Search

Unsaturated fatty acids, such as oleic acid (OA) and linoleic acid (LA), are promising antimicrobial and cytostatic agents. We modified OA and LA with thymol (TOA and TLA, respectively) to expand their bioavailability, stability, and possible applications, and encapsulated these derivatives in polymeric nanoparticles (TOA-NPs and TLA-NPs, respectively). Prior to synthesis, we performed mathematical simulations with PASS and ADMETlab 2.0 to predict the biological activity and pharmacokinetics of TOA and TLA. TOA and TLA were synthesized via esterification in the presence of catalysts. Next, we formulated nanoparticles using the single-emulsion solvent evaporation technique. We applied dynamic light scattering, Uv-vis spectroscopy, release studies under gastrointestinal (pH 1.2–6.8) and blood environment simulation conditions (pH 7.4), and in vitro biological activity testing to characterize the nanoparticles. PASS revealed that TOA and TLA have antimicrobial and anticancer therapeutic potential. ADMETlab 2.0 provided a rationale for TOA and TLA encapsulation. The nanoparticles had an average size of 212–227 nm, with a high encapsulation efficiency (71–93%), and released TOA and TLA in a gradual and prolonged mode. TLA-NPs possessed higher antibacterial activity against B. cereus and S. aureus and pronounced cytotoxic activity against MCF-7, K562, and A549 cell lines compared to TOA-NPs. Our findings expand the biomedical application of fatty acids and provide a basis for further in vivo evaluation of designed derivatives and formulations.

Upon oxidative stress, mammalian cells rapidly reprogram their translation. This is accompanied by the formation of stress granules (SGs), cytoplasmic ribonucleoprotein condensates containing untranslated mRNA molecules, RNA-binding proteins, 40S ribosomal subunits, and a set of translation initiation factors. Here we show that arsenite-induced stress causes a dramatic increase in the stop-codon readthrough rate and significantly elevates translation reinitiation levels on uORF-containing and bicistronic mRNAs. We also report the recruitment of translation termination factors eRF1 and eRF3, as well as ribosome recycling and translation reinitiation factors ABCE1, eIF2D, MCT-1, and DENR to SGs upon arsenite treatment. Localization of these factors to SGs may contribute to a rapid resumption of mRNA translation after stress relief and SG disassembly. It may also suggest the presence of post-termination, recycling, or reinitiation complexes in SGs. This new layer of translational control under stress conditions, relying on the altered spatial distribution of translation factors between cellular compartments, is discussed.

Changes in metabolic pathways are often associated with the development of various pathologies including cancer, inflammatory diseases, obesity and metabolic syndrome. Identification of the particular metabolic events that are dysregulated may yield strategies for pharmacologic intervention. However, such studies are hampered by the use of classic cell media that do not reflect the metabolite composition that exists in blood plasma and which cause non-physiological adaptations in cultured cells. In recent years two groups presented media that aim to reflect the composition of human plasma, namely human plasma-like medium (HPLM) and Plasmax. Here we describe that, in four different mammalian cell lines, Plasmax enhances mitochondrial respiration. This is associated with the formation of vast mitochondrial networks and enhanced production of reactive oxygen species (ROS). Interestingly, cells cultivated in Plasmax displayed significantly less lysosomes than when any standard media were used. Finally, cells cultivated in Plasmax support replication of various RNA viruses, such as hepatitis C virus (HCV) influenza A virus (IAV), severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and several others, albeit at lower levels and with delayed kinetics. In conclusion, studies of metabolism in the context of viral infections, especially those concerning mitochondria, lysosomes, or redox systems, should be performed in Plasmax medium.

Objective. To estimate performance characteristics and diagnostic value of immunofluorescent islet cell antibody (ICA) assay, immunoradiometric glutamic acid decarboxylase antibody (GADA) assay, and ELISA tyrosine phosphatase IA-2 antibody (IA-2A) and insulin antibody (IA) assays.Research Design and Methods. Antibodies were tested in 438 children and adolescents with newly diagnosed diabetes mellitus (DM) type 1, and in 891 subjects without DM type 1. ICA were determined by the classic indirect immunofluorescent method recommended by the Juvenile Diabetes Foundation International, GADA were determined with the Immunotech IRMA Anti-GAD kit, and IA-2A and IA were determined with Medizym Anti-IA2 and Orgentec Anti-Insulin ELISA kits, respectively. Sensitivity (Se), specificity (Sp), positive predictive value (PPV) and negative predictive value (NPV) of the tests were estimated with contingency tables. Diagnostic accuracy was estimated from areas under receiver operating curves (AUC).Results. ICA test was of the greatest diagnostic value (Se=88%, Sp=96%, PPV=96%, NPV=94%, AUC=0,94), followed by IA-2A (Se=66%, Sp=98%, PPV=98%, NPV=59%, AUC=0,82) and GADA (Se=73%, Sp=84%, PPV=75%, NPV=83%, AUC=0,79). IA test exhibited a very low Se (4,3%) and lacked diagnostic accuracy (AUC=0,5).Conclusions. We recommend to use ICA, IA-2A and GADA tests surveyed in our study for diagnosis of DM type 1 and differential diagnosis of DM. We don’t recommend IA testing with an Orgentec Anti-Insulin ELISA kit for usage in clinical practice.

Pediatric cancers represent a wide variety of different tumors, though they have unique features that distinguish them from adult cancers. Receptor tyrosine kinases KIT and TrkA functions in AML and NB, respectively, are well-characterized. Though expression of these receptors is found in both tumors, little is known about KIT function in NB and TrkA in AML. By combining gene enrichment analysis with multidimensional scaling we showed that pediatric AMLs with t(8;21) or inv16 and high KIT expression levels stand out from other AML subtypes as they share prominent transcriptomic features exclusively with KIT-overexpressing NBs. We showed that AML cell lines had a predominant expression of an alternative TrkAIII isoform, which reportedly has oncogenic features, while NB cell lines had dominating TrkAI-II isoforms. NB cells, on the other hand, had an abnormal ratio of KIT isoforms as opposed to AML cells. Both SCF and NGF exerted protective action against doxorubicin and cytarabine for t(8;21) AML and NB cells. We identified several gene sets both unique and common for pediatric AML and NB, and this expression is associated with KIT or TrkA levels. NMU, DUSP4, RET, SUSD5, NOS1, and GABRA5 genes are differentially expressed in NBs with high KIT expression and are associated with poor survival in NB. We identified HOXA10, BAG3, and MARCKS genes that are connected with TrkA expression and are marker genes of poor outcome in AML. We also report that SLC18A2, PLXNC1, and MRPL33 gene expression is associated with TrkA or KIT expression levels in both AML and NB, and these genes have a prognostic value for both cancers. Thus, we have provided a comprehensive characterization of TrkA and KIT expression along with the oncogenic signatures of these genes across two pediatric tumors.

Recent evidence suggests that fibrotic liver injury in patients with chronic hepatitis C correlates with cellular senescence in damaged liver tissue. However, it is still unclear how senescence can affect replication of the hepatitis C virus (HCV). In this work, we report that an inhibitor of cyclin-dependent kinases 4/6, palbociclib, not only induced in hepatoma cells a pre-senescent cellular phenotype, including G1 arrest in the cell cycle, but also accelerated viral replicon multiplication. Importantly, suppression of HCV replication by direct acting antivirals (DAAs) was barely affected by pre-senescence induction, and vice versa, the antiviral activities of host-targeting agents (HTAs), such as inhibitors of human histone deacetylases (HDACi), produced a wide range of reactions—from a dramatic reduction to a noticeable increase. It is very likely that under conditions of the G1 arrest in the cell cycle, HDACi exhibit their actual antiviral potency, since their inherent anticancer activity that complicates the interpretation of test results is minimized.

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.

Densovirus genome replication and capsid assembly take place in the nucleus of the infected cells. However, the mechanisms underlying such processes as the delivery of virus proteins to the nucleus and the export of progeny virus from the nucleus remain elusive. It is evident that nuclear transport signals should be involved in these processes. We performed an in silico search for the putative nuclear localization signal (NLS) and nuclear export signal (NES) motifs in the capsid proteins of the Blattella germanica Densovirus 1 (BgDV1) densovirus. A high probability NLS motif was found in the common C-terminal of capsid proteins together with a NES motif in the unique N-terminal of VP2. We also performed a global search for the nuclear traffic signals in the densoviruses belonging to five Densovirinae genera, which revealed high diversity in the patterns of NLSs and NESs. Using a heterologous system, the HeLa mammalian cell line expressing GFP-fused BgDV1 capsid proteins, we demonstrated that both signals are functionally active. We suggest that the NLS shared by all three BgDV1 capsid proteins drives the trafficking of the newly-synthesized proteins into the nucleus, while the NES may play a role in the export of the newly-assembled BgDV1 particles into the cytoplasm through nuclear pore complexes.

We have earlier shown that the typical Didinium nasutum nucleolus is a complex convoluted branched domain, comprising a dense fibrillar component located at the periphery of the nucleolus and a granular component located in the central part. Here our main interest was to study quantitatively the spatial distribution of nucleolar chromatin structures in these convoluted nucleoli. There are no "classical" fibrillar centers in D.nasutum nucleoli. The spatial distribution of nucleolar chromatin bodies, which play the role of nucleolar organizers in the macronucleus of D.nasutum, was studied using 3D reconstructions based on serial ultrathin sections. The relative number of nucleolar chromatin bodies was determined in macronuclei of recently fed, starved D.nasutum cells and in resting cysts. This parameter is shown to correlate with the activity of the nucleolus. However, the relative number of nucleolar chromatin bodies in different regions of the same convoluted nucleolus is approximately the same. This finding suggests equal activity in different parts of the nucleolar domain and indicates the existence of some molecular mechanism enabling it to synchronize this activity in D. nasutum nucleoli. Our data show that D. nasutum nucleoli display bipartite structure. All nucleolar chromatin bodies are shown to be located outside of nucleoli, at the periphery of the fibrillar component.

RNases are currently studied as non-mutagenic alternatives to the harmful DNA-damaging anticancer drugs commonly used in clinical practice. Many mammalian RNases are not potent toxins due to the strong inhibition by ribonuclease inhibitor (RI) presented in the cytoplasm of mammalian cells.In search of new effective anticancer RNases we studied the effects of barnase, a ribonuclease from Bacillus amyloliquefaciens, on human cancer cells. We found that barnase is resistant to RI. In MTT cell viability assay, barnase was cytotoxic to human carcinoma cell lines with half-inhibitory concentrations (IC(50)) ranging from 0.2 to 13 microM and to leukemia cell lines with IC(50) values ranging from 2.4 to 82 microM. Also, we characterized the cytotoxic effects of barnase-based immunoRNase scFv 4D5-dibarnase, which consists of two barnase molecules serially fused to the single-chain variable fragment (scFv) of humanized antibody 4D5 that recognizes the extracellular domain of cancer marker HER2. The scFv 4D5-dibarnase specifically bound to HER2-positive cells and was internalized via receptor-mediated endocytosis. The intracellular localization of internalized scFv 4D5-dibarnase was determined by electronic microscopy. The cytotoxic effect of scFv 4D5-dibarnase on HER2-positive human ovarian carcinoma SKOV-3 cells (IC(50) = 1.8 nM) was three orders of magnitude greater than that of barnase alone. Both barnase and scFv 4D5-dibarnase induced apoptosis in SKOV-3 cells accompanied by internucleosomal chromatin fragmentation, membrane blebbing, the appearance of phosphatidylserine on the outer leaflet of the plasma membrane, and the activation of caspase-3.These results demonstrate that barnase is a potent toxic agent for targeting to cancer cells.