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.

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.

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.

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.

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.

RNA-dependent disassembly of nuclear bodies.

Nuclear bodies are membraneless organelles that play important roles in genome functioning. A specific type of nuclear bodies known as interphase prenucleolar bodies (iPNBs) are formed in the nucleoplasm after hypotonic stress from partially disassembled nucleoli. iPNBs are then disassembled, and the nucleoli are reformed simultaneously. Here, we show that diffusion of B23 molecules (also known as nucleophosmin, NPM1) from iPNBs, but not fusion of iPNBs with the nucleoli, contributes to the transfer of B23 from iPNBs to the nucleoli. Maturation of pre-ribosomal RNAs (rRNAs) and the subsequent outflow of mature rRNAs from iPNBs led to the disassembly of iPNBs. We found that B23 transfer was dependent on the synthesis of pre-rRNA molecules in nucleoli; these pre-rRNA molecules interacted with B23 and led to its accumulation within nucleoli. The transfer of B23 between iPNBs and nucleoli was accomplished through a nucleoplasmic pool of B23, and increased nucleoplasmic B23 content retarded disassembly, whereas B23 depletion accelerated disassembly. Our results suggest that iPNB disassembly and nucleolus assembly might be coupled through RNA-dependent exchange of nucleolar proteins, creating a highly dynamic system with long-distance correlations between spatially distinct processes.

Novel 5'-Norcarbocyclic Pyrimidine Derivatives as Antibacterial Agents.

A series of novel 5'-norcarbocyclic derivatives of 5-alkoxymethyl or 5-alkyltriazolyl-methyl uracil were synthesized and the activity of the compounds evaluated against both Gram-positive and Gram-negative bacteria. The growth of Mycobacterium smegmatis was completely inhibited by the most active compounds at a MIC99 of 67 µg/mL (mc²155) and a MIC99 of 6.7⁻67 µg/mL (VKPM Ac 1339). Several compounds also showed the ability to inhibit the growth of attenuated strains of Mycobacterium tuberculosis ATCC 25177 (MIC99 28⁻61 µg/mL) and Mycobacterium bovis ATCC 35737 (MIC99 50⁻60 µg/mL), as well as two virulent strains of M. tuberculosis; a laboratory strain H37Rv (MIC99 20⁻50 µg/mL) and a clinical strain with multiple drug resistance MS-115 (MIC99 20⁻50 µg/mL). Transmission electron microscopy (TEM) evaluation of M. tuberculosis H37Rv bacterial cells treated with one of the compounds demonstrated destruction of the bacterial cell wall, suggesting that the mechanism of action for these compounds may be related to their interactions with bacteria cell walls.

Differential scanning calorimetric study of antibiotic distamycin A binding with chromatin within isolated rat liver nuclei.

CONTEXT: Natural oligopeptide antibiotic distamycin A (Dst) biosynthesized by Streptomyces distallicus is traditionally used in medical practice as an anti-inflammatory and antitumour drug. OBJECTIVE: Dst was investigated for its effect on the structural components of native chromatin directly within isolated rat liver nuclei in the presence of physiologically significant cations (magnesium or spermine and spermidine). MATERIALS AND METHODS: Differential scanning calorimetry (DSC) was used to study the Dst action at molar ratio Dst/DNA = 0.1 and 0.15 mM Dst on the melting profile of nuclei suspension in different conditions. RESULTS: Results showed that the thermodynamic parameters of control nuclei in the presence of polyamines or Mg2+ were different. The incubation of nuclei with Dst raised transition temperatures of relaxed (peak II) and topologically constrained DNA (peak III) by 6-8 °C and decreased by 2-4 °C that of core-histones (peak I). The total excess transition enthalpy (ΔHexc) in buffer with polyamines (24.7 kJ/mol DNA nucleotides) increased by1.5 times versus control but in buffer with Mg2+, the value of ΔHexc (35.8 kJ/mol DNA nucleotides) remained unchanged. CONCLUSIONS: The association of Dst with chromatin in the nucleus weakens histone-DNA contacts and causes additional strengthening of interaction between two complementary DNA chains. Our results contribute towards validation of DSC to test drug ability to modulate chromatin structure in the physiological environment and to clarify the mechanism of these modulations.

GC-Rich DNA Fragments and Oxidized Cell-Free DNA Have Different Effects on NF-kB and NRF2 Signaling in MSC.

It has been established that cell-free DNA circulating in the bloodstream affects cells. The characteristics of cfDNA depend on the physiological state of the organism. As we showed previously, diseases can cause either GC-enrichment of the cell-free DNA pool or its oxidation. Thus, in cases of cerebral atherosclerosis, heart attack and rheumatic arthritis the cell-free DNA pool is GC-enriched and, in the case of cancer, both GC-enriched and oxidized. Herein we investigated the time-dependent effect of oxidized and GC-rich cell-free DNA on NF-kB and NRF2 signaling pathways in human mesenchymal stem cells and showed that they affect cells in different ways. Oxidized DNA drastically increases expression of NRF2 in a short period of time, but the effect does not last long. GC-rich DNA causes a prolonged increase in mRNA levels of NF-kB and NRF2 which lasts 48 and 24 h, respectively.

Irrepressible MONOPTEROS/ARF5 promotes de novo shoot formation.

In vitro regeneration of complete organisms from diverse cell types is a spectacular property of plant cells. Despite the great importance of plant regeneration for plant breeding and biotechnology, its molecular basis is still largely unclear and many important crop plants have remained recalcitrant to regeneration. Hormone-exposure protocols to trigger the de novo formation of either roots or shoots from callus tissue demonstrate the importance of auxin and cytokinin signaling pathways, and genetic differences in these pathways may contribute to the highly divergent responsiveness of plant species to regeneration protocols. In this study, we show that signaling through MONOPTEROS (MP)/AUXIN RESPONSE FACTOR 5 is necessary for the formation of shoots from Arabidopsis calli. Most strikingly, an irrepressible variant of MP, MPΔ, is sufficient for promoting de novo shoot formation through pathways involving the genetically downstream functions of SHOOT MERISTEMLESS (STM) and CYTOKININ RESPONSE FACTOR2 (CRF2). We conclude that the MPΔ genotype can promote de novo shoot formation and can be used to probe corresponding signaling pathways.

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.

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.

Inhibition of Mycobacterium tuberculosis strains H37Rv and MDR MS-115 by a new set of C5 modified pyrimidine nucleosides.

Two sets of pyrimidine nucleoside derivatives bearing extended alkyloxymethyl or alkyltriazolidomethyl substituents at position 5 of the nucleobase were synthesized and evaluated as potential antituberculosis agents. The impact of modifications at 3'- and 5'-positions of the carbohydrate moiety on the antimycobacterial activity and cytotoxicity was studied. The highest effect was shown for 5-dodecyloxymethyl-2'-deoxyuridine, 5-decyltriazolidomethyl-2'-deoxyuridine, and 5-dodecyltriazolidomethyl-2'-deoxycytidine. They effectively inhibited the growth of two Mycobacterium tuberculosis strains in vitro, laboratory H37Rv (MIC99=20, 10, and 20µg/mL, respectively) and clinical MDR MS-115 resistant to five top antituberculosis drugs (МIC99=50, 10, and 10µg/mL, respectively).

Contribution of CXCL12 secretion to invasion of breast cancer cells.

INTRODUCTION: Neu (HER2/ErbB2) is overexpressed in 25% to 30% of human breast cancer, correlating with a poor prognosis. Researchers in previous studies who used the mouse mammary tumor virus Neu-transgenic mouse model (MMTV-Neu) demonstrated that the Neu-YB line had increased production of CXCL12 and increased metastasis, whereas the Neu-YD line had decreased metastasis. In this study, we examined the role of increased production of CXCL12 in tumor cell invasion and malignancy. METHODS: We studied invasion in the tumor microenvironment using multiphoton intravital imaging, in vivo invasion and intravasation assays. CXCL12 signaling was altered by using the CXCR4 inhibitor AMD3100 or by increasing CXCL12 expression. The role of macrophage signaling in vivo was determined using a colony-stimulating factor 1 receptor (CSF-1R) blocking antibody. RESULTS: The Neu-YD strain was reduced in invasion, intravasation and metastasis compared to the Neu-YB and Neu deletion mutant (activated receptor) strains. Remarkably, in the Neu-YB strain, in vivo invasion to epidermal growth factor was dependent on both CXCL12-CXCR4 and CSF1-CSF-1R signaling. Neu-YB tumors had increased macrophage and microvessel density. Overexpression of CXCL12 in rat mammary adenocarcinoma cells increased in vivo invasion as well as microvessel and macrophage density. CONCLUSIONS: Expression of CXCL12 by tumor cells results in increased macrophage and microvessel density and in vivo invasiveness.

In vivo invasion of head and neck squamous cell carcinoma cells does not require macrophages.

Invasion of tumor cells into the local stroma is an important component in cancer progression. Here we report studies of the in vivo invasion of head and neck squamous cell carcinoma (HNSCC) cells in response to applied gradients of a growth factor [epidermal growth factor (EGF)] and a chemokine (CXCL12), using orthotopic floor-of-mouth models. Analysis of the invading cells indicated that >75% of them were tumor cells, about 15% macrophages, and <10% were unidentified. Surprisingly, although macrophages invaded together with tumor cells, macrophage contributions were not required for HNSCC invasion. CXCL12-induced in vivo invasion of HNSCC cells was also observed and found to occur via a unidirectional transactivation of epidermal growth factor receptor (EGFR) through CXCR4. Inhibition of tumor necrosis factor-α-converting enzyme using TNF-α protease inhibitor-2 selectively inhibited CXCL12-induced invasion but not EGF-induced invasion, consistent with CXCL12 activation of EGFR via release of EGFR ligands.

Method of synthesized phase objects for pattern recognition: matched filtering.

To solve the pattern recognition problem, a method of synthesized phase objects is suggested. The essence of the suggested method is that synthesized phase objects are used instead of real amplitude objects. The former is object-dependent phase distributions calculated using the iterative Fourier-transform (IFT) algorithm. The method is experimentally studied with a Vander Lugt optical-digital 4F-correlator. We present the comparative analysis of recognition results using conventional and proposed methods, estimate the sensitivity of the latter to distortions of the structure of objects, and determine the applicability limits. It is demonstrated that the proposed method allows one: (а) to simplify the procedure of choice of recognition signs (criteria); (b) to obtain one-type δ-like recognition signals irrespective of the type of objects; (с) to improve signal-to-noise ratio (SNR) for correlation signals by 20 - 30 dB on average. The spatial separation of the Fourier-spectra of objects and optical noises of the correlator by means of the superposition of the phase grating on recognition objects at the recording of holographic filters and at the matched filtering has additionally improved SNR (>10 dB) for correlation signals. To introduce recognition objects in the correlator, we use a SLM LC-R 2500 device. Matched filters are recorded on a self-developing photopolymer.

The synthesis and antituberculosis activity of 5'-nor carbocyclic uracil derivatives.

A series of new carbocyclic uracil derivatives were synthesized and evaluated as potential antituberculosis agents. Racemic 1-[4'-hydroxy-2'-cyclopenten-1'-yl]-5-tetradecynyluracil completely inhibited the growth of Mycobacterium tuberculosis H37Rv in vitro at a concentration of 10 µg/ml. Individual (+) and (-) isomers of the above uracil derivative were isolated and showed the same level of activity against two strains of Mycobacterium tuberculosis: laboratory sensitive (H37Rv) and clinical resistant to five top antituberculosis drugs (MS-115).

Role of extracellular DNA oxidative modification in radiation induced bystander effects in human endotheliocytes.

The development of the bystander effect induced by low doses of irradiation in human umbilical vein endothelial cells (HUVECs) depends on extracellular DNA (ecDNA) signaling pathway. We found that the changes in the levels of ROS and NO production by human endothelial cells are components of the radiation induced bystander effect that can be registered at a low dose. We exposed HUVECs to X-ray radiation and studied effects of ecDNA(R) isolated from the culture media conditioned by the short-term incubation of irradiated cells on intact HUVECs. Effects of ecDNA(R) produced by irradiated cells on ROS and NO production in non-irradiated HUVECs are similar to bystander effect. These effects at least partially depend on TLR9 signaling. We compared the production of the nitric oxide and the ROS in human endothelial cells that were (1) irradiated at a low dose; (2) exposed to the ecDNA(R) extracted from the media conditioned by irradiated cells; and (3) exposed to human DNA oxidized in vitro. We found that the cellular responses to all three stimuli described above are essentially similar. We conclude that irradiation-related oxidation of the ecDNA is an important component of the ecDNA-mediated bystander effect.

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.