High dynamic range proteome imaging with the structured illumination gel imager

A current challenge for proteomics is detecting proteins over the large concentration ranges found in complex biological samples such as whole‐cell extracts. Currently, no unbiased, whole‐proteome analysis scheme is capable of detecting the full range of cellular proteins. This is due in part to the limited dynamic range of the detectors used to sense proteins or peptides. We present a new technology, structured illumination (SI) gel imager, which detects fluorescently labeled proteins in electrophoretic gels over a 1 000 000‐fold concentration range. SI uses computer‐generated masks to attenuate the illumination of highly abundant proteins, allowing for long exposures of low‐abundance proteins, thus avoiding detector saturation. A series of progressively masked gel images are assembled into a single, very high dynamic range image. We demonstrate that the SI imager can detect proteins over a concentration range of approximately 1 000 000‐fold, making it a useful tool for comprehensive, unbiased proteome‐wide surveys.     

2DE‐based approach for estimation of number of protein species in a cell

Insufficient sensitivity of methods for detection of proteins at a single molecule level does not yet allow obtaining the whole image of human proteome. But to go further, we need at least to know the proteome size, or how many different protein species compose this proteome. This is the task that could be at least partially realized by the method described in this article. The approach used in our study is based on detection of protein spots in 2DE after staining by protein dyes with various sensitivities. As the different protein spots contain different protein species, counting the spots opens a way for estimation of number of protein species. The function representing the dependence of the number of protein spots on sensitivity or LOD of protein dyes was generated. And extrapolation of this function curve to theoretical point of the maximum sensitivity (detection of a single smallest polypeptide) allowed to counting the number of different molecules (polypeptide species) at the concentration level of a single polypeptide per proteome. Using this approach, it was estimated that the minimal numbers of protein species for model objects, Escherichia coli and Pirococcus furiosus, are 6200 and 3400, respectively. We expect a single human cell (HepG2) to contain minimum 70 000 protein species.     

A concise approach to CF3-containing furan-3-ones, (bis)pyrazoles from novel fluorinated building blocks based on 2,3-butanedione

1,1,1-Trifluoro-4,5,5-trimethoxyhex-3-en-2-one and lithium (2Z)-1,1,1-trifluoro-5,5-dialkoxy-4-oxohex-2-en-2-olate were synthesized for the first time via direct trifluoroacetylation of 2,3-butanedione acetal derivatives. A simple and effective approach to acetal or acyl substituted CF₃-pyrazoles, 5,5′-bis(trifluoromethyl)-3,3′-bipyrazole, and, to 5-trifluoromethylfuran-3-ones is presented.     

Application of positron spectroscopy for investigation of threshold space of oil-gas rocks

This article considers the possibility of applying the positron annihilation spectroscopy method for investigating the pore space of rocks from oil-gas and methane-coal deposits. The diagnostics of the structure was performed using the method of spectrometry of angular correlation of annihilation rays (ACAR). Using the samples of porous silicon, the authors have shown the applicability of the ACAR method for determination of the average dimensions of spherical and cylindrical nanosized objects and their concentration      

New Luminescent Lanthanide Tetrakis-Complexes NEt4[LnL4] Based on Dimethyl-N-Benzoylamidophosphate

New lanthanide dimethyl-N-benzoylamidophosphate (HL) based tetrakis-complexes NEt₄[LnL₄] (Ln³⁺=La, Nd, Sm, Eu, Gd, Tb, Dy) are reported. The complexes are characterized by means of NMR, IR, absorption, and luminescent spectroscopy as well as by elemental, X-Ray, and thermal gravimetric analyses. The phenyl groups of the four ligands of the complex anion are directed towards one side, while the methoxy groups are directed in the opposite side, which makes the complexes under consideration structurally similar to calixarenes. The effect of changing the alkali metal counterion to the organic cation NEt₄⁺ on the structure and properties of the tetrakis-complex [LnL4]^- is analyzed. The complexes exhibit bright characteristic for respective lanthanides luminescence. Rather high intensity of the band of ⁵D₀→⁷F₄ transition, observed in the luminescence spectrum of NEt₄[EuL₄], is discussed based on theoretical calculations.     

Antiprotozoal and Antibacterial Activity of Ravenelin,a Xanthone Isolated from the Endophytic Fungus Exserohilum rostratum

The natural compound ravenelin was isolated from the biomass extracts of Exserohilum rostratum fungus, and its antimicrobial, antiplasmodial, and trypanocidal activities were evaluated. Ravenelin was isolated by column chromatography and HPLC and identified by NMR and MS. The susceptibility of Gram-positive and Gram-negative bacteria strains to ravenelin was determined by microbroth dilution assay. Cytotoxicity was evaluated in hepatocarcinoma cells (HepG2) and BALB/c peritoneal macrophages by using MTT. SYBR Green I-based assay was used in the asexual stages of Plasmodium falciparum. Trypanocidal activity was tested against the epimastigote and intracellular amastigote forms of Trypanosoma cruzi. Ravenelin was active against Gram-positive bacteria strains, with emphasis on Bacillus subtilis (MIC value of 7.5 µM). Ravenelin’s antiparasitic activities were assessed against both the epimastigote (IC₅₀ value of 5 ± 1 µM) and the intracellular amastigote forms of T. cruzi (IC₅₀ value of 9 ± 2 µM), as well as against P. falciparum (IC₅₀ value of 3.4 ± 0.4 µM). Ravenelin showed low cytotoxic effects on both HepG2 (CC₅₀ > 50 µM) and peritoneal macrophage (CC₅₀ = 185 ± 1 µM) cells with attractive selectivity for the parasites (SI values > 15). These findings indicate that ravenelin is a natural compound with both antibacterial and antiparasitic activities, and considerable selectivity indexes. Therefore, ravenelin is an attractive candidate for hit-to-lead development.     

Synthesis and Neurotropic Activity of New Heterocyclic Systems: Pyridofuro[3,2-d]pyrrolo[1,2-a]pyrimidines,Pyridofuro[3,2-d]pyrido[1,2-a]pyrimidines and Pyridofuro[3′,2′:4,5]pyrimido[1,2-a]azepines

Background: Neurotic disturbances, anxiety, neurosis-like disorders, and stress situations are widespread. Benzodiazepine tranquillizers have been found to be among the most effective antianxiety drugs. The pharmacological action of benzodiazepines is due to their interaction with the supra-molecular membrane GABA-a-benzodiazepine receptor complex, linked to the Cl-ionophore. Benzodiazepines enhance GABA-ergic transmission and this has led to a study of the role of GABA in anxiety. The search for anxiolytics and anticonvulsive agents has involved glutamate-ergic, 5HT-ergic substances and neuropeptides. However, each of these well-known anxiolytics, anticonvulsants and cognition enhancers (nootropics) has repeatedly been reported to have many adverse side effects, therefore there is an urgent need to search for new drugs able to restore damaged cognitive functions without causing significant adverse reactions. Objective: Considering the relevance of epilepsy diffusion in the world, we have addressed our attention to the discovery of new drugs in this field Thus our aim is the synthesis and study of new compounds with antiepileptic (anticonvulsant) and not only, activity. Methods: For the synthesis of compounds classical organic methods were used and developed. For the evaluation of biological activity some anticonvulsant and psychotropic methods were used. Results: As a result of multistep reactions 26 new, five-membered heterocyclic systems were obtained. PASS prediction of anticonvulsant activity was performed for the whole set of the designed molecules and probability to be active Pa values were ranging from 0.275 to 0.43. The studied compounds exhibit protection against pentylenetetrazole (PTZ) seizures, anti-thiosemicarbazides effect as well as some psychotropic effect. The biological assays evidenced that some of the studied compounds showed a high anticonvulsant activity by antagonism with pentylenetetrazole. The toxicity of compounds is low and they do not induce muscle relaxation in the studied doses. According to the study of psychotropic activity it was found that the selected compounds have an activating behavior and anxiolytic effects on the models of “open field” and “elevated plus maze” (EPM). The data obtained indicate the anxiolytic (anti-anxiety) activity of the derivatives of pyrimidines, especially pronounced in compounds 6n, 6b, and 7c. The studied compounds increase the latent time of first immobilization on the model of “forced swimming” (FST) and exhibit some antidepressant effect similarly to diazepam. Docking studies revealed that compound 6k bound tightly in the active site of GABA_A receptor with a value of the scoring function that estimates free energy of binding (ΔG) at −7.95 kcal/mol, while compound 6n showed the best docking score and seems to be dual inhibitor of SERT transporter as well as 5-HT_1A receptor. Conclusions: Тhe selected compounds have an anticonvulsant, activating behavior and anxiolytic effects, at the same time exhibit some antidepressant effect.     

Metabolomics Insights of the Immunomodulatory Activities of Phlorizin and Phloretin on Human THP-1 Macrophages

Dihydrochalcones, phlorizin (PZ) and its aglycone phloretin (PT), have evidenced immunomodulatory effects through several mechanisms. However, the differential metabolic signatures that lead to these properties are largely unknown. Since macrophages play an important role in the immune response, our study aimed to characterise human THP-1 macrophages under PZ and PT exposure. A multiplatform-based untargeted metabolomics approach was used to reveal metabolites associated with the anti-inflammatory mechanisms triggered by the dihydrochalcones in LPS-stimulated macrophages, for the first time. Results showed differential phenotypic response in macrophages for all treatments. Dihydrochalcone treatment in LPS-stimulated macrophages mimics the response under normal conditions, suggesting inhibition of LPS response. Antagonistic effects of dihydrochalcones against LPS was mainly observed in glycerophospholipid and sphingolipid metabolism besides promoting amino acid biosynthesis. Moreover, PT showed greater metabolic activity than PZ. Overall, the findings of this study yielded knowledge about the mechanisms of action PZ and PT at metabolic level in modulating inflammatory response in human cells.     

Synthesis and antiherpetic activity of novel purine conjugates with 7,8-difluoro-3-methyl-3,4-dihydro-2H-1,4-benzoxazine

Chemistry of Heterocyclic Compounds - A method for the synthesis of novel purine conjugates with 7,8-difluoro-3-methyl-3,4-dihydro-2H-1,4-benzoxazine containing fragments of ω-amino acids with...     

A survey of electrokinetically-driven microfluidics for cancer cells manipulation

Cancer is one of the leading causes of annual deaths worldwide, accounting for nearly 10 million deaths each year. Metastasis, the process by which cancer spreads across the patient's body, is the main cause of death in cancer patients. Because the rising trend observed in statistics of new cancer cases and cancer-related deaths does not allow for an optimistic viewpoint on the future—in relation to this terrible disease—the scientific community has sought methods to enable early detection of cancer and prevent the apparition of metastatic tumors. One such method is known as liquid biopsy, wherein a sample is taken from a bodily fluid and analyzed for the presence of CTCs or other cancer biomarkers (e.g., growth factors). With this objective, interest is growing by year in electrokinetically-driven microfluidics applied for the concentration, capture, filtration, transportation, and characterization of CTCs. Electrokinetic techniques—electrophoresis, dielectrophoresis, electrorotation, and electrothermal and EOF—have great potential for miniaturization and integration with electronic instrumentation for the development of point-of-care devices, which can become a tool for early cancer diagnostics and for the design of personalized therapeutics. In this contribution, we review the state of the art of electrokinetically-driven microfluidics for cancer cells manipulation.