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Cold physical plasma is a partially ionized gas operated at body temperature and utilized for heat-sensitive technical and medical purposes. Physical plasma is a multi-component system consisting of, e.g., reactive species, ions and electrons, electric fields, and UV light. Therefore, cold plasma technology is an interesting tool for introducing biomolecule oxidative modifications. This concept can be extended to anticancer drugs, including prodrugs, which could be activated in situ to enhance local anticancer effects. To this end, we performed a proof-of-concept study on the oxidative prodrug activation of a tailor-made boronic pinacol ester fenretinide treated with the atmospheric pressure argon plasma jet kINPen operated with either argon, argon–hydrogen, or argon–oxygen feed gas. Fenretinide release from the prodrug was triggered via Baeyer–Villiger-type oxidation of the boron–carbon bond based on hydrogen peroxide and peroxynitrite, which were generated by plasma processes and chemical addition using mass spectrometry. Fenretinide activation led to additive cytotoxic effects in three epithelial cell lines in vitro compared to the effects of cold plasma treatment alone regarding metabolic activity reduction and an increase in terminal cell death, suggesting that cold physical plasma-mediated prodrug activation is a new direction for combination cancer treatment studies.

We demonstrate the use of an acoustic device to actively encapsulate single red blood cells into individual droplets in a T-junction. We compare the active encapsulation with the passive encapsulation depending on the number of loaded cells as well as the created droplet volumes. This method overcomes the Poisson limitation statistical loading of cells for the passive encapsulation. In our experiments we reach a single cell encapsulation efficiency of 97.9 ± 2.1% at droplet formation rates exceeding 15 Hz.

Saliva is an attractive sampling matrix for measuring various endogenous and exogeneous substances but requires sample treatment prior to chromatographic analysis. Exploiting supercritical CO2 for both extraction and chromatography simplifies sample preparation, reduces organic solvent consumption, and minimizes exposure to potentially infectious samples, but has not yet been applied to oral fluid. Here, we demonstrate the feasibility and benefits of online supercritical fluid extraction coupled to supercritical fluid chromatography and single-quadrupole mass spectrometry for monitoring the model salivary tracer caffeine. A comparison of 13 C- and 32 S-labeled internal standards with external standard calibration confirmed the superiority of stable isotope-labeled caffeine over nonanalogous internal standards. As proof of concept, the validated method was applied to saliva from a magnetic resonance imaging study of gastric emptying. After administration of 35 mg caffeine via ice capsule, salivary levels correlated with magnetic resonance imaging data, corroborating caffeine's usefulness as tracer of gastric emptying (R2 = 0.945). In contrast to off-line methods, online quantification required only minute amounts of organic solvents and a single manual operation prior to online bioanalysis of saliva, thus demonstrating the usefulness of CO2 -based extraction and separation techniques for potentially infective biomatrices.

Droplet microfluidics allows one to address the ever-increasing demand to screen large libraries of biological samples. Absorbance spectroscopy complements the golden standard of fluorescence detection by label free target identification and providing more quantifiable data. However, this is limited by speed and sensitivity. In this paper we increase the speed of sorting by including acoustofluidics, achieving sorting rates of target droplets of 1 kHz. We improved the devices design for detection of absorbance using fibre-based interrogation of samples with integrated lenses in the microfluidic PDMS device for focusing and collimation of light. This optical improvement reduces the scattering and refraction artefacts, improving the signal quality and sensitivity. The novel design allows us to overcome limitations based on dielectrophoresis sorting, such as droplet size dependency, material and dielectric properties of samples. Our acoustic activated absorbance sorter removes the need for offset dyes or matching oils and sorts about a magnitude faster than current absorbance sorter.

Reduced river discharge and flow regulation are significant threats to freshwater biodiversity. An accurate representation of potential damage of water consumption on freshwater biodiversity is required to quantify and compare the environmental impacts of global value chains. The effect of discharge reduction on fish species richness was previously modeled in life cycle impact assessment, but models were limited by the restricted geographical scope of underlying species-discharge relationships and the small number of species data. Here, we propose a model based on a novel regionalized species-discharge relationship (SDR). Our SDR-based model covers 88 % of the global landmass (2320 river basins worldwide excluding deserts and permanently frozen areas) and is based on a global dataset of 11,450 riverine fish species, simulated river discharge, elevation, and climate zones. We performed 10-fold cross-validation to select the best set of predictors and validated the obtained SDRs based on observed discharge data. Our model performed better than previous SDRs employed in life cycle impact assessment (Kling-Gupta efficiency coefficient about 4 times larger). We provide both marginal and average models with their uncertainty ranges for assessing scenarios of small and large-scale water consumption, respectively, and include regional and global species loss. We conducted an illustrative case study to showcase the method's applicability and highlight the differences with the currently used approach. Our models are useful for supporting sustainable water consumption and riverine fish biodiversity conservation decisions. They enable a more specific, reliable, and complete impact assessment by differentiating impacts on regional riverine fish species richness and irreversible global losses, including up-to-date species data, and providing spatially explicit values with high geographical coverage. info:eu-repo/semantics/publishedVersion

The pathogenesis of autoimmune complications triggered by SARS‐CoV2 has not been completely elucidated. Here, we performed an analysis of the cellular immune status, cell ratios, and monocyte populations of patients with COVID‐19 treated in the intensive care unit (ICU) (cohort 1, N = 23) and normal care unit (NCU) (cohort 2, n = 10) compared with control groups: patients treated in ICU for noninfectious reasons (cohort 3, n = 30) and patients treated in NCU for infections other than COVID‐19 (cohort 4, n = 21). Patients in cohort 1 presented significant differences in comparison with the other cohorts, including reduced frequencies of lymphocytes, reduced CD8+T‐cell count, reduced percentage of activated and intermediate monocytes and an increased B/T8 cell ratio. Over time, patients in cohort 1 who died presented with lower counts of B, T, CD4+T, CD8+T‐lymphocytes, NK cells, and activated monocytes. The B/T8 ratio was significantly lower in the group of survivors. In cohort 1, significantly higher levels of IgG1 and IgG3 were found, whereas cohort 3 presented higher levels of IgG3 compared to controls. Among many immune changes, an elevated B/T8‐cell ratio and a reduced rate of activated monocytes were mainly observed in patients with severe COVID‐19. Both parameters were associated with death in cohort 1., Patients with severe COVID‐19 present a particular set of immune changes in comparison to patients with mild disease and controls. These include consumption of certain monocyte and lymphocyte populations and an elevated B/T8 Ratio. COVID‐19 patients in general share a proinflammatory immunoglobulin profile with elevated proportions of IgG1 and/or IgG3.

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We engineered the cytochrome P450 monooxygenase CYP107D1 (OleP) from Streptomyces antibioticus for the stereo‐ and regioselective 7β‐hydroxylation of lithocholic acid (LCA) to yield ursodeoxycholic acid (UDCA). OleP was previously shown to hydroxylate testosterone at the 7β‐position but LCA is exclusively hydroxylated at the 6β‐position, forming murideoxycholic acid (MDCA). Structural and 3DM analysis, and molecular docking were used to identify amino acid residues F84, S240, and V291 as specificity‐determining residues. Alanine scanning identified S240A as a UDCA‐producing variant. A synthetic “small but smart” library based on these positions was screened using a colorimetric assay for UDCA. We identified a nearly perfectly regio‐ and stereoselective triple mutant (F84Q/S240A/V291G) that produces 10‐fold higher levels of UDCA than the S240A variant. This biocatalyst opens up new possibilities for the environmentally friendly synthesis of UDCA from the biological waste product LCA., We report engineering of a P450 monooxygenase for the stereo‐ and regioselective 7β‐hydroxylation of lithocholic acid to produce ursodeoxycholic acid (UDCA). Structural and 3DM analysis, and molecular docking, identified selectivity‐influencing residues. A “small but smart” mutant library was then screened with a selective colorimetric assay. The best mutant has nearly perfect regio‐ and stereoselectivity, enabling a new route for UDCA synthesis.

Invasive infections caused by carbapenemase-producing bacteria are associated with excess mortality. We applied a rapid diagnostic test (RDT) on clinical samples with an elevated likelihood of carbapenemase-producing bacteria and documented its impact on antibiotic treatment decisions. Among 38 patients, twelve tested positive for infections caused by carbapenemase-producing bacteria (31.6%), mainly in blood cultures. KPC (n = 10) was more frequent than OXA-48 (n = 2). RDT-based carbapenemase detection led to a treatment modification to ceftazidime/avibactam-containing regimens in all patients before detailed antibiotic testing results became available. Eleven patients (92%) survived the acute infection, whereas one patient with a ceftazidime/avibactam- and colistin-resistant OXA-48-positive isolate died.

Methylation of free hydroxyl groups is an important modification for flavonoids. It not only greatly increases absorption and oral bioavailability of flavonoids, but also brings new biological activities. Flavonoid methylation is usually achieved by a specific group of plant O‐methyltransferases (OMTs) which typically exhibit high substrate specificity. Here we investigated the effect of several residues in the binding pocket of the Clarkia breweri isoeugenol OMT on the substrate scope and regioselectivity against flavonoids. The mutation T133M, identified as reported in our previous publication, increased the activity of the enzyme against several flavonoids, namely eriodictyol, naringenin, luteolin, quercetin and even the isoflavonoid genistein, while a reduced set of amino acids at positions 322 and 326 affected both, the activity and the regioselectivity of the methyltranferase. On the basis of this work, methylated flavonoids that are rare in nature were produced in high purity.

Human histone demethylases are known to play an important role in the development of several tumor types. Consequently, they have emerged as important medical targets for the treatment of human cancer. Herein, structural studies on tetrazolylhydrazide inhibitors as a new scaffold for a certain class of histone demethylases, the JmjC proteins, are reported. A series of compounds are structurally described and their respective binding modes to the KDM4D protein, which serves as a high‐resolution model to represent the KDM4 subfamily in crystallographic studies, are examined. Similar to previously reported inhibitors, the compounds described herein are competitors for the natural KDM4 cofactor, 2‐oxoglutarate. The tetrazolylhydrazide scaffold fills an important gap in KDM4 inhibition and newly described, detailed interactions of inhibitor moieties pave the way to the development of compounds with high target‐binding affinity and increased membrane permeability, at the same time., Bring on the subs! Tetrazolylhydrazide compounds are isosteres of 2‐oxoglutarate, which is the natural cofactor of KDM4 proteins. It is believed that substitution of the carboxylic groups with tetrazole and hydrazide moieties will lead to KDM4 inhibitors with improved physicochemical properties. The binding mode of various such ligands in the active site of KDM4D is elucidated and movements of the interacting amino acid residues upon ligand binding are described.

Supercritical fluid chromatography (SFC) holds the potential to become an orthogonal method to HPLC/UHPLC in xenobiotic metabolism studies, due to its outstanding capacity to simultaneously separate highly similar (as HPLC) and physicochemically different analytes (problematic using HPLC). Paucity of guideline-conform validation, however, has been a major obstacle to clinical application of SFC, even in cases where biotransformation yields chemically dissimilar metabolites that require more than one HPLC method for comprehensive analysis. Here, a method based on supercritical fluid chromatography coupled to single quadrupole MS detection was developed to simultaneously quantify the divisive analgesic flupirtine and its acidic and basic metabolites, represented by 4-fluorohippuric acid (4-FHA) and the active metabolite D-13223 respectively, using custom-made synthetic internal standards. Experimental data on the fundamental retention mechanisms under supercritical conditions, indicating the importance of halogen and π-π-bonding for specific retention on polysaccharide-based stationary-phases, is discussed. Compared to previous HPLC methods, the novel method offers higher versatility in terms of the target metabolite range (addressing both acidic and basic metabolites within a singular method), faster analysis (7.5 min), and compliance with green chemistry principles. Validation was performed according to EMA criteria on bioanalytical method validation, demonstrating selectivity, carry-over, calibration curve parameters (LLOQ, range, and linearity), within- and between-run accuracy and precision, dilution integrity, matrix effect and stability. For proof-of-concept, the SFC method was applied to clinical samples of human urine obtained after single intravenous (100 mg), single oral (100 mg), and repeated oral administration (400 mg). Flupirtine, D-13223, and 4-FHA could be quantified, shedding light on the extent of oxidative flupirtine metabolism in humans in the context of the unresolved biotoxification that has led to the withdrawal of specific neuronal KV7 openers.

The 2018 summer drought in Europe was particularly extreme in terms of intensity and impact due to the combination of low rainfall and high temperatures. However, it remains unclear how this drought developed in time and space in such an extreme way. In this study we aimed to get a better understanding of the role of land–atmosphere interactions. More specifically, we investigated whether there was a change in water vapor originating from land, if that caused a reduction in rainfall, and by this mechanism possibly the propagation and intensification of the drought in Europe. Our first step was to use remote sensing products for soil moisture content (SMC) and the normalized difference vegetation index (NDVI) to see where the 2018 drought started and how it developed in time and space. Our SMC and NDVI analysis showed that the 2018 drought started to impact the soil and vegetation state in June in Scandinavia and the British Isles. After that it moved towards the west of Europe where it intensified in July and August. In September, it started to decay. In October, drought was observed in Southeast Europe as well. Based on the observed patterns we divided Europe into six regions of similar spatiotemporal characteristics of SMC and NDVI. Then, we used a global gridded dataset of the fate of land evaporation (i.e., where it ends up as precipitation) to investigate whether the drought intensification and propagation was impacted by the reduction in water vapor transported from the regions that first experienced the drought. This impact was investigated by identifying the anomalies in the water vapor originating from land recycling, imports, and exports within Europe during the spring, summer, and autumn seasons. From these regions we identified four drought regions and investigated the changes in water vapor originating from source regions on the development of drought in those regions. It was found that during the onset phase of the 2018 drought in Europe that the water vapor originating from land played an important role in mitigating the precipitation anomalies as, for example, the share of land evaporation contributing to precipitation increased from 27% (normal years) to 38% (2018) during July in the west of Europe. Land evaporation played a minor role in amplifying it during the intensification phase of the drought as the share of land evaporation contribution to precipitation decreased from 23% (normal years) to 21% (2018) during August in the west of Europe. These findings are somewhat in contrast to similar studies in other continents that found the land surface to play a strong amplifying role for drought development. Subsequently, we found that the relative increase in the amount of land water vapor originating from eastern half of Europe played a role in delaying the onset and accelerating the decay of the 2018 drought in the west of Europe.

The mechanical properties of cells provide key insights into the type, differentiation and/or pathology of a cell. The mechanical analysis of a cell population, such as a blood sample, enables meaningful biological and medical interpretation for the diagnosis and monitoring of diseases. There are number of diseases, which alter the mechanical properties of human red blood cells (RBCs). Here, an acoustic method to probe both the viscous and elastic mechanics of single RBCs by SAW in a microfluidic device is presented. The device operates by exciting a surface acoustic wave in a microfluidic channel creating a stationary acoustic wave field of nodes and antinodes. RBCs are attracted to the nodes and are deformed. Using a stepwise increasing and periodically oscillating acoustic field, the static and dynamic deformation of individual red blood cells one by one was studied and the deformation by the Taylor deformation index D and relaxation times were quantified. The precision of the measurement allows to distinguish between individual cells in the suspension and provides a quantitative viscoelastic fingerprint of the blood sample with a resolution of a single cell. The method overcomes limitations of other techniques that provide averaged values and has the potential for high-throughput screening.

Hyperinflammation is frequently observed in patients with severe COVID-19. Inadequate and defective IFN type I responses against SARS-CoV-2, associated with autoantibodies in a proportion of patients, lead to severe courses of disease. In addition, hyperactive responses of the humoral immune system have been described. In the current study we investigated a possible role of neutralizing autoantibodies against anti-inflammatory mediators. Plasma from adult patients with severe and critical COVID-19 was screened by ELISA for antibodies against PGRN, IL-1-Ra, IL-10, IL-18BP, IL-22BP, IL-36-Ra, CD40, IFN-α2, IFN-γ, IFN-ω and serpinB1. Autoantibodies were characterized and the antigens were analyzed for immunogenic alterations. In a discovery cohort with severe to critical COVID-19 high titers of PGRN-autoantibodies were detected in 11 of 30 (36.7%), and of IL-1-Ra-autoantibodies in 14 of 30 (46.7%) patients. In a validation cohort of 64 patients with critical COVID-19 high-titer PGRN-Abs were detected in 25 (39%) and IL-1-Ra-Abs in 32 of 64 patients (50%). PGRN-Abs and IL-1-Ra-Abs belonged to IgM and several IgG subclasses. In separate cohorts with non-critical COVID-19, PGRN-Abs and IL-1-Ra-Abs were detected in low frequency (i.e. in < 5% of patients) and at low titers. Neither PGRN- nor IL-1-Ra-Abs were found in 40 healthy controls vaccinated against SARS-CoV-2 or 188 unvaccinated healthy controls. PGRN-Abs were not cross-reactive against SARS-CoV-2 structural proteins nor against IL-1-Ra. Plasma levels of both free PGRN and free IL-1-Ra were significantly decreased in autoantibody-positive patients compared to Ab-negative and non-COVID-19 controls. In vitro PGRN-Abs from patients functionally reduced PGRN-dependent inhibition of TNF-α signaling, and IL-1-Ra-Abs from patients reduced IL-1-Ra- or anakinra-dependent inhibition of IL-1ß signaling. The pSer81 hyperphosphorylated PGRN isoform was exclusively detected in patients with high-titer PGRN-Abs; likewise, a hyperphosphorylated IL-1-Ra isoform was only found in patients with high-titer IL-1-Ra-Abs. Thr111 was identified as the hyperphophorylated amino acid of IL-1-Ra. In longitudinally collected samples hyperphosphorylated isoforms of both PGRN and IL-1-Ra emerged transiently, and preceded the appearance of autoantibodies. In hospitalized patients, the presence of IL-1-Ra-Abs or IL-1-Ra-Abs in combination with PGRN-Abs was associated with a higher morbidity and mortality.To conclude, neutralizing autoantibodies to IL-1-Ra and PGRN occur in a significant portion of patients with critical COVID-19, with a concomitant decrease in circulating free PGRN and IL-1-Ra, indicative of a misdirected, proinflammatory autoimmune response. The break of self-tolerance is likely caused by atypical hyperphosphorylated isoforms of both antigens, whose appearances precede autoantibody induction. Our data suggest that these immunogenic secondary modifications are induced by the SARS-CoV-2-infection itself or the inflammatory environment evoked by the infection and predispose for a critical course of COVID-19.

Martin Kulke,1 Felix Nagel,1 Lukas Schulig,2 Norman Geist,1 Marcel Gabor,1 Julia Mayerle,3 Markus M Lerch,4 Andreas Link,2 Mihaela Delcea1 1Institute of Biochemistry, University of Greifswald, Greifswald, Germany; 2Institute of Pharmacy, University of Greifswald, Greifswald, Germany; 3Department of Medicine II, Ludwig-Maximilian University of Munich, Munich, Germany; 4Department of Medicine a, University Medicine Greifswald, Greifswald, GermanyCorrespondence: Mihaela Delcea; Martin KulkeInstitute of Biochemistry, University of Greifswald, Felix-Hausdorff-Straße 4, Greifswald, 17487, GermanyTel +49 3834 420 4423Fax +49 3834 420 4377Email delceam@uni-greifswald.de; makulke@web.dePurpose: Although strongly related, the pathophysiological effect of the N34S mutation in the serine protease inhibitor Kazal type 1 (SPINK1) in chronic pancreatitis is still unknown. In this study, we investigate the conformational space of the human cationic trypsin-serine protease inhibitor complex.Methods: Simulations with molecular dynamics, replica exchange, and transition pathway methods are used.Results: Two main binding states of the inhibitor to the complex were found, which explicitly relate the influence of the mutation site to conformational changes in the active site of trypsin.Conclusion: Based on our result, a hypothesis is formulated that explains the development of chronic pancreatitis through accelerated digestion of the mutant by trypsin.Keywords: trypsin, molecular dynamics simulations, replica exchange, transition path sampling, umbrella sampling

The use of light as an external trigger to change ligand shape and as a result its bioactivity, allows the probing of pharmacologically relevant systems with spatiotemporal resolution. A hetero-stilbene lead resulting from the screening of a compound that was originally designed as kinase inhibitor served as a starting point for the design of photoswitchable sirtuin inhibitors. Because the original stilbenoid structure exerted unfavourable photochemical characteristics it was remodelled to its heteroarylic diazeno analogue. By this intramolecular azologization, the shape of the molecule was left unaltered, whereas the photoswitching ability was improved. As anticipated, the highly analogous compound showed similar activity in its thermodynamically stable stretched-out (E)-form. Irradiation of this isomer triggers isomerisation to the long-lived (Z)-configuration with a bent geometry causing a considerably shorter end‐to‐end distance. The resulting affinity shifts are intended to enable real‐time photomodulation of sirtuins in vitro.

The potassium channel openers flupirtine and retigabine have proven to be valuable analgesics or antiepileptics. Their recent withdrawal due to occasional hepatotoxicity and tissue discoloration, respectively, leaves a therapeutic niche unfilled. Metabolic oxidation of both drugs gives rise to the formation of electrophilic quinones. These elusive, highly reactive metabolites may induce liver injury in the case of flupirtine and blue tissue discoloration after prolonged intake of retigabine. We examined which structural features can be altered to avoid the detrimental oxidation of the aromatic ring and shift oxidation toward the formation of more benign metabolites. Structure-activity relationship studies were performed to evaluate the KV 7.2/3 channel opening activity of 45 derivatives. Sulfide analogues were identified that are devoid of the risk of quinone formation, but possess potent KV 7.2/3 opening activity. For example, flupirtine analogue 3-(3,5-difluorophenyl)-N-(6-(isobutylthio)-2-(pyrrolidin-1-yl)pyridin-3-yl)propanamide (48) has 100-fold enhanced activity (EC50 =1.4 nm), a vastly improved toxicity/activity ratio, and the same efficacy as retigabine in vitro.

The highly structurally similar drugs flupirtine and retigabine have been regarded as safe and effective for many years but lately they turned out to exert intolerable side effects. While the twin molecules share the mode of action, both stabilize the open state of voltage-gated potassium channels, the form and severity of adverse effects is different. The analgesic flupirtine caused drug-induced liver injury in rare but fatal cases, whereas prolonged use of the antiepileptic retigabine led to blue tissue discoloration. Because the adverse effects seem unrelated to the mode of action, it is likely, that both drugs that occupied important therapeutic niches, could be replaced. Reasons for the clinically relevant toxicity will be clarified and future substitutes for these drugs presented in this review.

Single-use solid oral dosage forms have to be compounded for pediatric patients due to a lack of pediatric-licensed formulations or for small scale studies. Low content uniformity of such dosage forms, especially capsules, is a cause of variation of e.g. steroid doses in children. Besides quantification, identification of active pharmaceutical ingredients in different hard-shell capsules using different excipients can play a role, especially when ensuring the blinding effectiveness of investigational medical products or when rapid unblinding in case of emergency is necessary. Non-destructive quality control of compounded closed hard shell capsules containing very low doses is therefore highly rewarding. The individual control of a whole batch of pediatric capsules intended to be administered is possible, due to near-infrared spectroscopy (NIRS) being a rapid and non-destructive technique. FT-NIR spectroscopy with investigational sample preparation and custom-designed, 3D-printed sample holders were used to examine the capsules. This method ensures the results to be reproducible. Different chemometric approaches, which use the Mahalanobis distance, were applied to the data. Partial least squares regression (PLSR) was used to achieve the quantification of the content of hydrocortisone acetate in the capsules. While the identification of captopril, hydrochlorothiazide, and sildenafil citrate mixed with various excipients in closed hard shell capsules could be achieved, non-destructive quantification of very low doses of main real-life example hydrocortisone acetate remains an issue.