Dried blood spots via remote testing as a possible future application in the doping control process.

The use of DBS as biological matrix in combination with a novel technical remote testing app specifically developed for the doping control process shows that testing is becoming easier and variable through DBS. Supplemented by the remote testing solution, the system could significantly increase in the unpredictability of test dates and sample quantity.

Neoadjuvant Radiochemotherapy Alters the Immune and Metabolic Microenvironment in Oral Cancer-Analyses of CD68, CD163, TGF-ß1, GLUT-1 and HIF-1α Expressions.

BACKGROUND: The first-line treatment of oral squamous cell carcinoma (OSCC) involves surgical tumor resection, followed by adjuvant radio(chemo)therapy (R(C)T) in advanced cases. Neoadjuvant radio- and/or chemotherapy has failed to show improved survival in OSCC. Recently, neoadjuvant immunotherapy has shown promising therapeutic efficacy in phase 2 trials. In this context, the addition of radio- and chemotherapy is being reconsidered. Therefore, a better understanding of the tumor-biologic effects of neoadjuvant RCT would be beneficial. The current study was conducted on a retrospective cohort of patients who received neoadjuvant RCT for the treatment of oral cancer. The aim of the study was to evaluate the influence of neoadjuvant RCT on the immunological tumor microenvironment (TME) and hypoxic and glucose metabolisms. METHODS: A cohort of 45 OSSC tissue samples from patients were analyzed before and after RCT (total 50.4 Gy; 1.8 Gy 5× weekly; Cisplatin + 5-Fluorouracil). Immunohistochemistry for CD68, CD163, TGF-ß, GLUT-1 and HIF-1α was performed using tissue microarrays and automated cell counting. Differences in expression before and after RCT and associations with histomorphological parameters (T-status, N-status) were assessed using the Mann-Whitney U test. RESULTS: Tumor resection specimens after neoadjuvant RCT showed a significant decrease in CD68 infiltration and a significant increase in CD163 cell density. The CD68/CD163 ratio was significantly lower after RCT, indicating a shift toward M2 polarization. The GLUT-1 and HIF-1α expressions were significantly lower after RCT. Larger tumors (T3/T4) showed a lower GLUT-1 expression. Other biomarkers were not associated with the T- and N-status. CONCLUSIONS: Neoadjuvant RCT with 50.4 Gy induced a shift toward the M2 polarization of macrophages in the TME. This change in immune composition is not favorable and may be prognostically negative and counteract immunotherapeutic approaches. In addition, the decreased expressions in GLUT-1 and HIF-1α indicate reductions in the glucose metabolism and hypoxic energy metabolism in response to "high dose" neoadjuvant RCT, which may be therapeutically desirable.

Carbon dioxide and trace oxygen concentrations impact growth and product formation of the gut bacterium Phocaeicola vulgatus.

BACKGROUND: The promising yet barely investigated anaerobic species Phocaeicola vulgatus (formerly Bacteroides vulgatus) plays a vital role for human gut health and effectively produces organic acids. Among them is succinate, a building block for high-value-added chemicals. Cultivating anaerobic bacteria is challenging, and a detailed understanding of P. vulgatus growth and metabolism is required to improve succinate production. One significant aspect is the influence of different gas concentrations. CO2 is required for the growth of P. vulgatus. However, it is a greenhouse gas that should not be wasted. Another highly interesting aspect is the sensitivity of P. vulgatus towards O2. In this work, the effects of varying concentrations of both gases were studied in the in-house developed Respiratory Activity MOnitoring System (RAMOS), which provides online monitoring of CO2, O2, and pressure under gassed conditions. The RAMOS was combined with a gas mixing system to test CO2 and O2 concentrations in a range of 0.25-15.0 vol% and 0.0-2.5 vol%, respectively. RESULTS: Changing the CO2 concentration in the gas supply revealed a CO2 optimum of 3.0 vol% for total organic acid production and 15.0 vol% for succinate production. It was demonstrated that the organic acid composition changed depending on the CO2 concentration. Furthermore, unrestricted growth of P. vulgatus up to an O2 concentration of 0.7 vol% in the gas supply was proven. The viability decreased rapidly at concentrations larger than or equal to 1.3 vol% O2. CONCLUSIONS: The study showed that P. vulgatus requires little CO2, has a distinct O2 tolerance and is therefore well suited for industrial applications.

Suboptimal Dosing of Benzodiazepines and Levetiracetam in a Cohort of Status Epilepticus Patients and Outcomes Associated with Inadequate Dosing.

Background: Status epilepticus (SE) is a neurologic emergency that can result in serious morbidity and mortality. Recent studies have suggested underdosing of both benzodiazepines (BZDs) and antiseizure medications (ASM) which may result in poorer outcomes. Objectives: This study aims to determine the dose of BZDs and levetiracetam given in our emergency department for episodes of SE and determine the outcomes associated with this dosing. Methods: We conducted a retrospective cohort study of all adult patients with SE admitted to our hospital from 2017 to 2020. We collected demographic data, type of SE, dose of BZD and levetiracetam, and outcomes which included mortality and a calculated Glasgow outcome scale (GOS). We compared outcomes of patients with SE who received adequate dosing (according to practice guidelines) to those who did not. Results: 111 adult patients were included of whom 91% were seen initially in our emergency department. 75% had convulsive SE on presentation. Approximately 55% and 68% of patients did not receive an appropriate dose of BZD or levetiracetam, respectively. Inadequate dosing of BZD was associated with worse clinical outcomes based on GOS (43.6% favorable outcome vs 62.5% with adequate dosing P = .046 (95% CI, 1.01-4.60)) and inadequate dosing of both drugs was also associated with a worse GOS outcome (HR, 2.91 (95% CI, 1.05-9.67, P = .02). No difference was found in length of stay or mortality alone. Conclusion: Our study found inadequate dosing of drugs to treat SE in adults was common in our institution and was associated with worse outcomes.

Comparison of the performance of a clinical classification tree versus clinical gestalt in predicting sepsis with extended-spectrum beta-lactamase-producing gram-negative rods.

A clinical decision tree was developed using point-of-care characteristics to identify patients with culture-proven sepsis due to extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-PE). We compared its performance with the clinical gestalt of emergency department (ED) clinicians and hospital-based clinicians. The developed tree outperformed ED-based clinicians but was comparable to inpatient-based clinicians.

MRI Radiogenomics of Pediatric Medulloblastoma: A Multicenter Study.

Background Radiogenomics of pediatric medulloblastoma (MB) offers an opportunity for MB risk stratification, which may aid therapeutic decision making, family counseling, and selection of patient groups suitable for targeted genetic analysis. Purpose To develop machine learning strategies that identify the four clinically significant MB molecular subgroups. Materials and Methods In this retrospective study, consecutive pediatric patients with newly diagnosed MB at MRI at 12 international pediatric sites between July 1997 and May 2020 were identified. There were 1800 features extracted from T2- and contrast-enhanced T1-weighted preoperative MRI scans. A two-stage sequential classifier was designed-one that first identifies non-wingless (WNT) and non-sonic hedgehog (SHH) MB and then differentiates therapeutically relevant WNT from SHH. Further, a classifier that distinguishes high-risk group 3 from group 4 MB was developed. An independent, binary subgroup analysis was conducted to uncover radiomics features unique to infantile versus childhood SHH subgroups. The best-performing models from six candidate classifiers were selected, and performance was measured on holdout test sets. CIs were obtained by bootstrapping the test sets for 2000 random samples. Model accuracy score was compared with the no-information rate using the Wald test. Results The study cohort comprised 263 patients (mean age ± SD at diagnosis, 87 months ± 60; 166 boys). A two-stage classifier outperformed a single-stage multiclass classifier. The combined, sequential classifier achieved a microaveraged F1 score of 88% and a binary F1 score of 95% specifically for WNT. A group 3 versus group 4 classifier achieved an area under the receiver operating characteristic curve of 98%. Of the Image Biomarker Standardization Initiative features, texture and first-order intensity features were most contributory across the molecular subgroups. Conclusion An MRI-based machine learning decision path allowed identification of the four clinically relevant molecular pediatric medulloblastoma subgroups. © RSNA, 2022 Online supplemental material is available for this article. See also the editorial by Chaudhary and Bapuraj in this issue.

Machine Assist for Pediatric Posterior Fossa Tumor Diagnosis: A Multinational Study.

BACKGROUND: Clinicians and machine classifiers reliably diagnose pilocytic astrocytoma (PA) on magnetic resonance imaging (MRI) but less accurately distinguish medulloblastoma (MB) from ependymoma (EP). One strategy is to first rule out the most identifiable diagnosis. OBJECTIVE: To hypothesize a sequential machine-learning classifier could improve diagnostic performance by mimicking a clinician's strategy of excluding PA before distinguishing MB from EP. METHODS: We extracted 1800 total Image Biomarker Standardization Initiative (IBSI)-based features from T2- and gadolinium-enhanced T1-weighted images in a multinational cohort of 274 MB, 156 PA, and 97 EP. We designed a 2-step sequential classifier - first ruling out PA, and next distinguishing MB from EP. For each step, we selected the best performing model from 6-candidate classifier using a reduced feature set, and measured performance on a holdout test set with the microaveraged F1 score. RESULTS: Optimal diagnostic performance was achieved using 2 decision steps, each with its own distinct imaging features and classifier method. A 3-way logistic regression classifier first distinguished PA from non-PA, with T2 uniformity and T1 contrast as the most relevant IBSI features (F1 score 0.8809). A 2-way neural net classifier next distinguished MB from EP, with T2 sphericity and T1 flatness as most relevant (F1 score 0.9189). The combined, sequential classifier was with F1 score 0.9179. CONCLUSION: An MRI-based sequential machine-learning classifiers offer high-performance prediction of pediatric posterior fossa tumors across a large, multinational cohort. Optimization of this model with demographic, clinical, imaging, and molecular predictors could provide significant advantages for family counseling and surgical planning.

CRISPRi-Library-Guided Target Identification for Engineering Carotenoid Production by Corynebacterium glutamicum.

Corynebacterium glutamicum is a prominent production host for various value-added compounds in white biotechnology. Gene repression by dCas9/clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) allows for the identification of target genes for metabolic engineering. In this study, a CRISPRi-based library for the repression of 74 genes of C. glutamicum was constructed. The chosen genes included genes encoding enzymes of glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle, regulatory genes, as well as genes of the methylerythritol phosphate and carotenoid biosynthesis pathways. As expected, CRISPRi-mediated repression of the carotenogenesis repressor gene crtR resulted in increased pigmentation and cellular content of the native carotenoid pigment decaprenoxanthin. CRISPRi screening identified 14 genes that affected decaprenoxanthin biosynthesis when repressed. Carotenoid biosynthesis was significantly decreased upon CRISPRi-mediated repression of 11 of these genes, while repression of 3 genes was beneficial for decaprenoxanthin production. Largely, but not in all cases, deletion of selected genes identified in the CRISPRi screen confirmed the pigmentation phenotypes obtained by CRISPRi. Notably, deletion of pgi as well as of gapA improved decaprenoxanthin levels 43-fold and 9-fold, respectively. The scope of the designed library to identify metabolic engineering targets, transfer of gene repression to stable gene deletion, and limitations of the approach were discussed.

Acute Skeletal Muscle Contractions Orchestrate Signaling Mechanisms to Trigger Nuclear NFATc1 Shuttling and Epigenetic Histone Modifications.

BACKGROUND/AIMS: Calcium (Ca²âº) coordinates skeletal muscle functions by controlling contractions as well as signaling pathways and transcriptional properties. The ryanodine receptor 1 (RyR1), its phosphorylation site (pRyR1Ser²84°) and its stabilizers navigate Ca²âº oscillations to command muscle signaling cascades and transcriptional activities. While chronic exercise increases pRyR1Ser²84°, investigations on acute exercise's effects on RyR1 and Ca²âº-dependent modifications of skeletal muscle are rare. The aim of this study was to examine molecular events leading to RyR1 phosphorylation in a physiological model of acute exercise. We hypothesized that exercise-induced RyR1 phosphorylation is associated with altered Ca²âº-dependent physiological phenotypes. METHODS: We analyzed pRyR1Ser²84°, its stabilizers, involved signaling pathways, and Ca²âº-sensitive muscle-determining factors (i.e. NFATc1 and epigenetic histone H3 modifications) in rat muscles upon one single running bout of either concentric or eccentric contractions. RESULTS: Both acute exercises significantly increased pRyRSer²84° levels in muscles, which was accompanied by dissociations of stabilizers from RyR1. Additionally, RyR1 phosphorylation-inducing signaling cascades PTEN/CaMKII/ PKA were significantly activated upon exercise. Further, RyR1 phosphorylations were associated with increased Ca²âº-dependent NFATc1 nuclear abundances as well as increased Ca²âº-dependent epigenetic H3 acetylations pointing to a pRyR1Ser²84°-dependent rapid and novel Ca²âº equilibrium upon exercise. CONCLUSION: Our data report synergistic actions of several distinct pathways to modify RyR1 function to govern physiological phenotypes, here expressed as increased nuclear NFATc1 abundances and epigenetic H3 modifications.

Hypoglycemia but Not Hyperglycemia Is Associated with Mortality in Critically Ill Patients with Diabetes.

BACKGROUND: Both severe hyperglycemia (> 200 mg/dL) and hypoglycemia (≤70 mg/dL) are known to be associated with increased mortality in critically ill patients. Therefore, we investigated associations of a single episode of blood glucose deviation (concentration either ≤70 mg/dL and/or > 200 mg/dL) during an intensive care unit (ICU) stay with mortality in these patients. METHODS: A total of 4,986 patients (age 65 ± 15 years; 39% female; 14% type 2 diabetes [T2DM] based on medical records) admitted to a German ICU in a tertiary care hospital were investigated retrospectively. The intra-ICU and long-term mortality of patients between 4 and 7 years after their ICU submission were assessed. RESULTS: A total 62,659 glucose measurements were analyzed. A single glucose deviation was associated with adverse outcomes compared to patients without a glucose deviation, represented by both intra-ICU mortality (22 vs. 10%; OR 2.62; 95% CI 2.23-3.09; p < 0.001) and long-term mortality (HR 2.01; 95% CI 1.81-2.24; p < 0.001). In patients suffering from T2DM hypoglycemia (30 vs. 13%; OR 2.94; 95% CI 2.28-3.80; p < 0.001) but not hyperglycemia (16 vs. 14%; OR 1.05; 95% CI 0.68-1.62; p = 0.84) was associated with mortality. CONCLUSION: In patients with dia-betes, hypo- but not hyperglycemia was associated with increased mortality, whereas in patients without diabetes, both hyper- and hypoglycemia were associated with adverse outcome. Blood glucose concentration might need differential approaches depending on concomitant diseases.

Analysis of human microcirculation in weightlessness: Study protocol and pre-study experiments.

BACKGROUND: In weightlessness, alterations in organ systems have been reported. The microcirculation consists of a network of blood vessels with diameters of a few µm. It is considered the largest part of the circulatory system of the human body and essential for exchange of gas, nutrients and waste products. An investigation of the microcirculation in weightlessness seems warranted but has not yet been performed. OBJECTIVE: In this paper, we outline a study in which we will investigate the possible interrelations between weightlessness and microcirculation. We will induce weightlessness in the course of parabolic flight maneuvers, which will be conducted during a parabolic flight campaign. In this study protocol also an evaluation of a possible influence of parabolic flight premedication on microcirculation will be described. METHODS: The microcirculation will be investigated by sublingual intravital measurements applying sidestream darkfield microscopy. Parameters of macrocirculation such as heart rate, blood pressure and blood oxygenation will also be investigated. RESULTS: In our pre-study experiments, neither dimenhydrinate nor scopolamine altered microcirculation. CONCLUSIONS: As the application of motion sickness therapy did not alter microcirculation, it will be applied during the parabolic flight maneuvers of the campaign. Our results might deepen the understanding of microcirculation on space missions and on earth.

High peak PaO2 values associated with adverse outcome in patients treated with noninvasive ventilation for acute cardiogenic pulmonary edema and pneumonia.

BACKGROUND: Noninvasive ventilation (NIV) has a sigificant impact on mortality in acute respiratory failure (ARF). Predictive parameters for mortality are of high interest. METHODS: We retrospectively analyzed 3759 blood gas analysis and clinical parameters of 475 patients presenting with ARF based on acute cardiogenic pulmonary edema and/or pneumonia. The influence of peak arterial oxygen partial pressure levels (PaO2) with respect to its predictive value for in-hopital and long-term mortality was investigated. RESULTS: Overall intra-hospital mortality was 24%. Peak PaO2 levels in kPa were significantly higher in non-survivors (20.01±10.11) compared to survivors (15.65±6.79, P<0.001). A univariate Cox proportional-hazards analysis for long-term mortality revealed associations with maximum PaO2 levels (overall cohort: HR= 1.02; 95% CI: 1.007-1.03; P=0.003; CPE: HR= 1.02; 95% CI: 0.99-1.04, P=0.05, pneumonia: HR= 1.02; 95% CI: 1-1.4, P=0.02). A PaO2 cut-off value of 13 kiloPascal (kPa) was calculated by means of Youden Index and remained true even after correction for APACHE 2 Score (HR= 1.50; 95% CI: 1.00-2.25; P=0.05) and for PaCO2 (HR= 1.63; 95% CI: 1.14-2.33; P=0.01). CONCLUSIONS: Peak PaO2 levels were associated with worse in-hopital and long-term mortality in patients treated with NIV due to ARF. These findings may indicate that application of high oxygen may be detrimental in such patients.

Dynamic regulation of PGC-1alpha localization and turnover implicates mitochondrial adaptation in calorie restriction and the stress response.

There is increasing evidence that longevity and stress resistance are connected, but the mechanism is unclear. We report that mitochondria are regulated in response to oxidative stress and calorie restriction through a shared mechanism involving peroxisome proliferator-activated receptor-gamma co-activator 1alpha (PGC-1alpha). We demonstrate that PGC-1alpha subcellular distribution is regulated, and its transcriptional activity is promoted through SIRT1-dependent nuclear accumulation. In addition, the duration of PGC-1alpha activity is regulated by glycogen synthase kinase beta (GSK3beta), which targets PGC-1alpha for intranuclear proteasomal degradation. This mechanism of regulation permits the rapidity and persistence of PGC-1alpha activation to be independently controlled. We provide evidence that this pathway of PGC-1alpha regulation occurs in vivo in mice, both in the oxidative stress response and with calorie restriction. Our data show how mitochondrial function may be adapted in response to external stimuli, and support the concept that such adaptation is critically involved in cellular survival and in lifespan extension by calorie restriction.