Cell Line T84 Research Articles

Antibody-Free Fluorine-Assisted Metabolic Sequencing of RNA N4-Acetylcytidine.

N4-Acetylcytidine (ac4C) has been found to affect a variety of cellular and biological processes. For a mechanistic understanding of the roles of ac4C in biology and disease, we present an antibody-free, fluorine-assisted metabolic sequencing method to detect RNA ac4C, called "FAM-seq". We successfully applied FAM-seq to profile ac4C landscapes in human 293T, HeLa, and MDA cell lines in parallel with the reported acRIP-seq method. By comparison with the classic ac4C antibody sequencing method, we found that FAM-seq is a convenient and reliable method for transcriptome-wide mapping of ac4C. Because this method holds promise for detecting nascent RNA ac4C modifications, we further investigated the role of ac4C in regulating chemotherapy drug resistance in chronic myeloid leukemia. The results indicated that drug development or combination therapy could be enhanced by appreciating the key role of ac4C modification in cancer therapy.

Downregulation of miR-144 blocked the proliferation and invasion of nerve cells in Hirschsprung disease by regulating Transcription Factor AP 4 (TFAP4).

Hirschsprung's disease (HSCR) is characterized by a dysfunction of enteric neural crest cells (ENCCs) proliferation, migration and premature apoptosis during embryonic development, resulting in aganglionic colon. Our aim is to explore the role of miR-144 with its target gene Transcription Factor AP 4 (TFAP4) in nerve cells in HSCR. The relative expression levels of miR-144 in HSCR colon samples were detected by quantitative real-time PCR (RT-qPCR). Western blot assays were conducted to investigate the TFAP4 protein expressing level. The interaction of miR-144 and TFAP4 was predicted with bioinformatics analysis and examined with luciferase reporter assays. Overexpression or knockdown of miR-144 and TFAP4 in 293T and SH-SY5Y cell lines was applied. Cell proliferation, migration and invasion were detected by CCK-8 assays, Transwell migration and invasion assays. Cell cycle and apoptosis was examined by flow cytometric analysis. Downregulation of miR-144 and upregulation of TFAP4 were shown in HSCR. Luciferase reporter assay indicated that miR-144 reduced luciferase activity in 293T and SH-SY5Y transfected with TFAP4-WT-3UTR luciferase reporter and confirmed TFAP4 was the downstream target gene of miR-144. Data showed that miR-144 promoted the cell proliferation, migration and invasion of 293T and SH-SY5Y, while TFAP4 blocked the cell proliferation, migration and invasion. TFAP4 overexpression reversed the miR-144-mediated cell proliferation, migration and invasion of 293T and SH-SY5Y. Downregulation of miR-144 blocked the cell proliferation and migration of nerve cells via targeting TFAP4 and contributed to the pathogenesis of HSCR. This provides an innovative and candidate target for treatment of HSCR.

Membrane Domain Anti-Registration Induces an Intrinsic Transmembrane Potential.

Plasma membrane segregation into various nanoscale membrane domains is driven by distinct interactions between diverse lipids and proteins. Among them, liquid-ordered (Lo) membrane domains are defined as "lipid rafts" and liquid-disordered (Ld) ones as "lipid non-rafts". Using model membrane systems, both intra-leaflet and inter-leaflet dynamics of these membrane domains are widely studied. Nevertheless, the biological impact of the latter, which is accompanied by membrane domain registration/anti-registration, is far from clear. Hence, in this work, we studied the biological relevance of the membrane domain anti-registration using both all-atom molecular dynamics (MD) simulations and confocal fluorescence microscopy. All-atom MD simulations suggested an intrinsic transmembrane potential for the case of the membrane anti-registration (Lo/Ld). Meanwhile, confocal fluorescence microscopy experiments of HeLa and 293T cell lines indicated that membrane cholesterol depletion could significantly alter the transmembrane potential of cells. Considering differences in the cholesterol content between Lo and Ld membrane domains, our confocal fluorescence microscopy experiments are consistent with our all-atom MD simulations. In short, membrane domain anti-registration induces local membrane asymmetry and, thus, an intrinsic transmembrane potential.

Identification of two Bacillus thuringiensis Cry3Aa toxin-binding aminopeptidase N from Rhynchophorus ferrugineus (Coleoptera: Curculionidae).

Rhynchophorus ferrugineus is a quarantine pest that mainly damages plants in tropical regions, which are essential economic resources. Cry3Aa has been used to control coleopteran pests and is known to be toxic to R. ferrugineus. The binding of the Cry toxin to specific receptors on the target insect plays a crucial role in the toxicological mechanism of Cry toxins. However, in the case of R. ferrugineus, the nature and identity of the receptor proteins involved remain unknown. In the present study, pull-down assays and mass spectrometry were used to identify two proteins of aminopeptidase N proteins (RfAPN2a and RfAPN2b) in the larval midguts of R. ferrugineus. Cry3Aa was able to bind to RfAPN2a (Kd = 108.5 nM) and RfAPN2b (Kd = 68.2 nM), as well as midgut brush border membrane vesicles (Kd = 482.5 nM). In silico analysis of both RfAPN proteins included the signal peptide and anchored sites for glycosyl phosphatidyl inositol. In addition, RfAPN2a and RfAPN2b were expressed in the human embryonic kidney 293T cell line, and cytotoxicity assays showed that the transgenic cells were not susceptible to activated Cry3Aa. Our results show that RfAPN2a and RfAPN2b are Cry3Aa-binding proteins involved in the Cry3Aa toxicity of R. ferrugineus. This study deepens our understanding of the action mechanism of Cry3Aa in R. ferrugineus larvae.

Usnic Acid-Loaded Magnetite Nanoparticles-A Comparative Study between Synthesis Methods.

Since cancer is a continuously increasing concern for the general population, more efficient treatment alternatives ought to be developed. In this regard, a promising direction is represented by the use of magnetite nanoparticles (MNPs) to act both as a nanocarrier for the targeted release of antitumoral drugs and as hyperthermia agents. Thus, the present study focused on improving the control upon the outcome properties of MNPs by using two synthesis methods, namely the co-precipitation and microwave-assisted hydrothermal method, for the incorporation of usnic acid (UA), a natural lichen-derived metabolite with proven anticancer activity. The obtained UA-loaded MNPs were thoroughly characterized regarding their morpho-structural and physicochemical properties through X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS) and zeta potential, scanning electron microscopy (SEM), and vibrating sample magnetometry (VSM). Results demonstrated the formation of magnetite as the unique mineralogical phase through both types of synthesis, with increased uniformity regarding the drug loading efficiency, size, stability, and magnetic properties obtained through the microwave-assisted hydrothermal method. Furthermore, the cytotoxicity of the nanostructures against the HEK 293T cell line was investigated through the XTT assay, which further proved their potential for anticancer treatment applications.

Gynura Cusimba‐Derived Nitrogen‐Doped Carbon Dots/ZnO Nanocomposite: A Biocompatible Alternative for Detecting Latent Fingerprints

AbstractTo address the present need in forensic security perceptions, including the ability to detect latent fingerprints, designing and development of low‐cost fluorescent materials becomes inevitable. This work illustrates the preparation of ZnO and its nanocomposite, Zn−CD with nitrogen doped carbon dots (CD−GC) with all components being generated from a single natural precursor, “Gynura Cusimba”. Cell viability experiment carried out on HEK 293T cell line revealed that CD−GC, pure ZnO nanoparticles, and Zn−CD_0.8 were all biocompatible after being suitably evaluated by various analytical methods. Zn−CD_0.8 nanopowder was useful in detecting level 1–3 features of latent fingerprint developed on several substrates, which displayed good contrast and visibility. The work also highlights the application of Zn−CD_0.8 nanopowder for real time detection of latent fingerprints by demonstrating its use to identify aged fingerprints and those immersed in water. The results indicate that the potential of Zn−CD_0.8 to be a reasonably priced alternative to the existing commercial powders.

Cold atmospheric nitrogen plasma induces metal-initiated cell death by cell membrane rupture and mitochondrial perturbation.

Cold atmospheric plasma (CAP) is a novel biomedical tool used for cancer therapy. A device using nitrogen gas (N2 CAP) produced CAP that induced cell death through the production of reactive nitrogen species and an increase in intracellular calcium. In this study, we investigated the effect of N2 CAP-irradiation on cell membrane and mitochondrial function in human embryonic kidney cell line 293T. We investigated whether iron is involved in N2 CAP-induced cell death, as deferoxamine methanesulfonate (an iron chelator) inhibits this process. We found that N2 CAP induced cell membrane disturbance and loss of mitochondrial membrane potential in an irradiation time-dependent manner. BAPTA-AM, a cell-permeable calcium chelator, inhibited N2 CAP-induced loss of mitochondrial membrane potential. These results suggest that disruption of intracellular metal homeostasis was involved in N2 CAP-induced cell membrane rupture and mitochondrial dysfunction. Moreover, N2 CAP irradiation generated a time-dependent production of peroxynitrite. However, lipid-derived radicals are unrelated to N2 CAP-induced cell death. Generally, N2 CAP-induced cell death is driven by the complex interaction between metal movement and reactive oxygen and nitrogen species produced by N2 CAP.

Functionalized Cyclopentenones with Low Electrophilic Character as Anticancer Agents.

In this study were synthesized non-Michael acceptor cyclopentenones (CP) from biomass derivative furfural as anticancer agents. Cyclic enones, both from natural sources and synthetic analogues, have been described as cytotoxic agents. Most of these agents were unsuccessful in becoming valuable therapeutic agents due to toxicity problems derived from unselective critical biomacromolecule alkylation. This may be caused by Michael addition to the enone system. Ab initio studies revealed that 2,4-substituted CPs are less prone to Michael additions, and as such were tested three families of those derivatives. We prepare the new CPs from furfural through a tandem furan ring opening/Nazarov electrocyclization and further functionalization. Experimentally the 2,4-substituted CPs exhibited no reactivity towards sulphur nucleophiles, while maintaining cytotoxicity against HT-29, MCF-7, NCI-H460, HCT-116 and MDA-MB 231 cells lines. Moreover, the selected CP are non-toxic against healthy HEK 293T cell lines and present proper calculated drug-like properties.

MYO5B levels are linked to microRNAs and UNC45A in colorectal cancer

Background: Colorectal cancer (CRC) is currently the third most common cancer worldwide and the second most deadly cancer. CRC is characterized by the loss of cellular architecture and altered polarity; features which can be controlled by cell-trafficking. In the intestine key cell trafficking pathways are controlled by Myosin 5b (MYO5B). Loss of functional MYO5B leads to profound changes in gastrointestinal epithelial cells including increased proliferation, alterations in tight junction proteins, and decreased differentiation. For MYO5B to be functional, it must be folded into the correct structure. This folding is accomplished by the chaperone UNC45A. Recent work indicates that MYO5B is decreased in gastric cancer and during colorectal tumor progression. However, the mechanism resulting in altered MYO5B expression in colorectal cancer is currently unknown. Hypothesis: We hypothesize that microRNAs regulate the expression of UNC45A which in turn can impact MYO5B function to promote colorectal cancer. Methods & Results: Analysis of high-throughput RNA-sequencing data in the Cancer Genome Atlas revealed that the MYO5B gene was hyper-methylated in CRC tumors (n=286) compared to controls (n=41). In gastric cancer, hyper-methylation leads to silencing of MYO5B gene expression. Consistent with findings in gastric cancer, we found that MYO5B gene expression was significantly reduced in CRC tumors. Immunostaining of CRC tumor arrays and analysis of the Clinical Proteomic Tumor Analysis Consortium demonstrated that MYO5B was also reduced at the protein level. For comparison, we examined MYO5B levels in normal human colonic organoid monolayers, immortalized human colon NCM-460 cells, and colon cancer cell lines T84 and HT29-MTX cells. By qPCR, we found that MYO5B gene expression was significantly reduced in the cancer cells compared to the human colonic organoids, confirming our database analysis. We reasoned that even if MYO5B was generated in CRC, it might not be functional if UNC45A was not present. Using the same databases, we found that UNC45A levels were unchanged at the level of gene expression, but significantly reduced at the level of protein. Analysis of miRMap data revealed that multiple microRNAs can target UNC45A. The top 10 UN45A-targeting microRNAs were all found to be increased in CRC compared to normal control tissue; suggesting that UNC45A protein may be diminished due to microRNA mediated cleavage. The functional significance of decreased MYO5B in CRC was highlighted by the fact that patients with lower levels of MYO5B had decreased survival compared to those with higher levels of MYO5B. Additionally, loss of MYO5B correlated with increased ribosome biogenesis; a key driver of tumor proliferation. Conclusions: These data indicate the MYO5B is likely decreased in CRC due to gene methylation and improper protein folding by UNC45A. Loss of MYO5B is significant since it is associated with a worse prognosis and c This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A Missense Mutation c.1132G > A in Fumarate Hydratase (FH) Leads to Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) Syndrome and Insights into Clinical Management in Uterine Leiomyomata

HLRCC syndrome is a hereditary cancer predisposition syndrome caused by heterozygous germline pathogenic variant of the fumarate hydratase (FH) gene and characterized by cutaneous leiomyomas (CL), uterine leiomyomas (UL), and renal cell carcinoma (RCC). Loss of function variant of FH gene inactivates the Kreb's cycle enzyme activity and predisposes individuals with such variant to the development of HLRCC. Next-generation sequencing (NGS) and Sanger confirmation were given to family members accessible. Following that, a functional study in vitro was performed to further confirm the pathogenicity of the variant. FH-Wild type (FH-WT) and FH-mutant (FH-MUT) (E378K) plasmid were constructed and transfected into 293T and uterine leiomyoma cell lines, respectively. Proliferation assessment was executed to show how this mutation affects the growth of uterine leiomyoma. qPCR and Western blotting were performed to investigate the change of transcription and translation of FH with mutation (E378K), and FH enzyme assay activity were tested in 293T cells with mutation and wild-type plasmids. Here, we presented two families with the same missense variant (c.1132G > A) that has not been reported as a germline mutation in hereditary uterine leiomyomas before and classified as VUS in gene databases. Our in vitro experiments supported the pathogenicity of this missense variant, especially in uterine leiomyomata. According to the American College of Medical Genetics (ACMG) guideline, the E378K variant was classified as likely pathogenic (with evidence PS4_support, PS3_support, PM2_support, PP1, PP3 and PP4 evidence). Further insights into clinical management in uterine leiomyomata were discussed and should be practiced in gynecological clinical settings.

Comparative Analyses of Human Exosome Proteomes.

Exosomes are responsible for cell-to-cell communication and serves as a valuable drug delivery vehicle. However, exosome heterogeneity, non-standardized isolation methods and proteomics/bioinformatics approaches limit its clinical applications. To better understand exosome heterogeneity, biological function and molecular mechanism of its biogenesis, secretion and uptake, techniques in proteomics or bioinformatics were applied to investigate human embryonic kidney cell (293T cell line)-derived exosome proteome and enable an integrative comparison of exosomal proteins and protein-protein interaction (PPI) networks of eleven exosome proteomes extracted from diverse human samples, including 293T (two datasets), dermal fibroblast, mesenchymal stem cell, thymic epithelial primary cell, breast cancer cell line (MDA-MB-231), patient neuroblastoma cell, plasma, saliva, serum and urine. Mapping of exosome biogenesis/secretion/uptake-related proteins onto exosome proteomes highlights exosomal origin-specific routes of exosome biogenesis/secretion/uptake and exosome-dependent intercellular communication. The finding provides insight into comparative exosome proteomes and its biogenesis, secretion and uptake, and potentially contributes to clinical applications.

Arthrocolins Synergizing with Fluconazole Inhibit Fluconazole-Resistant Candida albicans by Increasing Riboflavin Metabolism and Causing Mitochondrial Dysfunction and Autophagy.

Our previous study reported that seminaturally occurring arthrocolins A to C with unprecedented carbon skeletons could restore the antifungal activity of fluconazole against fluconazole-resistant Candida albicans. Here, we showed that arthrocolins synergized with fluconazole, reducing the fluconazole minimum and dramatically augmenting the survivals of 293T human cells and nematode Caenorhabditis elegans infected with fluconazole-resistant C. albicans. Mechanistically, fluconazole can induce fungal membrane permeability to arthrocolins, leading to the intracellular arthrocolins that were critical to the antifungal activity of the combination therapy by inducing abnormal cell membranes and mitochondrial dysfunctions in the fungus. Transcriptomics and reverse transcription-quantitative PCR (qRT-PCR) analysis indicated that the intracellular arthrocolins induced the strongest upregulated genes that were involved in membrane transports while the downregulated genes were responsible for fungal pathogenesis. Moreover, riboflavin metabolism and proteasomes were the most upregulated pathways, which were accompanied by inhibition of protein biosynthesis and increased levels of reactive oxygen species (ROS), lipids, and autophagy. Our results suggested that arthrocolins should be a novel class of synergistic antifungal compounds by inducing mitochondrial dysfunctions in combination with fluconazole and provided a new perspective for the design of new bioactive antifungal compounds with potential pharmacological properties. IMPORTANCE The prevalence of antifungal-resistant Candida albicans, which is a common human fungal pathogen causing life-threatening systemic infections, has become a challenge in the treatment of fungal infections. Arthrocolins are a new type of xanthene obtained from Escherichia coli fed with a key fungal precursor toluquinol. Different from those artificially synthesized xanthenes used as important medications, arthrocolins can synergize with fluconazole against fluconazole-resistant Candida albicans. Fluconazole can induce the fungal permeability of arthrocolins into fungal cells, and then the intracellular arthrocolins exerted detrimental effects on the fungus by inducing fungal mitochondrial dysfunctions, leading to dramatically reduced fungal pathogenicity. Importantly, the combination of arthrocolins and fluconazole are effective against C. albicans in two models, including human cell line 293T and nematode Caenorhabditis elegans. Arthrocolins should be a novel class of antifungal compounds with potential pharmacological properties.

Transcriptional Regulation of Liver-Type OATP1B3 (Lt-OATP1B3) and Cancer-Type OATP1B3 (Ct-OATP1B3) Studied in Hepatocyte-Derived and Colon Cancer-Derived Cell Lines

Due to alternative splicing, the SLCO1B3 gene encodes two protein variants; the hepatic uptake transporter liver-type OATP1B3 (Lt-OATP1B3) and the cancer-type OATP1B3 (Ct-OATP1B3) expressed in several cancerous tissues. There is limited information about the cell type-specific transcriptional regulation of both variants and about transcription factors regulating this differential expression. Therefore, we cloned DNA fragments from the promoter regions of the Lt-SLCO1B3 and the Ct-SLCO1B3 gene and investigated their luciferase activity in hepatocellular and colorectal cancer cell lines. Both promoters showed differences in their luciferase activity depending on the used cell lines. We identified the first 100 bp upstream of the transcriptional start site as the core promoter region of the Ct-SLCO1B3 gene. In silico predicted binding sites for the transcription factors ZKSCAN3, SOX9 and HNF1α localized within these fragments were further analyzed. The mutagenesis of the ZKSCAN3 binding site reduced the luciferase activity of the Ct-SLCO1B3 reporter gene construct in the colorectal cancer cell lines DLD1 and T84 to 29.9% and 14.3%, respectively. In contrast, using the liver-derived Hep3B cells, 71.6% residual activity could be measured. This indicates that the transcription factors ZKSCAN3 and SOX9 are important for the cell type-specific transcriptional regulation of the Ct-SLCO1B3 gene.

P4HA2 induces hepatic ductular reaction and biliary fibrosis in chronic cholestatic liver diseases.

Prolyl-4-hydroxylases (P4Hs) are key enzymes in collagen synthesis. The P4HA subunit (P4HA1, P4HA2, and P4HA3) contains a substrate binding and catalyzation domain. We postulated that P4HA2 would play a key role in the cholangiocyte pathology of cholestatic liver diseases. We studied humans with primary biliary cholangitis (PBC) and Primary sclerosing cholangitis (PSC), P4HA2 -/- mice injured by DDC, and P4HA2 -/- /MDR2 -/- double knockout mice. A parallel study was performed in patients with PBC, PSC, and controls using immunohistochemistry and immunofluorescence. In the murine model, the level of ductular reaction and biliary fibrosis were monitored by histology, qPCR, immunohistochemistry, and Western blotting. Expression of Yes1 Associated Transcriptional Regulator (YAP) phosphorylation was measured in isolated mouse cholangiocytes. The mechanism of P4HA2 was explored in RBE and 293T cell lines by using qPCR, Western blot, immunofluorescence, and co-immunoprecipitation. The hepatic expression level of P4HA2 was highly elevated in patients with PBC or PSC. Ductular reactive cholangiocytes predominantly expressed P4HA2. Cholestatic patients with more severe liver injury correlated with levels of P4HA2 in the liver. In P4HA2 -/- mice, there was a significantly reduced level of ductular reaction and fibrosis compared with controls in the DDC-induced chronic cholestasis. Decreased liver fibrosis and ductular reaction were observed in P4HA2 -/- /MDR2 -/- mice compared with MDR2 -/- mice. Cholangiocytes isolated from P4HA2 -/- /MDR2 -/- mice displayed a higher level of YAP phosphorylation, resulting in cholangiocytes proliferation inhibition. In vitro studies showed that P4HA2 promotes RBE cell proliferation by inducing SAV1 degradation, eventually resulting in the activation of YAP. P4HA2 promotes hepatic ductular reaction and biliary fibrosis by regulating the SAV1-mediated Hippo signaling pathway. P4HA2 is a potential therapeutic target for PBC and PSC.

Evaluation of cytotoxicity, reactive oxygen species and nitrous oxide of nanochitosan from shrimp shell

This work aims to synthesize, characterize and evaluate the biological activity of nanochitosan (NQ) prepared from shrimp, showing an innovative character and correlating with sustainable development, in promoting an alternative to the solid waste (shrimp) shell and a biological application of the novel nanomaterial. The NQ synthesis was carried out by the alkaline deacetylation process of chitin obtained of the demineralization, deproteinization and deodorization steps from shrimp shells. NQ was characterized by X-ray Powder Diffraction (XRD), Fourier Transform infrared spectroscopy (FTIR), Scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDS), N2 porosimetry (BET/BJH methods), zeta potential (ZP) and zero charge point (pHZCP). To evaluate the safety profile was carried out the cytotoxicity, DCFHA and NO tests in 293T and HaCat cell lines. Regarding the cell viability, NQ did not show toxicity for the tested cell lines. In the evaluation of the ROS production and NO tests, there was no increase in the levels of free radicals and between the negative control, respectively. Therefore, NQ does not present cytotoxicity in the cell lines tested (10, 30, 100 and 300 μg mL−1), proposing new perspectives on the use of NQ as a potential nanomaterial for biomedical applications.

Nucleopeptide-Coupled Injectable Bioconjugated Guanosine-Quadruplex Hydrogel with Inherent Antibacterial Activity.

The multicomponent reaction-directed self-assembled hydrogels offer the opportunities to fabricate materials with ubiquitous properties which sometimes are not possible to generate from single components. Therefore, multicomponent-derived hydrogels have enormous applications in biomedical fields, and the number of such systems is increasing day by day. Herein, the multicomponent self-assembly techniques have been employed to develop a biomimetic low-molecular-weight G-quadruplex hydrogel under physiological conditions. The bioconjugation of guanosine, 4-formylphenylboronic acid, and cytosine-functionalized nucleopeptide (NP) is important to generate the multicomponent self-assembled dynamic imino-boronate ester-mediated bioconjugated G-quadruplex hydrogels. Using thioflavin T fluorescence assay, powder X-ray diffraction, and circular dichroism spectroscopic techniques, we confirm the existence of a G-quartet-like structure as the key parameter for the formation of nanofibrillar hydrogels. The multicomponent self-assembled G-quadruplex hydrogel possesses excellent inherent antibacterial activity against a broad range of bacterial species. The in vitro cytocompatibility of the synthesized hydrogel was evaluated on MCF-7 and HEK 293T cell lines to study the biocompatibility of the hydrogel. The proposed injectable, biocompatible, and NP-coupled G-quadruplex hydrogel with inherent antibacterial efficiency holds promising importance to prevent localized bacterial infections.

Polymyxin B Conjugates with Bio-Inspired Synthetic Polymers of Different Nature.

The emergence and growth of bacterial resistance to antibiotics poses an enormous threat to humanity in the future. In this regard, the discovery of new antibiotics and the improvement of existing ones is a priority task. In this study, we proposed the synthesis of new polymeric conjugates of polymyxin B, which is a clinically approved but limited-use peptide antibiotic. In particular, three carboxylate-bearing polymers and one synthetic glycopolymer were selected for conjugation with polymyxin B (PMX B), namely, poly(α,L-glutamic acid) (PGlu), copolymer of L-glutamic acid and L-phenylalanine (P(Glu-co-Phe)), copolymer of N-vinyl succinamic acid and N-vinylsuccinimide (P(VSAA-co-VSI)), and poly(2-deoxy-2-methacrylamido-D-glucose) (PMAG). Unlike PGlu and PMAG, P(Glu-co-Phe) and P(VSAA-co-VSI) are amphiphilic and form nanoparticles in aqueous media. A number of conjugates with different polymyxin B loading were synthesized and characterized. In addition, the complex conjugates of PGLu or PMAG with polymyxin B and deferoxamine (siderophore) were obtained. A release of PMX B from Schiff base and amide-linked polymer conjugates was studied in model buffer media with pH 7.4 and 5.8. In both cases, a more pronounced release was observed under slightly acidic conditions. The cytotoxicity of free polymers and PMX B as well as their conjugates was examined in human embryonic kidney cells (HEK 293T cell line). All conjugates demonstrated reduced cytotoxicity compared to the free antibiotic. Finally, the antimicrobial efficacy of the conjugates against Pseudomonas aeruginosa was determined and compared. The lowest values of minimum inhibitory concentrations (MIC) were observed for polymyxin B and polymyxin B/deferoxamine conjugated with PMAG. Among the polymers tested, PMAG appears to be the most promising carrier for delivery of PMX B in conjugated form due to the good preservation of the antimicrobial properties of PMX B and the ability of controlled drug release.

Rheumatoid factor recognizes specific domains of the IgG heavy chain complexed with HLA class II molecules.

We previously reported that RF recognized the IgG heavy chain (IgGH)/RA-susceptible HLA class II molecule complex. In the present study, we investigated the molecular mechanisms underlying HLA binding to and the RF recognition of IgGH. We synthesized various types of IgGH segments, including VH, CH1, CH2 and CH3, and transfected them with or without HLA class II molecules into the Human Embryonic Kidney 293T cell line. IgGH single domains linked with the HLA-Cw3 peptide, which binds to the binding groove of the HLA class II molecule, were also synthesized. The expression of IgGH domains on the cell surface and their recognition by RF were examined using flow cytometry. Flag-tagged IgGH segments containing CH1 (CH1, VH-CH1, CH1-CH2, VH-CH1-CH2, CH1-CH2-CH3 and VH-CH1-CH2-CH3) were clearly presented on the cell surface by HLA-DR4, while segments without the CH1 domain were expressed at a low level, and the CH3 single domain was only weakly detected on the cell surface, even with HLA-DR4. We then transfected IgGH single domains linked to the Cw3 peptide together with HLA-DR4 and showed that RF-containing sera from RA patients only recognized the CH3 domain and none of the other single domains. When various segments without the Cw3 peptide were transfected with HLA-DR4, only the CH1-CH2-CH3 segment and full-length IgGH were detected by the sera of RA patients. The CH1 domain of IgGH binds to the RA-susceptible HLA-DR molecule and is expressed on the cell surface. RF specifically recognizes the CH3 domain of the IgGH/HLA-DR4 complex.

A Comparative Analysis of Polyphenol Content and Biological Potential of Quercus petraea Matt. and Q. pubescens Willd. Bark Extracts

Quercus wood (oak wood) is a valuable resource, which has led to the intense processing of oak trees by the forestry industry. As a result, large amounts of forestry by-products (bark and leaves) are left in the woods, considered valueless. Thus, the aim of this study was to evaluate and compare the phytochemical profile and potential biological activities of Q. petreaea Matt. and Q. pubescens Willd. bark extracts. The extracts were obtained by microwave (MAE) and ultrasound-assisted extraction (UAE) with water (100%) and ethanol:water (70:30). These extracts were then characterized in terms of the total polyphenolic and tannin contents using the Folin–Ciocâlteu method. Their antioxidant properties were determined by observing the neutralizing effects of the extracts against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radicals. The antimicrobial effect was tested on Gram-positive and Gram-negative bacterial strains and three fungi from the Candida genus. Cellular counts were measured to determine the cytotoxic effects of the extracts on HEK 293T cell lines. Moreover, spectrophotometrical assays were performed to assess the inhibitory effects of the extracts against the enzymatic activity of α-glucosidase, tyrosinase, and acetylcholinesterase. The MAE resulted in higher yields of polyphenolic compounds and tannins compared to the UAE bark extracts. All of the experimental variants exhibited free-radical-neutralizing properties, especially Q. petraea extracts. Q. petraea extracts also had a more efficient antibacterial effect, especially against Gram-positive bacteria and K. pneumoniae. Antifungal activity was highlighted against C. krusei. Cell counts indicated a cytotoxic effect of the tested extracts against HEK 293T cells. The tested extracts inhibited the activity of α-glucosidase, tyrosinase, and acetylcholinesterase, indicating the potential use of these extracts as antidiabetic, neuroprotective, and skin-protecting agents. These findings highlight the untapped therapeutic potential of the bioactive compounds found in the bark of Q. petraea and Q. pubescens.

Small RNA interfering effects in chicken embryonic fibroblasts using two sites 8664/p201 and 2122/pll3.7 of chicken s1p1/edg 1 gene

Introduction: Currently, short interfering RNA (siRNA) is an experimental tool in molecular biology to study gene function in cell culture and in vivo in model of organisms (mouse and chicken embryos, etc.) Method: Lentivirus production technique by human embryonic kidney 293T cell lines (HEK293T) with two (2) sites 8664/P201 and 2122/Pll.3.7 of the sphingosine 1 phosphate receptor gene and endothelial differentiation 1 gene (S1P1/ Chicken EDG1) was used to show the effects of small interfering RNA in chicken embryonic fibroblasts during embryogenesis. After preparation of plasmids 8664/P201 and 2122/Pll3.7, lentivirus packaging, transfection with 2xHBS, virus titration and chicken egg preparation, microinjection of lentivirus was performed in the embryos chicken. Results: The results obtained after the microinjection of lentivirus into chicken embryos from the second day to the fourth day of incubation showed the effects of small interfering RNA in the embryonic development of chicken materialized by bleeding in the yolk sac of embryos therefore a decrease in gene function. Conclusion: Previous studies have already proven that expression of small interfering RNA decreases in embryos of certain vertebrates such as mammals.