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Herein, we explore the generation and characterization of the radical cations of glycylphenylalanylglycine, or [GFG]•+, formed via dissociative electron-transfer reaction from the tripeptide to copper(II) within a ternary complex. A comprehensive investigation employing isotopic labeling, infrared multiple-photon dissociation (IRMPD) spectroscopy, and density functional theory (DFT) calculations elucidated the details and energetics in formation of the peptide radical cations as well as their dissociation products. Unlike conventional aromatic-containing peptide radical cations that primarily form canonical π-radicals, our findings reveal that 75% of the population of the experimentally produced [GFG]•+ precursors are [GFα•G]+, where the radical resides on the middle α-carbon of the phenylalanyl residue. This unexpected isomeric ion has an enthalpy of 6.8 kcal/mol above the global minimum, which has an N-terminal captodative structure, [Gα•FG]+, comprising 25% of the population. The [b₂-H]•+ product ions are also present in a ratio of 75/25 from [GFα•G]+/ [Gα•FG]+, the results of which are obtained from matches between the IRMPD action spectrum and predicted IR absorption spectra of the [b₂-H]•+ candidate structures, as well as from IRMPD isomer population analyses.

[This corrects the article DOI: 10.1371/journal.pone.0254880.].

ObjectiveTo determine the association between infant non-nutritive suck (NNS) dynamics at 3 months and developmental outcomes at 12 months of age in full-term infants. We hypothesized that infants with more mature NNS at 3 months, as evidence by shorter burst duration, fewer cycles per burst, cycles per minute, higher amplitude, and more bursts, would have higher (better) scores on the developmental outcomes at 12 months.MethodsThis was a prospective study that utilized objective and self-report measures. A five-minute NNS sample was collected from 67 infants (54% male) at 3 months of age (average age 2.99 (0.27) months). At 12 months (average age 11.91 (0.26) months), the Development Profile-3 was administered through caregiver interview.ResultsInfant NNS burst duration, cycles per burst, and cycles per minute were significantly negatively associated with the Development Profile-3 cognitive domain and general scores at 12 months. This is consistent with our hypothesis that infants who have more efficient NNS (fewer bursts and cycles) at 3 months would have higher (better) scores on the Development Profile-3 at 12 months.ConclusionsFindings from this work complement emerging research linking infant NNS with subsequent neurodevelopmental outcomes. This is the first time that these associations have been examined using a quantitative and physiologic-based measure of NNS. These results seem to indicate that specific NNS metrics, which demonstrate maturation of this complex skill, may be useful predictors of neurodevelopment later in life.

An integrated botanical and chemical approach is used to study surface residues on Funnel Beaker ceramics from the site of Oldenburg LA 77, in northern Germany. Organic residues were discovered adhering to fragments of thick-walled, undecorated ceramic vessels (n = 19) and ceramic discs (n = 2). The surface residues were studied using scanning electron microscopy (SEM), to examine remains of cereals and other plant tissues that survived food preparation and cooking, and using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and direct time-resolved mass spectrometry (DTMS), to chemically identify specific food components. The SEM results show a reoccurring presence of cereal grain (emmer and barley) and one case of co-occurrence of emmer and fat-hen seeds. The SEM evidence for the use of sprouted emmer grain and milk-ripe barley from the Oldenburg residues greatly enhances our understanding of Neolithic foodways in northwestern Europe. The ATR-FTIR results showed that roughly a third of the surface residues contain traces of the original foods prepared or processed and DTMS results confirm that most of the residues primarily contain polysaccharides and a minimal amount of plant protein and that they lack lipids. Only one residue presents minor indications for a (partly) animal origin. The ceramic vessels were thus used almost exclusively for the processing or cooking of cereal grains. This study offers an intimate view of the cuisine and cooking practices (and in some cases their seasonal timing) in an early agricultural village located in a marginal farming region on the south coast of the Baltic Sea.

Gait is a large component and indicator of health. Many factors affect gait including age, disease, and even mood disorders. Few studies have looked at the influence of emotional states on gait. This study aimed to investigate the influence of emotional states on walking performance to understand whether an emotional state may be an important factor to consider when evaluating gait. Thirty-six young adults were recruited (23F, 13M) and performed a neutral baseline condition of walking which included six passes of walking across an 8m walkway (a total of 48m of walking). Participants then completed 6 pseudo-randomized emotional state induction conditions while immersive 360-degree videos were used to induce the following emotional conditions: happiness, excitement, sadness, fear, and anger. Participants viewed the emotion elicitation videos using a virtual reality head-mounted display (HMD), then rated their emotional state using self-assessment manikins and walked (without the HMD) over a pressure sensor walkway. One-way repeated measures ANOVA and pairwise comparisons were used to examine differences in gait parameters across the emotional conditions. Participants walked with significantly reduced step length and speed during the sadness condition compared to the other emotional conditions and the neutral condition. Furthermore, participants adjusted the timing of their walking during the sadness condition and walked with significantly increased step, stance, and swing times compared to other emotional conditions, but not the neutral condition. Step time was significantly reduced during the conditions of excitement and fear compared to the neutral condition. Emotions may impact variety of gait parameters involving pace and rhythm, however have little influence on gait variability and postural control. These results indicate that perhaps the emotions of sadness and excitement should be taken into account as potential confounds for future gait analysis.

BackgroundDiagnosing concomitant pulmonary embolism (PE) in COVID-19 patients remains challenging. As such, PE may be overlooked. We compared the diagnostic yield of systematic PE-screening based on the YEARS-algorithm to PE-screening based on clinical gestalt in emergency department (ED) patients with COVID-19.MethodsWe included all ED patients who were admitted because of COVID-19 between March 2020 and February 2021. Patients already receiving anticoagulant treatment were excluded. Up to April 7, 2020, the decision to perform CT-pulmonary angiography (CTPA) was based on physician's clinical gestalt (clinical gestalt cohort). From April 7 onwards, systematic PE-screening was performed by CTPA if D-dimer level was ≥1000 ug/L, or ≥500 ug/L in case of ≥1 YEARS-item (systematic screening cohort).Results1095 ED patients with COVID-19 were admitted. After applying exclusion criteria, 289 were included in the clinical gestalt and 574 in the systematic screening cohort. The number of PE diagnoses was significantly higher in the systematic screening cohort compared to the clinical gestalt cohort: 8.2% vs. 1.0% (3/289 vs. 47/574; p100 mg/L (OR 2.78, 95%CI 1.37-5.66, p = 0.005) were independently associated with PE.ConclusionIn ED patients with COVID-19, the number of PE diagnosis was significantly higher in the cohort that underwent systematic PE screening based on the YEARS-algorithm in comparison with the clinical gestalt cohort, with a number needed to test of 7.1 CTPAs to detect one PE.

Standardized Inka tunics, or unku, were created under the auspices of the state as symbolic expressions of its expansionist power. To ensure these textiles acquired the status of effective insignias of power and territorial control, the Inka established and imposed technical and stylistic canons for their production (techne) by means of highly-skilled state weavers. In the provinces, social groups that came under imperial rule, local expert weaving agents adopted the conventions of the state and included meaningful symbolic elements of the idiosyncrasies, traditions, and experiential knowledge of the local community (metis). We therefore propose that this was not a unidirectional process and that the Caleta Vitor Inka unku (hereon referred to as the CV unku), presented here, reflects a syncretism promoted by local weavers. In terms of methods, we have developed a decoding tool for the unku, with the aim of distinguishing state from local hallmarks, thereby revealing the syncretic complexity of these iconic tunics. This methodological tool is based on a series of standard analytical parameters and attributes linked to morphological, technological, and stylistic features, which we applied to the CV unku. Unlike others, this unku does come from a looted tomb but was scientifically excavated in a cemetery located in the Caleta Vitor Bay in northern Chile. By deconstructing the CV unku we determined the steps in the chaîne opératoire at which local technical and stylistic elements were incorporated, thus affecting or transforming, in part, its emblematic imperial imagery. This study also marks a step forward in our understanding of a syncretic landscape that combines the state worldview and organized production system (imperial Inka) with craft-production practices that were rooted in provincial and local communities (provincial Inka).

The Cancer Genetic Markers of Susceptibility genome-wide association study (GWAS) originally identified a single nucleotide polymorphism (SNP) rs11249433 at 1p11.2 associated with breast cancer risk. To fine-map this locus, we genotyped 92 SNPs in a 900kb region (120,505,799-121,481,132) flanking rs11249433 in 45,276 breast cancer cases and 48,998 controls of European, Asian and African ancestry from 50 studies in the Breast Cancer Association Consortium. Genotyping was done using iCOGS, a custom-built array. Due to the complicated nature of the region on chr1p11.2: 120,300,000-120,505,798, that lies near the centromere and contains seven duplicated genomic segments, we restricted analyses to 429 SNPs excluding the duplicated regions (42 genotyped and 387 imputed). Per-allelic associations with breast cancer risk were estimated using logistic regression models adjusting for study and ancestry-specific principal components. The strongest association observed was with the original identified index SNP rs11249433 (minor allele frequency (MAF) 0.402; per-allele odds ratio (OR) = 1.10, 95% confidence interval (CI) 1.08-1.13, P = 1.49 x 10-21). The association for rs11249433 was limited to ER-positive breast cancers (test for heterogeneity P≤8.41 x 10-5). Additional analyses by other tumor characteristics showed stronger associations with moderately/well differentiated tumors and tumors of lobular histology. Although no significant eQTL associations were observed, in silico analyses showed that rs11249433 was located in a region that is likely a weak enhancer/promoter. Fine-mapping analysis of the 1p11.2 breast cancer susceptibility locus confirms this region to be limited to risk to cancers that are ER-positive.

Deficits in sensorimotor gating measured by prepulse inhibition (PPI) of the startle have been known as characteristics of patients with schizophrenia and related neuropsychiatric disorders. PPI disruption is thought to rely on the activity of the mesocorticolimbic dopaminergic system and is inhibited by most antipsychotic drugs. These drugs however act also at the nigrostriatal dopaminergic pathway and exert adverse locomotor responses. Finding a way to inhibit the mesocorticolimbic- without affecting the nigrostriatal-dopaminergic pathway may thus be beneficial to antipsychotic therapies. The melanin-concentrating hormone (MCH) system has been shown to modulate dopamine-related responses. Its receptor (MCH1R) is expressed at high levels in the mesocorticolimbic and not in the nigrostriatal dopaminergic pathways. Interestingly a genomic linkage study revealed significant associations between schizophrenia and markers located in the MCH1R gene locus. We hypothesize that the MCH system can selectively modulate the behavior associated with the mesocorticolimbic dopamine pathway. Using mice, we found that central administration of MCH potentiates apomorphine-induced PPI deficits. Using congenic rat lines that differ in their responses to PPI, we found that the rats that are susceptible to apomorphine (APO-SUS rats) and exhibit PPI deficits display higher MCH mRNA expression in the lateral hypothalamic region and that blocking the MCH system reverses their PPI deficits. On the other hand, in mice and rats, activation or inactivation of the MCH system does not affect stereotyped behaviors, dopamine-related responses that depend on the activity of the nigrostriatal pathway. Furthermore MCH does not affect dizocilpine-induced PPI deficit, a glutamate related response. Thus, our data present the MCH system as a regulator of sensorimotor gating, and provide a new rationale to understand the etiologies of schizophrenia and related psychiatric disorders.

Recent advances in DNA sequencing methods revolutionized biology by providing highly accurate reads, with high throughput or high read length. These read data are being used in many biological and medical applications. Modern DNA sequencing methods have no equivalent in protein sequencing, severely limiting the widespread application of protein data. Recently, several optical protein sequencing methods have been proposed that rely on the fluorescent labeling of amino acids. Here, we introduce the reprotonation-deprotonation protein sequencing method. Unlike other methods, this proposed technique relies on the measurement of an electrical signal and requires no fluorescent labeling. In reprotonation-deprotonation protein sequencing, the terminal amino acid is identified through its unique protonation signal, and by repeatedly cleaving the terminal amino acids one-by-one, each amino acid in the peptide is measured. By means of simulations, we show that, given a reference database of known proteins, reprotonation-deprotonation sequencing has the potential to correctly identify proteins in a sample. Our simulations provide target values for the signal-to-noise ratios that sensor devices need to attain in order to detect reprotonation-deprotonation events, as well as suitable pH values and required measurement times per amino acid. For instance, an SNR of 10 is required for a 61.71% proteome recovery rate with 100 ms measurement time per amino acid.

PURPOSE:The aim was to assess personalised contrast media (CM) protocols-based on patient's blood volume (BV) and automated tube voltage selection (ATVS)-in coronary computed tomography angiography (CCTA). METHODS:A total of 114 consecutive patients received an ECG-triggered or ECG-gated helical scan on a 3rd-generation dual-source CT with 70-120kV (ATVS) and 330mAsqual.ref. CM was adapted to BV, scan time (s) and kV. Image quality (IQ) was assessed in a 17-segment coronary model using attenuation values (HU), contrast-to-noise (CNR), signal-to-noise ratio (SNR) (objective IQ) and a Likert scale (subjective IQ: 1 = poor/2 = sufficient/3 = good/4 = excellent). ig. RESULTS:Patient distribution was: n = 60 for 70kV, n = 37 80kV and n = 17 90kV. Mean BV was 5.4±0.6L for men and 4.1±0.6L for women. Mean CM volume (300 mg I/mL) and flow rate were: 30.9±6.4mL and 3.3±0.5mL/s (70kV); 40.8±7.1mL and 4.5±0.6mL/s (80kV); 53.6±8.6mL and 5.7±0.6mL/s (90kV). Overall mean HU was >300HU in 98.2% (112/114) of patients. Overall mean attenuation was below 300HU in two scans (70kV) due to late scan timing. Of 1.661 segments, 95.4% was assessable. Mean CNR was 14±4(70kV), 13±3(80kV) and 14±4(90kV); mean SNR was 10±2(both 70kV+80kV) and 9±2(90kV). Objective IQ was comparable between kV settings, protocols and sex. Subjective IQ was diagnostic in all scans and excellent-sufficient in 95.4% of segments. CONCLUSIONS:Personalisation of CCTA CM injection protocols to BV and ATVS is a promising technique to tailor CM administration to the individual patient, while maintaining diagnostic IQ.

Drug-related sinusoidal dilatation (SD) is a common form of hepatotoxicity associated with oxaliplatin-based chemotherapy used prior to resection of colorectal liver metastases (CRLM). Recently, hepatic SD has also been associated with anti-delta like 4 (DLL4) cancer therapies targeting the NOTCH pathway. To investigate the hypothesis that NOTCH signaling plays an important role in drug-induced SD, gene expression changes were examined in livers from anti-DLL4 and oxaliplatin-induced SD in non-human primate (NHP) and patients, respectively. Putative mechanistic biomarkers of bevacizumab (bev)-mediated protection against oxaliplatin-induced SD were also investigated. RNA was extracted from whole liver sections or centrilobular regions by laser-capture microdissection (LCM) obtained from NHP administered anti-DLL4 fragment antigen-binding (F(ab')2 or patients with CRLM receiving oxaliplatin-based chemotherapy with or without bev. mRNA expression was quantified using high-throughput real-time quantitative PCR. Significance analysis was used to identify genes with differential expression patterns (false discovery rate (FDR) < 0.05). Eleven (CCL2, CCND1, EFNB2, ERG, ICAM1, IL16, LFNG, NOTCH1, NOTCH4, PRDX1, and TGFB1) and six (CDH5, EFNB2, HES1, IL16, MIK67, HES1 and VWF) candidate genes were differentially expressed in the liver of anti-DLL4- and oxaliplatin-induced SD, respectively. Addition of bev to oxaliplatin-based chemotherapy resulted in differential changes in hepatic CDH5, HEY1, IL16, JAG1, MMP9, NOTCH4 and TIMP1 expression. This work implicates NOTCH and IL16 pathways in the pathogenesis of drug-induced SD and further explains the hepato-protective effect of bev in oxaliplatin-induced SD observed in CRLM patients.

Neochloris oleoabundans is an oleaginous microalgal species that can be cultivated in fresh water as well as salt water. Using salt water gives the opportunity to reduce production costs and the fresh water footprint for large scale cultivation. Production of triacylglycerols (TAG) usually includes a biomass growth phase in nitrogen-replete conditions followed by a TAG accumulation phase under nitrogen-deplete conditions. This is the first report that provides insight in the saline resistance mechanism of a fresh water oleaginous microalgae. To better understand the osmoregulatory mechanism of N. oleoabundans during growth and TAG accumulating conditions, the transcriptome was sequenced under four different conditions: fresh water nitrogen-replete and -deplete conditions, and salt water (525 mM dissolved salts, 448mM extra NaCl) nitrogen-replete and -deplete conditions. In this study, several pathways are identified to be responsible for salt water adaptation of N. oleoabundans under both nitrogen-replete and -deplete conditions. Proline and the ascorbate-glutathione cycle seem to be of importance for successful osmoregulation in N. oleoabundans. Genes involved in Proline biosynthesis were found to be upregulated in salt water. This was supported by Nuclear magnetic resonance (NMR) spectroscopy, which indicated an increase in proline content in the salt water nitrogen-replete condition. Additionally, the lipid accumulation pathway was studied to gain insight in the gene regulation in the first 24 hours after nitrogen was depleted. Oil accumulation is increased under nitrogen-deplete conditions in a comparable way in both fresh and salt water. The mechanism behind the biosynthesis of compatible osmolytes can be used to improve N. oleoabundans and other industrially relevant microalgal strains to create a more robust and sustainable production platform for microalgae derived products in the future.

Recently, studies in the United States and European countries have shown that the degree of attachment is associated with the attribution of emotions to companion animals. These studies imply that investigating the degree of attachment to companion animals is a good way for researchers to explore animal emotions and then improve animal welfare. Although a promising area of study, in Japan, no empirical studies have examined the correlation between the degree of attachment and the attribution of emotions to companion animals. In this research, we aimed to assess companion animal owners' attribution of six primary (anger, joy, sadness, disgust, fear and surprise) and four secondary (shame, jealousy, disappointment and compassion) emotions to their dogs and cats, as well as how the degree of attachment related to such attribution of emotions from a Japanese cultural perspective. The "Pet Bonding Scale" (PBS), which is used to determine the level of bonding between humans and animals, was introduced to measure respondents' degree of attachment to their companion animals. The results of a questionnaire (N = 546) distributed throughout Japan showed that respondents attributed a wide range of emotions to their animals. Companion animals' primary emotions, compared to secondary emotions, were more commonly attributed by their owners. The attribution of compassion and jealousy was reported at a high level (73.1% and 56.2%, respectively), which was surprising as compassion and jealousy are generally defined as secondary emotions. All participants were highly attached to their companion animals, and this attachment was positively associated with the attribution of emotions (9/10) to companion animals (all p < 0.05). This study is one of the first to investigate animal emotions by analyzing the bonding between companion animals and owners in Japan, and it can therefore provide knowledge to increase Japanese people's awareness of animal welfare.

Variability in spider major ampullate (MA) silk properties at different scales has proven difficult to determine and remains an obstacle to the development of synthetic fibers mimicking MA silk performance. A multitude of techniques may be used to measure multiscale aspects of silk properties. Here we fed five species of Araneoid spider solutions that either contained protein or were protein deprived and performed silk tensile tests, small and wide-angle X-ray scattering (SAXS/WAXS), amino acid composition analyses, and silk gene expression analyses, to resolve persistent questions about how nutrient deprivation induces variations in MA silk mechanical properties across scales. Our analyses found that the properties of each spider's silk varied differently in response to variations in their protein intake. We found changes in the crystalline and non-crystalline nanostructures to play specific roles in inducing the property variations we found. Across treatment MaSp expression patterns differed in each of the five species. We found that in most species MaSp expression and amino acid composition variations did not conform with our predictions based on a traditional MaSp expression model. In general, changes to the silk's alanine and proline compositions influenced the alignment of the proteins within the silk's amorphous region, which influenced silk extensibility and toughness. Variations in structural alignment in the crystalline and non-crystalline regions influenced ultimate strength independent of genetic expression. Our study provides the deepest insights thus far into the mechanisms of how MA silk properties vary from gene expression to nanostructure formations to fiber mechanics. Such knowledge is imperative for promoting the production of synthetic silk fibers.

Gelatinase B/matrix metalloproteinase-9 (MMP-9) triggers multiple sclerosis (MS) and the animal model of experimental autoimmune encephalomyelitis (EAE) by the breakdown of the blood-brain barrier. Interestingly, MMP-9 is beneficial in systemic autoimmunity caused by Fas-deficiency. Fas-deficient (faslpr) and Fas-ligand-deficient mice are protected against EAE. We here investigated the interaction between Fas and MMP-9 in the setting of induction of EAE and compared short- and long-term effects. We provoked EAE with myelin oligodendrocyte glycoprotein (MOG) peptide and compared EAE development in four genotypes (wild-type (WT), single knockout mmp-9-/-, faslpr, and mmp-9-/-/faslpr) and monitored leukocytes, cytokines and chemokines as immunological parameters. As expected, faslpr mice were resistant against EAE induction, whereas MMP-9 single knockout mice were not. In the double mmp-9-/-/ faslpr mice the effects on disease scores pointed to independent rather than interrelated disease mechanisms. On a short term, after EAE induction leukocytes infiltrated into the brain and cytokine and chemokine levels were significantly higher in all the four genotypes studied, even in the faslpr and mmp-9-/-/faslpr, which did not develop clinical disease. The levels of MMP-9 but not of MMP-2 were increased in the brain and in the peripheral organs after EAE induction. After 40 days all the animals recovered and did not show signs of EAE. However, the absence of MMP-9 in the remission phase suggested a protective role of MMP-9 in the late phase of the disease, because single mmp-9-/- mice presented a delayed remission in comparison with WT animals suggesting a phase-dependent role of MMP-9 in the disease. Nevertheless, the levels of some cytokines and chemokines remained higher than in control animals even 100 days after EAE induction, attesting to a prolonged state of immune activation. We thus yielded new insights and useful markers to monitor this activated immune status. Furthermore, MMP-9 but not MMP-2 levels remained increased in the brains and, to a higher extend, in the spleens of the WT mice even during the remission phase, which is in line with the role of MMP-9 as a useful marker and a protective factor for EAE in the remission phase.

The United States Virgin Islands are comprised of two separate insular platforms separated by the deep water Anegada Passage. Although managed by the same regulations, as one fishery, several physical and spatial differences exist between the two northern shelf islands, St. Thomas and St. John, and isolated St. Croix. Based on two long-term fisheries independent datasets, collected by the U.S. Virgin Islands Territorial Coral Reef Monitoring Program and the National Oceanographic and Atmospheric Administration Center for Coastal Monitoring and Assessment, there were significant differences between the northern USVI and St. Croix in both the occurrence and size of several species of large and commercially important reef fishes. These fishes are primarily apex piscivores and generally the first species over-exploited in small-scale fisheries. The disparities between the fish communities on the two island shelves cannot be explained solely by differences in habitat (coral cover, rugosity) or fisheries management, such as relative amount of marine protected area in local waters. They are instead probably caused by a combination of several other interrelated factors including water depth, fishing methodology, fishable area, and the presence or absence of viable fish spawning areas. This study considers those aspects, and illustrates the need for management of island artisanal fisheries that is tailored to the physical and spatial constraints imposed by insular platforms.

This study aimed to identify the barriers female sex workers (FSWs) in Bangladesh face with regard to accessing sexual and reproductive health (SRH) care, and assess the satisfaction with the healthcare received.Data were collected from coverage areas of four community-based drop-in-centers (DICs) in Dhaka where sexually transmitted infection (STI) and human immunovirus (HIV) prevention interventions have been implemented for FSWs. A total of 731 FSWs aged 15-49 years were surveyed. In addition, in-depth interviews (IDIs) were conducted with 14 FSWs and 9 service providers. Respondent satisfaction was measured based on recorded scores on dignity, privacy, autonomy, confidentiality, prompt attention, access to social support networks during care, basic amenities, and choice of institution/care provider.Of 731 FSWs, 353 (51%) reported facing barriers when seeking sexual and reproductive healthcare. Financial problems (72%), shame about receiving care (52.3%), unwillingness of service providers to provide care (39.9%), unfriendly behavior of the provider (24.4%), and distance to care (16.9%) were mentioned as barriers. Only one-third of the respondents reported an overall satisfaction score of more than fifty percent (a score of between 9 and16) with formal healthcare. Inadequacy or lack of SRH services and referral problems (e.g., financial charge at referral centers, unsustainable referral provision, or unknown location of referral) were reported by the qualitative FSWs as the major barriers to accessing and utilizing SRH care.These findings are useful for program implementers and policy makers to take the necessary steps to reduce or remove the barriers in the health system that are preventing FSWs from accessing SRH care, and ultimately meet the unmet healthcare needs of FSWs.

BACKGROUND:Arrhythmogenic cardiomyopathy is an inherited heart muscle disorder leading to ventricular arrhythmias and heart failure, mainly as a result of mutations in cardiac desmosomal genes. Desmosomes are cell-cell junctions mediating adhesion of cardiomyocytes; however, the molecular and cellular mechanisms underlying the disease remain widely unknown. Desmocollin-2 is a desmosomal cadherin serving as an anchor molecule required to reconstitute homeostatic intercellular adhesion with desmoglein-2. Cardiac specific lack of desmoglein-2 leads to severe cardiomyopathy, whereas overexpression does not. In contrast, the corresponding data for desmocollin-2 are incomplete, in particular from the view of protein overexpression. Therefore, we developed a mouse model overexpressing desmocollin-2 to determine its potential contribution to cardiomyopathy and intercellular adhesion pathology. METHODS AND RESULTS:We generated transgenic mice overexpressing DSC2 in cardiac myocytes. Transgenic mice developed a severe cardiac dysfunction over 5 to 13 weeks as indicated by 2D-echocardiography measurements. Corresponding histology and immunohistochemistry demonstrated fibrosis, necrosis and calcification which were mainly localized in patches near the epi- and endocardium of both ventricles. Expressions of endogenous desmosomal proteins were markedly reduced in fibrotic areas but appear to be unchanged in non-fibrotic areas. Furthermore, gene expression data indicate an early up-regulation of inflammatory and fibrotic remodeling pathways between 2 to 3.5 weeks of age. CONCLUSION:Cardiac specific overexpression of desmocollin-2 induces necrosis, acute inflammation and patchy cardiac fibrotic remodeling leading to fulminant biventricular cardiomyopathy.

BACKGROUND:Induced pluripotent stem cells (iPSC) can be differentiated into cardiomyocytes and represent a possible autologous cell source for myocardial repair. We analyzed the engraftment and functional effects of murine iPSC-derived cardiomyocytes (iPSC-CMs) in a murine model of myocardial infarction. METHODS AND RESULTS:To maximize cardiomyocyte yield and purity a genetic purification protocol was applied. Murine iPSCs were genetically modified to express a Zeocin™ resistance gene under control of the cardiac-specific α-myosin heavy chain (α-MHC, MYH6) promoter. Thus, CM selection was performed during in vitro differentiation. iPSC-CM aggregates ("cardiac bodies", CBs) were transplanted on day 14 after LAD ligation into the hearts of previously LAD-ligated mice (800 CBs/animal; 2-3x106 CMs). Animals were treated with placebo (PBS, n = 14) or iPSC-CMs (n = 35). Myocardial remodeling and function were evaluated by magnetic resonance imaging (MRI), conductance catheter (CC) analysis and histological morphometry. In vitro and in vivo differentiation was investigated. Follow up was 28 days (including histological assessment and functional analysis). iPSC-CM purity was >99%. Transplanted iPSC-CMs formed mature grafts within the myocardium, expressed cardiac markers and exhibited sarcomeric structures. Intramyocardial transplantation of iPSC-CMs significantly improved myocardial remodeling and left ventricular function 28 days after LAD-ligation. CONCLUSIONS:We conclude that iPSCs can effectively be differentiated into cardiomyocytes and genetically enriched to high purity. iPSC derived cardiomyocytes engraft within the myocardium of LAD-ligated mice and contribute to improve left ventricular function.

Gelatinase B or matrix metalloproteinase-9 (MMP-9) (EC 3.4.24.35) is increased in inflammatory processes and cancer, and is associated with disease progression. In part, this is due to MMP-9-mediated degradation of extracellular matrix, facilitating influx of leukocytes into inflamed tissues and invasion or metastasis of cancer cells. MMP-9 is produced as proMMP-9 and its propeptide is subsequently removed by other proteases to generate proteolytically active MMP-9. The significance of MMP-9 in pathologies triggered the development of specific inhibitors of this protease. However, clinical trials with synthetic inhibitors of MMPs in the fight against cancer were disappointing. Reports on active compounds which inhibit MMP-9 should be carefully examined in this regard. In a considerable set of recent publications, two antibiotics (minocycline and azythromycin) and the proteasome inhibitor bortezomib, used in cancers, were reported to inhibit MMP-9 at different stages of its expression, activation or activity. The current study was undertaken to compare and to verify the impact of these compounds on MMP-9. With exception of minocycline at high concentrations (>100 μM), the compounds did not affect processing of proMMP-9 into MMP-9, nor did they affect direct MMP-9 gelatinolytic activity. In contrast, azithromycin specifically reduced MMP-9 mRNA and protein levels without affecting NF-κB in endotoxin-challenged monocytic THP-1 cells. Bortezomib, although being highly toxic, had no MMP-9-specific effects but significantly upregulated cyclooxygenase-2 (COX-2) activity and PGE2 levels. Overall, our study clarified that azithromycin decreased the levels of MMP-9 by reduction of gene and protein expression while minocycline inhibits proteolytic activity at high concentrations.

Previous studies demonstrated that circulating microRNA-375 (miR-375) is a suitable plasma biomarker for real-time detection of beta cell death. The present study evaluated the use of this biomarker to assess the beta cytoprotective effect of phenylpropenoic acid glucoside (PPAG), which was previously demonstrated to protect beta cells against various types of injury, and of exendin-4, which is an established antidiabetic drug.PPAG or exendin-4 were administered in mice treated with streptozotocin (STZ) to acutely induce beta cell death. Beta cell mass and apoptotic death were measured in pancreatic tissue sections. Circulating miR-375 was measured in blood plasma by RT-qPCR. The release of miR-375 was also measured in vitro by MIN-6 beta cells.Administration of STZ resulted in measurable circulating levels of miR-375, a decrease in beta cell mass and increase in frequency of apoptotic beta cells. In vitro, there was a good correlation between miR-375 release and the extent of beta cell death. Treatment of mice with PPAG or exendin-4 significantly attenuated STZ-induced loss of beta cell mass and beta cell apoptosis, and normalized the blood level of miR-375.These findings show the potential use of serological miR-375 measurements to evaluate the beta cytoprotective effect of (potential) antidiabetic drugs in vivo.

BACKGROUND:APOBEC3B was recently identified as a gain-of-function enzymatic source of mutagenesis, which may offer novel therapeutic options with molecules that specifically target this enzyme. In primary breast cancer, APOBEC3B mRNA is deregulated in a substantial proportion of cases and its expression is associated with poor prognosis. However, its expression in breast cancer metastases, which are the main causes of breast cancer-related death, remained to be elucidated. PATIENTS AND METHODS:RNA was isolated from 55 primary breast cancers and paired metastases, including regional lymph node (N = 20) and distant metastases (N = 35). APOBEC3B mRNA levels were measured by RT-qPCR. Expression levels of the primary tumors and corresponding metastases were compared, including subgroup analysis by estrogen receptor (ER/ESR1) status. RESULTS:Overall, APOBEC3B mRNA levels of distant metastases were significantly higher as compared to the corresponding primary breast tumor (P = 0.0015), an effect that was not seen for loco-regional lymph node metastases (P = 0.23). Subgroup analysis by ER-status showed that increased APOBEC3B levels in distant metastases were restricted to metastases arising from ER-positive primary breast cancers (P = 0.002). However, regarding ER-negative primary tumors, only loco-regional lymph node metastases showed increased APOBEC3B expression when compared to the corresponding primary tumor (P = 0.028). CONCLUSION:APOBEC3B mRNA levels are significantly higher in breast cancer metastases as compared to the corresponding ER-positive primary tumors. This suggests a potential role for APOBEC3B in luminal breast cancer progression, and consequently, a promising role for anti-APOBEC3B therapies in advanced stages of this frequent form of breast cancer.

The gastrin releasing peptide receptor (GRPR) and the somatostatin receptor 2 (SSTR2) are overexpressed on primary breast cancer (BC), making them ideal candidates for receptor-mediated nuclear imaging and therapy. The aim of this study was to determine whether these receptors are also suitable targets for metastatic BC.mRNA expression of human BC samples were studied by in vitro autoradiography and associated with radioligand binding. Next, GRPR and SSTR2 mRNA levels of 60 paired primary BCs and metastases from different sites were measured by quantitative reverse transcriptase polymerase chain reaction. Receptor mRNA expression levels were associated with clinico-pathological factors and expression levels of primary tumors and corresponding metastases were compared.Binding of GRPR and SSTR radioligands to tumor tissue correlated significantly with receptor mRNA expression. High GRPR and SSTR2 mRNA levels were associated with estrogen receptor (ESR1)-positive tumors (p

This study aimed to investigate the effects of nanomaterial (NM) exposure on DNA methylation.Intra-tracheal administration of NM: gold nanoparticles (AuNPs) of 5-, 60- and 250-nm diameter; single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) at high dose of 2.5 mg/kg and low dose of 0.25 mg/kg for 48 h to BALB/c mice. Study showed deregulations in immune pathways in NM-induced toxicity in vivo. NM administration had the following DNA methylation effects: AuNP 60 nm induced CpG hypermethylation in Atm, Cdk and Gsr genes and hypomethylation in Gpx; Gsr and Trp53 showed changes in methylation between low- and high-dose AuNP, 60 and 250 nm respectively, and AuNP had size effects on methylation for Trp53.Epigenetics may be implicated in NM-induced disease pathways.

Overall and splice specific expression of Spleen Tyrosine Kinase (SYK) has been posed as a marker predicting both poor and favorable outcome in various epithelial malignancies. However, its role in colorectal cancer is largely unknown. The aim of this study was to explore the prognostic role of SYK in three cohorts of colon cancer patients.Total messenger RNA (mRNA) expression of SYK, SYK(T), and mRNA expression of its two splice variants SYK short (S) and SYK long (L) were measured using quantitative reverse transcriptase (RT-qPCR) in 240 primary colon cancer patients (n = 160 patients with chemonaive lymph node negative [LNN] and n = 80 patients with adjuvant treated lymph node positive [LNP] colon cancer) and related to microsatellite instability (MSI), known colorectal cancer mutations, and disease-free (DFS), hepatic metastasis-free (HFS) and overall survival (OS). Two independent cohorts of patients with respectively 48 and 118 chemonaive LNN colon cancer were used for validation.Expression of SYK and its splice variants was significantly lower in tumors with MSI, and in KRAS wild type, BRAF mutant and PTEN mutant tumors. In a multivariate Cox regression analysis, as a continuous variable, increasing SYK(S) mRNA expression was associated with worse HFS (Hazard Ratio[HR] = 1.83; 95% Confidence Interval[CI] = 1.08-3.12; p = 0.026) in the LNN group, indicating a prognostic role for SYK(S) mRNA in patients with chemonaive LNN colon cancer. However, only a non-significant trend between SYK(S) and HFS in one of the two validation cohorts was observed (HR = 4.68; 95%CI = 0.75-29.15; p = 0.098).In our cohort, we discovered SYK(S) as a significant prognostic marker for HFS for patients with untreated LNN colon cancer. This association could however not be confirmed in two independent smaller cohorts, suggesting that further extensive validation is needed to confirm the prognostic value of SYK(S) expression in chemonaive LNN colon cancer.

Acceptor substrate specificity of Streptomyces roseochromogenes prenyltransferase SrCloQ was investigated using different non-genuine phenolic compounds. RP-UHPLC-UV-MSn was used for the tentative annotation and quantification of the prenylated products. Flavonoids, isoflavonoids and stilbenoids with different types of substitution were prenylated by SrCloQ, although with less efficiency than the genuine substrate 4-hydroxyphenylpyruvate. The isoflavan equol, followed by the flavone 7,4'-dihydroxyflavone, were the best non-genuine acceptor substrates. B-ring C-prenylation was in general preferred over A-ring C-prenylation (ratio 5:1). Docking studies of non-genuine acceptor substrates with the B-ring oriented towards the donor substrate dimethylallyl pyrophosphate, showed that the carbonyl group of the C-ring was able to make stabilizing interactions with the residue Arg160, which might determine the preference observed for B-ring prenylation. No reaction products were formed when the acceptor substrate had no phenolic hydroxyl groups. This preference can be explained by the essential hydrogen bond needed between a phenolic hydroxyl group and the residue Glu281. Acceptor substrates with an additional hydroxyl group at the C3' position (B-ring), were mainly O3'-prenylated (> 80% of the reaction products). This can be explained by the proximity of the C3' hydroxyl group to the donor substrate at the catalytic site. Flavones were preferred over isoflavones by SrCloQ. Docking studies suggested that the orientation of the B-ring and of the phenolic hydroxyl group at position C7 (A-ring) of flavones towards the residue Tyr233 plays an important role in this observed preference. Finally, the insights obtained on acceptor substrate specificity and regioselectivity for SrCloQ were extended to other prenyltransferases from the CloQ/NhpB family.

Lipid production in microalgae is highly dependent on the applied light intensity. However, for the EPA producing model-diatom Phaeodactylum tricornutum, clear consensus on the impact of incident light intensity on lipid productivity is still lacking. This study quantifies the impact of different incident light intensities on the biomass, TAG and EPA yield on light in nitrogen starved batch cultures of P. tricornutum. The maximum biomass concentration and maximum TAG and EPA contents were found to be independent of the applied light intensity. The lipid yield on light was reduced at elevated light intensities (>100 μmol m-2 s-1). The highest TAG yield on light (112 mg TAG molph-1) was found at the lowest light intensity tested (60 μmol m-2 s-1), which is still relatively low to values reported in literature for other algae. Furthermore, mass balance analysis showed that the EPA fraction in TAG may originate from photosynthetic membrane lipids.

Individuals from different taxa, including songbirds, differ consistently in behaviour and personality when facing different situations. Although our understanding of animal behaviour has increased, knowledge about between-individual differences in cognitive abilities is still limited. By using an experimental approach and a free-living songbird (Parus major) as a model, we attempted to understand between-individual differences in habituation to playbacks (as a proxy of learning speed), by investigating the role of personality, age and reproductive investment (clutch size). Pre-breeding males were tested for exploration (a proxy of personality) in standardized conditions. In addition, the same individuals were exposed to three playbacks in the field during incubation. Birds significantly moved less, stayed further away and overlapped less the playback with successive playback stimulation. While a decrease in the locomotor behaviour can be explained by personality, differences in habituation of overlapping were predicted by both reproductive investment and personality. Fast explorers habituated less. Moreover, males paired to females with larger clutches did not vary the intensity of overlapping. Since habituation requires information for recognition of non-threatening signals, personality may bias information gathering. While fast explorers may collect less information from the environment, slow explorers (reactive birds) seem to pay attention to environmental clues and collect detailed information. We provided evidence that the rate of habituation of behavioural responses, a proxy of cognitive abilities, may be affected by different factors and in a complex way.

Concussion is a serious health concern. Concussion in athletes is of particular interest with respect to the relationship of concussion exposure to risk of chronic traumatic encephalopathy (CTE), a neurodegenerative condition associated with altered cognitive and psychiatric functions and profound tauopathy. However, much remains to be learned about factors other than cumulative exposure that could influence concussion pathogenesis. Approximately 20% of CTE cases report a history of substance use including androgenic-anabolic steroids (AAS). How acute, chronic, or historical AAS use may affect the vulnerability of the brain to concussion is unknown. We therefore tested whether antecedent AAS exposure in young, male C57Bl/6 mice affects acute behavioral and neuropathological responses to mild traumatic brain injury (TBI) induced with the CHIMERA (Closed Head Impact Model of Engineered Rotational Acceleration) platform. Male C57Bl/6 mice received either vehicle or a cocktail of three AAS (testosterone, nandrolone and 17α-methyltestosterone) from 8-16 weeks of age. At the end of the 7th week of treatment, mice underwent two closed-head TBI or sham procedures spaced 24 h apart using CHIMERA. Post-repetitive TBI (rTBI) behavior was assessed for 7 d followed by tissue collection. AAS treatment induced the expected physiological changes including increased body weight, testicular atrophy, aggression and downregulation of brain 5-HT1B receptor expression. rTBI induced behavioral deficits, widespread axonal injury and white matter microgliosis. While AAS treatment did not worsen post-rTBI behavioral changes, AAS-treated mice exhibited significantly exacerbated axonal injury and microgliosis, indicating that AAS exposure can alter neuronal and innate immune responses to concussive TBI.

OBJECTIVES:Choosing the right mentor is crucial for effective mentorship. Yet, many medical students have difficulties finding a suitable mentor. Thus we developed mentoring speed dating (MSD) as a promising matching tool to connect students and faculty mentors successfully. The purpose of this study was to explore mentees' and mentors' experience with MSD and investigate the impact of MSD on the perceived mentorship quality and continuance of the mentoring relationship. METHODS:The authors completed a mixed methods study at the University Medical Center Hamburg-Eppendorf, Germany, between June 2011 and March 2014. They conducted four focus groups with mentees and mentors who participated in a mentoring speed dating event and analyzed transcripts using conventional content analysis with inductive categorizing. In addition, three mentoring cohorts (two matched via MSD, one matched via conventional online profiles) were surveyed on mentorship satisfaction and the 1-year continuance of their mentorship was monitored. Fifteen mentees and fifteen mentors participated in the focus groups. The authors identified several themes such as short and long term benefits of MSD and fulfillment of expectations. Benefits included finding out about the personal connection, matching expectations, providing an efficient overview of candidates. The survey was completed by 93 students (n = 29 without MSD; n = 64 with MSD). Independent t-tests and multivariate analysis of variance were used to analyze the impact of MSD on student's mentorship satisfaction. RESULTS:There were significant differences in responses to the items "Commitment of mentor" (p = .019) and "Constructive feedback" (p = .038) among the students who attended MSD and the students without MSD. After one year far more mentoring relationships existed among those mentees who participated in MSD in comparison to the "no MSD group". CONCLUSION:MSD is a valuable matching tool with beneficial effects on the mentorship quality. It enhances essential factors in the mentoring relationship such as commitment and satisfaction.

Low levels of insulin-like growth factor-1 (IGF-1), an essential neurotrophic factor, have been associated with worse cognitive function in older adults. However, few studies have assessed the prospective association of serum IGF-1 with cognitive function. We aimed to determine the association between serum IGF-1 on concurrent and prospective cognitive function in a population sample of men aged 40-80 years. Blood samples were assessed for IGF-1 levels at baseline and neuropsychological assessments were performed at baseline (n = 400) and at follow-up after a mean duration of 8.3 years (n = 286). Linear regression analyses were carried out to determine the associations between quintiles of IGF-1 and cognitive function at the baseline and follow-up visits. Results showed that those in the top quintile of IGF-1 had lower processing capacity and global cognition scores at follow-up after controlling for cognitive function at baseline and other confounding factors. Additional analyses exploring associations with IGF-1 separately in middle-aged and older participants, and with quartiles of IGF-1 produced similar results. In those older than 60 years, high IGF-1 levels were also associated with lower baseline processing capacity. These results suggest that high IGF-1 levels are associated with worse long-term cognition in men. Together with past studies, we suggest that both, high and low levels of IGF-1 may be associated with poor cognitive function and that optimum levels of IGF-1 (quintile 2 and 3 in current study) may be associated with better cognitive function.

Increased APOBEC3B mRNA levels are associated with a hypermutator phenotype and poor prognosis in ER-positive breast cancer patients. In addition, a 29.5 kb deletion polymorphism of APOBEC3B, resulting in an APOBEC3A-B hybrid transcript, has been associated with an increased breast cancer risk and the hypermutator phenotype. Here we evaluated whether the APOBEC3B deletion polymorphism also associates with clinical outcome of breast cancer. Copy number analysis was performed by quantitative PCR (qPCR) in primary tumors of 1,756 Dutch breast cancer patients. The APOBEC3B deletion was found in 187 patients of whom 16 carried a two-copy deletion and 171 carried a one-copy deletion. The prognostic value of the APOBEC3B deletion for the natural course of the disease was evaluated among 1,076 lymph-node negative (LNN) patients who did not receive adjuvant systemic treatment. No association was found between APOBEC3B copy number values and the length of metastasis-free survival (MFS; hazard ratio (HR) = 1.00, 95% confidence interval (CI) = 0.90-1.11, P = 0.96). Subgroup analysis by ER status also did not reveal an association between APOBEC3B copy number values and the length of MFS. The predictive value of the APOBEC3B deletion was assessed among 329 ER-positive breast cancer patients who received tamoxifen as the first-line therapy for recurrent disease and 226 breast cancer patients who received first-line chemotherapy for recurrent disease. No association between APOBEC3B copy number values and the overall response rate (ORR) to either tamoxifen (odds ratio (OR) = 0.88, 95% CI = 0.69-1.13, P = 0.31) or chemotherapy (OR = 0.97, 95% CI = 0.71-1.33, P = 0.87) was found. Thus, in contrast to APOBEC3B mRNA levels, the APOBEC3B deletion polymorphism has neither a prognostic nor a predictive value for breast cancer patients. Although a correlation exists between APOBEC3B copy number and mRNA expression, it is relatively weak. This suggests that other mechanisms exist that may affect and therefore determine the prognostic value of APOBEC3B mRNA levels.

Sporopollenin is the main constituent of the exine layer of spore and pollen walls. The anther-specific chalcone synthase-like (ASCL) enzyme of Physcomitrella patens, PpASCL, has previously been implicated in the biosynthesis of sporopollenin, the main constituent of exine and perine, the two outermost layers of the moss spore cell wall. We made targeted knockouts of the corresponding gene, PpASCL, and phenotypically characterized ascl sporophytes and spores at different developmental stages. Ascl plants developed normally until late in sporophytic development, when the spores produced were structurally aberrant and inviable. The development of the ascl spore cell wall appeared to be arrested early in microspore development, resulting in small, collapsed spores with altered surface morphology. The typical stratification of the spore cell wall was absent with only an abnormal perine recognisable above an amorphous layer possibly representing remnants of compromised intine and/or exine. Equivalent resistance of the spore walls of ascl mutants and the control strain to acetolysis suggests the presence of chemically inert, defective sporopollenin in the mutants. Anatomical abnormalities of late-stage ascl sporophytes include a persistent large columella and an air space incompletely filled with spores. Our results indicate that the evolutionarily conserved PpASCL gene is needed for proper construction of the spore wall and for normal maturation and viability of moss spores.

INTRODUCTION:Physical activity is beneficial for individual health, but endurance sport is associated with the development of arrhythmias like atrial fibrillation. The underlying mechanisms leading to this increased risk are still not fully understood. MicroRNAs are important mediators of proarrhythmogenic remodeling and have potential value as biomarkers in cardiovascular diseases. Therefore, the objective of our study was to determine the value of circulating microRNAs as potential biomarkers for atrial remodeling in marathon runners (miRathon study). METHODS:30 marathon runners were recruited into our study and were divided into two age-matched groups depending on the training status: elite (ER, ≥55 km/week, n = 15) and non-elite runners (NER, ≤40 km/week, n = 15). All runners participated in a 10 week training program before the marathon. MiRNA plasma levels were measured at 4 time points: at baseline (V1), after a 10 week training period (V2), immediately after the marathon (V3) and 24h later (V4). Additionally, we obtained clinical data including serum chemistry and echocardiography at each time point. RESULTS:MiRNA plasma levels were similar in both groups over time with more pronounced changes in ER. After the marathon miR-30a plasma levels increased significantly in both groups. MiR-1 and miR-133a plasma levels also increased but showed significant changes in ER only. 24h after the marathon plasma levels returned to baseline. MiR-26a decreased significantly after the marathon in elite runners only and miR-29b showed a non-significant decrease over time in both groups. In ER miRNA plasma levels showed a significant correlation with LA diameter, in NER miRNA plasma levels did not correlate with echocardiographic parameters. CONCLUSION:MiRNAs were differentially expressed in the plasma of marathon runners with more pronounced changes in ER. Plasma levels in ER correlate with left atrial diameter suggesting that circulating miRNAs could potentially serve as biomarkers of atrial remodeling in athletes.

BACKGROUND:The reduced ability to identify a second target when it is presented in close temporal succession of a first target is called the attentional blink (AB). Studies have shown large individual differences in AB task performance, where lower task performance has been associated with more reversed order reports of both targets if these were presented in direct succession. In order to study the suggestion that reversed order reports reflect loss of temporal information, in the current study, we investigated whether individuals with a larger AB have a higher tendency to temporally integrate both targets into one visual event by using an AB paradigm containing symbol target stimuli. METHODOLOGY/PRINCIPAL FINDINGS:Indeed, we found a positive relation between the tendency to temporally integrate information and individual AB magnitude. In contrast to earlier work, we found no relation between order reversals and individual AB magnitude. The occurrence of temporal integration was negatively related to the number of order reversals, indicating that individuals either integrated or separated and reversed information. CONCLUSION:We conclude that individuals with better AB task performance use a shorter time window to integrate information, and therefore have higher preservation of temporal information. Furthermore, order reversals observed in paradigms with alphanumeric targets indeed seem to at least partially reflect temporal integration of both targets. Given the negative relation between temporal integration and 'true' order reversals observed with the current symbolic target set, these two behavioral outcomes seem to be two sides of the same coin.

The Cancer Genetic Markers of Susceptibility genome-wide association study (GWAS) originally identified a single nucleotide polymorphism (SNP) rs11249433 at 1p11.2 associated with breast cancer risk. To fine-map this locus, we genotyped 92 SNPs in a 900kb region (120,505,799-121,481,132) flanking rs11249433 in 45,276 breast cancer cases and 48,998 controls of European, Asian and African ancestry from 50 studies in the Breast Cancer Association Consortium. Genotyping was done using iCOGS, a custom-built array. Due to the complicated nature of the region on chr1p11.2: 120,300,000-120,505,798, that lies near the centromere and contains seven duplicated genomic segments, we restricted analyses to 429 SNPs excluding the duplicated regions (42 genotyped and 387 imputed). Per-allelic associations with breast cancer risk were estimated using logistic regression models adjusting for study and ancestry-specific principal components. The strongest association observed was with the original identified index SNP rs11249433 (minor allele frequency (MAF) 0.402; per-allele odds ratio (OR) = 1.10, 95% confidence interval (CI) 1.08-1.13, P = 1.49 x 10-21). The association for rs11249433 was limited to ER-positive breast cancers (test for heterogeneity P≤8.41 x 10-5). Additional analyses by other tumor characteristics showed stronger associations with moderately/well differentiated tumors and tumors of lobular histology. Although no significant eQTL associations were observed, in silico analyses showed that rs11249433 was located in a region that is likely a weak enhancer/promoter. Fine-mapping analysis of the 1p11.2 breast cancer susceptibility locus confirms this region to be limited to risk to cancers that are ER-positive.

Based on controlled 36 h experiments a higher dietary protein intake causes a positive protein balance and a negative fat balance. A positive net protein balance may support fat free mass accrual. However, few data are available on the impact of more prolonged changes in habitual protein intake on whole-body protein metabolism and basal muscle protein synthesis rates.To assess changes in whole-body protein turnover and basal muscle protein synthesis rates following 12 weeks of adaptation to a low versus high dietary protein intake.A randomized parallel study was performed in 40 subjects who followed either a high protein (2.4 g protein/kg/d) or low protein (0.4 g protein/kg/d) energy-balanced diet (30/35/35% or 5/60/35% energy from protein/carbohydrate/fat) for a period of 12 weeks. A subgroup of 7 men and 8 women (body mass index: 22.8±2.3 kg/m2, age: 24.3±4.9 y) were selected to evaluate the impact of prolonged adaptation to either a high or low protein intake on whole body protein metabolism and basal muscle protein synthesis rates. After the diet, subjects received continuous infusions with L-[ring-2H5]phenylalanine and L-[ring-2H2]tyrosine in an overnight fasted state, with blood samples and muscle biopsies being collected to assess post-absorptive whole-body protein turnover and muscle protein synthesis rates in vivo in humans.After 12 weeks of intervention, whole-body protein balance in the fasted state was more negative in the high protein treatment when compared with the low protein treatment (-4.1±0.5 vs -2.7±0.6 μmol phenylalanine/kg/h;P

Freezing of gait (FOG) is arguably the most severe symptom associated with Parkinson's disease (PD), and often occurs while performing dual tasks or approaching narrowed and cluttered spaces. While it is well known that visual cues alleviate FOG, it is not clear if this effect may be the result of cognitive or sensorimotor mechanisms. Nevertheless, the role of vision may be a critical link that might allow us to disentangle this question. Gaze behaviour has yet to be carefully investigated while freezers approach narrow spaces, thus the overall objective of this study was to explore the interaction between cognitive and sensory-perceptual influences on FOG. In experiment #1, if cognitive load is the underlying factor leading to FOG, then one might expect that a dual-task would elicit FOG episodes even in the presence of visual cues, since the load on attention would interfere with utilization of visual cues. Alternatively, if visual cues alleviate gait despite performance of a dual-task, then it may be more probable that sensory mechanisms are at play. In compliment to this, the aim of experiment#2 was to further challenge the sensory systems, by removing vision of the lower-limbs and thereby forcing participants to rely on other forms of sensory feedback rather than vision while walking toward the narrow space. Spatiotemporal aspects of gait, percentage of gaze fixation frequency and duration, as well as skin conductance levels were measured in freezers and non-freezers across both experiments. Results from experiment#1 indicated that although freezers and non-freezers both walked with worse gait while performing the dual-task, in freezers, gait was relieved by visual cues regardless of whether the cognitive demands of the dual-task were present. At baseline and while dual-tasking, freezers demonstrated a gaze behaviour that neglected the doorway and instead focused primarily on the pathway, a strategy that non-freezers adopted only when performing the dual-task. Interestingly, with the combination of visual cues and dual-task, freezers increased the frequency and duration of fixations toward the doorway, compared to non-freezers. These results suggest that although increasing demand on attention does significantly deteriorate gait in freezers, an increase in cognitive demand is not exclusively responsible for freezing (since visual cues were able to overcome any interference elicited by the dual-task). When vision of the lower limbs was removed in experiment#2, only the freezers' gait was affected. However, when visual cues were present, freezers' gait improved regardless of the dual-task. This gait behaviour was accompanied by greater amount of time spent looking at the visual cues irrespective of the dual-task. Since removing vision of the lower-limbs hindered gait even under low attentional demand, restricted sensory feedback may be an important factor to the mechanisms underlying FOG.

Orobates pabsti, a basal diadectid from the lower Permian, is a key fossil for the understanding of early amniote evolution. Quantitative analysis of anatomical information suffers from fragmentation of fossil bones, plastic deformation due to diagenetic processes and fragile preservation within surrounding rock matrix, preventing further biomechanical investigation. Here we describe the steps taken to digitally reconstruct MNG 10181, the holotype specimen of Orobates pabsti, and subsequently use the digital reconstruction to assess body mass, position of the centre of mass in individual segments as well as the whole animal, and study joint mobility in the shoulder and hip joints. The shape of most fossil bone fragments could be recovered from micro-focus computed tomography scans. This also revealed structures that were hitherto hidden within the rock matrix. However, parts of the axial skeleton had to be modelled using relevant isolated bones from the same locality as templates. Based on the digital fossil, mass of MNG 10181 was estimated using a model of body shape that was varied within a plausible range to account for uncertainties of the dimension. In the mean estimate model the specimen had an estimated mass of circa 4 kg. Varying of the mass distribution amongst body segments further revealed that Orobates carried most of its weight on the hind limbs. Mostly unrestricted joint morphology further suggested that MNG 10181 was able to effectively generate propulsion with the pelvic limbs. The digital reconstruction is made available for future biomechanical studies.

The variability of electromyographic (EMG) recordings between and within participants is a complex problem, rarely studied in swimming. The importance of signal normalization has long been recognized, but the method used might influence variability. The aims of this study were to: (i) assess the intra-individual variability of the EMG signal in highly skilled front crawl swimmers, (ii) determine the influence of two methods of both amplitude and time normalization of the EMG signal on intra-individual variability and of time normalization on muscle activity level and (iii) describe the muscle activity, normalized using MVIC, in relation to upper limb crawl stroke movements. Muscle activity of rectus abdominis and deltoideus medialis was recorded using wireless surface EMG in 15 adult male competitive swimmers during three trials of 12.5 m front crawl at maximal speed without breathing. Two full upper limb cycles were analyzed from each of the swimming trials, resulting in six full cycles used for the intra-individual variability assessment, quantified with the coefficient of variation (CV), coefficient of quartile variation (CQV) and the variance ratio (VR). The results of this study support previous findings on EMG patterns of deltoideus medialis and rectus abdominis as prime mover during the recovery (45% activity relative to MVIC), and stabilizer of the trunk during the pull (14.5% activity) respectively. The intra-individual variability was lower (VR of 0.34-0.47) when compared to other cyclic movements. No meaningful differences were found between variability measures CV or VR when applying either of the amplitude or the time normalization methods. In addition to reporting the mean amplitude and standard deviation, future EMG studies in swimming should also report the intra-individual variability, preferably using VR as it is independent of peak amplitude, provides a good measure of repeatability and is insensitive to mean EMG amplitude and the degree of smoothing applied.

Single-channel optical density measurements of population growth are the dominant large scale phenotyping methodology for bridging the gene-function gap in yeast. However, a substantial amount of the genetic variation induced by single allele, single gene or double gene knock-out technologies fail to manifest in detectable growth phenotypes under conditions readily testable in the laboratory. Thus, new high-throughput phenotyping technologies capable of providing information about molecular level consequences of genetic variation are sorely needed. Here we report a protocol for high-throughput Fourier transform infrared spectroscopy (FTIR) measuring biochemical fingerprints of yeast strains. It includes high-throughput cultivation for FTIR spectroscopy, FTIR measurements and spectral pre-treatment to increase measurement accuracy. We demonstrate its capacity to distinguish not only yeast genera, species and populations, but also strains that differ only by a single gene, its excellent signal-to-noise ratio and its relative robustness to measurement bias. Finally, we illustrated its applicability by determining the FTIR signatures of all viable Saccharomyces cerevisiae single gene knock-outs corresponding to lipid biosynthesis genes. Many of the examined knock-out strains showed distinct, highly reproducible FTIR phenotypes despite having no detectable growth phenotype. These phenotypes were confirmed by conventional lipid analysis and could be linked to specific changes in lipid composition. We conclude that the introduced protocol is robust to noise and bias, possible to apply on a very large scale, and capable of generating biologically meaningful biochemical fingerprints that are strain specific, even when strains lack detectable growth phenotypes. Thus, it has a substantial potential for application in the molecular functionalization of the yeast genome.

Attention is restricted for the second of two targets when it is presented within 200-500 ms of the first target. This attentional blink (AB) phenomenon allows one to study the dynamics of temporal selective attention by varying the interval between the two targets (T1 and T2). Whereas the AB has long been considered as a robust and universal cognitive limitation, several studies have demonstrated that AB task performance greatly differs between individuals, with some individuals showing no AB whatsoever.Here, we studied these individual differences in AB task performance in relation to differences in attentional timing. Furthermore, we investigated whether AB magnitude is predictive for the amount of attention allocated to T1. For both these purposes pupil dilation was measured, and analyzed with our recently developed deconvolution method. We found that the dynamics of temporal attention in small versus large blinkers differ in a number of ways. Individuals with a relatively small AB magnitude seem better able to preserve temporal order information. In addition, they are quicker to allocate attention to both T1 and T2 than large blinkers. Although a popular explanation of the AB is that it is caused by an unnecessary overinvestment of attention allocated to T1, a more complex picture emerged from our data, suggesting that this may depend on whether one is a small or a large blinker.The use of pupil dilation deconvolution seems to be a powerful approach to study the temporal dynamics of attention, bringing us a step closer to understanding the elusive nature of the AB. We conclude that the timing of attention to targets may be more important than the amount of allocated attention in accounting for individual differences.

Gene expression profiling of tumors is a successful tool for the discovery of new cancer biomarkers and potential targets for the development of new therapeutic strategies. Reliable profiling is preferably performed on fresh frozen (FF) tissues in which the quality of nucleic acids is better preserved than in formalin-fixed paraffin-embedded (FFPE) material. However, since snap-freezing of biopsy materials is often not part of daily routine in pathology laboratories, one may have to rely on archival FFPE material. Procedures to retrieve the RNAs from FFPE materials have been developed and therefore, datasets obtained from FFPE and FF materials need to be made compatible to ensure reliable comparisons are possible.To develop an efficient method to compare gene expression profiles obtained from FFPE and FF samples using the same platform.Twenty-six FFPE-FF sample pairs of the same tumors representing various cancer types, and two FFPE-FF sample pairs of breast cancer cell lines, were included. Total RNA was extracted and gene expression profiling was carried out using Illumina's Whole-Genome cDNA-mediated Annealing, Selection, extension and Ligation (WG-DASL) V3 arrays, enabling the simultaneous detection of 24,526 mRNA transcripts. A sample exclusion criterion was created based on the expression of 11 stably expressed reference genes. Pearson correlation at the probe level was calculated for paired FFPE-FF, and three cut-off values were chosen. Spearman correlation coefficients between the matched FFPE and FF samples were calculated for three probe lists with varying levels of significance and compared to the correlation based on all measured probes. Unsupervised hierarchical cluster analysis was performed to verify performance of the included probe lists to compare matched FPPE-FF samples.Twenty-seven FFPE-FF pairs passed the sample exclusion criterion. From the profiles of 27 FFPE and FF matched samples, the best correlating probes were identified for various levels of significance (Pearson P

Consumption of meals with different macronutrient contents, especially high in carbohydrates, may influence the stress-induced physiological and psychological response. The objective of this study was to investigate effects of consumption of a high-protein vs. high-carbohydrate meal on the physiological cortisol response and psychological mood response. Subjects (n = 38, 19 m/19f, age =25 ± 9 yrs, BMI = 25.0 ± 3.3 kg/m²) came to the university four times, fasted, for either condition: rest-protein, stress-protein, rest-carbohydrate, stress-carbohydrate (randomized cross-over design). Stress was induced by means of a psychological computer-test. The test-meal was either a high-protein meal (En% P/C/F 65/5/30) or a high-carbohydrate meal (En% P/C/F 6/64/30), both meals were matched for energy density (4 kJ/g) and daily energy requirements (30%). Per test-session salivary cortisol levels, appetite profile, mood state and level of anxiety were measured. High hunger, low satiety (81 ± 16, 12 ± 15 mm VAS) confirmed the fasted state. The stress condition was confirmed by increased feelings of depression, tension, anger, anxiety (AUC stress vs. rest p < 0.02). Consumption of the high-protein vs. high-carbohydrate meal did not affect feelings of depression, tension, anger, anxiety. Cortisol levels did not differ between the four test-sessions in men and women (AUC nmol·min/L p > 0.1). Consumption of the test-meals increased cortisol levels in men in all conditions (p < 0.01), and in women in the rest-protein and stress-protein condition (p < 0.03). Men showed higher cortisol levels than women (AUC nmol·min/L p < 0.0001). Consumption of meals with different macronutrient contents, i.e. high-protein vs. high-carbohydrate, does not influence the physiological and psychological response differentially. Men show a higher meal-induced salivary cortisol response compared with women.

Highly lethal terrorist attacks, which we define as those killing 21 or more people, account for 50% of the total number of people killed in all terrorist attacks combined, yet comprise only 3.5% of terrorist attacks. Given the disproportionate influence of these incidents, uncovering systematic patterns in attacks that precede and anticipate these highly lethal attacks may be of value for understanding attacks that exact a heavy toll on life. Here we examined whether the activity of terrorist groups escalates--both in the number of people killed per attack and in the frequency of attacks--leading up to highly lethal attacks. Analyses of terrorist attacks drawn from a state-of-the-art international terrorism database (The Global Terrorism Database) showed evidence for both types of escalation leading up to highly lethal attacks, though complexities to the patterns emerged as well. These patterns of escalation do not emerge among terrorist groups that never commit a highly lethal attack.

Bone marrow failure syndromes and MDS represent a heterogenous group of diseases, characterized by ineffective myelopoiesis, the risk of clonal evolution and a generally poor response to chemotherapy-based treatment regimen. Nitrostyrene derivatives have been studied as protein phosphatase inhibitors in various tumor models. Pharmacological studies have identified nitrostyrene as the structural core underlying a pro-apoptotic effect in tumor cells, yet their effects on normal cells, including those of the hematopoietic system, are largely unknown. In this study, utilizing umbilical cord blood-derived myeloid progenitor cells, patient-derived bone marrow cells, and a (BALB/c) mouse model; we investigated the effects of treatment with two nitrostyrene derivatives (NTS1 and NTS2) on myeloid development. We demonstrate that these compounds stimulate the expansion and differentiation of myeloid progenitors in vitro and improve myeloid reconstitution after chemotherapy-induced bone marrow depletion in vitro and in vivo. These effects were accompanied by increased C/EBPα expression and activity and inhibition of the p38MAPK signalling pathway. Together, our data suggest that nitrostyrenes improve myelopoiesis and represent potential new treatment strategies for patients suffering from bone marrow failure syndromes, hypocellular myelodysplastic syndrome and chemotherapy-induced aplasia.

BACKGROUND: Glucose effects on beta cell survival and DNA-synthesis suggest a role as regulator of beta cell mass but data on beta cell numbers are lacking. We examined outcome of these influences on the number of beta cells isolated at different growth stages in their population. METHODS: Beta cells from neonatal, young-adult and old rats were cultured serum-free for 15 days. Their number was counted by automated whole-well imaging distinguishing influences on cell survival and on proliferative activity. RESULTS: Elevated glucose (10-20 versus 5 mmol/l) increased the number of living beta cells from 8-week rats to 30%, following a time- and concentration-dependent recruitment of quiescent cells into DNA-synthesis; a glucokinase-activator lowered the threshold but did not raise total numbers of glucose-recruitable cells. No glucose-induced increase occurred in beta cells from 40-week rats. Neonatal beta cells doubled in number at 5 mmol/l involving a larger activated fraction that did not increase at higher concentrations; however, their higher susceptibility to glucose toxicity at 20 mmol/l resulted in 20% lower living cell numbers than at start. None of the age groups exhibited a repetitively proliferating subpopulation. CONCLUSIONS: Chronically elevated glucose levels increased the number of beta cells from young-adult but not from old rats; they interfered with expansion of neonatal beta cells and reduced their number. These effects are attributed to age-dependent differences in basal and glucose-induced proliferative activity and in cellular susceptibility to glucose toxicity. They also reflect age-dependent variations in the functional heterogeneity of the rat beta cell population.

Individuals with Parkinson's disease (PD) commonly experience freezing of gait under time constraints, in narrow spaces, and in the dark. One commonality between these different situations is that they may all provoke anxiety, yet anxiety has never been directly examined as a cause of FOG. In this study, virtual reality was used to induce anxiety and evaluate whether it directly causes FOG. Fourteen patients with PD and freezing of gait (Freezers) and 17 PD without freezing of gait (Non-Freezers) were instructed to walk in two virtual environments: (i) across a plank that was located on the ground (LOW), (ii) across a plank above a deep pit (HIGH). Multiple synchronized motion capture cameras updated participants' movement through the virtual environment in real-time, while their gait was recorded. Anxiety levels were evaluated after each trial using self-assessment manikins. Freezers performed the experiment on two separate occasions (in their ON and OFF state). Freezers reported higher levels of anxiety compared to Non-Freezers (p < 0.001) and all patients reported greater levels of anxiety when walking across the HIGH plank compared to the LOW (p < 0.001). Freezers experienced significantly more freezing of gait episodes (p = 0.013) and spent a significantly greater percentage of each trial frozen (p = 0.005) when crossing the HIGH plank. This finding was even more pronounced when comparing Freezers in their OFF state. Freezers also had greater step length variability in the HIGH compared to the LOW condition, while the step length variability in Non-Freezers did not change. In conclusion, this was the first study to directly compare freezing of gait in anxious and non-anxious situations. These results present strong evidence that anxiety is an important mechanism underlying freezing of gait and supports the notion that the limbic system may have a profound contribution to freezing in PD.

BACKGROUND:The limited effectiveness of cardiac cell therapy has generated concern regarding its clinical relevance. Experimental studies show that cell retention and engraftment are low after injection into ischemic myocardium, which may restrict therapy effectiveness significantly. Surgical aspects and mechanical loss are suspected to be the main culprits behind this phenomenon. As current techniques of monitoring intramyocardial injections are complex and time-consuming, the aim of the study was to develop a fast and simple model to study cardiac retention and distribution following intramyocardial injections. For this purpose, our main hypothesis was that macroscopic fluorescence imaging could adequately serve as a detection method for intramyocardial injections. METHODS AND RESULTS:A total of 20 mice underwent ligation of the left anterior descending artery (LAD) for myocardial infarction. Fluorescent microspheres with cellular dimensions were used as cell surrogates. Particles (5 × 10(5)) were injected into the infarcted area of explanted resting hearts (Ex vivo myocardial injetions EVMI, n = 10) and in vivo into beating hearts (In vivo myocardial injections IVMI, n = 10). Microsphere quantification was performed by fluorescence imaging of explanted organs. Measurements were repeated after a reduction to homogenate dilutions. Cardiac microsphere retention was 2.78 × 10(5) ± 0.31 × 10(5) in the EVMI group. In the IVMI group, cardiac retention of microspheres was significantly lower (0.74 × 10(5) ± 0.18 × 10(5); p