Macrophage migration inhibitory factor blockade reprograms macrophages and disrupts prosurvival signaling in acute myeloid leukemia.

The malignant microenvironment plays a major role in the development of resistance to therapies and the occurrence of relapses in acute myeloid leukemia (AML). We previously showed that interactions of AML blasts with bone marrow macrophages (MΦ) shift their polarization towards a protumoral (M2-like) phenotype, promoting drug resistance; we demonstrated that inhibiting the colony-stimulating factor-1 receptor (CSF1R) repolarizes MΦ towards an antitumoral (M1-like) phenotype and that other factors may be involved. We investigated here macrophage migration inhibitory factor (MIF) as a target in AML blast survival and protumoral interactions with MΦ. We show that pharmacologically inhibiting MIF secreted by AML blasts results in their apoptosis. However, this effect is abrogated when blasts are co-cultured in close contact with M2-like MΦ. We next demonstrate that pharmacological inhibition of MIF secreted by MΦ, in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF), efficiently reprograms MΦ to an M1-like phenotype that triggers apoptosis of interacting blasts. Furthermore, contact with reprogrammed MΦ relieves blast resistance to venetoclax and midostaurin acquired in contact with CD163+ protumoral MΦ. Using intravital imaging in mice, we also show that treatment with MIF inhibitor 4-IPP and GM-CSF profoundly affects the tumor microenvironment in vivo: it strikingly inhibits tumor vasculature, reduces protumoral MΦ, and slows down leukemia progression. Thus, our data demonstrate that MIF plays a crucial role in AML MΦ M2-like protumoral phenotype that can be reversed by inhibiting its activity and suggest the therapeutic targeting of MIF as an avenue towards improved AML treatment outcomes.

Comprehensive spectroscopic, metabolomic, and proteomic liquid biopsy in the diagnostics of hepatocellular carcinoma.

Liquid biopsy is a very topical issue in clinical diagnostics research nowadays. In this study, we explored and compared various analytical approaches to blood plasma analysis. Finally, we proposed a comprehensive procedure, which, thanks to the utilization of multiple analytical techniques, allowed the targeting of various biomolecules in blood plasma reflecting diverse biological processes underlying disease development. The potential of such an approach, combining proteomics, metabolomics, and vibrational spectroscopy along with preceding blood plasma fractionation, was demonstrated on blood plasma samples of patients suffering from hepatocellular carcinoma in cirrhotic terrain (n = 20) and control subjects with liver cirrhosis (n = 20) as well as healthy subjects (n = 20). Most of the applied methods allowed the classification of the samples with an accuracy exceeding 80.0 % and therefore have the potential to be used as a stand-alone method in clinical diagnostics. Moreover, a final panel of 48 variables obtained by a combination of the utilized analytical methods enabled the discrimination of the hepatocellular carcinoma samples from cirrhosis with 94.3 % cross-validated accuracy. Thus, this study, although limited by the cohort size, clearly demonstrated the benefit of the multimethod approach in clinical diagnosis.

New Flexible Analogues of 8-Aza-7-deazapurine Nucleosides as Potential Antibacterial Agents.

A variety of ribo-, 2'-deoxyribo-, and 5'-norcarbocyclic derivatives of the 8-aza-7-deazahypoxanthine fleximer scaffolds were designed, synthesized, and screened for antibacterial activity. Both chemical and chemoenzymatic methods of synthesis for the 8-aza-7-deazainosine fleximers were compared. In the case of the 8-aza-7-deazahypoxanthine fleximer, the transglycosylation reaction proceeded with the formation of side products. In the case of the protected fleximer base, 1-(4-benzyloxypyrimidin-5-yl)pyrazole, the reaction proceeded selectively with formation of only one product. However, both synthetic routes to realize the fleximer ribonucleoside (3) worked with equal efficiency. The new compounds, as well as some 8-aza-7-deazapurine nucleosides synthesized previously, were studied against Gram-positive and Gram-negative bacteria and M. tuberculosis. It was shown that 1-(ß-D-ribofuranosyl)-4-(2-aminopyridin-3-yl)pyrazole (19) and 1-(2',3',4'-trihydroxycyclopent-1'-yl)-4-(pyrimidin-4(3H)-on-5-yl)pyrazole (9) were able to inhibit the growth of M. smegmatis mc2 155 by 99% at concentrations (MIC99) of 50 and 13 µg/mL, respectively. Antimycobacterial activities were revealed for 4-(4-aminopyridin-3-yl)-1H-pyrazol (10) and 1-(4'-hydroxy-2'-cyclopenten-1'-yl)-4-(4-benzyloxypyrimidin-5-yl)pyrazole (6). At concentrations (MIC99) of 40 and 20 µg/mL, respectively, the compounds resulted in 99% inhibition of M. tuberculosis growth.

Proteotypic peptides of hairs for the identification of common European domestic and wild animal species revealed by in-sample protein digestion and mass spectrometry analysis.

The aim of this work is to offer an alternative or complementary analytical tool to the time-consuming and expensive methods commonly used for the recognition of animal species according to their hair. The paper introduces a simple and fast way for species differentiation of animal hairs called in-sample digestion. A total of 10 European animal species, including cat, cow, common degu, dog, fallow deer, goat, horse, sika deer, rabbit, roe deer, and 17 different breeds of dogs were examined using specific tryptic cleavage directly in hair followed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography-electrospray ionization quadrupole time of flight. Principal component analysis was used for the subsequent mass spectrometric data evaluation. This novel approach demonstrates the ability to distinguish among individual animal species, which is supported by finding characteristic m/z values obtained by the mass spectrometry for each animal species. The approach was successfully tested on two "blind" samples. On the other hand, the attempt to distinguish among hairs of different dog breeds has not been successful due to the very similar protein composition and their amino acid sequences.

Acute and chronic blood serum proteome changes in patients with methanol poisoning.

Twenty-four blood serum samples from patients with acute methanol poisoning (M) from the mass methanol poisoning outbreak in the Czech Republic in 2012 were compared with 46 patient samples taken four years after poisoning (S) (overlap of 10 people with group M) and with a control group (C) of 24 samples of patients with a similar proportion of chronic alcohol abuse. When comparing any two groups, tens to hundreds of proteins with a significant change in concentration were identified. Fifteen proteins showed significant changes when compared between any two groups. The group with acute methanol poisoning showed significant changes in protein concentrations for at least 64 proteins compared to the other groups. Among the most important identified proteins closely related to intoxication are mainly those involved in blood coagulation, metabolism of vitamin A (increased retinol-binding protein), immune response (e.g., increased complement factor I, complement factors C3 and C5), and lipid transport (increased apolipoprotein A I, apolipoprotein A II, adiponectin). For blood coagulation, the most affected proteins with significant changes in the methanol poisoning group were von Willebrand factor, carboxypeptidase N, alpha-2-antiplasmin (all increased), inter-alpha-trypsin inhibitor heavy chain H4, kininogen-1, plasma serine protease inhibitor, plasminogen (all decreased). However, heparin administration used for the methanol poisoning group could have interfered with some of the changes in their concentrations. Data are available via ProteomeXchange with the identifier PXD035726.

Pharmacogenetic Study of the Impact of ABCB1 Single Nucleotide Polymorphisms on the Response to Cyclosporine in Psoriasis Patients.

Psoriasis is a chronic, T cell-mediated skin disease affecting 2-3% of the Caucasian population. Cyclosporine A is a calcineurin inhibitor that acts selectively on T cells. The cyclosporine A treatment response has been suggested to be modulated by single-nucleotide polymorphisms (SNPs) in the ABCB1 gene. The aim of this research was to evaluate the effect of ABCB1 genetic variants that could affect the response to a cyclosporine treatment in Russian psoriasis patients with the ABCB1 genotype status. The ABCB1 T-129C, G1199A, C1236T, G2677T/A and C3435T SNPs in the 168 patients with psoriasis were genotyped by PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) and TaqMan SNP genotyping assays. The ABCB1 C1236T, G2677T/A and C3435T SNPs were significantly associated with a negative response to cyclosporine therapy. A very strong association was evident for the C3435T SNP in the ABCB1 gene in the allele, dominant and recessive models (OR = 2.58, OR = 4.01, OR = 2.50, respectively). ABCB1 C1236T and G2677T/A polymorphisms were significantly associated with a negative response to the cyclosporine therapy in the codominant, dominant and recessive models (p ˂ 0.05). Additionally, the haplotype analysis identified that the TGC haplotype is significantly associated with a negative response to cyclosporine therapy in psoriasis patients (p ˂ 0.05). The current study to the best of our knowledge is the first of its kind to be performed in the Russian population. In conclusion, the present results suggest an association between the ABCB1 genetic variants and unresponsiveness to cyclosporine in the Russian population. Further, larger studies are necessary to confirm our findings and replicate them in various ethnic populations before its implementation in the clinical practice.

PLGA Carriers for Controlled Release of Levofloxacin in Anti-Tuberculosis Therapy.

Levofloxacin (LFX) is a highly effective anti-tuberculosis drug with a pronounced bactericidal activity against Mycobacterium tuberculosis (Mtb). In this work, an "organic solvent-free" approach has been used for the development of polylactic-co-glycolic acid (PLGA) microparticles and scaffolds containing LFX at a therapeutically significant concentration, providing for its sustained release. To achieve the target, both nonpolar supercritical carbon dioxide and polar supercritical trifluoromethane have been used. By changing the composition, surface morphology, size, and internal structure of the polymer carriers, one can control the kinetics of the LFX release into phosphate buffered saline solutions and physiological media, providing for its acceptable burst and desirable concentration in the prolonged phase. The biocompatibility and bactericidal efficacy of PLGA/LFX carriers assessed both in vitro (against Mtb phagocytosed by macrophages) and in vivo (against inbred BALB/c mice aerogenically infected with Mtb) demonstrated their anti-tuberculosis activity comparable with that of the standard daily intragastric levofloxacin administration. These results make it possible to consider the developed compositions as a promising candidate for anti-tuberculosis control release formulations providing for the further evaluation of their activity against Mtb and their metabolism in vivo over long periods of tuberculosis infection.

Spectral and Angular Characteristics of the High-Contrast Dielectric Grating under the Resonant Interaction of a Plane Wave and a Gaussian Beam.

The resonant interaction of a plane wave and a one-dimensional Gaussian beam with a high-contrast dielectric grating was analyzed. Rigorous coupled wave analysis (RCWA) was used to numerically model the diffraction of a plane wave by the grating. RCWA, a discrete Fourier transform at the fulfillment (of the conditions) of the sampling theorem, was used to study diffraction of the Gaussian beam. The grating can be considered as a one-dimensional photonic crystal along which the waveguide mode propagates under resonance. The corresponding photonic crystal has both allowed and forbidden photonic bands for the propagating waveguide mode under resonance due to the high-contrast dielectric permittivity. There is no significant difference between the spectral and angular characteristics under the interaction of the plane wave or the Gaussian beam with grating, if the waveguide mode is in the forbidden photonic bandgap. The reflection coefficient from the grating is practically equal to unity for both cases. Resonant spectral and angular characteristics become wider at the Gaussian beam diffraction compared to the resonance curves for the plane wave in the case when the waveguide mode is in the allowed photon bandgap. The reflection coefficient from the grating becomes less than unity and its value tends to unity when the Gaussian beam width increases.

LSSmScarlet, dCyRFP2s, dCyOFP2s and CRISPRed2s, Genetically Encoded Red Fluorescent Proteins with a Large Stokes Shift.

Genetically encoded red fluorescent proteins with a large Stokes shift (LSSRFPs) can be efficiently co-excited with common green FPs both under single- and two-photon microscopy, thus enabling dual-color imaging using a single laser. Recent progress in protein development resulted in a great variety of novel LSSRFPs; however, the selection of the right LSSRFP for a given application is hampered by the lack of a side-by-side comparison of the LSSRFPs' performance. In this study, we employed rational design and random mutagenesis to convert conventional bright RFP mScarlet into LSSRFP, called LSSmScarlet, characterized by excitation/emission maxima at 470/598 nm. In addition, we utilized the previously reported LSSRFPs mCyRFP1, CyOFP1, and mCRISPRed as templates for directed molecular evolution to develop their optimized versions, called dCyRFP2s, dCyOFP2s and CRISPRed2s. We performed a quantitative assessment of the developed LSSRFPs and their precursors in vitro on purified proteins and compared their brightness at 488 nm excitation in the mammalian cells. The monomeric LSSmScarlet protein was successfully utilized for the confocal imaging of the structural proteins in live mammalian cells and multicolor confocal imaging in conjugation with other FPs. LSSmScarlet was successfully applied for dual-color two-photon imaging in live mammalian cells. We also solved the X-ray structure of the LSSmScarlet protein at the resolution of 1.4 Å that revealed a hydrogen bond network supporting excited-state proton transfer (ESPT). Quantum mechanics/molecular mechanics molecular dynamic simulations confirmed the ESPT mechanism of a large Stokes shift. Structure-guided mutagenesis revealed the role of R198 residue in ESPT that allowed us to generate a variant with improved pH stability. Finally, we showed that LSSmScarlet protein is not appropriate for STED microscopy as a consequence of LSSRed-to-Red photoconversion with high-power 775 nm depletion light.

CSF1R Inhibition Combined with GM-CSF Reprograms Macrophages and Disrupts Protumoral Interplays with AML Cells.

Relapse is a major issue in acute myeloid leukemia (AML) and while the contribution of gene mutations in developing drug resistance is well established, little is known on the role of macrophages (MΦs) in an AML cell microenvironment. We examined whether myeloblasts could educate MΦs to adopt a protumoral orientation supporting myeloblast survival and resistance to therapy. Flow cytometry analyses demonstrated that M2-like CD163+ MΦs are abundantly present, at diagnosis, in the bone marrow of AML patients. We showed that myeloblasts, or their conditioned medium, polarize monocytes to M2-like CD163+ MΦs, induce the secretion of many protumoral factors, and promote myeloblast survival and proliferation as long as close intercellular contacts are maintained. Importantly, pharmacologic inhibition of the CSF1 receptor (CSF1R), in the presence of GM-CSF, reprogrammed MΦ polarization to an M1-like orientation, induced the secretion of soluble factors with antitumoral activities, reduced protumoral agonists, and promoted the apoptosis of myeloblasts interacting with MΦs. Furthermore, myeloblasts, which became resistant to venetoclax or midostaurin during their interplay with protumoral CD163+ MΦs, regained sensitivity to these targeted therapies following CSF1R inhibition in the presence of GM-CSF. These data reveal a crucial role of CD163+ MΦ interactions with myeloblasts that promote myeloblast survival and identify CSF1R inhibition as a novel target for AML therapy.

Evaluation of a new assay for nontuberculous mycobacteria species identification in diagnostic material and cultures.

The purpose of the present study was to evaluate a real-time PCR system for 12 nontuberculous mycobacteria (NTM) species identification developed by Central Tuberculosis Research Institute (CTRI; Moscow, Russia) in cooperation with Syntol LLC (Moscow, Russia). NTM cultures (210 strains, 19 species), Mycobacterium tuberculosis complex (MTBC) cultures (21 strains, 2 species), non-mycobacterial microorganisms (18 strains, 13 species) were used for the first stage of the assay evaluation. Clinical samples (sputum, N = 973) positive for smear microscopy and MTBC/NTM DNA by a PCR-based screening assay collected from 819 patients were used for specificity and sensitivity evaluation. Sensitivity for determining the NTM species directly from diagnostic material was 99.71%, with the specificity of 100%. The sensitivity and specificity for NTM species identification in cultures was 99.67% and 100%, respectively. Both sensitivity and specificity for determining MTBC in cultures was 100%.

Comparison of proteomic approaches used for the detection of potential biomarkers of Alzheimer's disease in blood plasma.

At present, Alzheimer's disease is detected mainly using psychological tests, which can only confirm the disease in its more advanced phases. Therefore, bioanalytical possibilities for detecting this disease earlier are being investigated. To date, the results of analyses, which focus mainly on the study of lipids and proteins either in cerebrospinal fluid or much less often in blood plasma, do not provide satisfactory results. In addition, cerebrospinal fluid sampling is uncomfortable for the patients and involves many health risks. In this work, we deal with proteomic analysis using Matrix-Assisted Laser Desorption/Ionisation-Time of Flight and Liquid Chromatography coupled to tandem Mass Spectrometry of blood plasma with a focus on various ways of preanalytical sample treatments. This should lead to results improvement and facilitate the subsequent evaluation using principal component analysis and partial least squares discriminant analysis. The obtained results indicate the direction of further research, namely the study of interactions between proteins and lipids contained in blood plasma. These substances may be regarded as potential biomarkers allowing for the diagnosis of Alzheimer´s disease even in its early stages.

Methylene blue can act as an antidote to pesticide poisoning of bumble bee mitochondria.

The population of bumble bees and other pollinators has considerably declined worldwide, probably, due to the toxic effect of pesticides used in agriculture. Inexpensive and available antidotes can be one of the solutions for the problem of pesticide toxicity for pollinators. We studied the properties of the thiazine dye Methylene blue (MB) as an antidote against the toxic action of pesticides in the bumble bee mitochondria and found that MB stimulated mitochondrial respiration mediated by Complex I of the electron transport chain (ETC) and increased respiration of the mitochondria treated with mitochondria-targeted (chlorfenapyr, hydramethylnon, pyridaben, tolfenpyrad, and fenazaquin) and non-mitochondrial (deltamethrin, metribuzin, and penconazole) pesticides. MB also restored the mitochondrial membrane potential dissipated by the pesticides affecting the ETC. The mechanism of MB action is most probably related to its ability to shunt electron flow in the mitochondrial ETC.

Efficient Confirmation of Plant Viral Proteins and Identification of Specific Viral Strains by nanoLC-ESI-Q-TOF Using Single-Leaf-Tissue Samples.

Plant viruses are important pathogens that cause significant crop losses. A plant protein extraction protocol that combines crushing the tissue by a pestle in liquid nitrogen with subsequent crushing by a roller-ball crusher in urea solution, followed by RuBisCO depletion, reduction, alkylation, protein digestion, and ZipTip purification allowed us to substantially simplify the sample preparation by removing any other precipitation steps and to detect viral proteins from samples, even with less than 0.2 g of leaf tissue, by a medium resolution nanoLC-ESI-Q-TOF. The presence of capsid proteins or polyproteins of fourteen important viruses from seven different families (Geminiviridae, Luteoviridae, Bromoviridae, Caulimoviridae, Virgaviridae, Potyviridae, and Secoviridae) isolated from ten different economically important plant hosts was confirmed through many identified pathogen-specific peptides from a protein database of host proteins and potential pathogen proteins assembled separately for each host and based on existing online plant virus pathogen databases. The presented extraction protocol, combined with a medium resolution LC-MS/MS, represents a cost-efficient virus protein confirmation method that proved to be effective at identifying virus strains (as demonstrated for PPV, WDV) and distinct disease species of BYDV, as well as putative new viral protein sequences from single-plant-leaf tissue samples. Data are available via ProteomeXchange with identifier PXD022456.

Resonant and Sensing Performance of Volume Waveguide Structures Based on Polymer Nanomaterials.

Organic-inorganic photocurable nanocomposite materials are a topic of intensive research nowadays. The wide variety of materials and flexibility of their characteristics provide more freedom to design optical elements for light and neutron optics and holographic sensors. We propose a new strategy of nanocomposite application for fabricating resonant waveguide structures (RWS), whose working principle is based on optical waveguide resonance. Due to their resonant properties, RWS can be used as active tunable filters, refractive index (RI) sensors, near-field enhancers for spectroscopy, non-linear optics, etc. Our original photocurable organic-inorganic nanocomposite was used as a material for RWS. Unlike known waveguide structures with corrugated surfaces, we investigated the waveguide gratings with the volume modulation of the RI fabricated by a holographic method that enables large-size structures with high homogeneity. In order to produce thin photosensitive waveguide layers for their subsequent holographic structuring, a special compression method was developed. The resonant and sensing properties of new resonant structures were experimentally examined. The volume waveguide gratings demonstrate narrow resonant peaks with a bandwidth less than 0.012 nm. The Q-factor exceeds 50,000. The sensor based on waveguide volume grating provides detection of a minimal RI change of 1 × 10-4 RIU. Here we also present the new theoretical model that is used for analysis and design of developed RWS. Based on the proposed model, fairly simple analytical relationships between the parameters characterizing the sensor were obtained.

The GAR domain integrates functions that are necessary for the proper localization of fibrillarin (FBL) inside eukaryotic cells.

Fibrillarin (FBL) is an essential nucleolar protein that participates in pre-rRNA methylation and processing. The methyltransferase domain of FBL is an example of an extremely well-conserved protein domain in which the amino acid sequence was not substantially modified during the evolution from Archaea to Eukaryota. An additional N-terminal glycine-arginine-rich (GAR) domain is present in the FBL of eukaryotes. Here, we demonstrate that the GAR domain is involved in FBL functioning and integrates the functions of the nuclear localization signal and the nucleolar localization signal (NoLS). The methylation of the arginine residues in the GAR domain is necessary for nuclear import but decreases the efficiency of nucleolar retention via the NoLS. The presented data indicate that the GAR domain can be considered an evolutionary innovation that integrates several functional activities and thereby adapts FBL to the highly compartmentalized content of the eukaryotic cell.

Interaction of 5-substituted pyrimidine nucleoside analogues and M.Tuberculosis: A view through an electron microscope.

The data of transmission electron microscopy (TEM) on morphology of M. tuberculosis H37Rv bacterial cells treated with four analogues of pyrimidine nucleosides with different substituents at 5 position of base are presented. We showed that the growth of M. tuberculosis H37Rv cells effectively inhibited by each of these compounds. This process is accompanied with the accumulation of lipid intracellular vacuole-like inclusions in the cells, appearance of deep protrusions and indentations on the surface, partial and/or complete destruction of the three-layered cell envelope. The exact molecular mechanism of action of 5-substituted pyrimidine nucleosides on M. tuberculosis cells remains to be proved. However, one can suggest that mechanism of action for these compounds is related either to their direct interactions with bacteria cell walls or to interactions with enzymes participating in the process of cell wall formation.

Optical and Nonlinear Properties of Photonic Polymer Nanocomposites and Holographic Gratings Modified with Noble Metal Nanoparticles.

Nanocomposites based on transparent polymer matrices containing nanoparticles (NPs) of noble metals are modern-day materials that can be specially designed for photonics, linear and nonlinear optics, laser physics and sensing applications. We present the improved photosensitive nanocomposites doped with Au and Ag NPs allowing fabrication of high effective submicrometer dimensional diffraction structures using holographic method. A general approach for the fabrication of holographic structures using a two-component mixture of the monomers of different reactivity was developed. Two different methods, ex situ and in situ, were studied to introduce Au and Ag NPs in the polymer matrix. The diffusion model of the grating formation upon holographic exposure as well as the process of Ag NP synthesis in a polymer matrix is considered. The influence of the NP size on the polymerization process, material dynamic range and nonlinear properties were investigated. The mechanisms and characteristics of the nanocomposite nonlinear optical response are discussed.

Verification of cheeses authenticity by mass spectrometry.

Cheeses are a group of fermented dairy products that are produced all over the world in various forms and flavours. Milk, especially sheep or goat milk, is still regarded as an expensive raw material in the world, which makes milk and milk products highly attractive as a fraud target. Most often, such fraud includes partial or complete substitution with cheaper sorts of milk (e.g. bovine milk). The aim of this work was to verify the authenticity of 27 cheeses commonly emerging on the Czech food market. The cheeses were distinguished on the basis of milk animal species origin. For this purpose, two mass spectrometry techniques were used: matrix-assisted laser desorption/ionization with time of flight mass spectrometry together with principal component analysis method and liquid chromatography coupled with electrospray ionization and quadrupole time-of-flight mass spectrometry. The results were a partial success, because the cheeses could only be partially distinguished with the first mass spectrometry technique probably because of the influence of some protein additive materials in cheeses. The second technique allowed for collecting higher quality results and thus appears to be highly suitable for the research task.

Investigation of 5'-Norcarbocyclic Nucleoside Analogues as Antiprotozoal and Antibacterial Agents.

Carbocyclic nucleosides have long played a role in antiviral, antiparasitic, and antibacterial therapies. Recent results from our laboratories from two structurally related scaffolds have shown promising activity against both Mycobacterium tuberculosis and several parasitic strains. As a result, a small structure activity relationship study was designed to further probe their activity and potential. Their synthesis and the results of the subsequent biological activity are reported herein.