Simultaneous analysis of antihyperglycemic small molecule drugs and peptide drugs by means of dual liquid chromatography high-resolution mass spectrometry.

OBJECTIVES: The study aimed to evaluate dual liquid chromatography (LC) coupled to high-resolution mass spectrometry (HRMS) for the simultaneous analysis of small and large molecule drugs by development and application of a validated bioanalytical method. METHODS: The oral antihyperglycemic drugs (OAD) dapagliflozin, empagliflozin, glibenclamide, glimepiride, metformin, pioglitazone, repaglinide, saxagliptin, sitagliptin, and vildagliptin, as well as the antihyperglycemic peptides exenatide, human insulin, insulin aspart, insulin degludec, insulin detemir, insulin glargine, insulin glulisine, insulin lispro, and semaglutide were included in the analytical procedure. Analytes were extracted using a combination of protein precipitation and solid-phase extraction. Two identical reversed-phase columns were used for separation followed by Orbitrap high-resolution mass spectrometry. The whole procedure was validated according to international recommendations. RESULTS: Different MS parameters had to be used for the two analyte groups, but dual LC separation allowed elution of all analytes within 12 min using the same column type. The analytical procedure was accurate and precise for most of the compounds except for exenatide, semaglutide, and insulin glargine, which were included qualitatively in the method. Analysis of proof-of-concept samples revealed OAD concentrations mostly within their therapeutic range, insulins could be detected in five cases but at concentrations below the lower limit of quantification except for one case. CONCLUSIONS: Dual LC in combination with HRMS was shown to be a suitable platform to analyze small and large molecules in parallel and the current method allowed the determination of a total of 19 antihyperglycemic drugs in blood plasma within 12 min.

Effects of rapid fluid infusion on hemoglobin concentration: a systematic review and meta-analysis.

BACKGROUND: Rapid fluid administration may decrease hemoglobin concentration (Hb) by a diluting effect, which could limit the increase in oxygen delivery (DO2) expected with a positive response to fluid challenge in critically ill patients. Our aim was to quantify the decrease in Hb after rapid fluid administration. METHODS: Our protocol was registered in PROSPERO (CRD42020165146). We searched PubMed, the Cochrane Database, and Embase from inception until February 15, 2022. We selected studies that reported Hb before and after rapid fluid administration (bolus fluid given over less than 120 min) with crystalloids and/or colloids in adults. Exclusion criteria were studies that included bleeding patients, or used transfusions or extracorporeal circulation procedures. Studies were divided according to whether they involved non-acutely ill or acutely ill (surgical/trauma, sepsis, circulatory shock or severe hypovolemia, and mixed conditions) subjects. The mean Hb difference and, where reported, the DO2 difference before and after fluid administration were extracted. Meta-analyses were conducted to assess differences in Hb before and after rapid fluid administration in all subjects and across subgroups. Random-effect models, meta-regressions and subgroup analyses were performed for meta-analyses. Risk of bias was assessed using the Cochrane Risk of Bias Assessment Tool. Inconsistency among trial results was assessed using the I2 statistic. RESULTS: Sixty-five studies met our inclusion criteria (40 in non-acutely ill and 25 in acutely ill subjects), with a total of 2794 participants. Risk of bias was assessed as "low" for randomized controlled trials (RCTs) and 'low to moderate' for non-RCTs. Across 63 studies suitable for meta-analysis, the Hb decreased significantly by a mean of 1.33 g/dL [95% CI - 1.45 to - 1.12; p < 0.001; I2 = 96.88] after fluid administration: in non-acutely ill subjects, the mean decrease was 1.56 g/dL [95% CI - 1.69 to - 1.42; p < 0.001; I2 = 96.71] and in acutely ill patients 0.84 g/dL [95% CI - 1.03 to - 0.64; p = 0.033; I2 = 92.91]. The decrease in Hb was less marked in patients with sepsis than in other acutely ill patients. The DO2 decreased significantly in fluid non-responders with a significant decrease in Hb. CONCLUSIONS: Hb decreased consistently after rapid fluid administration with moderate certainty of evidence. This effect may limit the positive effects of fluid challenges on DO2 and thus on tissue oxygenation.

Comparison of magnetic resonance angiography and 18F-fluorodeoxyglucose positron emission tomography in large-vessel vasculitis.

OBJECTIVES: To assess agreement between interpretation of magnetic resonance angiography (MRA) and 18F-fluorodeoxyglucose positron emission tomography (PET) for disease extent and disease activity in large-vessel vasculitis (LVV) and determine associations between imaging and clinical assessments. METHODS: Patients with giant cell arteritis (GCA), Takayasu's arteritis (TAK) and comparators were recruited into a prospective, observational cohort. Imaging and clinical assessments were performed concurrently, blinded to each other. Agreement was assessed by per cent agreement, Cohen's kappa and McNemar's test. Multivariable logistic regression identified MRA features associated with PET scan activity. RESULTS: Eighty-four patients (GCA=35; TAK=30; comparator=19) contributed 133 paired studies. Agreement for disease extent between MRA and PET was 580 out of 966 (60%) arterial territories with Cohen's kappa=0.22. Of 386 territories with disagreement, MRA demonstrated disease in more territories than PET (304vs82, p<0.01). Agreement for disease activity between MRA and PET was 90 studies (68%) with Cohen's kappa=0.30. In studies with disagreement, MRA demonstrated activity in 23 studies and PET in 20 studies (p=0.76). Oedema and wall thickness on MRA were independently associated with PET scan activity. Clinical status was associated with disease activity by PET (p<0.01) but not MRA (p=0.70), yet 35/69 (51%) patients with LVV in clinical remission had active disease by both MRA and PET. CONCLUSIONS: In assessment of LVV, MRA and PET contribute unique and complementary information. MRA better captures disease extent, and PET scan is better suited to assess vascular activity. Clinical and imaging-based assessments often do not correlate over the disease course in LVV. TRIAL REGISTRATION NUMBER: NCT02257866.

Two consecutive large outbreaks of Salmonella Muenchen linked to pig farming in Germany, 2013 to 2014: Is something missing in our regulatory framework?

In 2013, raw pork was the suspected vehicle of a large outbreak (n = 203 cases) of Salmonella Muenchen in the German federal state of Saxony. In 2014, we investigated an outbreak (n = 247 cases) caused by the same serovar affecting Saxony and three further federal states in the eastern part of Germany. Evidence from epidemiological, microbiological and trace-back investigations strongly implicated different raw pork products as outbreak vehicles. Trace-back analysis of S. Muenchen-contaminated raw pork sausages narrowed the possible source down to 54 pig farms, and S. Muenchen was detected in three of them, which traded animals with each other. One of these farms had already been the suspected source of the 2013 outbreak. S. Muenchen isolates from stool of patients in 2013 and 2014 as well as from food and environmental surface swabs of the three pig farms shared indistinguishable pulsed-field gel electrophoresis patterns. Our results indicate a common source of both outbreaks in the primary production of pigs. Current European regulations do not make provisions for Salmonella control measures on pig farms that have been involved in human disease outbreaks. In order to prevent future outbreaks, legislators should consider tightening regulations for Salmonella control in causative primary production settings.

Deep Learning-Based Detection of Glottis Segmentation Failures.

Medical image segmentation is crucial for clinical applications, but challenges persist due to noise and variability. In particular, accurate glottis segmentation from high-speed videos is vital for voice research and diagnostics. Manual searching for failed segmentations is labor-intensive, prompting interest in automated methods. This paper proposes the first deep learning approach for detecting faulty glottis segmentations. For this purpose, faulty segmentations are generated by applying both a poorly performing neural network and perturbation procedures to three public datasets. Heavy data augmentations are added to the input until the neural network's performance decreases to the desired mean intersection over union (IoU). Likewise, the perturbation procedure involves a series of image transformations to the original ground truth segmentations in a randomized manner. These data are then used to train a ResNet18 neural network with custom loss functions to predict the IoU scores of faulty segmentations. This value is then thresholded with a fixed IoU of 0.6 for classification, thereby achieving 88.27% classification accuracy with 91.54% specificity. Experimental results demonstrate the effectiveness of the presented approach. Contributions include: (i) a knowledge-driven perturbation procedure, (ii) a deep learning framework for scoring and detecting faulty glottis segmentations, and (iii) an evaluation of custom loss functions.

Being out in high school: Positive implications for well-being in three U.S. cohorts of sexual minority adults.

For lesbian, gay, bisexual, or queer (i.e., sexual minority [SM]) youth, coming out is an important developmental milestone and is typically associated with positive well-being. However, coming out in high school may entail a higher risk of school-based victimization. Due to the greater risk of homophobic bullying, the implications of being out in adolescence and well-being later in adulthood remain unclear. Using data from a national probability survey (Generations Study) of three distinct age cohorts of SM adults (N = 1,474) in the United States, this study (a) examined how being out at school in adolescence affects general well-being in adulthood and (b) SM-specific well-being in adulthood, and (c) examined if these associations differ by cohort. Results from multivariate regression analyses demonstrated that being out in adolescence was not significantly associated with general well-being, but was significantly associated with SM-specific well-being: higher rates of identity centrality and community connectedness, and lower rates of internalized homophobia. There were no cohort differences in the associations between outness in high school, general well-being, and SM well-being. The findings from this national probability sample of SM adults provide novel insight into implications of being out across the life course, including the positive implications of being out at school in adolescence for SM-specific well-being in adulthood. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Mapping discontinuous protein-binding sites via structure-based peptide libraries: combining in silico and in vitro approaches.

To perform their various functions, protein surfaces often have to interact with each other in a specific way. Usually, only parts of a protein are accessible and can act as binding sites. Because proteins consist of polypeptide chains that fold into complex three-dimensional shapes, binding sites can be divided into two different types: linear sites that follow the primary amino acid sequence and discontinuous binding sites, which are made up of short peptide fragments that are adjacent in spatial proximity. Such discontinuous binding sites dominate protein-protein interactions, but are difficult to identify. To meet this challenge, we combined a computational, structure-based approach and an experimental, high-throughput method. SUPERFICIAL is a program that uses protein structures as input and generates peptide libraries to represent the protein's surface. A large number of the predicted peptides can be simultaneously synthesised applying the SPOT technology. The results of a binding assay subsequently help to elucidate protein-protein interactions; the approach is applicable to any kind of protein. The crystal structure of the complex of hen egg lysozyme with the well-characterised murine IgG1 antibody HyHEL-5 is available, and the complex is known to have a discontinuous binding site. Using SUPERFICIAL, the entire surface of lysozyme was translated into a peptide library that was synthesised on a cellulose membrane using the SPOT technology and tested against the HyHEL-5 antibody. In this way, it was possible to identify two peptides (longest common sequence and peptide 19) that represented the discontinuous epitope of lysozyme.

Is there a typical germinal center? A large-scale immunohistological study on the cellular composition of germinal centers during the hapten-carrier-driven primary immune response in mice.

Germinal centers (GCs) are complex, multicell-type, transient structures that form in secondary lymphatic tissues in response to T cell-dependent stimulation. This process is crucial to the adaptive immune response because it is the source of affinity maturation and long-lived B cell memory. Our previous studies showed that the growth of murine splenic GCs is nonsynchronized, involving broad-volume distributions of individual GCs at any time. This raises the question whether such a thing as a typical GC exists. To address this matter, we acquired large-scale confocal data on GCs throughout the course of the 2-phenyl-5-oxazolone chicken serum albumin-driven primary immune response in BALB/c mice. Semiautomated image analysis of 3457 GC sections revealed that, although there is no typical GC in terms of size, GCs have a typical cellular composition in that the cell ratios of resident T cells, macrophages, proliferating cells, and apoptotic nuclei are maintained during the established phase of the response. Moreover, our data provide evidence that the dark zone (DZ) and light zone (LZ) compartments of GCs are about the same size and led us to estimate that the minimal cell loss rate in GCs is 3% per hour. Furthermore, we found that the population of GC macrophages is larger and more heterogeneous than previously thought, and that despite enrichment of T cells in the LZ, the DZ of murine splenic GCs is not poor in T cells. DZ and LZ differ in the T cell-to-macrophage ratio rather than in the density of T cells.

Trace-back and trace-forward tools developed ad hoc and used during the STEC O104:H4 outbreak 2011 in Germany and generic concepts for future outbreak situations.

The Shiga toxin-producing Escherichia coli O104:H4 outbreak in Germany in 2011 required the development of appropriate tools in real-time for tracing suspicious foods along the supply chain, namely salad ingredients, sprouts, and seeds. Food commodities consumed at locations identified as most probable site of infection (outbreak clusters) were traced back in order to identify connections between different disease clusters via the supply chain of the foods. A newly developed relational database with integrated consistency and plausibility checks was used to collate these data for further analysis. Connections between suppliers, distributors, and producers were visualized in network graphs and geographic projections. Finally, this trace-back and trace-forward analysis led to the identification of sprouts produced by a horticultural farm in Lower Saxony as vehicle for the pathogen, and a specific lot of fenugreek seeds imported from Egypt as the most likely source of contamination. Network graphs have proven to be a powerful tool for summarizing and communicating complex trade relationships to various stake holders. The present article gives a detailed description of the newly developed tracing tools and recommendations for necessary requirements and improvements for future foodborne outbreak investigations.

Sexual and gender minority adverse childhood experiences (SGM-ACEs), perceived social support, and adult mental health.

BACKGROUND: Building on decades of research into the long-term developmental impacts of adverse childhood experiences (ACEs), researchers have called for expanding the ACEs framework to include experiences specific to minoritized identities. Recent empirical research has led to the development of a measure of sexual and gender minority adverse childhood experiences (SGM-ACEs). Within the SGM-specific ACEs framework, research on the long-term impact to adult mental health and the role of perceived social support are not well studied. OBJECTIVE: The purpose of this paper was two-fold. First, examine whether SGM-ACEs adversely impact mental health in SGM adults. Second, examine the role of perceived social support in the association between SGM-ACEs and adult mental health. PARTICIPANTS AND SETTING: Data were collected using a multifaceted sampling strategy. In total, 1819 self-identified SGM Texans completed an online survey inquiring about ACEs, SGM-ACEs, mental health, and demographic characteristics. RESULTS: We estimated two competing structural equation models (SEM) examining the associations between SGM-ACEs and anxiety and depressive symptoms, and whether perceived social support may moderate or mediate his association. While both models demonstrated that SGM-ACEs were significantly associated with higher anxiety and depressive symptoms. However, we found more support for the mediation model such that SGM-ACEs had direct effects on anxiety and depressive symptoms, and an indirect effect on anxiety symptoms through family support. CONCLUSIONS: Our findings confirmed that exposure to SGM-ACE is associated with poorer adult mental health. Additionally, SGM-ACEs exposure undermines SGM individuals' perceptions of family support, which increases symptoms of anxiety.

Lightweight Techniques to Improve Generalization and Robustness of U-Net Based Networks for Pulmonary Lobe Segmentation.

Lung lobe segmentation in chest CT is relevant to a wide range of clinical applications. However, existing segmentation pipelines often exhibit vulnerabilities and performance degradations when applied to external datasets. This is usually attributed to the size of the available dataset or model. We show that it is possible to enhance generalizability without huge resources by carefully curating the dataset and combining machine learning with medical expertise. Multiple machine learning techniques (self-supervision (SSL), attention (A), and data augmentation (DA)) are used to train a fast and fully-automated lung lobe segmentation model based on 2D U-Net. Our study involved evaluating these techniques on a diverse dataset collected under the RACOON project, encompassing 100 CT chest scans from patients with bacterial, viral, or SARS-CoV2 infections. We compare our model to a baseline U-Net trained on the same dataset. Our approach significantly improved segmentation accuracy (Dice score of 92.8% vs. 82.3%, p < 0.001). Moreover, our model achieved state-of-the-art performance (Dice score of 92.8% vs. 90.8% for the literature's state-of-the-art, p = 0.102) with reduced training examples (69 vs. 231 CT Scans). Among the techniques, data augmentation with expert knowledge displayed the most significant impact, enhancing the Dice score by +0.056. Notably, these enhancements are not limited to lobe segmentation but can be seamlessly integrated into various medical imaging segmentation tasks, demonstrating their versatility and potential for broader applications.

The Sexual and Gender Minority Adverse Childhood Experiences Scale.

INTRODUCTION: Sexual and gender minority (SGM) adverse childhood experiences (ACEs) are identity-based forms of early life adversity. Exposure to SGM ACEs is associated with increased odds of depression, anxiety, and post-traumatic stress disorder in SGM adults. The purpose of this study was to further test a revised version of the measure in a U.S. sample with more robust and clinically relevant mental health outcomes. METHODS: In May and June 2022, a national sample of SGM adults (N=4,445) was recruited from a Qualtrics Panel to complete a 20-minute online survey that included questions regarding ACEs, SGM ACEs, depression, anxiety, and post-traumatic stress disorder. Exploratory factor analysis and confirmatory factor analysis were used to examine factor structure. Multivariable regression was used to assess criterion validity, and a sensitivity analysis was conducted. Data were analyzed in February 2023. RESULTS: Respondents indicate that vicarious trauma (81%) and school bullying (67%) were the most common experiences and that all SGM ACEs were frequently occurring before adulthood. Confirmatory factor analysis determined a 1-factor solution. Participants with more SGM adverse childhood experiences exposure had worse anxiety, depression, and post-traumatic stress disorder symptoms (ß=0.16, ß=0.18, ß=0.26, respectively, p<0.0001) after controlling for ACEs exposure and demographic factors. A sensitivity analysis indicated that estimates were similar in terms of magnitude and direction. CONCLUSIONS: SGM ACEs commonly and frequently occur before adulthood and impact adult SGM mental health. Overall, the measure had good-to-excellent psychometric properties. Future research should consider integrating SGM ACEs and Minority Stress Theory.

Understanding the Experiences of Latinx LGBTQ Texans at the Beginning of the COVID-19 Pandemic.

Marginalized communities have been disproportionately affected by COVID-19, including both racial/ethnic minority and sexual minority populations. To date, there has been little research examining the impact of the COVID-19 pandemic at the intersections of marginalized identities. Furthermore, available national data on COVID-19 outcomes may obscure our understanding of region-specific outcomes, particularly in the U.S. South. Using an intersectional approach, we explore differences in worries over COVID-19, preventative behaviors, and COVID-19 outcomes in the early months of the pandemic in a diverse sample of LGBTQ people (N = 1076) living in Texas. Our findings indicated that LGBTQ Latinx people in Texas reported more COVID-19 related worries and adverse outcomes than non-Latinx LGBTQ people. These findings are in line with previous research that found that the increased risk to Latinx and LGBTQ populations in public health crises is often overlooked and can be attributed to many factors such as socioeconomic status, occupational propensity, disparities in physical health, and barriers to healthcare access. Furthermore, our findings suggest the necessity of utilizing an intersectional approach when examining the disproportionate burden marginalized communities face in public health crises.

Affinity maturation of B cells involves not only a few but a whole spectrum of relevant mutations.

Affinity maturation of B lymphocytes within germinal centers involves both diversification of their B-cell receptors (BCRs) by somatic hypermutation (SHM) and a crucial receptor-mediated selection step. However, in contrast to recent advances in revealing the molecular mechanism of SHM, the fundamentals of the selection process are still poorly understood, i.e. it is often not clear how and how many mutations contribute to improving a BCR during the response against a given antigen. A general drawback in assessing the mutations relevant to the selection process is the difficult task of rating the relative contributions of selection and intrinsic biases to the experimentally observed mutation patterns of BCRs. The approach proposed here is premised on statistical comparison of the frequency distributions of nucleotide substitutions as observed in datasets of hypermutated BCRs against their frequency distribution expected under the null hypothesis of no selection. Thereby, we show that the spectrum of mutations relevant to maturation of canonical anti-(4-hydroxy-3-nitrophenyl)acetyl BCRs is much broader than previously acknowledged, going beyond the scope of single key mutations. Moreover, our results suggest that maturation not only involves selection by means of affinity but likewise expression and stabilization of BCRs.

Broad volume distributions indicate nonsynchronized growth and suggest sudden collapses of germinal center B cell populations.

Immunization with a T cell-dependent Ag leads to the formation of several hundred germinal centers (GCs) within secondary lymphoid organs, a key process in the maturation of the immune response. Although prevailing perceptions about affinity maturation intuitively assume simultaneous seeding, growth, and decay of GCs, our previous mathematical simulations led us to hypothesize that their growth might be nonsynchronized. To investigate this, we performed computer-aided three-dimensional reconstructions of splenic GCs to measure size distributions at consecutive time points following immunization of BALB/c mice with a conjugate of 2-phenyl-oxazolone and chicken serum albumin. Our analysis reveals a broad volume distribution of GCs, indicating that individual GCs certainly do not obey the average time course of the GC volumes and that their growth is nonsynchronized. To address the cause and implications of this behavior, we compared our empirical data with simulations of a stochastic mathematical model that allows for frequent and sudden collapses of GCs. Strikingly, this model succeeds in reproducing the empirical average kinetics of GC volumes as well as the underlying broad size distributions. Possible causes of GC B cell population collapses are discussed in the context of the affinity-maturation process.

Future parenting aspirations and minority stress in U.S. sexual minority adults.

Parenthood is an aspiration shared by a majority of U.S. adults. However, previous research has found that sexual minority adults (e.g., lesbian, gay, bisexual [LGB]) are less likely than heterosexual counterparts to be parents or desire to become parents in the future. To date, few studies have examined how minority stress (i.e., everyday discrimination [ED], felt stigma [FS], and internalized homophobia [IH]) influences parenting desire, parenting likelihood, and expectation a gap between the two. Even fewer studies have examined the parenting aspirations of sexual minority individuals following nationwide legalization of same-sex marriage in the United States. We analyzed data from 487 childfree lesbian, gay, bisexual, and queer participants from two cohorts ("equality cohort": ages 18-25; "visibility" cohort: ages 34-41) from the Generations study, a national probability sample of sexual minority adults in the United States. As expected, we found that the visibility cohort adults had significantly lower parenting desire and parenting likelihood, and a greater parenting expectation gap than the equality cohort adults. Additionally, while everyday discrimination (ED) did not predict parenting aspirations, IH and FS predicted higher parenting desire and lower parenting likelihood, respectively. We found a cohort by FS interaction such that FS predicted even lower parenting likelihood in the visibility cohort adults. Our findings contribute to a growing body of research on sexual minority parenthood and may inform community practice and clinical support for sexual minority adults who pursue parenthood. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Development and psychometric properties of the sexual and gender minority adverse childhood experiences (SGM-ACEs): Effect on sexual and gender minority adult mental health.

BACKGROUND: Research has consistently shown a relationship between adverse childhood experiences (ACEs) and poor mental health outcomes, and recent research shows that sexual and gender minority (SGM) individuals are at increased risk for ACEs. Moreover, SGM individuals may experience unique ACEs. Increased risk for exposure to traditional and SGM-specific ACEs are related to heterosexism. OBJECTIVE: The purpose of this paper is two-fold. First, establish the need for an SGM specific ACEs framework that accounts for exposure to heterosexism. Second, assess the psychometric properties of the SGM-ACEs scale. PARTICIPANTS AND SETTING: Data were collected using a multifaceted sampling strategy. In total, 1725 self-identified SGM Texans completed an online survey about ACEs, SGM-ACEs, mental health diagnoses, and demographic characteristics. RESULTS: The most commonly reported SGM-ACEs were seeing or hearing of other SGM being physical harmed (71.2%), being bullied in school for being SGM (67.9%), and hearing religious leaders say homophobic, biphobic, or transphobic things (60.8%). The EFA showed that 7-items loaded onto a single factor and the CFA indicated a good model fit, with items showing a significant factor loading higher than 0.60. SGM-ACE showed adequate to good psychometric properties and predicted depression (AOR = 1.49, CI = 1.20, 1.86), anxiety (AOR = 1.61, CI = 1.25, 2.00), and PTSD (AOR = 1.97, CI = 1.47, 2.66), when controlling for ACEs and demographic factors. CONCLUSIONS: The 7-item SGM-ACEs measure is a psychometrically sound and unidimensional measure that can be quickly used to assess common adverse childhood experiences related to heterosexism.

The coxsackievirus-adenovirus receptor reveals complex homophilic and heterophilic interactions on neural cells.

The coxsackievirus-adenovirus receptor (CAR) is a member of the Ig superfamily strongly expressed in the developing nervous system. Our histological investigations during development reveal an initial uniform distribution of CAR on all neural cells with a concentration on membranes that face the margins of the nervous system (e.g., the basal laminae and the ventricular side). At more advanced stages, CAR becomes downregulated and restricted to specific regions including areas rich in axonal and dendritic surfaces. To study the function of CAR on neural cells, we used the fiber knob of the adenovirus, extracellular CAR domains, blocking antibodies to CAR, as well as CAR-deficient neural cells. Blocking antibodies were found to inhibit neurite extension in retina organ and retinal explant cultures, whereas the application of the recombinant fiber knob of the adenovirus subtype Ad2 or extracellular CAR domains promoted neurite extension and adhesion to extracellular matrices. We observed a promiscuous interaction of CAR with extracellular matrix glycoproteins, which was deduced from analytical ultracentrifugation experiments, affinity chromatography, and adhesion assays. The membrane proximal Ig domain of CAR, termed D2, was found to bind to a fibronectin fragment, including the heparin-binding domain 2, which promotes neurite extension of wild type, but not of CAR-deficient neural cells. In contrast to heterophilic interactions, homophilic association of CAR involves both Ig domains, as was revealed by ultracentrifugation, chemical cross-linking, and adhesion studies. The results of these functional and binding studies are correlated to a U-shaped homodimer of the complete extracellular domains of CAR detected by x-ray crystallography.

Development, validation, and application of a fast and simple GC-MS method for determination of some therapeutic drugs relevant in emergency toxicology.

BACKGROUND: To date, immunoassays are commercially available for quantification of valproic acid, salicylic acid, paracetamol, phenobarbital, phenytoin, and primidone. As they are no longer available, a fast, simple, and cost-effective quantitative gas chromatography-mass spectrometry (GC-MS) method was developed and fully validated for these drugs. METHODS: After simple and fast liquid-liquid extraction, the samples were analyzed by GC-MS using the selected ion monitoring mode. The method was validated including the parameters selectivity, calibration model, precision, accuracy, and extraction efficiency. RESULTS: The above-mentioned analytes were separated within 8.5 minutes and sensitively detected. No interfering peaks were observed in blank samples from 8 different sources. The linearity ranges were 20-200 mg/L for valproic acid, 100-1200 mg/L for salicylic acid, 10-200 mg/L for paracetamol, 10-200 mg/L for phenobarbital, 4-20 mg/L for primidone, and 2.5-30 mg/L for phenytoin. Generally accepted criteria for accuracy and precision were fulfilled for all analytes using 6-point calibration. Even 1-point calibration was applicable for all analytes. The assay was successfully applied to analysis of real plasma samples and proficiency testing material. CONCLUSIONS: The assay described allowed fast and reliable determination of analytes relevant in the diagnosis of poisonings. Furthermore, time- and cost-saving 1-point calibration was shown to be suitable for daily routine work, especially in emergency cases.

A validated GC-MS procedure for fast, simple, and cost-effective quantification of glycols and GHB in human plasma and their identification in urine and plasma developed for emergency toxicology.

Methods developed for use in emergency toxicology have to be fast and simple. Additionally, such methods should be multi-analyte procedures because they allow monitoring of analytes of different drug classes in one single body sample. This is important because often only a limited amount of sample is available and the results have to be reported as fast as possible. Therefore, we describe the improvement of an existing method published by van Hee at al. The new method is fast and simple and designed for the simultaneous determination of ethylene glycol, 1,2-propylene glycol, lactic acid, glycolic acid, gamma-hydroxybutyric acid (GHB), diethylene glycol, triethylene glycol, and tetraethylene glycol in human plasma or urine. A 50-µL aliquot of sample was deproteinized and 20 µl of the diluted specimen were derivatized using bis-N,O-trimethylsilyl trifluoroacetamide and the catalyst dimethylformamide. After microwave-assisted derivatization, an aliquot was injected into the gas chromatograph and analyzed with electron ionization mass spectrometry in selective ion monitoring mode. All compounds are separated within 12 min and detected with a limit of quantification of 0.05 and 0.01 g/L for glycols and GHB, respectively. Calibration was linear from 0.05 to 1.0 g/L for glycols and 0.01 to 0.2 g/L for GHB. Validation criteria were shown to be in the required limits with exception of lactic acid. Average analysis time from starting sample preparation until quantitative plasma results of approximately 35 min was achieved. This turnaround time is considered most appropriate for emergency cases.