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Aim: To determine a recommended Phase II dose of the oral fluoropyrimidine trifluridine/tipiracil (FTD/TPI) combined with the multi-kinase inhibitor regorafenib (REG) in refractory metastatic colorectal cancer patients. Materials & methods: A conventional 3 + 3 dose finding design was used. FTD/TPI was administered on days 1–5 and 8–12 of a 28-day cycle, REG on days 2–22. Two dose levels were used: FTD/TPI 25 mg/m2 b.i.d. + REG 120 mg/d, then escalated to FTD/TPI 35 mg/m2 b.i.d. + REG 120 mg/d. Results: In total, 12 patients were treated at two dose levels. Three dose-limiting toxicities were observed; all were grade 3 hypertension causally attributed to REG. Recommended Phase II dose is FTD/TPI 25 mg/m2 b.i.d. + REG 120 mg/d. Median progression-free survival was 3.81 months (95% CI: 1.51–5.29), median OS 11.1 months (95% CI: 2.3–18.2). Conclusion: The combination of REG and FTD/TPI is feasible and safe. Efficacy signals exceed that of the single agents at acceptable toxicity levels and are clinically meaningful.

The quantitative analysis of substances in dried blood spots (DBS) has gained vast popularity in the past decade. The World Anti-Doping Agency (WADA) also recently committed to implementing DBS. Currently, DBS sampling mainly has focused on various volumetric sampling devices such as Hemaxis, Capitainer, and Mitra. These devices are designed to collect a specific sample volume, independent of the hematocrit (HCT), to enable quantitative DBS analysis. Here, we present an automated solution that makes the necessity of volumetric sampling for quantitative DBS analysis obsolete. Combining automated reflectance-based HCT correction in combination with fully automated DBS LC-MS/MS analysis, the novel strategy permits high-throughput analysis in combination with HCT independence. Studying the model compound phosphatidylethanol 16:0/18:1, which is HCT-dependent due to incorporation into red blood cells, an implementation of DBS HCT normalization is presented. First, the performance of the automated HCT module with DBS is demonstrated compared to standardized HCT analysis from whole blood using a centrifuge. Second, the HCT dependency of fully automated PEth analysis from DBS is evaluated. Third, a solution to correct for the HCT dependency of PEth using the HCT scanner is presented. The study demonstrates that as soon as the HCT dependence of an analyte is known, a correction factor can be applied for the normalization of HCT levels. In the context of PEth, a linear increase in PEth concentration was observed, as the analyte is primarily located within the cellular fraction. Based on the obtained results, the use of a common correction factor for PEth DBS is possible.

OBJECTIVE In recent decades, significant numbers of pharmaceutical trials have gradually been relocated to low- and middle-income countries. However, there is little evidence regarding the transferability of trial outcomes across countries. Analysing duloxetine randomised controlled trials (RCTs) conducted in different countries, we investigated whether per capita gross national income (GNI) and healthcare expenditure (HE) are associated with pre-post mean changes of depression severity and differences in duloxetine-placebo effect sizes. METHOD Meta-analyses included RCTs investigating duloxetine efficacy in patients with depression. Individual participant data (IPD) from multi-centre duloxetine trials were provided by the manufacturer. Data extracted from published reports also entered analyses in case of trials conducted in only one country. A meta-regression approach was applied to analyse associations of GNI and HE with standardised pre-post mean change using raw score standardisation (SMCR) and comparative effect size, that is, the mean differences (MD) in pre-post effect size between duloxetine and placebo treatment. RESULTS 23 trials with 8417 randomised participants entered analyses. Regression coefficients indicated a negative linear relationship of SMCR with GNI (z-standardised β = -3.61, R2 = 14.8%, p < 0.001) and HE (β = -4.72, R2 = 24.8%, p < 0.001) for participants treated with duloxetine. Similar associations were found for placebo treatment (GNI: β = -3.52, R2 = 23.8%, p < 0.001; β = -3.34, R2 = 21.0% p < 0.001). Neither GNI nor HE was associated with the MD between duloxetine and placebo pre-post differences. CONCLUSIONS Findings challenge the idea of the universal transferability of antidepressant trial outcomes across countries. Understanding the results of antidepressant RCTs demands more sophisticated clarification of context factors involved in determining effectiveness of antidepressant medication and should be discussed with a view to socio-economic context in their countries of origin.

Purpose This executive summary of a national living guideline aims to provide rapid evidence based recommendations on the role of drug interventions in the treatment of hospitalized patients with COVID-19. Methods The guideline makes use of a systematic assessment and decision process using an evidence to decision framework (GRADE) as recommended standard WHO (2021). Recommendations are consented by an interdisciplinary panel. Evidence analysis and interpretation is supported by the CEOsys project providing extensive literature searches and living (meta-) analyses. For this executive summary, selected key recommendations on drug therapy are presented including the quality of the evidence and rationale for the level of recommendation. Results The guideline contains 11 key recommendations for COVID-19 drug therapy, eight of which are based on systematic review and/or meta-analysis, while three recommendations represent consensus expert opinion. Based on current evidence, the panel makes strong recommendations for corticosteroids (WHO scale 5–9) and prophylactic anticoagulation (all hospitalized patients with COVID-19) as standard of care. Intensified anticoagulation may be considered for patients with additional risk factors for venous thromboembolisms (VTE) and a low bleeding risk. The IL-6 antagonist tocilizumab may be added in case of high supplemental oxygen requirement and progressive disease (WHO scale 5–6). Treatment with nMABs may be considered for selected inpatients with an early SARS-CoV-2 infection that are not hospitalized for COVID-19. Convalescent plasma, azithromycin, ivermectin or vitamin D3 should not be used in COVID-19 routine care. Conclusion For COVID-19 drug therapy, there are several options that are sufficiently supported by evidence. The living guidance will be updated as new evidence emerges.

Liver transplantation is a complex procedure that requires multiple evaluations, including abstinence monitorization. While literature assessing the impact of different variables on relapse, survival, and graft loss exists, little is known about the predictive capacity of direct alcohol biomarkers. The primary aim of this study was to evaluate the prediction capacity of direct alcohol biomarkers regarding patient survival and clinical relapse. We hypothesized that patients screening positive for any of the experimental biomarkers would show an increased risk of clinical alcohol relapse and death. We conducted a retrospective data recollection from medical files of patients awaiting liver transplantation, who were at baseline screened with Peth, EtG in hair and urine, and EtS. We tested the prediction capacity of the biomarkers with two Cox-regression models. A total of 50 patients were included (84% men, mean age 59 years (SD = 6)). Biomarkers at baseline were positive in 18 patients. The mean follow-up time for this study was 26 months (SD = 10.4). Twelve patients died, liver transplantation was carried out in 12 patients, and clinical relapse was observed in eight patients. The only significant covariate in the Cox-regression models was age with clinical relapse, with younger patients being at greater risk of relapse. This study could not find a significant prediction capacity of direct alcohol biomarkers for mortality or clinical relapse during follow-up. Higher sample sizes might be needed to detect statistically significant differences. All in all, we believe that direct alcohol biomarkers should be widely used in liver transplantation settings due to their high sensitivity for the detection of recent drinking.

Niobium is an important material for high temperature applications, in space, in superconductors or in chemical process constructions. Laser-based powder bed fusion of niobium (PBF-LB/M/Nb) offers new opportunities in design, though it is still an expensive technique. The build-up rate is an important factor for economical manufacturing using PBF-LB/M/Nb. It is largely influenced by variation of process parameters, affecting the heat flow during the manufacturing process. In this work, an empirical model for PBF-LB/M/Nb is developed. Based on this model, manufacturing parameter sets using different volume build-up rates are predicted and confirmed. They enable the manufacture of parts with homogeneous and crack-free microstructure with more than 99.9% relative density. Tensile and hardness tests of specimens, which were manufactured using different parameter sets, are performed to determine the effects of the build-up rate—and thus the heat flow during manufacturing—on different mechanical properties. The ultimate tensile strength and yield strength of as-manufactured specimens reach values up to 525 MPa and 324 MPa, respectively, while the elongation at break ranges between approximately 8 and 16%. The Vickers hardness of all specimens was in the range of 149 ± 8 HV0.1. In addition, the microstructure of the manufactured samples is investigated by means of light as well as scanning electron microscopy.

Phosphatidylethanol (PEth), which is formed by enzymatic reaction between ethanol and phosphatidylcholine, is a direct marker for alcohol usage. PEth has a long elimination half-life (~5-10 d) and specimens can be sampled using minimally invasive microsampling strategies. In combination with rapid analysis procedures PEth has proved to be advantageous for the detection of abstinence over other direct (e.g., ethyl glucuronide in blood, urine or hair) and indirect (e.g., carbohydrate-deficient transferrin in serum) alcohol markers. Although PEth determination is widely applied around the world, laboratory protocols are not standardized. Here we provide general guidelines for the analysis of PEth in dried blood spots (DBSs), including reference material evaluation, synthesis of a deuterated internal standard, preparation of calibration samples (reference material in teetotaller blood), and analyte separation and detection. The protocol contains information to extract the DBSs either manually or with a fully automated autosampler. Extraction of the analytes from DBS filter paper cards is performed using an organic extraction, followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). For accurate and reliable measurement of PEth, the two most abundant analogs, PEth 16:0/18:1 and PEth 16:0/18:2, are quantified. We show data that provide guidelines on how to interpret the results for both demographic studies and forensic applications. The described protocol can be applied by experienced laboratory staff with basic LC-MS/MS knowledge and takes 2 d to perform.

This work aimed to study the dysregulated network of galectins in OA chondrocyte pellets, and to assess whether their recently discovered activity as molecular switches of functional biomarkers results in degradation of extracellular matrix in vitro. Scaffold-free 3D pellet cultures were established of human OA chondrocytes. Expression and secretion of galectin(Gal)-1, -3, and -8 were monitored relative to 2D cultures or clinical tissue sections by RT-qPCR, immunohistochemistry and ELISAs. Exposure of 2D and 3D cultures to an in vivo-like galectin mixture (Gal-1 and Gal-8: 5 µg/ml, Gal-3: 1 µg/ml) was followed by the assessment of pellet size, immunohistochemical matrix staining, and/or quantification of MMP-1, -3, and -13. Application of inhibitors of NF-κB activation probed into the potential of intervening with galectin-induced matrix degradation. Galectin profiling revealed maintained dysregulation of Gal-1, -3, and -8 in pellet cultures, resembling the OA situation in situ. The presence of the galectin mixture promoted marked reduction of pellet size and loss of collagen type II-rich extracellular matrix, accompanied by the upregulation of MMP-1, -3, and -13. Inhibition of p65-phosphorylation by caffeic acid phenethyl ester effectively alleviated the detrimental effects of galectins, resulting in downregulated MMP secretion, reduced matrix breakdown and augmented pellet size. This study suggests that the dysregulated galectin network in OA cartilage leads to extracellular matrix breakdown, and provides encouraging evidence of the feasible inhibition of galectin-triggered activities. OA chondrocyte pellets have the potential to serve as in vitro disease model for further studies on galectins in OA onset and progression. Electronic supplementary material The online version of this article (10.1007/s00223-020-00774-4) contains supplementary material, which is available to authorized users.

The multikinase inhibitor sorafenib was the only approved systemic therapy in advanced hepatocellular carcinoma (HCC) for about a decade. In recent years, the number of approved agents has increased significantly as a result of a number of positive phase iii clinical trials. Lenvatinib as a first-line treatment, and regorafenib, cabozantinib, and ramucirumab in the second-line setting are now approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency. In phase II studies, immunotherapy with nivolumab and monotherapy using pembrolizumab yielded impressive results for overall survival in therapy-naïve and pretreated patients, leading to the accelerated approval by the FDA of nivolumab and pembrolizumab for second-line treatment. However, phase III trials of nivolumab in the first line and pembrolizumab in the second line as single agents failed to reach statistical significance, although clinical benefit for a subset of patients with long durations of response could be demonstrated. Despite that setback, immunotherapy for HCC is a promising therapeutic approach, and the combination of immunotherapy with other treatment modalities such as monoclonal antibodies, tyrosine kinase inhibitors, or local therapies has the potential to increase the overall response rate and survival. Recently, the results of a phase III trial of combination atezolizumab–bevacizumab compared with sorafenib showed a highly significant survival benefit and median overall survival that was not reached in the immunotherapy arm, making the combination the preferred standard of care in first-line therapy. Despite the impressive results and generally good toxicity profile of immunotherapy, patients who respond to therapy constitute only a subset of the overall population, and response rates are still limited. This review focuses on the currently reported results and ongoing clinical trials of checkpoint inhibitor–based immunotherapy in HCC.

Objectives In patients with LN, clinical and histological responses to treatment have been shown to be discordant. We investigated whether per-protocol repeat kidney biopsies are predictive of LN relapses and long-term renal function impairment. Methods Forty-two patients with incident biopsy-proven active proliferative (class III/IV±V) LN from the database of the UCLouvain were included in this retrospective study. Per-protocol repeat biopsies were performed after a median [interquartile range (IQR)] time of 24.3 (21.3–26.2) months. The National Institutes of Health activity index (AI) and chronicity index (CI) scores were assessed in all biopsies. Results Despite a moderate correlation between urinary protein/creatinine ratios (UPCR) and AI scores at repeat biopsy (r = 0.48; P = 0.001), 10 patients (23.8%) with UPCR 3). High AI scores (continuous) in repeat biopsies were associated with an increased probability and/or shorter time to renal relapse (n = 11) following the repeat biopsy [hazard ratio (HR) = 1.2, 95% CI: 1.1, 1.3; P = 0.007], independently of proteinuria levels. High CI scores (continuous) in repeat biopsies were associated with a sustained increase in serum creatinine levels corresponding to ≥120% of the baseline value (HR = 1.8, 95% CI: 1.1, 2.9; P = 0.016) through a median (IQR) follow-up time of 131.5 (73.8–178.2) months, being also the case for acute tubulointerstitial inflammation and interstitial fibrosis/tubular atrophy in repeat but not baseline biopsies. Conclusion Our results highlight the usefulness of per-protocol repeat biopsies, herein performed after a median time of 24 months from baseline, as an integral part of the treatment evaluation, also in patients showing adequate clinical response.

Phosphatidylethanol (PEth) in human blood samples is a marker for alcohol usage. Typically, PEth is detected by reversed-phase liquid chromatography coupled with negative ion tandem mass spectrometry, investigating the fatty acyl anions released from the precursor ion upon collision-induced dissociation (CID). It has been established that in other classes of asymmetric glycerophospholipids, the unimolecular fragmentation upon CID is biased depending on the relative position (known as sn-position) of each fatty acyl chain on the glycerol backbone. As such, the use of product ions in selected-reaction-monitoring (SRM) transitions could be prone to variability if more than one regioisomer is present in either the reference materials or the sample. Here, we have investigated the regioisomeric purity of three reference materials supplied by different vendors, labeled as PEth 16:0/18:1. Using CID coupled with ozone-induced dissociation, the regioisomeric purity (% 16:0 at sn-1) was determined to be 76, 80 and 99%. The parallel investigation of the negative ion CID mass spectra of standards revealed differences in product ion ratios for both fatty acyl chain product ions and ketene neutral loss product ions. Furthermore, investigation of the product ion abundances in CID spectra of PEth within authentic blood samples appears to indicate a limited natural variation in isomer populations between samples, with the cannonical, PEth 16:0/18:1 (16:0 at sn-1) predominant in all cases. Different reference material isomer distributions led to variation in fully automated quantification of PEth in 56 authentic dried blood spot (DBS) samples when a single quantifier ion was used. Our results suggest caution in ensuring that the regioisomeric compositions of reference materials are well-matched with those of the authentic blood samples.

Display of short peptides on the surface of adeno-associated viruses (AAVs) is a powerful technology for the generation of gene therapy vectors with altered cell specificities and/or transduction efficiencies. Following its extensive prior use in the best characterized AAV serotype 2 (AAV2), recent reports also indicate the potential of other AAV isolates as scaffolds for peptide display. In this study, we systematically explored the respective capacities of 13 different AAV capsid variants to tolerate 27 peptides inserted on the surface followed by production of reporter-encoding vectors. Single-round screening in pre-arrayed 96-well plates permitted rapid and simple identification of superior vectors in >90 cell types, including T cells and primary cells. Notably, vector performance depended not only on the combination of capsid, peptide, and cell type, but also on the position of the inserted peptide and the nature of flanking residues. For optimal data availability and accessibility, all results were assembled in a searchable online database offering multiple output styles. Finally, we established a reverse-transduction pipeline based on vector pre-spotting in 96- or 384-well plates that facilitates high-throughput library panning. Our comprehensive illustration of the vast potential of alternative AAV capsids for peptide display should accelerate their in vivo screening and application as unique gene therapy vectors.

Spatially and temporally resolved information on thermochemical conditions in hydrocarbon/air flames requires concentration and temperature data obtained by spontaneous Raman/Rayleigh scattering. During the combustion of renewable and more complex fuels, such as ethanol, partially oxidized hydrocarbons are created as intermediates in concentrations large enough to have an influence on the turbulence-chemistry interaction. To obtain concentrations and temperature in flames at atmospheric pressure with a high spatial and temporal resolution, information on the temperature dependent shape and scattering cross section of the major as well as the minor intermediate species is required. Therefore, a novel gas heater based on mixing the hydrocarbons with helium at high temperatures in combination with a multipass Raman spectrometer were developed, able to characterize ro-vibrational Raman scattering of gaseous ethanol in the gaseous phase up to high temperatures and in different spectral regions. The setup is described, its performance evaluated and Raman spectra of gaseous ethanol at atmospheric pressure up to high temperatures is demonstrated.

Background and aim Safety of regorafenib in hepatocellular carcinoma (HCC) recurrence after liver transplantation (LT) has been recently demonstrated. We aimed to assess the survival benefit of regorafenib compared to best supportive care (BSC) in LT-patients after sorafenib discontinuation. Methods This observational multicenter retrospective study included LT-patients with HCC-recurrence who discontinued first-line sorafenib. Group-1 was constituted by regorafenib-treated patients, while control group was selected among patients treated with best supportive care (BSC) due to unavailability of second-line options at the time of sorafenib discontinuation and who were sorafenib-tolerant progressors (group-2). Primary endpoint was overall survival (OS) of group-1 compared to group-2. Secondary endpoints were safety and OS of sequential treatment sorafenib+regorafenib/BSC. Results Among 132 LT-patients who discontinued sorafenib included in the study, 81 patients were sorafenib-tolerant: 36 received regorafenib (group-1) and 45 (group-2) received BSC. Overall, 24 (67%) patients died in group-1 and 40 (89%) in group-2: the median OS was significantly longer in group-1 than in group-2 (13.1 vs 5.5 months; p=0.002). Regorafenib treatment was an independent predictor of reduced mortality (HR 0.37, 95%CI 0.16-0.89, p=0.02). Median treatment duration with regorafenib was 7.0 (95%CI 5.5-8.5) months; regorafenib dose was reduced in 22 (61%) patients for adverse events and discontinued for tumor progression in 93%. The median OS calculated from sorafenib start was 28.8 months (95%CI: 17.6-40.1) in group-1 vs 15.3 months (95%CI: 8.8-21.7) in group-2 (p=0.002). Conclusions Regorafenib is an effective second-line treatment after sorafenib in patients with HCC-recurrence after LT.

Objective Functional cartilage repair requires the new formation of organized hyaline cartilaginous matrix to avoid the generation of fibrous repair tissue. The potential of mesenchymal progenitors was used to assemble a 3-dimensional structure in vitro, reflecting the zonation of collagen matrix in hyaline articular cartilage. Design The 3-dimensional architecture of collagen alignment in pellet cultures of chondroprogenitors (CPs) was assessed with Picrosirius red staining analyzed under polarized light. In parallel assays, the trilineage capability was confirmed by calcium deposition during osteogenesis by alizarin S staining and alkaline phosphatase staining. Using reverse transcription–quantitative polymerase chain reaction (RT-qPCR), mRNA levels of ALP, RUNX2, and BGLAP were assessed after 21 days of osteoinduction. Lipid droplets were stained with oil red O and adipogenic differentiation was confirmed by RT-qPCR analysis of PPARG and LPL gene expression. Results Under conditions promoting the chondrogenic signature in self-assembling constructs, CPs formed an aligned extracellular matrix, positive for glycosaminoglycans and collagen type II, showing developing zonation of birefringent collagen fibers along the cross section of pellets, which reflect the distribution of collagen fibers in hyaline cartilage. Induced osteogenic and adipogenic differentiation confirmed the trilineage potential of CPs. Conclusion This model promotes the differentiation and self-organization of postnatal chondroprogenitors, resulting in the formation of zonally organized engineered hyaline cartilage comparable to the 3 zones of native cartilage.

Several studies showed that chronic ethanol exposure can cause color vision deficiencies. There has been no agreement about the axis of color defects due to alcohol misuse since changes in the red-green and the blue-yellow axis have been described in literature. The acute influence of alcohol on the blue-yellow color vision has not been studied as well. The aim of this study was to determine the effect of acute alcohol ingestion on blue-yellow color vision by using short wavelength automated perimetry (SWAP) and anomaloscopy with the Moreland equation. This is the first study evaluating that question by using SWAP and anomaloscopy. Sixteen healthy subjects without a history of alcohol-related and ophthalmological problems were examined by SWAP and anomaloscopy (Moreland equation) before and after alcohol ingestion. Mean sensitivity (MS), mean deviation (MD), loss of variance (LV), reliability factor (RF), and duration of examination were assessed for perimetry and match midpoint (MP), matching range (MR), and duration of examination for anomaloscopy. Blood alcohol concentrations (BAC) were determined by gas chromatography and phosphatidylethanol concentrations (marker of an alcohol misuse) by liquid-chromatography tandem-mass spectrometry in venous blood samples from a cubital vein. Mean blood BAC was 0.86 ± 0.20 g/kg while performing perimetry and 0.84 ± 0.20 g/kg while performing anomaloscopy (BAC: 0.1 g/kg ≈ 0.01 g/dL). MS, MD, RF, MP, MR, and duration of perimetry examination were not altered significantly after alcohol intake. LV showed a significant increase. The duration of anomaloscope testing was shortened significantly under the influence of alcohol. The subjects also revealed a significantly narrower matching range after alcohol intake. In the range of 0.8 g/kg BAC, no blue-yellow vision deficiencies could be demonstrated. In further studies, the effect of higher BAC on blue-yellow vision should be investigated by different methods.

The aim of the present study was to evaluate the cytotoxicity of five synthetic cannabinoids in two cell lines, for which both reference standards and herbal blends were available. An in-house smoking device was developed to produce smoke condensates. The cell viability of reference standards, herbal blend extracts and smoke condensates was measured using an MTT assay in the A549 lung carcinoma cell line and the TR146 buccal carcinoma cell line. Damiana extracts and damiana smoke condensates were also tested for cytotoxicity. For the reference standards of 5F-MDMB-PINACA, ADB-CHMINACA and MDMB-CHMICA, a significant concentration-dependent decrease in cell viability was observed. ADB-CHMINACA and MDMB-CHMICA extracts were more potent than the damiana extract, whereas the NM-2201 blend extract abrogated the effects of the damiana leaves. Compared with damiana smoke condensate, MDMB-CHMICA smoke condensate had higher sensitivity, while ADB-CHMINACA smoke condensate was less potent. This is the first study to investigate the comparative cytotoxicity of reference standards, herbal blend extracts and smoke condensates of synthetic cannabinoids. The data showed that the presence of damiana plant contributed to enhancing the cytotoxicity of 5C-AKB48, 5F-MDMB-PINACA, and MDMB-CHMICA. The plant effects were generally more marked for smoke condensates. MDMB-CHMICA was the most potent substance. The NM-2201 reference standard and extract led to an increase in cell viability as compared to the control. This is of special interest, because the NM-2201 herbal blend extract seemed to abrogate the effects of the damiana leaf extracts. Further studies would be necessary to assess potential procarcinogenic or tumorigenic effects of NM-2201.

Mineralocorticoid receptor antagonists have beneficial effects on left ventricular remodeling, cardiac fibrosis, and arrhythmia in heart failure, but efficacy and safety in dialysis patients is less clear. We evaluated the effect of spironolactone on left ventricular mass (LVM), an independent predictor of all-cause and cardiovascular mortality, in hemodialysis patients. In this placebo-controlled, parallel-group trial, 97 hemodialysis patients (23% female; mean age 60.3 years) were randomized to spironolactone 50 mg once daily (n=50) or placebo (n=47). The primary efficacy endpoint was change in LVM index (LVMi) from baseline to 40 weeks as determined by cardiac magnetic resonance imaging. Safety endpoints were development of hyperkalemia and change in residual renal function. There was no significant change in LVMi in participants randomized to spironolactone compared to placebo (-2.86±11.87 vs. 0.41±10.84 g/m2). There was also no difference in the secondary outcomes of mean 24-hour systolic or diastolic ambulatory blood pressure, left ventricular ejection fraction, 6-minute walk test distance, or New York Heart Association functional class. Moderate hyperkalemia (pre-dialysis potassium levels of 6.0-6.5 mmol/L) was more frequent with spironolactone treatment (155 vs. 80 events), but severe hyperkalemia (≥6.5 mmol/L) was not (14 vs. 24 events). Changes in residual urine volume and measured glomerular filtration rate did not differ between groups. There were no deaths in the spironolactone group and 4 deaths in the placebo group. Thus, treatment with 50 mg spironolactone did not change left ventricular mass index, cardiac function, or blood pressure in hemodialysis patients. Spironolactone increased the frequency of moderate hyperkalemia, but did not increase severe hyperkalemia.

The transition from silenced heterochromatin to a biologically active state and vice versa is a fundamental part of the implementation of cell type-specific gene expression programs. To reveal structure-function relationships and dissect the underlying mechanisms, experiments that ectopically induce transcription are highly informative. In particular, the approach to perturb chromatin states by recruiting fusions of the catalytically inactive dCas9 protein in a sequence-specific manner to a locus of interest has been used in numerous applications. Here, we describe how this approach can be applied to activate pericentric heterochromatin (PCH) in mouse cells as a prototypic silenced state by providing protocols for the following workflow: (a) Recruitment of dCas9 fusion constructs with the strong transcriptional activator VPR to PCH. (b) Analysis of the resulting changes in chromatin compaction, epigenetic marks, and active transcription by fluorescence microscopy-based readouts. (c) Automated analysis of the resulting images with a set of scripts in the R programming language. Furthermore, we discuss how parameters for chromatin decondensation and active transcription are extracted from these experiments and can be combined with other readouts to gain insights into PCH activation.

Cosmic Ray Energetics And Mass for the International Space Station (ISS-CREAM) was designed to study high-energy cosmic rays up to PeV and recorded data from August 22nd, 2017 to February 12th, 2019 on the ISS. In this analysis, the Silicon Charge Detector (SCD), CALorimeter (CAL), and Top and Bottom Counting Detectors (TCD/BCD) are used. The SCD is composed of four layers and provides the measurement of cosmic-ray charges with a resolution of $\sim$0.2e. The CAL comprises 20 interleaved tungsten plates and scintillators, measures the incident cosmic-ray particles' energies, and provides a high energy trigger. The TCD/BCDs consist of photodiode arrays and plastic scintillators and provide a low-energy trigger. In this analysis, the SCD top layer is used for charge determination. Here, we present the heavy nuclei analysis using the ISS-CREAM instrument.

DNA-encoded libraries (DEL) are increasingly being used to identify new starting points for medicinal chemistry in drug discovery. Herein, we discuss the development of methods that allow the conversion of both primary amines and anilines, attached to DNA, to their corresponding azides in excellent yields. The scope of these diazo-transfer reactions was investigated, and a proof-of-concept has been devised to allow for the synthesis of macrocycles on DNA.

Purpose N-Glycan profiling provides an indicator of the cellular potential for functional pairing with tissue lectins. Following the discovery of galectin expression by chondrocytes as a factor in osteoarthritis pathobiology, mapping of N-glycans upon their phenotypic dedifferentiation in culture and in fibroblast-like synoviocytes is a step to better understand glycobiological contributions to disease progression. Experimental design The profiles of cellular N-glycans of human osteoarthritic chondrocytes and fibroblast-like synoviocytes were characterized by mass spectrometry. RT-qPCR experiments determined mRNA levels of 16 glycosyltransferases. Responsiveness of cells to galectins was quantified by measuring the mRNA level for interleukin-1β. Results The shift of chondrocytes to a fibroblastic phenotype (dedifferentiation) is associated with changes in N-glycosylation. The N-glycan profile of chondrocytes at passage 4 reflects characteristics of synoviocytes. Galectins-1 and -3 enhance expression of interleukin-1β mRNA in both cell types, most pronounced in primary culture. Presence of interleukin-1β leads to changes in sialylation in synoviocytes that favor galectin binding. Conclusions and clinical relevance N-Glycosylation reflects phenotypic changes of osteoarthritic cells in vitro. Like chondrocytes, fibroblast-like synoviocytes express N-glycans that are suited to bind galectins, and these proteins serve as inducers of pro-inflammatory markers in these cells. Synoviocytes can thus contribute to disease progression in osteoarthritis in situ. This article is protected by copyright. All rights reserved.

Considering the high clinical and forensic relevance of pharmaco-adherence during lisdexamphetamine (LDX) treatment for attention-deficit/hyperactivity disorder (ADHD), the aim here was to evaluate hair analysis as a tool for monitoring compliance in patients currently undergoing long term treatment with LDX, by detecting possible interruptions of medication intake or changes in dosage. For this purpose, a total of 24 patients from an outpatient clinic for ADHD were recruited. Hair and urine samples were taken after three consecutive therapy sessions over a 7-month period and analyzed for amphetamine (AMP) enantiomers and other drugs, using chiral and achiral liquid chromatography-tandem mass spectrometry (LC-MS/MS). Participants also provided information on the condition of their hair, the consumption of illegal psychotropic substances and the regularity of taking LDX. Two participants withdrew from the study early. Urine analyses were positive for D-AMP in all urine samples and therapy sessions, except in two patients who did not take LDX on a daily basis. D-AMP was detected in all hair samples; however, no correlation was found between prescribed dose/day and D-AMP concentrations in proximal hair segments. Qualitative interpretation of hair analysis showed that 18 of the 22 study completers were compliant concerning the intake of LDX without additional consumption of illegal D,L-AMP. Analysis of urine taken during the therapy sessions showed no correlation between D-AMP concentrations and prescribed dosage, with or without normalization for creatinine. In conclusion, chiral LC-MS/MS hair analysis might represent a non-invasive way to confirm LDX use within the approximate period covered by the hair segment tested, but it does not allow for quantitative therapeutic drug monitoring because of interindividual variability of concentrations in hair. Drug concentrations in hair at different stages of long-term treatment should thus be interpreted with caution by clinicians and forensic experts alike when making assessments of treatment adherence.

Alcohol-related disorders are common, expensive in their entire course, and often underdiagnosed. To facilitate early diagnosis and therapy of alcohol-related disorders and thus prevent later complications, questionnaires and biomarkers are useful. Indirect state markers such as gamma-glutamyl transpeptidase (GGT), mean corpuscular volume (MCV), and carbohydrate deficiency transferrin (CDT) are influenced by age, gender, various substances, and nonalcohol-related illnesses and do not cover the entire timeline for alcohol consumption. Direct state markers such as ethyl glucuronide (EtG), phosphatidylethanol (PEth), and fatty acid ethyl esters (FAEEs) have gained enormous interest in the last decades, as they are metabolites of alcohol becoming only positive in the presence of alcohol. As biomarkers with high sensitivity and specificity covering the complimentary timeline, they are already routinely in use and contribute to new perspectives in prevention, interdisciplinary cooperation, diagnosis, and therapy of alcohol-related disorders.

BACKGROUND Alcohol contributes to numerous annual deaths and various societal problems not just in adult, but also in adolescent, populations. Therefore, it is vital to find methods for reliably detecting alcohol use for early preventative measures. Research has shown phosphatidylethanol (PEth) to be superior to self-report instruments and indirect biomarkers for alcohol consumption in adult populations. However, the transferability onto an adolescent population has not yet been investigated. METHODS N = 106 adolescents and young adults aged between 13 and 21 years were included. PEth analysis using high-pressure liquid chromatography-tandem mass spectrometry was performed on dried blood spot samples. Self-report questionnaires for alcohol consumption (Alcohol Use Disorders Identification Test-Consumption, AUDIT-C, and Timeline Followback, TLFB) and drug and alcohol consumption (Detection of Alcohol and Drug Problems in Adolescents, DEP-ADO) were completed by each participant. RESULTS AUDIT-C scores showed large correlations with PEth 16:0/18:1 (rs = 0.732) and PEth 16:0/18:2 (rs = 0.661) concentrations. AUDIT-C with a cutoff value ≥3 was largely correlated with PEth 16:0/18:1 (η = 0.411) and showed a medium-sized correlation with PEth 16:0/18:2 (η = 0.397) concentrations. Using an AUDIT-C cutoff value ≥5 showed large correlations with both PEth 16:0/18:1 (η = 0.510) and PEth 16:0/18:2 (η = 0.497) concentrations, respectively. ROC curves indicated higher PEth concentrations are a good model for detecting positive AUDIT-C cutoff values (AUROC range: 0.800 to 0.849). PEth concentrations showed medium to large correlations with DEP-ADO and TLFB subscales (range rs = 0.469 to 0.746). CONCLUSION The results suggest that PEth is a reliable and objective marker for quantifying alcohol consumption in adolescents and young adults. This could be of importance for early preventative measures against hazardous alcohol consumption, which is increasingly common at younger ages.

The assessment of alcohol consumption behavior in hair is well established in forensic toxicology. The Society of Hair Testing (SoHT) recommends the direct alcohol markers ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEE) for the detection of past alcohol consumption. In this study, we investigated if detox shampoos which are sold online can have an impact on EtG or FAEE concentrations in hair. According to customer reviews, positive drug testing results could be avoided after long-term incubation with shampoo under a swimming cap. To evaluate the potential of four detox shampoos, we incubated distal hair samples from three subjects, obtained during a standard haircut, for 2.5, 5, 7.5, and 10 hours. For EtG, three shampoos performed similar to deionized water. The fourth shampoo showed additional heavy washout effects with a decrease of up to 86% after 2.5 hours. For the apolar FAEE, no washout was observed. Incubation with two shampoos resulted in increases in FAEE concentrations due to FAEE being present as shampoo ingredients. Further investigation of EtG washout in proximal forensic hair samples (n = 9) with the most potent shampoo showed a mean decrease in deionized water of 23% ± 25%, and a decrease by the use of detox shampoo of 73% ± 12%, compared to non-incubated hair after 8 hours. In conclusion, detox shampoos proved to have the potential to alter EtG and FAEE concentrations in hair during in vitro experiments.

Highly potent cannabis concentrates obtained by butane or by supercritical carbon dioxide-extraction are gaining popularity. These extracts called butane hash oil (BHO) with Δ9-tetrahydrocannabinolic acid A (THCA) contents above 60% are consumed by flash vaporization on a glowing titanium nail, followed by inhalation of the resulting vapor through a water pipe in a single puff — a technique referred to as “dabbing”. We herein investigated the decarboxylation rate of THCA during artificial smoking of cannabis plant material and simulated dabbing, and the lung availability of Δ9-tetrahydrocannabinol (THC) which we define as the recovery of THC in the smoke and vapor condensates. Preliminary smoking and dabbing experiments were performed using an apparatus built in-house. Due to availability of cannabidiol (CBD)-rich hemp in Switzerland, we included a sample of CBD flowers in our experiments and investigated the decarboxylation and recovery of cannabidiolic acid (CBDA) and CBD, respectively. Decarboxylation of THCA and CBDA during combustion of the plant material and vaporization of the BHO, respectively, was complete. The high recovery of total THC (75.5%) by dabbing cannot be achieved by smoking marijuana. Lung availability ranged from 12% for mixed cannabis material with a rather low THC content, to approximately 19–27% for marijuana flowers, similar for THC in marijuana as for CBD in CBD-rich marijuana. In reality, when smoking a joint, further losses in recovery must be assumed by additional sidestream smoke. The rather high lung availability of THC via dabbing can explain the increased psychoactive and adverse effects associated with this new trend of cannabis consumption.

Direct and indirect biomarkers are widely applied for the determination of alcohol consumption. They help to assess past or present alcohol consumption. Depending on the window of detection and sensitivity of the investigated marker, punctual alcohol consumption may remain undetected. In this study, different sampling strategies for the intermediary long-term marker phosphatidylethanol (PEth) are evaluated and compared to the determination of the short-term markers ethyl glucuronide (EtG) and ethyl sulfate (EtS) in urine. Samples from 19 patients undergoing alcohol use disorder treatment were collected during the withdrawal treatment and successive rehabilitation (33 ± 26 days (range: 3-74 days)). With liquid chromatography-tandem mass spectrometry (LC-MS/MS) EtG and EtS in urine, PEth in blood, PEth in dried blood spot (DBS) from venous blood, and PEth in DBS from capillary blood were quantified and compared. The use of volumetric capillary DBS, prepared from 20 μL of blood, provided the same results as the use of venous DBS (95% ± 10%, R 0.9899 for PEth 16:0/18:1). Capillary DBS sampling has the advantage that it can be performed without venipuncture. The use of PEth in DBS proved to prevent post-sampling degradation, providing a longer detection in comparison to PEth in liquid blood, which only showed 67% ± 24% of the PEth DBS 16:0/18:1 concentration. When compared with EtG and EtS in urine, PEth monitoring proved to be advantageous for the detection of relapse situations, as the accumulation of PEth in blood prolongs the detectability. In conclusion, volumetric capillary DBS sampling for PEth is a simple and useful tool for compliance monitoring, and avoids hematocrit issues.

Background The AOAC Stakeholder Panel on Strategic Food Analytical Methods approved Standard Method Performance Requirements (SMPR®) 2018.009 for lactose in low-lactose or lactose-free milk, milk products, and products containing dairy ingredients. The LactoSens®R Method is a biosensor assay kit developed for the determination of lactose in a variety of lactose-free or low-lactose milk, dairy, and infant formula products produced with yeast-neutral lactases. Objective In response to a call for methods, the LactoSensR method was validated in a single laboratory study with comparison to SMPR 2018.009. Method The LactoSensR method was evaluated for calibration, interference, repeatability, recovery, and robustness. In a method comparison study samples naturally containing low levels of lactose were evaluated using LactoSensR and an accredited high-performance anion-exchange chromatography with pulsed amperometric detection. Results Calibration with lactose standard solutions was shown to be linear and the method was shown to be free of interference from a variety of sugars, vitamins, alcohols, flavorings, and other compounds. Matrix studies, including 85 spiked materials, 55 products naturally containing lactose, and 13 reference materials, resulted in RSDr of 0–10.5% at 8–100 mg lactose/100 g and 0.2–5.4% at >100 mg lactose/100 g for milk and dairy products and 1.0–6.8% for infant formula, in compliance with SMPR 2018.009 with few exceptions. Recovery was 85.0–110.3% at 8–100 mg lactose/100 g and 85.6–109.7% at >100 mg lactose/100 g for milk and dairy products and 91.1–97.0% for infant formula, also meeting the performance requirements with few exceptions. The method was shown to be robust to changes in ambient temperature, sample temperature, and sample volume. Conclusions The LactoSensR method compares favorably with the requirements of SMPR 2018.009 and should be adopted as a First Action AOAC Official MethodSM. Highlights The LactoSensR method is a fast, easy-to-use method that meets the requirements of SMPR 2018.009.

Phosphatidylethanol (PEth) is an alcohol biomarker formed from phosphatidylcholine (PC) by the enzyme phospholipase D (PLD) in the presence of ethanol. A drinking study revealed individual differences in maximum PEth levels after drinking to a targeted blood alcohol concentration (BAC) of 0.1%. This seemed to be due to different PLD activities in the tested persons. Furthermore, post-sampling formation of PEth occurred in blood samples, still containing alcohol. Therefore, a standardized in vitro test for measuring individual PEth formation rates was developed. Two PLD inhibitors were tested for their potency to inhibit post-sampling PEth formation. PEth-negative blood samples were collected from a volunteer. Ethanol was added in different concentrations (0.01-0.3% BAC) directly after blood sampling. The specimens were incubated at 37 °C. Aliquots were taken at the start of the incubation, and every hour until 8 h after start of incubation, and one sample was taken on subsequent days over 1 week. PEth 16:0/18:1 and PEth 16:0/18:2 were determined by online SPE-LC-MS/MS. Furthermore, this test system was applied to blood samples of 12 volunteers. For the inhibition tests, fresh blood (spiked with 0.1% ethanol) was spiked with 30, 300, 3000, or 30,000 nM of either halopemide or 5-fluoro-2-indolyl-deschlorohalopemide (FIPI), and incubated at 37 °C. PEth concentrations were determined hourly over 5 h on the first day and once on day 2 and day 3. PEth formation was linear in the first 7 h of incubation and dependent on the alcohol concentration. The formation rates of PEth 16:0/18:1 were 0.002 μmol L-1 h-1 (0.01% BAC), 0.016 μmol L-1 h-1 (0.1% BAC), 0.025 μmol L-1 h-1 (0.2% BAC), and 0.029 μmol L-1 h-1 (0.3% BAC). For PEth 16:0/18:2, the formation rates were 0.002 μmol L-1 h-1 (0.01% BAC), 0.019 μmol L-1 h-1 (0.1% BAC), 0.025 μmol L-1 h-1 (0.2% BAC), and 0.030 μmol L-1 h-1 (0.3% BAC). Maximum concentrations reached 431 ng/mL (PEth 16:0/18:1) and 496 ng/mL (PEth 16:0/18:2) at 0.3% BAC after 3 days. Maximum velocity (vmax) was not reached under these conditions. PEth formation in blood of the 12 volunteers ranged between 0.011 and 0.025 μmol L-1 h-1 for PEth 16:0/18:1 and between 0.014 and 0.021 μmol L-1 h-1 for PEth 16:0/18:2. PEth formation in human blood was inhibited by halopemide in a concentration-dependent manner. However, a complete inhibition was not achieved by the applied maximum concentration of 30,000 nM. FIPI showed a better inhibition of PEth formation. A complete inhibition could be achieved by a concentration of 30,000 nM for the first 24 h (for PEth 16:0/18:1) and for 48 h (for PEth 16:0/18:2). Formation of PEth was found to be dependent on the BAC. As a consequence, it is essential to inhibit PLD activity after blood collection to avoid post-sampling formation of PEth in blood samples with a positive BAC. Inhibition of PEth formation was more effective using FIPI, compared to halopemide.

The sulfoximine group has been reported as a versatile and beneficial functionality for pharmaceutical or agrochemical entities. Herein, we report the Csp2–H sulfoximidation of electron-rich arenes ­under the irradiation of blue light using an organic acridinium photocatalyst and molecular oxygen or peroxodisulfates as terminal oxidants. The method allows for the late-stage introduction of various sulfoximines onto complex bioactive compounds showing high functional group compatibility without the need for prefunctionalization.

Orthopaedic implant materials are exposed to high mechanical loading. Conventional materials based on stainless steel or cobalt-chromium alloys display adequate mechanical strength, but also toxicological concerns due to release of toxic or allergenic elements (particles or ions) resulting in inflammatory reactions in the adjacent tissues. Metal alloys based on titanium, tantalum and niobium represent higher biocompatibility with appropriate mechanical properties for avoiding stress-shielding and consecutive implant loosening. Application of specifically designed spherical Ti/Nb(/Ta) alloy powders in additive manufacturing, i.e. 3D metal printing processes such as selective laser melting (SLM) or electron beam melting (EBM), enable the production of components with a high degree in freedom of design. Accordingly, SLM or EBM of Ti/Nb(/Ta) alloys offer the possibility to fabricate patient-specific orthopaedic implants. The present paper gives an overview on the development of bio-compatible Ti/Nb(/Ta) alloys especially designed for application in 3D metal printing. Furthermore, the additively manufactured 3D structures are mechanically tested and the influence of differently grained alloy powders and bulk parts on the viability and proliferation of human cells (fibroblasts, osteoblasts) are examined.

We report initial results from an ion mobility spectrometry (IMS)-based analysis of natural cannabis samples and explore the possibility of using this technique to distinguish medical marijuana from illegal forms of the drug, as defined by Swiss legislation. We analyzed cannabis extracts by electrospray ionization IMS-MS and found that high-resolution drift-tube IMS ( R > 150) can effectively isolate and quantify the controlled substance, Δ9-tetrahydrocannabinol (THC), even in the presence of other noncontrolled cannabinoid isomers including cannabidiol (CBD). We used this information to determine whether the THC content of a given sample surpassed the legal limit, which is 1% by weight in Switzerland. Our IMS-MS methodology produced equivalent quantification results to standard HPLC-based methods and offers the additional advantage of significantly shorter time requirements for the analysis. In addition, IMS-based analysis offers flexibility over HPLC in that it can be performed on portable devices. As such, these findings may have implications for cannabis testing in police laboratories.

The reading of glycan-encoded signals by tissue lectins is considered a major route of the flow of biological information in many (patho)physiological processes. The arising challenge for current research is to proceed from work on a distinct protein to family-wide testing of lectin function. Having previously identified homodimeric galectin-1 and chimera-type galectin-3 as molecular switches in osteoarthritis progression, we here provide proof-of-principle evidence for an intra-network cooperation of galectins with three types of modular architecture. We show that the presence of tandem-repeat-type galectin-8 significantly correlated with cartilage degeneration and that it is secreted by osteoarthritic chondrocytes. Glycan-inhibitable surface binding of galectin-8 to these cells increased gene transcription and the secretion of functional disease markers. The natural variant galectin-8 (F19Y) was less active than the prevalent form. Genome-wide array analysis revealed induction of a pro-degradative/inflammatory gene signature, largely under control of NF-κB signaling. This signature overlapped with respective gene-expression patterns elicited by galectins-1 and -3, but also presented supplementary features. Functional assays with mixtures of galectins that mimic the pathophysiological status unveiled cooperation between the three galectins. Our findings shape the novel concept to consider individual galectins as part of a so far not realized teamwork in osteoarthritis pathogenesis, with relevance beyond this disease. Electronic supplementary material The online version of this article (10.1007/s00018-018-2856-2) contains supplementary material, which is available to authorized users.

This study aimed to evaluate the chondroprotective and anti-inflammatory activity of brazilin in human osteoarthritic (OA) cartilage and chondrocytes with particular focus on the nuclear factor-kappa B (NF-κB) pathway. Therefore, brazilin was isolated from Caesalpinia sappan and identified using high performance liquid chromatography (HPLC). The effect of brazilin was assessed in cartilage explants treated with 10 ng/ml interleukin (IL)-1β and 10 ng/ml tumor necrosis factor (TNF)-α using histological and biochemical glycosaminoglycan (GAG) analyses and in primary chondrocytes treated with 10 ng/ml IL-1β using RT-qPCR, ELISA, and Western blot. The involvement of NF-κB signaling was examined using a human NF-κB signaling array and in silico pathway analysis. Brazilin was found to reduce the GAG loss from cartilage explants stimulated with IL-1β and TNF-α. NF-κB pathway analysis in chondrocytes revealed NFKB1/p50 as a central player regulating the anti-inflammatory activities of brazilin. Brazilin suppressed the IL-1β-mediated up-regulation of OA markers and the induction of NFKB1/p50 in chondrocytes. In conclusion, brazilin effectively attenuates catabolic processes in human OA cartilage and chondrocytes-at least in part due to the inhibition of NFKB1/p50-which indicates a chondroprotective potential of brazilin in OA. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2431-2438, 2018.

Researchers are intensifying efforts to understand the mechanisms by which changes in metabolic states influence differentiation programs. An emerging objective is to define how fluctuations in metabolites influence the epigenetic states that contribute to differentiation programs. This is because metabolites such as S-adenosylmethionine, acetyl-CoA, α-ketoglutarate, 2-hydroxyglutarate, and butyrate are donors, substrates, cofactors, and antagonists for the activities of epigenetic-modifying complexes and for epigenetic modifications. We discuss this topic from the perspective of specialized CD4+ T cells as well as effector and memory T cell differentiation programs. We also highlight findings from embryonic stem cells that give mechanistic insight into how nutrients processed through pathways such as glycolysis, glutaminolysis, and one-carbon metabolism regulate metabolite levels to influence epigenetic events and discuss similar mechanistic principles in T cells. Finally, we highlight how dysregulated environments, such as the tumor microenvironment, might alter programming events.

In the presence of alcohol, phosphatidylcholine (PC) is transformed to the direct alcohol biomarker phosphatidylethanol (PEth). This reaction is catalyzed by the enzyme phospholipase D (PLD) and dependent on substrate availability. As recent methods have solely focused on the determination of PEth, information about the PC composition was generally missing. To address this issue and monitor PC (16:0/18:1 and 16:0/18:2) and PEth (16:0/18:1 and 16:0/18:2) simultaneously, a reversed phase LC-MS/MS method based on a C8 core-shell column, coupled to a Sciex 5500 QTrap instrument was developed. By application of polarity switching, at first, PC was measured in ESI positive SRM mode, while PEth was determined at a later stage in ESI negative SRM mode. The PEth method was validated for human blood samples to show its robustness and subsequently applied for the investigation of systematic in vitro PEth formation in animal tissue samples (brain, kidney, liver, and blood) from a pig, a calf, and a goat. Homogenized tissue was incubated at 37°C with varying ethanol concentrations from 1 to 7g/kg (determined by HS-GC-FID) for 5h, whereby a sample was taken every 30min. For all tissue samples, an increase in PEth was measurable. PEth concentrations formed in blood remained below the LLOQ, in agreement with literature. Data analysis of Michaelis-Menten kinetics and PC within the tissue provided a detailed insight about PEth formation, as the occurrence of PEth species can be linked to the observed PC composition. The results of this study show that PEth formation rates vary from tissue to tissue and among different species. Furthermore, new recommendations for PEth analysis are presented.

We studied the influence of periodic growth interruptions during co-deposition of diindeno-perylene (DIP) and buckminsterfullerene (C60) in an equimolar mixing ratio. DIP and C60 are known to phase-separate when co-deposited, but the details and in particular the length scales depend on kinetic factors. Using X-ray scattering and atomic force microscopy, we demonstrate that the phase separation mechanism is in fact influenced by growth interruptions, with more pronounced effects if the deposition rates are low. For high deposition rates, growth interruptions have no appreciable effect. We discuss our proof of the concept investigation in the context of the relevant processes and their time scales.

To investigate the potential of N-acetyltaurine (NAcT) in blood as a biomarker for alcohol uptake, a previously published LC-MS/MS method for urine was modified to simultaneously detect NAcT and ethyl glucuronide (EtG). The method was applied in a drinking study and by analyzing 147 forensic case samples. In the drinking study, contrary to EtG, NAcT proved to be an endogenous substance, which was present at 22 ± 7 ng/mL (13-31 ng/mL) in the blood after 2 weeks of abstinence. A moderate increase in NAcT to 40 ± 10 ng/mL (27-57 ng/mL) was observed after drinking. Within 24 h, the NAcT concentrations declined to starting concentrations in seven out of eight subjects. Peak EtG concentrations (c¯max) of 445 ± 101 ng/mL (278-662 ng/mL) were reached. While EtG in blood can be used to detect alcohol consumption even if ethanol is already eliminated, some of the maximum NAcT concentrations after a single ethanol dose were in the range of endogenous levels detected prior to the start of drinking in other subjects. In the 147 blood samples, the following concentrations were found: blood alcohol concentration (BAC): 1.22 ± 0.95 g/kg (0-3.46 g/kg); NAcT: 37.8 ± 18.4 ng/mL (12.1-109 ng/mL); EtG: 1149 ± 1121 ng/mL (0-5950 ng/mL). ROC curve analysis for BAC thresholds at 0.8 and 1.6 g/kg were performed for EtG and NAcT. Due to the presence of endogenous NAcT levels resulting in a lower sensitivity and selectivity when compared to EtG, and due to a minor increase in concentration after alcohol uptake, the usefulness of NAcT as an alcohol biomarker in blood is very limited.

We have recently introduced a 1,4,7-triazacyclononane (TACN) based chelating system with additional five-membered azaheterocyclic substituents for complexation of radioactive Cu2+ ions. In this work, we investigated the complexation properties of these novel chelators with Ga3+. In labeling experiments, we could show that the penta- and hexadentate imidazole derivatives NODIA-Me 4 and NOTI-Me 1 can be labeled with 68Ga in specific activities up to ∼30 MBq nmol-1, while the corresponding thiazole derivative NOTThia 2 did not label satisfactorily under identical conditions. NMR studies on the Ga complexes of 1 and the model compound NODIA-Me-NH-Me 5 revealed formation of rigid 1:1 chelates with a slow macrocyclic interconversion and inert Ga-N bonds to the methylimidazole residues on the NMR time scale. The TACN-derived bifunctional chelator NODIA-Me was furthermore conjugated to a prostate-specific membrane antigen (PSMA) targeting moiety to give the corresponding bioconjugate NODIA-Me-PSMA 7. Serum stability and ligand challenge experiments of 68Ga-7 confirmed formation of a stable complex for up to 4 h. The remaining coordination site of five-coordinate Ga complexes was found to be occupied by monodentate ligands including hydroxide and chloride anions depending on the conditions. According to density functional theory calculations, coordination of monodentate ligands as well as of the amide group for the bioconjugated ligand are energetically plausible. Finally, the labeled bioconjugate 68Ga-7 exhibited rapid renal clearance in biodistribution studies performed by small animal PET imaging with no indication of transchelation/demetalation in vivo. Altogether, our results provide strong evidence for a stable Ga complexation of our novel TACN-based chelators bearing imidazole arms. Despite the formation of two complexes incorporating different monodentate ligands in vitro, the imidazole type ligands show promise as chelating agents for the future development of gallium based radiopharmaceuticals.

The adsorption mechanism for three peptides was studied under overloaded conditions through adsorption isotherm measurements in the presence of an ion-pairing reagent, trifluoroacetic acid (TFA), on an end-capped C18-bonded stationary phase. The overall aim of the study was to obtain a better understanding of how the acetonitrile and the TFA fractions in the eluent affected the overloaded elution profiles and the selectivity between peptides using mechanistic modelling and multivariate design of experiments. When studying the effect of TFA, direct evidence for ion pair formation between a peptide and TFA in acetonitrile-water solutions was provided by fluorine-proton nuclear Overhauser NMR enhancement experiments and the adsorption of TFA on the stationary phase was measured by frontal analysis. The adsorption isotherms for each peptide were then determined by the inverse method at eight TFA concentrations ranging from 2.6mM to 37.3mM (0.02-0.29vol-%) in isocratic elution. The equilibrium between the peptide ion and the peptide-TFA complex was modelled by coupling the mass-balance to reaction kinetics and determining separate adsorption isotherms for the two species. We found that a Langmuir isotherm described the elution profile of peptide-TFA complex well while the peptide ion was described by a bi-Langmuir adsorption isotherm since it exhibited strong secondary interactions. The elution profiles had an unfavorable shape at low TFA concentrations consisting of a spike in their front and a long tailing rear due to the secondary interactions for the peptide ion having very low saturation capacity. The acetonitrile dependence on the adsorption isotherms was studied by determination of adsorption isotherms directly from elution profiles obtained in gradient elution which enabled a broad acetonitrile interval to be studied. Here, it was found that the column saturation capacity was quickly reached at very low acetonitrile fractions and that there were significant variations in adsorption with the molecular weight. Finally, practical implications for method development are discussed based on an experimental design where gradient slope and TFA concentrations are used as factors.

Background In patients with Lupus Nephritis (LN), clinical response to treatment and renal histopathology have been shown to be discordant. We investigated whether per-protocol repeat renal biopsies are predictive of LN relapses and long-term impairment of renal function. Methods Forty-two patients with an incident biopsy-proven active proliferative (class III/IV ± V) LN from the LN database of the Universite catholique de Louvain were included in the present retrospective study. Per-protocol repeat kidney biopsies were performed in all patients after a median time of 24.3 (IQR: 21.3–26.2) months. The NIH activity index (AI) and chronicity index (CI) scores were assessed in both baseline and repeat biopsies. Results Despite a moderate correlation between urinary protein/creatinine (U-P/C) ratios and AI scores at repeat biopsy (r=0.48; P=0.001), ten patients (23.8%) with U-P/C ratios 3; figure 1). High AI scores in repeat biopsies were associated with an increased probability and/or shorter time to renal relapse (N=11) following the repeat biopsy (HR: 1.2; 95% CI: 1.1–1.3; P=0.007), independently of proteinuria levels. High NIH CI scores in repeat biopsies were associated with a sustained increase in serum creatinine levels corresponding to ≥120% of the baseline value (HR: 1.8; 95% CI: 1.1–2.9; P=0.016) through a median follow-up time of 131.5 (IQR: 73.8–178.2) months. Baseline AI/CI scores were not predictive of these outcomes. Conclusions Our results highlight the usefulness of per-protocol repeat biopsies as an integral part of the treatment evaluation, also in patients who have shown adequate clinical response.

The Mediator kinase module regulates eukaryotic transcription by phosphorylating transcription-related targets and by modulating the association of Mediator and RNA polymerase II. The activity of its catalytic core, cyclin-dependent kinase 8 (CDK8), is controlled by Cyclin C and regulatory subunit MED12, with its deregulation contributing to numerous malignancies. Here, we combine in vitro biochemistry, cross-linking coupled to mass spectrometry, and in vivo studies to describe the binding location of the N-terminal segment of MED12 on the CDK8/Cyclin C complex and to gain mechanistic insights into the activation of CDK8 by MED12. Our data demonstrate that the N-terminal portion of MED12 wraps around CDK8, whereby it positions an “activation helix” close to the T-loop of CDK8 for its activation. Intriguingly, mutations in the activation helix that are frequently found in cancers do not diminish the affinity of MED12 for CDK8, yet likely alter the exact positioning of the activation helix. Furthermore, we find the transcriptome-wide gene-expression changes in human cells that result from a mutation in the MED12 activation helix to correlate with deregulated genes in breast and colon cancer. Finally, functional assays in the presence of kinase inhibitors reveal that binding of MED12 remodels the active site of CDK8 and thereby precludes the inhibition of ternary CDK8 complexes by type II kinase inhibitors. Taken together, our results not only allow us to propose a revised model of how CDK8 activity is regulated by MED12, but also offer a path forward in developing small molecules that target CDK8 in its MED12-bound form.

In selective laser sintering (SLS) predominantly polyamide 12 powder (PA12) is used. Often, after the sintering process approximately 70 % of powder, which has not been sintered, remains. The flow properties of this used powder have been affected because of the thermal stress during the sintering process. Due to the high price of new PA12 powder, the used and aged powder must be recycled by refreshing with new powder. This powder mixture can be reused in a next sintering process. However, due to the repeating refreshing process of the used powder, mixtures are produced whose quality is difficult to define. They differ greatly in their flow properties, which in turn has a negative effect on the component quality. In order to generate a good and reproducible powder quality, the thermal aging behavior of PA12 powder has been examined in detail by the authors. Based on these results, a recycling process could be developed step by step, by which the thermal aging can be reversed. A chemical agent is added to the used powder which is able to cleave the long polymer chains of the used powder at its amide bonds. Depending on the kind of chemical agent and its concentration, the flow properties of the used powder can be adjusted selectively, which also makes the quality of these mixtures reproducible. By means of this recycling process, the used powder has better flow and sintering properties and thus, can be reused several times. Thereby, it contributes to material and cost efficiency and thus, leads to an increased cost effectiveness of selective laser sintering.

Liver transplantation remains an essential procedure for many patients suffering from alcoholic liver disease. Alcohol use monitoring remains paramount all through the stages of this complex process. Direct alcohol biomarkers, with improved specificity and sensibility, should replace traditional indirect markers. Phosphatidylethanol (PEth) has been recently tested in alcoholic liver disease patients, but more evidence is needed, especially in comparison with other direct biomarkers. We conducted an observational study among patients awaiting liver transplantation. We analyzed Peth in blood, ethylglucuronide (EtG) in hair and urine and ethylsulphate (EtS) in urine, using mass spectrometry methods. In addition, transaminases, and self-reports were analyzed. A total of 50 patients were included (84% men, mean age 59 years (SD = 6)). 18 patients (36%) screened positive for any marker. Self-reports were positive in 3 patients. EtS was the biomarker with more positive screens. It also was the most frequently exclusive biomarker, screening positive in 7 patients who were negative for all other biomarkers. PEth was positive in 5 patients, being the only positive biomarker in 2 patients. It showed a false negative in a patient admitting alcohol use the previous week and screening positive for EtG and EtS. Hair EtG was positive in 3 patients who had negative Peth, EtG. EtG did not provide any exclusive positive result.A combination of biomarkers seems to be the best option to fully ascertain abstinence in this population. Our study suggest EtS might also play a significant role.

The mRNA therapeutics is a new class of medicine to treat many various diseases. However, in vitro transcribed (IVT) mRNA triggers immune responses due to recognition by human endosomal and cytoplasmic RNA sensors, but incorporation of modified nucleosides have been shown to reduce such responses. Therefore, an assay signifying important aspects of the human immune system is still required. Here, we present a simple ex vivo method called 'RNA ImmunoGenic Assay' to measure immunogenicity of IVT-mRNAs in human whole blood. Chemically modified and unmodified mRNA are complexed with a transfection reagent (TransIT), and co-incubated in human whole blood. Specific cytokines are measured (TNF-α, INF-α, INF-γ, IL-6 and IL-12p70) using ELISAs. The qPCR analysis is performed to reveal the activation of specific immune pathways. The RNA ImmunoGenic Assay provides a simple and fast method to detect donor specific - immune response against mRNA therapeutics. Graphic abstract: Schematic representation of RNA ImmunoGenic Assay.

Cannabidiol (CBD) rich hemp and hemp products low in Δ9-tetrahydrocannabinol (THC) (less than 1%) are legally available in Switzerland. Besides herbs for smoking and oils, liquids (e-liquids) for smoking in electronic cigarettes (e-cigs) have recently appeared on the market. These e-liquids are available with different CBD concentrations and can be flavoured. The aim of the current study was to investigate 20 e-liquids legally available in Switzerland for their contents using Fourier-transform infrared spectroscopy (FTIR) as a preliminary step followed by gas-chromatography coupled to mass spectrometry to identify potential cannabinoids, natural plant compounds and flavours. Quantification of CBD, cannabidiol carboxylic acid (CBD-acid), cannabinol (CBN), Δ9-tetrahydrocannabinol (THC), and Δ9-tetrahydrocannabinol carboxylic acid A (THC-acid) was performed by a validated method with ultra-high-pressure-liquid chromatography coupled to a diode array detector (UHPLC-DAD). FTIR analysis could confirm that for all investigated samples the e-liquid matrix consisted of 1,2-propanediol and glycerol. The qualitative GC-MS could identify ten phytocannabinoids including the quantified analytes, six natural plant compounds and five flavours. All analysed samples had a total THC content below 0.1059% (by weight), hence meeting the legal requirements of both Switzerland (

Highlights • Validated tools are needed to evaluate alcohol-reduction interventions in low income countries. • Among young Ugandans ACASI-administered 30-day and 12-month-AUDIT have good diagnostic properties. • Self-reported AUDIT provides an efficient means of assessing alcohol misuse., Background Validated tools for assessing alcohol use among young people in low-income countries are needed to estimate prevalence and evaluate alcohol-reduction interventions. We validated Alcohol Use Disorders Identification Test (AUDIT) against Timeline Follow Back (TLFB), Diagnostic and Statistical Manual of Mental Disorders (DSM-5) and phosphatidylethanol (PEth); and the 30-day-AUDIT against the 12-months-AUDIT among young Ugandans. Methods In 2018, we collected retrospective data on 30-day and 12-month AUDIT, TLFB and DSM-5 in a cross-sectional study of 15–24 year old residents of Ugandan fishing communities. AUDIT was administered by Audio Computer Assisted Self-Interviewing (ACASI) and DSM-5 and TLFB by psychiatric nurses. We determined PEth16:0/18:1 levels from dried blood spots using liquid chromatography tandem-mass spectrometry (heavy usage, ≥210 ng/mL) and calculated sensitivity and specificity of AUDIT against the other measures. Results Among 1281 participants (52.7% male, mean age 20 years), half (n = 659; 51.4%) reported ever drinking alcohol, 19.4% had 12-month-AUDIT ≥ 8 (21.5% men; 17.0% women), and 24.2% had 30-day-AUDIT ≥ 8 (29.0% men; 18.9% women). Twenty percent of participants had detectable PEth with 55 (4.3%) classified as heavy drinkers; 50.7% reported ≥ 2 symptoms on DSM-5 and 6.3% reported binge drinking in the previous month based on TLFB (8.9% men, 3.5% women). The 30-day-AUDIT ≥ 8 had sensitivity 86.7%, 95%CI: 81.8%–90.7% and specificity 90.9%, 95%CI:89.0%–92.6% versus 12-month-AUDIT ≥ 8. Both 30-day and 12-month-AUDIT ≥ 8 were sensitive and specific markers of heavy drinking by PEth (12-month-AUDIT sensitivity = 80.0%; 95%CI:67.0%–89.6%; specificity = 83.3%; 95%CI:81.1%–85.3%). The 30-day-AUDIT was a sensitive and specific marker of binge drinking based on TLFB (sensitivity = 82.7%; 95%CI:72.7%–90.2%, specificity = 79.8%; 95%CI:77.4%–82.1%); 12-month-AUDIT had lower sensitivity. Both 30-day and 12-month AUDIT ≥ 8 were highly specific but insensitive markers of having DSM-5 ≥ 2 symptoms. Conclusion Among young people in Uganda, ACASI-administered 30-day and 12-month-AUDIT have good diagnostic properties compared to PEth, DSM-5 and TLFB. Self-reported AUDIT provides a quick and valid means of assessing alcohol misuse in these communities.

The interaction between arbuscular mycorrhizal fungi (AM fungi) and Pseudomonas sp. has received considerable attention. The presence of biochar may affect these microorganisms, with subsequent modification of the phosphorus uptake and root morphology, and plant biomass accumulation. This research sought to identify, in the presence or absence of biochar, the effects of the interactions of mycorrhizal fungi and Pseudomonas sp. on the responses of phosphorus (P) and nitrogen (N) uptake and the root length, surface area, and volume of celery plants with low and high P fertilization under different substrate and soil conditions. The results indicate that strong growth responses of celery plants were observed due to the combination of AM fungi, Pseudomonas sp., and biochar with low P fertilization. A strong linear relationship was found between the plant root length and P accumulation in the shoot fraction in the present study. Increased P and N uptake occurred in treatments combining these microorganisms rather than alone, and this increase especially occurred in the presence of biochar. The low availability of P was substantially recovered by the association of these three aspects. The root morphology was greatly influenced by the biochar additives and in combination with AM fungi and Pseudomonas sp. The root colonization rate of AM fungi was increased by the combination of the inoculation of Pseudomonas sp. and biochar rather than AM fungi and/or Pseudomonas sp. These results indicate an accumulating effect of AM fungi, Pseudomonas sp., and biochar exists on the plant growth response and nutrient uptake because of the increasing root length, surface area, and volume, rather than root biomass.