Despite the odds: formation of the SARS-CoV-2 methylation complex.

Coronaviruses modify their single-stranded RNA genome with a methylated cap during replication to mimic the eukaryotic mRNAs. The capping process is initiated by several nonstructural proteins (nsp) encoded in the viral genome. The methylation is performed by two methyltransferases, nsp14 and nsp16, while nsp10 acts as a co-factor to both. Additionally, nsp14 carries an exonuclease domain which operates in the proofreading system during RNA replication of the viral genome. Both nsp14 and nsp16 were reported to independently bind nsp10, but the available structural information suggests that the concomitant interaction between these three proteins would be impossible due to steric clashes. Here, we show that nsp14, nsp10, and nsp16 can form a heterotrimer complex upon significant allosteric change. This interaction is expected to encourage the formation of mature capped viral mRNA, modulating nsp14's exonuclease activity, and protecting the viral RNA. Our findings show that nsp14 is amenable to allosteric regulation and may serve as a novel target for therapeutic approaches.

Lipid droplets in mammalian eggs are utilized during embryonic diapause.

Embryonic diapause (ED) is a temporary arrest of an embryo at the blastocyst stage when it waits for the uterine receptivity signal to implant. ED used by over 100 species may also occur in normally "nondiapausing" mammals when the uterine receptivity signal is blocked or delayed. A large number of lipid droplets (LDs) are stored throughout the preimplantation embryo development, but the amount of lipids varies greatly across different mammalian species. Yet, the role of LDs in the mammalian egg and embryo remains unknown. Here, using a mouse model, we provide evidence that LDs play a crucial role in maintaining ED. By mechanical removal of LDs from zygotes, we demonstrated that delipidated embryos are unable to survive during ED. LDs are not essential for normal prompt implantation, without ED. We further demonstrated that with the progression of ED, the amount of intracellular lipid reduces, and composition changes. This decrease in lipid is caused by a switch from carbohydrate metabolism to lipid catabolism in diapausing blastocysts, which also exhibit increased release of exosomes reflecting elevated embryonic signaling to the mother. We have also shown that presence of LDs in the oocytes of various mammals positively corelates with their species-specific length of diapause. Our results reveal the functional role of LDs in embryonic development. These results can help to develop diagnostic techniques and treatment of recurrent implantation failure and will likely ignite further studies in developmental biology and reproductive medicine fields.

Matrix-assisted laser desorption/ionization mass spectrometry imaging sample preparation using wet-interface matrix deposition for lipid analysis.

RATIONALE: Sample preparation is one of the most crucial steps for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Scientists beginning their study with this technique may be overwhelmed by the variety of matrices, solvents, and concentrations; the methods of their applications; and the lack of widely available knowledge of the effect of these parameters on the results. Here we present in depth the aspects of matrix deposition, which will be helpful for the scientific community. METHODS: In this study, we tested several MALDI matrices, such as 2,5-dihydroxybenzoic acid (DHB), norharmane, N-(1-naphthyl)ethylenediamine dihydrochloride (NEDC), and 9-aminoacridine (9AA), using the SunCollect system: wet-interface matrix deposition in the context of lipid analysis. We optimized the number of matrix layers and nozzle settings in terms of spectral intensity and the overall quality of the obtained ion maps. RESULTS: Our research presents the effect of the number of matrix layers and nozzle settings on the results and allows for choosing the optimal parameters for the analyses. In positive ionization mode, DHB matrix could be chosen first. In the negative ionization mode, 1,5-diaminonaphthalene matrix produces a higher peak intensity in a lower mass range and seems to provide more information than 9AA. We recommend NEDC for particular processes such as glucose analysis. Compared to the remaining matrices, norharmane shows significant changes in the obtained ion maps. CONCLUSIONS: Such a large amount of data allow us to observe an interesting conclusion: the obtained ion image for a particular ion could differ dramatically with a change in the matrix, the solvent composition, or even the number of matrix layers. This must be considered when interpreting the result, impelling us to compare the results obtained with different matrices with caution.

Scalable algorithms for semiparametric accelerated failure time models in high dimensions.

Semiparametric accelerated failure time (AFT) models are a useful alternative to Cox proportional hazards models, especially when the assumption of constant hazard ratios is untenable. However, rank-based criteria for fitting AFT models are often nondifferentiable, which poses a computational challenge in high-dimensional settings. In this article, we propose a new alternating direction method of multipliers algorithm for fitting semiparametric AFT models by minimizing a penalized rank-based loss function. Our algorithm scales well in both the number of subjects and number of predictors, and can easily accommodate a wide range of popular penalties. To improve the selection of tuning parameters, we propose a new criterion which avoids some common problems in cross-validation with censored responses. Through extensive simulation studies, we show that our algorithm and software is much faster than existing methods (which can only be applied to special cases), and we show that estimators which minimize a penalized rank-based criterion often outperform alternative estimators which minimize penalized weighted least squares criteria. Application to nine cancer datasets further demonstrates that rank-based estimators of semiparametric AFT models are competitive with estimators assuming proportional hazards in high-dimensional settings, whereas weighted least squares estimators are often not. A software package implementing the algorithm, along with a set of auxiliary functions, is available for download at github.com/ajmolstad/penAFT.

Synthesis, Crystal Structures, Lipophilic Properties and Antimicrobial Activity of 5-Pyridylmethylidene-3-rhodanine-carboxyalkyl Acids Derivatives.

The constant increase in the resistance of pathogenic bacteria to the commonly used drugs so far makes it necessary to search for new substances with antibacterial activity. Taking up this challenge, we obtained a series of rhodanine-3-carboxyalkyl acid derivatives containing 2- or 3- or 4-pyridinyl moiety at the C-5 position. These compounds were tested for their antibacterial and antifungal activities. They showed activity against Gram-positive bacteria while they were inactive against Gram-negative bacteria and yeast. In order to explain the relationship between the activity of the compounds and their structure, for selected derivatives crystal structures were determined using the X-ray diffraction method. Modeling of the isosurface of electron density was also performed. For all tested compounds their lipophilicity was determined by the RP-TLC method and by calculation methods. On the basis of the carried-out research, it was found that the derivatives with 1.5 N···S electrostatics interactions between the nitrogen atom in the pyridine moiety and the sulfur atom in the rhodanine system showed the highest biological activity.

MASS SPECTROMETRY IN VIROLOGICAL SCIENCES.

Virology, as a branch of the life sciences, discovered mass spectrometry (MS) to be the pivotal tool around two decades ago. The technique unveiled the complex network of interactions between the living world of pro- and eukaryotes and viruses, which delivered "a piece of bad news wrapped in protein" as defined by Peter Medawar, Nobel Prize Laureate, in 1960. However, MS is constantly evolving, and novel approaches allow for a better understanding of interactions in this micro- and nanoworld. Currently, we can investigate the interplay between the virus and the cell by analyzing proteomes, interactomes, virus-cell interactions, and search for the compounds that build viral structures. In addition, by using MS, it is possible to look at the cell from the broader perspective and determine the role of viral infection on the scale of the organism, for example, monitoring the crosstalk between infected tissues and the immune system. In such a way, MS became one of the major tools for the modern virology, allowing us to see the infection in the context of the whole cell or the organism. © 2019 John Wiley & Sons Ltd. Mass Spec Rev.

Mass Spectrometry versus Conventional Techniques of Protein Detection: Zika Virus NS3 Protease Activity towards Cellular Proteins.

Mass spectrometry (MS) used in proteomic approaches is able to detect hundreds of proteins in a single assay. Although undeniable high analytical power of MS, data acquired sometimes lead to confusing results, especially during a search of very selective, unique interactions in complex biological matrices. Here, we would like to show an example of such confusing data, providing an extensive discussion on the observed phenomenon. Our investigations focus on the interaction between the Zika virus NS3 protease, which is essential for virus replication. This enzyme is known for helping to remodel the microenvironment of the infected cells. Several reports show that this protease can process cellular substrates and thereby modify cellular pathways that are important for the virus. Herein, we explored some of the targets of NS3, clearly shown by proteomic techniques, as processed during infection. Unfortunately, we could not confirm the biological relevance of protein targets for viral infections detected by MS. Thus, although mass spectrometry is highly sensitive and useful in many instances, also being able to show directions where cell/virus interaction occurs, we believe that deep recognition of their biological role is essential to receive complete insight into the investigated process.

The Study of Derivatization Prior MALDI MSI Analysis-Charge Tagging Based on the Cholesterol and Betaine Aldehyde.

Mass spectrometry imaging is a powerful tool for analyzing the different kinds of molecules in tissue sections, but some substances cannot be measured easily, due to their physicochemical properties. In such cases, chemical derivatization could be applied to introduce the charge into the molecule and facilitate its detection. Here, we study cholesterol derivatization with betaine aldehyde from tissue slices and evaluate how different sample preparation methods influence the signal from the derivatization product. In this study, we have tested different solutions for betaine aldehyde, different approaches to betaine aldehyde deposition (number of layers, deposition nozzle height), and different MALDI matrices for its analysis. As a result, we proved that the proposed approach could be used for the analysis of cholesterol in different tissues.

Fungal α-1,3-Glucan as a New Pathogen-Associated Molecular Pattern in the Insect Model Host Galleria mellonella.

Recognition of pathogen-associated molecular patterns (PAMPs) by appropriate pattern recognition receptors (PRRs) is a key step in activating the host immune response. The role of a fungal PAMP is attributed to ß-1,3-glucan. The role of α-1,3-glucan, another fungal cell wall polysaccharide, in modulating the host immune response is not clear. This work investigates the potential of α-1,3-glucan as a fungal PAMP by analyzing the humoral immune response of the greater wax moth Galleria mellonella to Aspergillus niger α-1,3-glucan. We demonstrated that 57-kDa and 61-kDa hemolymph proteins, identified as ß-1,3-glucan recognition proteins, bound to A. niger α-1,3-glucan. Other hemolymph proteins, i.e., apolipophorin I, apolipophorin II, prophenoloxidase, phenoloxidase activating factor, arylphorin, and serine protease, were also identified among α-1,3-glucan-interacting proteins. In response to α-1,3-glucan, a 4.5-fold and 3-fold increase in the gene expression of antifungal peptides galiomicin and gallerimycin was demonstrated, respectively. The significant increase in the level of five defense peptides, including galiomicin, corresponded well with the highest antifungal activity in hemolymph. Our results indicate that A. niger α-1,3-glucan is recognized by the insect immune system, and immune response is triggered by this cell wall component. Thus, the role of a fungal PAMP for α-1,3-glucan can be postulated.

Antibacterial properties of 5-substituted derivatives of rhodanine-3-carboxyalkyl acids. Part II.

Two series of rhodanine-3-acetic and rhodanine-3-propionic acids derivatives having benzylidene and cinnamylidene substituents with additional electron donating and withdrawing groups at the C-5 position, were synthesised. The structures of the obtained derivatives were confirmed by spectroscopic methods and their lipophilicity was screened. The crystal structures were determined for selected compounds. The antibacterial activity of the derivatives was depended on the type of carboxyalkyl group in the N-3 position and on the type of the substituent in the C-5 position. The derivatives of rhodanine-3-propionic acid demonstrated the highest activity against Gram-positive bacteria. However, none of tested derivatives showed activity against Gram-negative bacteria and yeast. We believe that the presence of the N,N-diethylamine group in the aromatic system and the number of carbon atoms in the carboxyalkyl group is more significant for the biological activity than the fact that the benzylidene or cinnamylidene substituent was present at the C-5 position.

Glycosylation Changes in Serum Proteins Identify Patients with Pancreatic Cancer.

After more than a decade of biomarker discovery using advanced proteomic and genomic approaches, very few biomarkers have been involved in clinical diagnostics. Most candidate biomarkers are focused on the protein component. Targeting post-translational modifications (PTMs) in combination with protein sequences will provide superior diagnostic information with regards to sensitivity and specificity. Glycosylation is one of the most common and functionally important PTMs. It plays a central role in many biological processes, including protein folding, host-pathogen interactions, immune response, and inflammation. Cancer-associated aberrant glycosylation has been identified in various types of cancer. Expression of cancer-specific glycan epitopes represents an excellent opportunity for diagnostics and potentially specific detection of tumors. Here, we report four proteins (LIFR, CE350, VP13A, HPT) found in sera from pancreatic cancer patients carrying aberrant glycan structures as compared to those of controls.

Proteomic Data in Morphine Addiction Versus Real Protein Activity: Metabolic Enzymes.

Drug dependence is an escalating problem worldwide and many efforts are being made to understand the molecular basis of addiction. The morphine model is widely used in these investigations. To date, at least 29 studies exploring the influence of morphine on mammals' proteomes have been published. Among various proteins indicated as up- or down-regulated, the expression changes of enzymes engaged in energy metabolism pathways have often been confirmed. To verify whether proteomics-indicated alterations in enzyme levels reflect changes in their activity, four enzymes: PK, MDH, Complex I, and Complex V were investigated in morphine addiction and abstinence models. After analyses of the rat brain mitochondria fraction in the model of morphine dependence, we found that one of the investigated enzymes (pyruvate kinase) showed statistically significant differences observed between morphine, control, and abstinence groups. J. Cell. Biochem. 118: 4323-4330, 2017. © 2017 Wiley Periodicals, Inc.

Brain lipidomic changes after morphine, cocaine and amphetamine administration - DESI - MS imaging study.

Drug addiction is a complex disorder, evoking significant changes in the proteome of the central nervous system. To check if there are also changes in the lipidomic profiles we used desorption electrospray-MS technique for imaging of the brain slices of rats exposed to morphine, cocaine and amphetamine. Our investigations showed alternative regulation of selected lipid's levels in the central nervous system structures, under the influence of applied drugs. Results of our investigations can show changes in the brain treated with drugs of abuse in the new light, indicating role of the lipids in the addiction development.

Imaging mass spectrometry: Instrumentation, applications, and combination with other visualization techniques.

Imaging Mass Spectrometry (IMS) is strengthening its position as a valuable analytical tool. It has unique ability to identify structures and to unravel molecular changes that occur in the precisely defined part of the sample. These unique features open new possibilities in the field of various aspects of biological research. In this review we briefly discuss the main imaging mass spectrometry techniques, as well as the nature of biological samples and molecules, which might be analyzed by such methodology. Moreover, a novel approach, where different analytical techniques might be combined with the results of IMS study, is emphasized and discussed. With such a fast development of IMS and related methods, we can foresee the promising future of this technique.

Antibacterial properties of 5-substituted derivatives of rhodanine-3-carboxyalkyl acids.

A series of rhodanine 3-carboxyalkanoic acid derivatives possessing 4'-(N,N-dialkyl-amino or diphenylamino)-benzylidene moiety as a substituent at the C-5 position were synthesised and their antibacterial activity was screened. All the rhodanine derivatives showed bacteriostatic or bactericidal activity to the reference gram-positive bacterial strains, but lack of activity to the reference Gram-negative bacterial strains and yeast strains was observed.

Protocol: MYTHBUSTERS: a universal procedure for sample preparation for mass spectrometry.

Improvements in proteomic strategies from the development of new and robust separation and identification techniques have led to broad applications of proteomics to solve numerous biological questions. For all analyses, sample quality is unquestionably a critical factor; therefore protein extraction is of outmost importance. The ideal extraction method should provide reproducible spectra of the most comprehensive repertoire of proteins, while minimizing sample loss and degradation. It is already known that to capture the whole proteome is an unenforceable task. Many protein extraction protocols have been described, yet there is no "one perfect procedure" taking into account the vast diversity of biological and physical properties of proteins, including their charge, size, hydrophobicity, interactions and sub-cellular localization. The research presented here reflects the main obstacle occurring in proteomic experimental design; i.e. the lack of reproducibility as a result of alterations in protein extraction methods. We have performed a series of experiments, aimed towards identification of the aptamer-binding partners in cancerous cells. Aptamers are chemically synthesized, short, single-stranded nucleic acids with a strictly defined three-dimensional structure, which allows them to interact with a target molecule with high affinity. The low immunogenicity and cellular- targeting properties of aptamers might facilitate design of suitable drugs with low side-effects. Aptamers can be used for identification of molecules associated with a pathogenic state of a cell. Aptamers can be considered as a powerful tool, since they possess unique properties to benefit cancer diagnosis, prevention and treatment. We have used different types of protein extraction methods prior to analyses of complex biological samples by mass spectrometry, based on slight changes of homogenization buffers, and have observed the changes in the identified compounds. These results should prove to be very useful for future proteomic studies and the design of studies in terms of sample preparation, especially sample homogenization and protein extraction.

Comparison of two freely available software packages for mass spectrometry imaging data analysis using brains from morphine addicted rats.

Data analysis from mass spectrometry imaging (MSI) imaging experiments is a very complex task. Most of the software packages devoted to this purpose are designed by the mass spectrometer manufacturers and, thus, are not freely available. Laboratories developing their own MS-imaging sources usually do not have access to the commercial software, and they must rely on the freely available programs. The most recognized ones are BioMap, developed by Novartis under Interactive Data Language (IDL), and Datacube, developed by the Dutch Foundation for Fundamental Research of Matter (FOM-Amolf). These two systems were used here for the analysis of images received from rat brain tissues subjected to morphine influence and their capabilities were compared in terms of ease of use and the quality of obtained results.

A comparative study of glycoproteomes in androgen-sensitive and -independent prostate cancer cell lines.

Prostate cancer is one of the most common malignancies in men and is predicted to be the second leading cause of cancer-related deaths. After 6-18 months, hormone ablation treatment results in androgen-independent growth of cancer cells, metastasis and progression. The mechanism of androgen-independent growth of prostatic carcinoma cells is still unknown. Identification of factors that facilitate the transition from androgen-dependent to independent states is crucial in designing future diagnostics and medication strategies. To understand the biochemical meaning of hormone dependency deprivation, glycoproteins enriched profiles were compared between DU145 (hormone non-responding) and LNCaP (hormone responding) prostate cancer cells. These results allow for anticipation on the important role of glycosylation in malignant transformation. Both Tn antigen and complex antennary N-oligosaccharides were recognized. Their occurrence might be involved in the development and progression of tumor, and failure of hormone ablation therapy. Among identified proteins in androgen-sensitive cells nucleolin (P19338) was found that is widely described as apoptosis inhibitor, and also transporter of molecules from the membrane to the cytoplasm or nucleus. In addition, 14-3-3 protein family (P27348, P31946, P61981, P63104, P62258, Q04917, and P31947) was investigated across available databases as it forms stable complexes with glycoproteins. Our studies indicate that isoforms: sigma and eta were found in androgen-dependent prostate cancer cells, while other isoforms were present in androgen non-responding cells. 14-3-3 binding partners are involved in cancer pathogenesis. These findings may contribute to a better understanding of prostate cancer tumorigenesis and to a more efficient prognosis and individual therapy in a future. However, it still remains to be revealed how important those changes are for androgen dependency loss in prostate cancer patients carried out on clinically relevant populations.

Desorption electrospray ionisation (DESI) for beginners--how to adjust settings for tissue imaging.

RATIONALE: Desorption electrospray ionisation (DESI) is the ambient technique used for surface imaging. Despite its simplicity, the proper use of this technique is not easy, and usually leads to discouragement, especially in the case of biological sample measurements. Here, we present some tips and tricks which may be helpful during a complex process of ion source optimisation to achieve the desired results. METHODS: Rat liver tissue as an example of a biological sample and a surface covered with rhodamine-containing marker were measured using a DESI ion source (OMNIspray source, Prosolia, Indianapolis, IN, USA) connected to a AmaZon ETD ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany). RESULTS: The geometry of the ion source (nebulisation capillary angle, its distance to the surface, and to the MS inlet), and other settings like nebulising gas pressure, solvent flow and capillary voltage, were changed during the optimisation process. The results obtained for different parameters are presented. CONCLUSIONS: Differences between the results and the method of optimisation for biological and non-biological samples were shown. The influence of different parameters on the quality of mass spectra was indicated. Optimal parameters for the tissue and non-biological sample analysis were suggested.

Electrochemical simulation of cocaine metabolism-a step toward predictive toxicology for drugs of abuse.

Knowledge of the metabolic pathways and biotransformation of the most popular drugs, such as cocaine, amphetamine, morphine and others, is crucial for the elucidation of their possible toxicity and mechanism of action in the human body. In vitro studies on metabolism are mainly based on the incubation of drugs with liver celL homogenate and utilizing Living animals. These methods need to be followed by isolation and detection of metabolic products, which makes these techniques time-consuming and technically demanding. We show here that the oxidative metabolism that occurs in the liver cells and is mainly caused by cytochrome P450 can be successfully mimicked with the electrochemical system [EC] connected on-line with electrospray ionization mass spectrometry. Cocaine was chosen as a model drug for these studies and was analyzed with a previously described system under various conditions using the boron-doped diamond working electrode. The results were compared with the number of metabolites generated by a standard procedure based on the reaction with the rat Liver microsomes. Two electrochemical products of cocaine oxidation were created, of which one was a natural metabolite of cocaine in the human body-norcocaine. The EC provides a promising platform for the screening of the addictive drug phase I metabolism. The metabolites can be directly analyzed by mass spectrometry or collected and separated by Liquid chromatog- raphy. No Liver cell homogenate or microsome is necessary to generate these metabolites, which simplifies separation of the mixtures and reduces time and costs of all experiments.