Systematic Analysis of the Effect of Genomic Knock-Out of Non-Essential Promiscuous HAD-Like Phosphatases YcsE, YitU and YwtE on Flavin and Adenylate Content in Bacillus Subtilis.

Studying the metabolic role of non-essential promiscuous enzymes is a challenging task, as genetic manipulations usually do not reveal at which point(s) of the metabolic network the enzymatic activity of such protein is beneficial for the organism. Each of the HAD-like phosphatases YcsE, YitU and YwtE of Bacillus subtilis catalyzes the dephosphorylation of 5-amino-6-ribitylamino-uracil 5'-phosphate, which is essential in the biosynthesis of riboflavin. Using CRISPR technology, we have found that the deletion of these genes, individually or in all possible combinations failed to cause riboflavin auxotrophy and did not result in significant growth changes. Analysis of flavin and adenylate content in B. subtilis knockout mutants showed that (i) there must be one or several still unidentified phosphatases that can replace the deleted proteins; (ii) such replacements, however, cannot fully restore the intracellular content of any of three flavins studied (riboflavin, FMN, FAD); (iii) whereas bacterial fitness was not significantly compromised by mutations, the intracellular balance of flavins and adenylates did show some significant changes.

Preparation and Immunochemical Characterization of a Water-Soluble Gluten Peptide Fraction for Improving the Diagnosis of Celiac Disease.

The diagnosis of celiac disease (CD) is complex and requires a multi-step procedure (symptoms, serology, duodenal biopsy, effect of a gluten-free diet, and optional genetic). The aim of the study was to contribute to the improvement of CD diagnosis by preparing a water-soluble gluten peptide fraction (called Solgluten) and by selecting gluten-specific enzyme-linked immunosorbent assays (ELISA) for the detection of gluten immunogenic gluten peptides (GIPs) in urine and blood serum spiked with Solgluten. Food-grade Solgluten was prepared by the extraction of a peptic digest of vital gluten with water, centrifugation, and freeze-drying. The process was relatively easy, repeatable, and cheap. The content of gliadin-derived GIPs was 491 mg/g. Solgluten was used as antigenic material to compare two competitive ELISA kits (R7021 and K3012) and two sandwich ELISA kits (M2114 and R7041) in their quality regarding the quantitation of GIPs in urine and blood serum. The quality parameters were the reactivity, sensitivity, coefficients of variation and determination, and curve shape. The evaluation of the kits showed a number of discrepancies in individual quality parameters measured in urine and serum. Due to the lowest limit of quantitation and the highest coefficient of determination, M2114 may be the first choice, while R7021 appeared to be less suitable because of the high coefficients of variation and unfavorable curve progression. The results set the stage for improving CD diagnosis by supplementing conventional blood tests with oral provocation with Solgluten and subsequent ELISA measurement of GIPs that could support the no-biopsy approach and by better assessing the effect of a gluten-free diet by monitoring adherence to the diet by measuring GIPs in urine and blood.

Successful microvascular surgery in patients with thrombophilia in head and neck surgery: a case series.

BACKGROUND: In this case series, a perioperative anticoagulation protocol for microvascular head and neck surgery in patients with thrombophilia is presented. Microvascular free-flap surgery is a standard procedure in head and neck surgery with high success rates. Nevertheless, flap loss-which is most often caused by thrombosis-can occur and has far-reaching consequences, such as functional impairment, prolonged hospitalization, and increased costs. The risk of flap loss owing to thrombosis is significantly increased in patients with thrombophilia. Therefore, perioperative anticoagulation is mandatory. To date, no perioperative anticoagulation protocol exists for these high-risk patients. CASE PRESENTATION: We present three exemplary male Caucasian patients aged 53-57 years with free flap loss owing to an underlying, hidden thrombophilia. CONCLUSION: We present a modified anticoagulation protocol for microvascular surgery in these high-risk patients, enabling successful microsurgical reconstruction.

The roles of brain lipids and polar metabolites in the hypoxia tolerance of deep-diving pinnipeds.

Lipids make up more than half of the human brain's dry weight, yet the composition and function of the brain lipidome is not well characterized. Lipids not only provide the structural basis of cell membranes, but also take part in a wide variety of biochemical processes. In neurodegenerative diseases, lipids can facilitate neuroprotection and serve as diagnostic biomarkers. The study of organisms adapted to extreme environments may prove particularly valuable in understanding mechanisms that protect against stressful conditions and prevent neurodegeneration. The brain of the hooded seal (Cystophora cristata) exhibits a remarkable tolerance to low tissue oxygen levels (hypoxia). While neurons of most terrestrial mammals suffer irreversible damage after only short periods of hypoxia, in vitro experiments show that neurons of the hooded seal display prolonged functional integrity even in severe hypoxia. How the brain lipidome contributes to the hypoxia tolerance of marine mammals has been poorly studied. We performed an untargeted lipidomics analysis, which revealed that lipid species are significantly modulated in marine mammals compared with non-diving mammals. Increased levels of sphingomyelin species may have important implications for efficient signal transduction in the seal brain. Substrate assays also revealed elevated normoxic tissue levels of glucose and lactate, which suggests an enhanced glycolytic capacity. Additionally, concentrations of the neurotransmitters glutamate and glutamine were decreased, which may indicate reduced excitatory synaptic signaling in marine mammals. Analysis of hypoxia-exposed brain tissue suggests that these represent constitutive mechanisms rather than an induced response towards hypoxic conditions.

A liquid chromatography-tandem mass spectrometry method for the quantification of ampicillin/sulbactam and clindamycin in jawbone, plasma, and platelet-rich fibrin: Application to patients with osteonecrosis of the jaw.

Ampicillin in combination with sulbactam is a widely used drug choice for infection prophylaxis, especially in oral and maxillofacial surgery. Clindamycin serves as an alternative in patients with known allergy to ß-lactam antibiotics. To ensure effective prophylaxis, it is important to achieve sufficiently high concentrations of active antibiotic substances in the tissues affected by the surgery. To this end, a LC-MS/MS method was developed and validated that allows the quantification of ampicillin, sulbactam and clindamycin in jawbone, plasma, and so-called platelet-rich fibrin (PRF). Validation was performed in accordance with the European Medicines Agency guidelines for bioanalytical method validation. For all matrices, sample processing was carried out by protein precipitation with acetonitrile or methanol 80%, containing the isotope labelled internal standards (IS) of the three drugs. Analytes were separated on a pentaflourophenyl column at 20 °C using gradient elution. Furthermore, detection was accomplished by electrospray ionisation in positive-ion mode (ampicillin, clindamycin and corresponding IS) and negative-ion mode (sulbactam and corresponding IS) in combination with multiple reaction monitoring. Depending on the analyte and the matrix under investigation, calibration curves ranged from 0.14 to 59.8 µg/g (jawbone - ampicillin), 2.0-1000 µg/mL (plasma - ampicillin), and 1.0-495 µg/mL (PRF - ampicillin). All analytes fulfilled the requirements of the guideline regarding sensitivity, linearity, selectivity, carryover, within-run and between run accuracy and precision, matrix effect and extraction recovery in all matrices. The method was successfully applied to measure concentrations of ampicillin, sulbactam and clindamycin in real-life samples obtained in routine clinical practice.

Drug-Membrane Interactions: Effects of Virus-Specific RNA-Dependent RNA Polymerase Inhibitors Remdesivir and Favipiravir on the Structure of Lipid Bilayers.

The two RNA-dependent RNA polymerase inhibitors remdesivir and favipiravir were originally developed and approved as broad-spectrum antiviral drugs for the treatment of harmful viral infections such as Ebola and influenza. With the outbreak of the global SARS-CoV-2 pandemic, the two drugs were repurposed for the treatment of COVID-19 patients. Clinical studies suggested that the efficacy of the drugs is enhanced in the case of an early or even prophylactic application. Because the contact between drug molecules and the plasma membrane is essential for a successful permeation process of the substances and therefore for their intracellular efficiency, drug-induced effects on the membrane structure are likely and have already been shown for other substances. We investigated the impact of remdesivir and favipiravir on lipid bilayers in model and cell membranes via several biophysical approaches. The measurements revealed that the embedding of remdesivir molecules in the lipid bilayer results in a disturbance of the membrane structure of the tested phospholipid vesicles. Nevertheless, in a cell-based assay, the presence of remdesivir induced only weak hemolysis of the treated erythrocytes. In contrast, no experimental indication for an effect on the structure and integrity of the membrane was detected in the case of favipiravir. Regarding potential prophylactic or accompanying use of the drugs in the therapy of COVID-19, the physiologically relevant impacts associated with the drug-induced structural modifications of the membrane might be important to understand side effects and/or low effectivities.

Food Authentication of Almonds (Prunus dulcis Mill.). Fast Origin Analysis with Laser Ablation Inductively Coupled Plasma Mass Spectrometry and Chemometrics.

Food fraud is a growing problem, especially misdeclaration due to regional price differences offering a wide field. Fast, powerful, and cost-effective analytical methods are therefore essential to counteract food fraud. The isotopolome is suitable for origin discrimination and was analyzed in this study using laser ablation inductively coupled plasma mass spectrometry (ICP-MS). A total of 250 almond samples from six countries and four crop years were analyzed and evaluated by chemometric methods. By using a ratio-based assessment, calibration problems were avoided and an origin predictive accuracy of 85.2 ± 1.2% was achieved. Compared to ICP-MS with solution nebulization, the analysis time could be reduced to about one-fifth.

Glass authentication: Laser ablation-inductively coupled plasma mass spectrometry (LA-ICP-MS) for origin discrimination of glass bottles.

Counterfeiting is an omnipresent issue, among others in the cosmetics industry or on the art market. Particularly in the case of very expensive perfumes or very valuable art objects, counterfeits are strongly represented and are steadily increasing. Typically, the content of perfumes is analyzed, but the bottle offers another level of authentication, as it is an essential part of the product. For art objects made of glass, glass is an essential part of the artwork and thus provides an important contribution to the authenticity of the object. In the present pilot study, we developed a laser ablation-inductively coupled plasma mass spectrometry (LA-ICP-MS) method to classify glass using perfume bottles manufactured at different production facilities, Germany, India, Peru and Poland as an example. Using minimally invasive laser ablation invisible to the eye, we were able to detect counterfeit flacons without having to open the vials. A total of 63 elements could be recorded during method development. After statistical evaluation (t-test, ANOVA, principal component analysis (PCA)), 15 (Li, Na, Al, Ti, V, Co, Rb, Sr, Mo, Ba, La, Ce, Pr, Er and Pb) significant marker elements were extracted from the data sets to differentiate the different glass origins. By using LDA, six different production sites from four different countries could be differentiated over a sample period of six months with a prediction accuracy of 100%.

Lactoyl leucine and isoleucine are bioavailable alternatives for canonical amino acids in cell culture media.

Increasing demands for protein-based therapeutics such as monoclonal antibodies, fusion proteins, bispecific molecules, and antibody fragments require researchers to constantly find innovative solutions. To increase yields and decrease costs of next generation bioprocesses, highly concentrated cell culture media formulations are developed but often limited by the low solubility of amino acids such as tyrosine, cystine, leucine, and isoleucine, in particular at physiological pH. This study sought to investigate highly soluble and bioavailable derivatives of leucine and isoleucine that are applicable for fed-batch processes. N-lactoyl-leucine and N-lactoyl-isoleucine sodium salts were tested in cell culture media and proved to be beneficial to increase the overall solubility of cell culture media formulations. These modified amino acids proved to be bioavailable for various Chinese hamster ovary (CHO) cells and were suitable for replacement of canonical amino acids in cell culture feeds. The quality of the final recombinant protein was studied in bioprocesses using the derivatives, and the mechanism of cleavage was investigated in CHO cells. Altogether, both N-lactoyl amino acids represent an advantageous alternative to canonical amino acids to develop highly concentrated cell culture media formulations to support next generation bioprocesses.

Excited State Vibrations of Isotopically Labeled FMN Free and Bound to a Light-Oxygen-Voltage (LOV) Protein.

Flavoproteins are important blue light sensors in photobiology and play a key role in optogenetics. The characterization of their excited state structure and dynamics is thus an important objective. Here, we present a detailed study of excited state vibrational spectra of flavin mononucleotide (FMN), in solution and bound to the LOV-2 (Light-Oxygen-Voltage) domain of Avena sativa phototropin. Vibrational frequencies are determined for the optically excited singlet state and the reactive triplet state, through resonant ultrafast femtosecond stimulated Raman spectroscopy (FSRS). To assign the observed spectra, vibrational frequencies of the excited states are calculated using density functional theory, and both measurement and theory are applied to four different isotopologues of FMN. Excited state mode assignments are refined in both states, and their sensitivity to deuteration and protein environment are investigated. We show that resonant FSRS provides a useful tool for characterizing photoactive flavoproteins and is able to highlight chromophore localized modes and to record hydrogen/deuterium exchange.

Interaction of the small-molecule kinase inhibitors tofacitinib and lapatinib with membranes.

Lapatinib and tofacitinib are small-molecule kinase inhibitors approved for the treatment of advanced or metastatic breast cancer and rheumatoid arthritis, respectively. So far, the mechanisms which are responsible for their activities are not entirely understood. Here, we focus on the interaction of these drug molecules with phospholipid membranes, which has not yet been investigated before in molecular detail. Owing to their lipophilic characteristics, quantitatively reflected by large differences of the partition equilibrium between water and octanol phases (expressed by logP values), rather drastic differences in the membrane interaction of both molecules have to be expected. Applying experimental (nuclear magnetic resonance, fluorescence and ESR spectroscopy) and theoretical (molecular dynamics simulations) approaches, we found that lapatinib and tofacitinib bind to lipid membranes and insert into the lipid-water interface of the bilayer. For lapatinib, a deeper embedding into the membrane bilayer was observed than for tofacitinib implying different impacts of the molecules on the bilayer structure. While for tofacitinib, no influence to the membrane structure was found, lapatinib causes a membrane disturbance, as concluded from an increased permeability of the membrane for polar molecules. These data may contribute to a better understanding of the cellular uptake mechanism(s) and the side effects of the drugs.

Keto leucine and keto isoleucine are bioavailable precursors of their respective amino acids in cell culture media.

Highly concentrated cell culture media formulations are essential to develop next generation bioprocesses used to produce therapeutic monoclonal antibodies, fusion proteins, bispecific molecules and mAb fragments. Although cysteine/cystine and tyrosine are the first components preventing the development of highly concentrated complex cell culture media, leucine and isoleucine were identified as the next critical amino acids due to their limited solubility. This work sought to investigate highly soluble and readily bioavailable derivatives of both amino acids that may be used in batch, fed-batch or perfusion processes. The α-keto acids of Leu and Ile, keto leucine and keto isoleucine sodium salts, were tested in cell culture media and proved to be beneficial to increase the overall solubility of cell culture media formulations. These keto acids were readily bioavailable for various CHO cells and can be used in both media and feeds. The quality of the final recombinant protein was studied in processes using the precursors and the mechanism of amination was investigated in CHO cells. Altogether, both keto acids represent an alternative to their respective amino acids to develop highly concentrated cell culture media formulations to support next generation bioprocesses.

The Influence of Met Receptor Level on HGF-Induced Glycolytic Reprogramming in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is known to overexpress a variety of receptor tyrosine kinases, such as the HGF receptor Met. Like other malignancies, HNSCC involves a mutual interaction between the tumor cells and surrounding tissues and cells. We hypothesized that activation of HGF/Met signaling in HNSCC influences glucose metabolism and therefore substantially changes the tumor microenvironment. To determine the effect of HGF, we submitted three established HNSCC cell lines to mRNA sequencing. Dynamic changes in glucose metabolism were measured in real time by an extracellular flux analyzer. As expected, the cell lines exhibited different levels of Met and responded differently to HGF stimulation. As confirmed by mRNA sequencing, the level of Met expression was associated with the number of upregulated HGF-dependent genes. Overall, Met stimulation by HGF leads to increased glycolysis, presumably mediated by higher expression of three key enzymes of glycolysis. These effects appear to be stronger in Methigh-expressing HNSCC cells. Collectively, our data support the hypothesized role of HGF/Met signaling in metabolic reprogramming of HNSCC.

Nonradioactive Cell Assay for the Evaluation of Modular Prostate-Specific Membrane Antigen Targeting Ligands via Inductively Coupled Plasma Mass Spectrometry.

The development of novel prostate-specific membrane antigen (PSMA)-targeted radioactive theranostic agents is currently limited to facilities capable of working with high-energy radioisotopes. Even preselection of lead structures in vitro relies mostly on radioactive assays with PSMA(+) LNCaP and PSMA(-) PC-3 cells. Assays utilizing radioisotopes are time consuming, costly, and limit discovery to a small group of scientists with special facilities. Nonradioactive alternatives are therefore needed in the field. In this paper, we describe an inductively coupled plasma mass spectrometry (ICP-MS)-based method for the evaluation of PSMA-targeting ligands conjugated to DOTA-chelates of Europium. This method is based on LNCaP and PC-3 cells and has been validated with the well-established targeting ligand PSMA-617.

Signal pattern plot: a simple tool for time-dependent metabolomics studies by 1H NMR spectroscopy.

We show an alternative way to visualize time course NMR data without the application of multivariate data analysis, based on the temporal change of the metabolome of hazelnuts after mold infestation. Fresh hazelnuts were inoculated with eight different natural mold species and the growth was studied over a period of 14 days. The data were plotted in a color-coded scheme showing metabolic changes as a function of chemical shift, which we named signal pattern plot. This plot graphically displays alteration (trend) of a respected signal over time and allows visual interpretation in a simple manner. Changes are compared with a reference sample stored under identical conditions as the infected nuts. The plot allows, at a glance, the recognition of individual landmarks specific to a sample group as well as common features of the spectra. Each sample reveals an individual signal pattern. The plot facilitates the recognition of signals that belong to biological relevant metabolites. Betaine and five signals were identified that specifically changed upon mold infestation. Graphical abstract.

Localising functionalised gold-nanoparticles in murine spinal cords by X-ray fluorescence imaging and background-reduction through spatial filtering for human-sized objects.

Accurate in vivo localisation of minimal amounts of functionalised gold-nanoparticles, enabling e.g. early-tumour diagnostics and pharmacokinetic tracking studies, requires a precision imaging system offering very high sensitivity, temporal and spatial resolution, large depth penetration, and arbitrarily long serial measurements. X-ray fluorescence imaging could offer such capabilities; however, its utilisation for human-sized scales is hampered by a high intrinsic background level. Here we measure and model this anisotropic background and present a spatial filtering scheme for background reduction enabling the localisation of nanoparticle-amounts as reported from small-animal tumour models. As a basic application study towards precision pharmacokinetics, we demonstrate specific localisation to sites of disease by adapting gold-nanoparticles with small targeting ligands in murine spinal cord injury models, at record sensitivity levels using sub-mm resolution. Both studies contribute to the future use of molecularly-targeted gold-nanoparticles as next-generation clinical diagnostic and pharmacokinetic tools.

Variation in LOV Photoreceptor Activation Dynamics Probed by Time-Resolved Infrared Spectroscopy.

The light, oxygen, voltage (LOV) domain proteins are blue light photoreceptors that utilize a noncovalently bound flavin mononucleotide (FMN) cofactor as the chromophore. The modular nature of these proteins has led to their wide adoption in the emerging fields of optogenetics and optobiology, where the LOV domain has been fused to a variety of output domains leading to novel light-controlled applications. In this work, we extend our studies of the subpicosecond to several hundred microsecond transient infrared spectroscopy of the isolated LOV domain AsLOV2 to three full-length photoreceptors in which the LOV domain is fused to an output domain: the LOV-STAS protein, YtvA, the LOV-HTH transcription factor, EL222, and the LOV-histidine kinase, LovK. Despite differences in tertiary structure, the overall pathway leading to cysteine adduct formation from the FMN triplet state is highly conserved, although there are slight variations in rate. However, significant differences are observed in the vibrational spectra and kinetics after adduct formation, which are directly linked to the specific output function of the LOV domain. While the rate of adduct formation varies by only 3.6-fold among the proteins, the subsequent large-scale structural changes in the full-length LOV photoreceptors occur over the micro- to submillisecond time scales and vary by orders of magnitude depending on the different output function of each LOV domain.

Phage Display on the Anti-infective Target 1-Deoxy-d-xylulose-5-phosphate Synthase Leads to an Acceptor-Substrate Competitive Peptidic Inhibitor.

Enzymes of the 2-C-methyl-d-erythritol-4-phosphate pathway for the biosynthesis of isoprenoid precursors are validated drug targets. By performing phage display on 1-deoxy-d-xylulose-5-phosphate synthase (DXS), which catalyzes the first step of this pathway, we discovered several peptide hits and recognized false-positive hits. The enriched peptide binder P12 emerged as a substrate (d-glyceraldehyde-3-phosphate)-competitive inhibitor of Deinococcus radiodurans DXS. The results indicate possible overlap of the cofactor- and acceptor-substrate-binding pockets and provide inspiration for the design of inhibitors of DXS with a unique and novel mechanism of inhibition.

Cold-induced conversion of cholesterol to bile acids in mice shapes the gut microbiome and promotes adaptive thermogenesis.

Adaptive thermogenesis is an energy-demanding process that is mediated by cold-activated beige and brown adipocytes, and it entails increased uptake of carbohydrates, as well as lipoprotein-derived triglycerides and cholesterol, into these thermogenic cells. Here we report that cold exposure in mice triggers a metabolic program that orchestrates lipoprotein processing in brown adipose tissue (BAT) and hepatic conversion of cholesterol to bile acids via the alternative synthesis pathway. This process is dependent on hepatic induction of cytochrome P450, family 7, subfamily b, polypeptide 1 (CYP7B1) and results in increased plasma levels, as well as fecal excretion, of bile acids that is accompanied by distinct changes in gut microbiota and increased heat production. Genetic and pharmacological interventions that targeted the synthesis and biliary excretion of bile acids prevented the rise in fecal bile acid excretion, changed the bacterial composition of the gut and modulated thermogenic responses. These results identify bile acids as important metabolic effectors under conditions of sustained BAT activation and highlight the relevance of cholesterol metabolism by the host for diet-induced changes of the gut microbiota and energy metabolism.

Femtosecond to Millisecond Dynamics of Light Induced Allostery in the Avena sativa LOV Domain.

The rational engineering of photosensor proteins underpins the field of optogenetics, in which light is used for spatiotemporal control of cell signaling. Optogenetic elements function by converting electronic excitation of an embedded chromophore into structural changes on the microseconds to seconds time scale, which then modulate the activity of output domains responsible for biological signaling. Using time-resolved vibrational spectroscopy coupled with isotope labeling, we have mapped the structural evolution of the LOV2 domain of the flavin binding phototropin Avena sativa (AsLOV2) over 10 decades of time, reporting structural dynamics between 100 fs and 1 ms after optical excitation. The transient vibrational spectra contain contributions from both the flavin chromophore and the surrounding protein matrix. These contributions are resolved and assigned through the study of four different isotopically labeled samples. High signal-to-noise data permit the detailed analysis of kinetics associated with the light activated structural evolution. A pathway for the photocycle consistent with the data is proposed. The earliest events occur in the flavin binding pocket, where a subpicosecond perturbation of the protein matrix occurs. In this perturbed environment, the previously characterized reaction between triplet state isoalloxazine and an adjacent cysteine leads to formation of the adduct state; this step is shown to exhibit dispersive kinetics. This reaction promotes coupling of the optical excitation to successive time-dependent structural changes, initially in the ß-sheet and then α-helix regions of the AsLOV2 domain, which ultimately gives rise to Jα-helix unfolding, yielding the signaling state. This model is tested through point mutagenesis, elucidating in particular the key mediating role played by Q513.