandemic conditions have not yet been investigated. Furthermore, there is a particular need for research among students in health and social professions programs, as these students are particularly affected by the pandemic. As theoretical basis, the study draws on the demands-resources-theory. It is hypothesized that pandemic-related study and personal resources drive engagement during the pandemic, and that pandemic-related demands negatively influence engagement., Method: The study uses a cross-sectional survey to explore the hypothesized effects. The sample consists of 559 university students of health and social professions in Germany. The study was carried out in July 2020, towards the end of the first digital semester and after the first peak in COVID-19 cases. Data are analyzed using linear multiple regression analysis., Results: The findings show that the demands-resources-theory is suitable to explain study engagement even under pandemic conditions. Suitable digital learning formats and social support are identified as important study resources for study engagement during major life events, while emotional resilience, active self-care and academic self-efficacy are identified as important personal resources., Conclusions: Under pandemic conditions academic institutions should focus on providing beneficial teaching formats and innovative ways to support students lacking social networks. Besides, they should consider developing means to help students structuring daily life as well as establishing initiatives to strengthen students' self-efficacy beliefs., Competing Interests: The authors have declared that no competing interests exist.

Background: Low plasma testosterone, either spontaneous or as a result of androgen deprivation therapy for prostate cancer, is associated with an increased risk of cardiovascular events. The underlying mechanism in humans is not understood. Experimental studies in mice have shown that castration facilitates atherogenesis and may increase signs of plaque vulnerability. Pigs used for translational atherosclerosis research have frequently been castrated for practical or commercial reasons, but the effect of castration on atherosclerosis has never been systematically evaluated in pigs., Objective: To study the effect of castration on atherosclerotic plaque burden and type in genetically modified minipigs with hypercholesterolemia., Methods: Newborn male Yucatan minipigs with transgenic overexpression of a human gain-of-function mutant of proprotein convertase subtilisin/kexin type 9 were randomized to undergo orchiectomy (n = 8) or serve as controls (n = 6). Minipigs were started on high-fat diet at 3 months of age and the amount and composition of atherosclerotic lesions were analyzed at 12 months of age. Plasma lipid profiles and behavioral parameters were also assessed., Results: Plasma lipids were slightly affected to a more atherogenic profile by orchiectomy, but atherosclerotic lesion size was unaltered in the LAD, thoracic aorta, abdominal aorta, and iliac arteries. The distribution of lesion types (xanthomas, pathological intimal thickening and fibroatheromas) were also not statistically different between groups in any of the examined vascular territories. The abdominal aorta developed the most advanced stages of disease with reproducible fibroatheroma formation, and here it was found that the area of necrotic core was significantly increased in orchiectomized pigs compared with controls. Orchiectomy also reduced aggressive behavior., Conclusions: Castration does not alter the burden of atherosclerosis in hypercholesterolemic Yucatan minipigs, but may increase necrotic core area in fibroatheromas., Competing Interests: JFB and CBS are inventors on a patent on PCSK9 transgenic minipigs owned by Aarhus University (US 8,546,643). This does not alter our adherence to PLOS ONE policies on sharing data and materials. The other authors have no disclosures to report.

The present study reports on observations carried out in the Tropical North Atlantic in summer and autumn 2017, documenting Sargassum aggregations using both ship-deck observations and satellite sensor observations at three resolutions (MSI-10 m, OLCI-300 m, VIIRS-750 m and MODIS-1 km). Both datasets reported that in summer, Sargassum aggregations were mainly observed off Brazil and near the Caribbean Islands, while they accumulated near the African coast in autumn. Based on in situ observations, we propose a five-class typology allowing standardisation of the description of in situ Sargassum raft shapes and sizes. The most commonly observed Sargassum raft type was windrows, but large rafts composed of a quasi-circular patch hundreds of meters wide were also observed. Satellite imagery showed that these rafts formed larger Sargassum aggregations over a wide range of scales, with smaller aggregations (of tens of m2 area) nested within larger ones (of hundreds of km2). Match-ups between different satellite sensors and in situ observations were limited for this dataset, mainly because of high cloud cover during the periods of observation. Nevertheless, comparisons between the two datasets showed that satellite sensors successfully detected Sargassum abundance and aggregation patterns consistent with in situ observations. MODIS and VIIRS sensors were better suited to describing the Sargassum aggregation distribution and dynamics at Atlantic scale, while the new sensors, OLCI and MSI, proved their ability to detect Sargassum aggregations and to describe their (sub-) mesoscale nested structure. The high variability in raft shape, size, thickness, depth and biomass density observed in situ means that caution is called for when using satellite maps of Sargassum distribution and biomass estimation. Improvements would require additional in situ and airborne observations or very high-resolution satellite imagery., Competing Interests: The authors have declared that no competing interests exist.

Five evacuation experiments were performed in a road tunnel in order to test how pedestrians react when exposed to reduced visibility, how the decision making process is carried out, and finally what is the impact of various circumstances like: different level of smokiness, competitive behavior or learning effect on an evacuation process. In four experiments pedestrians were exposed to artificial, non-toxic smoke. During evacuation of a group of people gathered in low and moderate level of smokiness (when Cs < 0.5m-1) we observed multi-line patterns created by pedestrians. Decision making was engaged in only by the first group of passengers, while under heavy smokiness Cs > 0.7m-1 we have observed decision making by small groups and characteristic double-lines patterns. In four experiments the same group of participants was involved, and a learning effect was observed: increasingly shorter pre-movement time and decreasing time required to leave the main tunnel. We show, that movement speed in smoke is influenced by the evacuees' attitude and familiarity with environment and evacuation procedures and not only by the visibility level., Competing Interests: The authors have declared that no competing interests exist.

Sensorineural hearing loss (SNHL) can be overcome by electrical stimulation of spiral ganglion neurons (SGNs) via a cochlear implant (CI). Restricted CI performance results from the spatial gap between the SGNs and the electrode, but the efficacy of CI is also limited by the degeneration of SGNs as one consequence of SHNL. In the healthy cochlea, the survival of SGNs is assured by endogenous neurotrophic support. Several applications of exogenous neurotrophic supply have been shown to reduce SGN degeneration in vitro and in vivo. In the present study, nanoporous silica nanoparticles (NPSNPs), with an approximate diameter of <100 nm, were loaded with the brain-derived neurotrophic factor (BDNF) to test their efficacy as long-term delivery system for neurotrophins. The neurotrophic factor was released constantly from the NPSNPs over a release period of 80 days when the surface of the nanoparticles had been modified with amino groups. Cell culture investigations with NIH3T3 fibroblasts attest a good general cytocompatibility of the NPSNPs. In vitro experiments with SGNs indicate a significantly higher survival rate of SGNs in cell cultures that contained BDNF-loaded nanoparticles compared to the control culture with unloaded NPSNPs (p<0.001). Importantly, also the amounts of BDNF released up to a time period of 39 days increased the survival rate of SGNs. Thus, NPSNPs carrying BDNF are suitable for the treatment of inner ear disease and for the protection and the support of SGNs. Their nanoscale nature and the fact that a direct contact of the nanoparticles and the SGNs is not necessary for neuroprotective effects, should allow for the facile preparation of nanocomposites, e.g., with biocompatible polymers, to install coatings on implants for the realization of implant-based growth factor delivery systems.

Human uterine fibroids, benign tumors derived from the smooth muscle layers of the uterus, impose a major health burden to up to 50% of premenopausal women in their daily life. To improve our understanding of this disease, we developed and characterized a patient-derived xenograft model by subcutaneous transplantation of pieces of human uterine fibroid tissue into three different strains of severe combined immunodeficient mice. Engrafted uterine fibroid tissue preserved the classical morphology with interwoven bundles of smooth muscle cells and an abundant deposition of collagenous matrix, similar to uterine fibroids in situ. The grafts expressed both estrogen receptor 1 and progesterone receptor. Additionally, both receptors were up-regulated by estrogen treatment. Growth of the fibroid grafts was dependent on 17β-estradiol and progesterone supplementation at levels similar to women with the disease and was studied for up to 60 days at maximum. Co-treatment with the antiprogestin mifepristone reduced graft growth (four independent donors, p<0.0001 two-sided t-test), as did treatment with the mTOR inhibitor rapamycin (three independent donors, p<0.0001 two-sided t-test). This in vivo animal model preserves the main histological and functional characteristics of human uterine fibroids, is amenable to intervention by pharmacological treatment, and can thus serve as an adequate model for the development of novel therapies.

Proteasomes are the major enzyme complexes for non-lysosomal protein degradation in eukaryotic cells. Mammals express two sets of catalytic subunits: the constitutive subunits β1, β2 and β5 and the immunosubunits LMP2 (β1i), MECL-1 (β2i) and LMP7 (β5i). The LMP7-propeptide (proLMP7) is required for optimal maturation of LMP2/MECL-1-containing precursors to mature immunoproteasomes, but can also mediate efficient integration into mixed proteasomes containing β1 and β2. In contrast, the β5-propeptide (proβ5) has been suggested to promote preferential integration into β1/β2-containing precursors, consequently favouring the formation of constitutive proteasomes. Here, we show that proβ5 predominantly promotes integration into LMP2/MECL-1-containing precursors in IFNγ-stimulated, LMP7-deficient cells and infected LMP7-deficient mice. This demonstrates that proβ5 does not direct preferential integration into β1/β2-containing precursors, but instead promotes the formation of mixed LMP2/MECL-1/β5 proteasomes under inflammatory conditions. Moreover, the propeptides substantially differ in their capacity to promote proteasome maturation, with proLMP7 showing a significantly higher chaperone activity as compared to proβ5. Increased efficiency of proteasome maturation mediated by proLMP7 is required for optimal MHC class I cell surface expression and is equally important as the catalytic activity of immunoproteasomes. Intriguingly, induction of LMP7 by infection not only results in rapid exchange of constitutive by immunosubunits, as previously suggested, but also increases the total proteasome abundance within the infected tissue. Hence our data identify a novel LMP7-dependend mechanism to enhance the activity of the proteasome system in infection, which is based on the high chaperone activity of proLMP7 and relies on accelerated maturation of active proteasome complexes.

Low levels of 25-hydroxy vitamin D (25(OH)D) are associated with cardiovascular diseases. Herein, we tested the hypothesis that vitamin D deficiency could be a causal factor in atherosclerotic vascular changes and vascular calcification. Aortic root sections of vitamin D receptor knockout (VDR(-/-)) mice that were stained for vascular calcification and immunostained for osteoblastic differentiation factors showed more calcified areas and a higher expression of the osteogenic key factors Msx2, Bmp2, and Runx2 than the wild-type mice (P<0.01). Data from LDL receptor knockout (LDLR(-/-)) mice that were fed western diet with either low (50 IU/kg), recommended (1,000 IU/kg), or high (10,000 IU/kg) amounts of vitamin D(3) over 16 weeks revealed increasing plasma concentrations of 25(OH)D (P<0.001) with increasing intake of vitamin D, whereas levels of calcium and phosphorus in plasma and femur were not influenced by the dietary treatment. Mice treated with the low vitamin D diet had more calcified lesions and a higher expression of Msx2, Bmp2, and Runx2 in aortic roots than mice fed recommended or high amounts of vitamin D (P<0.001). Taken together, these findings indicate vitamin D deficiency as a risk factor for aortic valve and aortic vessel calcification and a stimulator of osteogenic key factor expression in these vascular areas.