Human Cell Cultures Research Articles

Carbon nanotubes conjugated with cisplatin activate different apoptosis signaling pathways in 2D and 3D-spheroid triple-negative breast cancer cell cultures: a comparative study.

The type of experimental model for the in vitro testing of drug formulations efficiency represents an important tool in cancer biology, with great attention being granted to three-dimensional (3D) cultures as these offer a closer approximation of the clinical sensitivity of drugs. In this study, the effects induced by carboxyl-functionalized single-walled carbon nanotubes complexed with cisplatin (SWCNT-COOH-CDDP) and free components (SWCNT-COOH and CDDP) were compared between conventional 2D- and 3D-spheroid cultures of human breast cancer cells. The 2D and 3D breast cancer cultures were exposed to various doses of SWCNT-COOH (0.25-2μg/mL), CDDP (0.158-1.26μg/mL) and the same doses of SWNCT-COOH-CDDP complex for 24 and 48h. The anti-tumor activity, including modulation of cell viability, oxidative stress, proliferation, apoptosis, and invasion potential, was explored by spectrophotometric and fluorometric methods, immunoblotting, optical and fluorescence microscopy. The SWCNT-COOH-CDDP complex proved to have high anti-cancer efficiency on 2D and 3D cultures by inhibiting cell proliferation and activating cell death. A dose of 0.632μg/mL complex triggered different pathways of apoptosis in 2D and 3D cultures, by intrinsic, extrinsic, and reticulum endoplasmic pathways. Overall, the 2D cultures showed higher susceptibility to the action of complex compared to 3D cultures and SWCNT-COOH-CDDP proved enhanced anti-tumoral activity compared to free CDDP.

Nanoformulation of dasatinib cannot overcome therapy resistance of pancreatic cancer cells with low LYN kinase expression.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most difficult to treat tumors. The Src (sarcoma) inhibitor dasatinib (DASA) has shown promising efficacy in preclinical studies of PDAC. However, clinical confirmation could not be achieved. Overall, our aim was to deliver arguments for the possible reinitiating clinical testing of this compound in a biomarker-stratifying therapy trial for PDAC patients. We tested if the nanofunctionalization of DASA can increase the drug efficacy and whether certain Src members can function as clinical predictive biomarkers. Methods include manufacturing of poly(vinyl alcohol) stabilized gold nanoparticles and their drug loading, dynamic light scattering, transmission electron microscopy, thermogravimetric analysis, Zeta potential measurement, sterile human cell culture, cell growth quantification, accessing and evaluating transcriptome and clinical data from molecular tumor dataset TCGA, as well as various statistical analyses. We generated homo-dispersed nanofunctionalized DASA as an AuNP@PVA-DASA conjugate. The composite did not enhance the anti-growth effect of DASA on PDAC cell lines. The cell model with high LYN expression showed the strongest response to the therapy. We confirm deregulated Src kinetome activity as a prevalent feature of PDAC by revealing mRNA levels associated with higher malignancy grade of tumors. BLK (B lymphocyte kinase) expression predicts shorter overall survival of diabetic PDAC patients. Nanofunctionalization of DASA needs further improvement to overcome the therapy resistance of PDAC. LYN mRNA is augmented in tumors with higher malignancy and can serve as a predictive biomarker for the therapy resistance of PDAC cells against DASA. Studying the biological roles of BLK might help to identify underlying molecular mechanisms associated with PDAC in diabetic patients.

Electrospun fibers modified with polydopamine for enhancing human mesenchymal stem cell culture.

In this study, we coated electrospun polycaprolactone (PCL) fibers with polydopamine (PDA) to modify their hydrophobicity and fabricated a matrix for culturing mesenchymal stem cells (MSCs). Additionally, we incorporated Arg-Gly-Asp (RGD) peptides into PDA to enhance MSCs culture performance on PCL fibers. PDA and RGD were successfully coated in one step by immersing the electrospun fibers in a coating solution, without requiring an additional surface activation process. The characteristics of functionalized PCL fibers were analyzed by scanning electron microscope (SEM) with energy-dispersive X-ray (EDX) analysis, Fourier transform infrared (FTIR) spectroscopy, water contact angle measurement, and fluorescence measurements using a carboxylic-modified fluorescent microsphere. MSCs cultured on the modified PCL fibers demonstrated enhanced cell adhesion, proliferation, and osteogenic- and chondrogenic differentiation. This study provides insight into potential applications for scaffold fabrication in MSCs-based tissue engineering, wound dressing, implantation, and a deeper understanding of MSCs behavior in vitro.&#xD.

Proposal of Simplified Standardization of the Cell-Growth-Promoting Activity of Human Adipose Tissue Mesenchymal Stromal Cell Culture Supernatants

The conditioned medium (CM) obtained from mesenchymal stromal cell (MSC) culture has excellent cell growth-promoting activity and is used for cosmetics and healthcare products. Unlike pharmaceuticals, strict efficacy verification is not legally required for these products. However, their efficacy must be substantiated as commercial products. We attempted to simplify CM production and to standardize the evaluation of the growth-promoting activity of CM. CM was obtained through the culturing of two lines of commercially available human adipose tissue-derived MSCs using MEMα with or without 10% fetal bovine serum (FBS) for 24 h. Non-CM control media were produced by the same protocol without MSCs. Growth-promoting activities of the CM were estimated by [3H]-thymidine pulse. CM were subjected to molecular weight fractionation with ultrafiltration using 10 k-, 30 k-, 50 k-, and 100 k-membranes. The FBS-free CMs showed 1.34- to 1.85-fold increases and FBS-containing CMs showed 1.45- to 1.67-fold increases in proliferation-promoting activity compared with non-CM controls, regardless of the source of the cell. The thymidine incorporation levels were approximately three times higher in FBS-containing CMs. Aged cells also showed 1.67- to 2.48-fold increases in the activity due to FBS-containing CM, but not to FBS-free CM. The CM activities were sustained even after 1 year at 4 °C. Molecular weight fractionation showed that the activity was recovered in the fraction above 100 k. Clear and stable cell-growth-promoting activity was confirmed with CMs of commercially available adipose tissue MSCs. The activity was detected in the fraction over 100 k. We propose here the importance of standardizing the production and evaluation of CMs to indicate their specific action.

Air-liquid intestinal cell culture allows in situ rheological characterization of intestinal mucus.

Intestinal health heavily depends on establishing a mucus layer within the gut with physical properties that strike a balance between being sufficiently elastic to keep out harmful pathogens yet viscous enough to flow and turnover the contents being digested. Studies investigating dysfunction of the mucus layer in the intestines are largely confined to animal models, which require invasive procedures to collect the mucus fluid. In this work, we develop a nondestructive method to study intestinal mucus. We use an air-liquid interface culture of primary human intestinal epithelial cells that exposes their apical surface to allow in situ analysis of the mucus layer. Mucus collection is not only invasive but also disrupts the mucus microstructure, which plays a crucial role in the interaction between mucus and the gut microbiome. Therefore, we leverage a noninvasive rheology technique that probes the mechanical properties of the mucus without removal from the culture. Finally, to demonstrate biomedical uses for this cell culture system, we characterize the biochemical and biophysical properties of intestinal mucus due to addition of the cytokine IL-13 to recapitulate the gut environment of Nippostrongylus brasiliensis infection.

Chemoreactome screening of aquacobalamin and heptamethyl ester of cyanoaquacobyrinic acid cytotoxic effects on tumor cells with experimental confirmation on BT-474 and A549 cell

Background. The search for an effective and safe pharmacotherapy for tumor diseases includes the evaluation of the action of candidate molecules on various types of tumor cells. Vitamin B12 and its derivatives are promising molecules whose properties can be controlled through chemical modifications.Objective: conducting in silico chemoreactom screening and in vitro experimental study of aquacobalamin and heptamethyl ester of cyanoaquacobyrinic acid (HECСA).Material and methods. Chemoreactome screening was carried out on the basis of a problem-oriented theory of chemograph isomorphism analysis, which is an extension of the algebraic approach to machine learning and recognition problems. Trainable algorithms for calculating chemical distances between molecules were used, on the basis of which the values of half-maximal inhibitory concentration (IC50) were calculated. Screening was carried out for 470 cultures of human tumor cells, including SNB19 (astrocytoma), HCT116 (colon cancer), HeLa (cervical carcinoma), BT-474 (breast duct carcinoma), and A549 (lung carcinoma) cell lines. Dicyanocobyric acid heptamethyl ester ((CN)2Cby(OCH3)7) was obtained by boiling a solution of vitamin B12 in methanol with sulfuric acid (1.0 M) for 4 days. HECСA ((CN)(H2O)Cby(OCH3)7) was obtained by vacuum drying an aqueous solution of (CN)2Cby(OCH3)7 (pH 4.0 and 25 °С). The ester structure and purity were confirmed by 1H nuclear magnetic resonance data, elemental analysis, and MALDI-ToF (matrix-assisted laser desorption/ionization time of flight) mass spectroscopy. Experimental studies of tumor cell cultures were carried out using the MTT testwith aquacobalamin and HECСA on cell lines of immortalized (telomerized) fibroblasts (Fb-hTERT), lung carcinoma (A549), and breast duct cancer (BT-474).Results. Chemoreactome screening of the effects of molecules on tumor cells made it possible to obtain estimates of cell growth IC50 for 470 tumor cell lines. Depending on cell line and vitamin B12 derivative molecule, IC50 values varied in a fairly wide range: from 15 to 2000 nM. In vitro studies on cultures of two human tumor cell lines (BT-474 and A549) and telomerized Fb-hTERT fibroblasts confirmed the cytotoxic effect of aquacobalamin and its hydrophobic derivative HECСA. It was shown that aquacobalamin had weak cytotoxic properties in the concentration range of 3.125–200 µg/l (IC50 > 200 nM), and HECСA significantly reduces the survival of BT-474 and A549 tumor cell lines at high concentrations (100–200 µg/l, IC50 about 100 nM).Conclusion. Correspondence was shown between the results of in silico chemoreactome screening and in vitro cell culture studies: IC50 values for HECСA were significantly lower than for aquacobalamin, and the conversion factor from chemoreactome estimates to experimental ones was almost the same (2.64 for BT-474, and 2.63 for A549). The results of chemoreactome screening for other tumor cell lines can be used to plan further cell experiments with vitamin B12 derivatives.

Isolation and Culture of Primary Human Mammary Epithelial Cells

Isolation and Culture of Primary Human Mammary Epithelial Cells

Abstract PO1-24-12: Investigating the immunological function of alpha-2-glycoprotein 1, zinc-binding in regulating tumor response in the breast cancer microenvironment

Abstract Purpose: Tumor immunology has attracted considerable attention as an innovative therapeutic strategy for various types of cancer. Elucidating the unique immunoregulatory mechanisms in the breast cancer microenvironment will assist in the development of novel treatment strategies. Recently, we and other researchers have found that androgen receptor (AR) expression is associated with the immunosuppressive phenotype in the breast cancer microenvironment, suggesting some immunoregulatory function in breast cancer; however, the mechanism remains unclear. Here, we focused on AR-dependent secreted protein, alpha-2-glycoprotein 1, zinc-binding (ZAG) encoded by the AZGP1 gene in breast cancer, which is structurally similar to HLA class I and is implicated in immune regulation. In this study, we investigated the immunological function of AZGP1/ZAG in the breast cancer microenvironment. Methods: We performed a gene set enrichment analysis (GSEA) to screen the biological processes associated with AZGP1 expression using a gene expression profile dataset of the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC). Subsequently, we analyzed the correlation between AZGP1 expression and the immune cell composition in breast cancer tissues estimated with the CIBERSORTx using METABRIC and another dataset of The Sweden Cancerome Analysis Network-Breast. In our previous study of 45 breast cancer tissue samples, we evaluated the infiltration of 11 types of immune cells using flow cytometry (FCM). ZAG expression was further evaluated by immunohistochemistry, and the relationship between ZAG expression and the immune cell composition in breast cancer tissue was analyzed. Based on the results of this analysis (shown in the results section) we hypothesized that ZAG has some effect on the macrophage (Mφ). The action of ZAG in M1/M2 polarization models constructed using primary culture of human peripheral blood mononuclear cells (PBMC)-derived Mφ was assessed based on the expression of M1/M2 differentiation markers (CD86, CD80/CD163, MRC1) and HLA class I/II expression evaluated by FCM (n=15 each). Results: GSEA demonstrated that AZGP1 expression was negatively correlated with multiple gene sets representing immunological processes, including inflammatory response, allograft rejection, interferon gamma response, IL6/JAK/STAT3 signaling, complement, and IL2/STAT5 signaling. Analysis by CIBERSORTx showed that AZGP1 expression was negatively correlated with the absolute score (the absolute abundance of total immune cell infiltration), Mφ M1, NK cells activated, CD4+ T memory activated, and CD8+ T (r<-3 and p< 0.05). Analyses of our in-house dataset using breast cancer tissue showed that ZAG expression was associated with decreased infiltration of monocytes/macrophages, non-classical monocytes, and myeloid-derived suppressor cells in breast cancer tissues assessed by FCM. In the in vitro analyses, ZAG decreased the expression of CD80, CD163, MRC1, and HLA classes I and II in the M1 polarization model and the expression of CD163 and MRC1 in the M2 polarization model. Conclusion: AZGP1/ZAG was associated with an immunosuppressive phenotype and reduced infiltration of specific immune cell subsets, particularly Mφ, into breast cancer tissues. In the in vitro analysis, ZAG demonstrated some regulatory effects on the phenotypic change of Mφ. Our findings strongly suggest ZAG is a novel mediator of AR-dependent immunomodulation in the breast cancer microenvironment, laying the foundation for future studies to elucidate the immunological role of ZAG in breast cancer. Citation Format: Toru Hanamura, Kozue Yokoyama, Shigehisa Kitano, Hiroshi Kagamu, Makiko Yamashita, Mayako Terao, Takuho Okamura, Nobue Kumaki, Katsuto Hozumi, Takayuki Iwamoto, Chikako Honda, Sasagu Kurozumi, Jennifer Richer, Naoki Niikura. Investigating the immunological function of alpha-2-glycoprotein 1, zinc-binding in regulating tumor response in the breast cancer microenvironment [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-12.

Enhanced Electroactive Phases of Poly(vinylidene Fluoride) Fibers for Tissue Engineering Applications.

Nanofibrous materials generated through electrospinning have gained significant attention in tissue regeneration, particularly in the domain of bone reconstruction. There is high interest in designing a material resembling bone tissue, and many scientists are trying to create materials applicable to bone tissue engineering with piezoelectricity similar to bone. One of the prospective candidates is highly piezoelectric poly(vinylidene fluoride) (PVDF), which was used for fibrous scaffold formation by electrospinning. In this study, we focused on the effect of PVDF molecular weight (180,000 g/mol and 530,000 g/mol) and process parameters, such as the rotational speed of the collector, applied voltage, and solution flow rate on the properties of the final scaffold. Fourier Transform Infrared Spectroscopy allows for determining the effect of molecular weight and processing parameters on the content of the electroactive phases. It can be concluded that the higher molecular weight of the PVDF and higher collector rotational speed increase nanofibers' diameter, electroactive phase content, and piezoelectric coefficient. Various electrospinning parameters showed changes in electroactive phase content with the maximum at the applied voltage of 22 kV and flow rate of 0.8 mL/h. Moreover, the cytocompatibility of the scaffolds was confirmed in the culture of human adipose-derived stromal cells with known potential for osteogenic differentiation. Based on the results obtained, it can be concluded that PVDF scaffolds may be taken into account as a tool in bone tissue engineering and are worth further investigation.

Transcription factor dynamics, oscillation, and functions in human enteroendocrine cell differentiation.

Enteroendocrine cells (EECs) secrete serotonin (enterochromaffin [EC] cells) or specific peptide hormones (non-EC cells) that serve vital metabolic functions. The basis for terminal EEC diversity remains obscure. By forcing activity of the transcription factor (TF) NEUROG3 in 2D cultures of human intestinal stem cells, we replicated physiologic EEC differentiation and examined transcriptional and cis-regulatory dynamics that culminate in discrete cell types. Abundant EEC precursors expressed stage-specific genes and TFs. Before expressing pre-terminal NEUROD1, post-mitotic precursors oscillated between transcriptionally distinct ASCL1+ and HES6hi cell states. Loss of either factor accelerated EEC differentiation substantially and disrupted EEC individuality; ASCL1 or NEUROD1 deficiency had opposing consequences on EC and non-EC cell features. These TFs mainly bind cis-elements that are accessible in undifferentiated stem cells, and they tailor subsequent expression of TF combinations that underlie discrete EEC identities. Thus, early TF oscillations retard EEC maturation to enable accurate diversity within a medically important cell lineage.

Comparison of the efficacy of two-dimensional and three-dimensional cultures of human rotator cuff-derived mesenchymal stem cells for rotator cuff regeneration

Comparison of the efficacy of two-dimensional and three-dimensional cultures of human rotator cuff-derived mesenchymal stem cells for rotator cuff regeneration

Focal Adhesion and Actin Orientation Regulated by Cellular Geometry Determine Stem Cell Differentiation via Mechanotransduction

Tuning cell adhesion geometry can affect cytoskeleton organization and the distribution of cytoskeleton forces, which play critical roles in controlling cell functions. To elucidate the geometrical relationship with cytoskeleton force distribution, it is necessary to control cell morphology. In this study, a series of dextral vortex micropatterns were prepared to precisely control cell morphology for investigating the influence of the curvature degree of adhesion curves on intracellular force distribution and stem cell differentiation at a sub-cellular level. Peripherial actin filaments of micropatterned cells were assembled along the adhesion curves and showed different orientations, filament thicknesses and densities. Focal adhesion and cytoskeleton force distribution were dependent on the curvature degree. Intracellular force distribution was also regulated by adhesion curves. The cytoskeleton and force distribution affected the osteogenic differentiation of mesenchymal stem cells through a YAP/TAZ-mediated mechanotransduction process. Thus, regulation of cell adhesion curvature, especially at cytoskeletal filament level, is critical for cell function manipulation. Statement of SignificanceIn this study, a series of dextral micro-vortexes were prepared and used for the culture of human mesenchymal stem cells (hMSCs) to precisely control adhesive curvatures (0°, 30°, 60°, and 90°). The single MSCs on the micropatterns had the same size and shape but showed distinct focal adhesion (FA) and cytoskeleton orientations. Cellular nanomechanics were observed to be correlated with the curvature degrees, subsequently influencing nuclear morphological features. As a consequence, the localization of the mechanotransduction sensor and activator-YAP/TAZ was affected, influencing osteogenic differentiation. The results revealed the pivotal role of adhesive curvatures in the manipulation of stem cell differentiation via the machanotransduction process, which has rarely been investigated.

Do PFCAs drive the establishment of thyroid cancer microenvironment? Effects of C6O4, PFOA and PFHxA exposure in two models of human thyroid cells in primary culture

BackgroundExposure to environmental pollutants is suspected to be one of the potential causes accounting for the increase in thyroid cancer (TC) incidence worldwide. Among the ubiquitous pollutants, per-polyfluoroalkyl substances (PFASs), were demonstrated to exert thyroid disrupting effects. Perfluoroalkyl carboxylates (PFCAs) represent a subgroup of PFAS and include perfluoro carboxylic acids (PFOA and PFHxA) and perfluoropolyether carboxylic acid (C6O4).The potential relationship between exposure to PFCAs and TC was not yet fully elucidated.This in vitro study investigated whether certain PFCAs (C6O4, PFOA, and PFHxA) can influence the composition of TC microenvironment. MethodsTwo models of normal thyroid cells in primary cultures: Adherent (A-NHT) and Spheroids (S-NHT) were employed. A-NHT and S-NHT were exposed to C6O4, PFOA or PFHxA (0; 0.01; 0.1, 1; 10; 100; 1000 ng/mL) to assess viability (WST-1 and AV/PI assay), evaluate spherification index (SI) and volume specifically in S-NHT. CXCL8 and CCL2 (mRNA and protein), and EMT-related genes were assessed in both models after exposure to PFCAs. ResultsPFHxA reduced the viability of both A-NHT and S-NHT. None of the PFCAs interfered with the volume or spherification process in S-NHT. CXCL8 and CCL2 mRNA and protein levels were differently up-regulated by each PFCAs, being PFOA and PFHxA the stronger inducers. Moreover, among the tested PFCAs, PFHxA induced a more consistent increase in the mRNA levels of EMT-related genes. Conclusions: This is the first evaluation of the effects of exposure to PFCAs on factors potentially involved in establishing the TC microenvironment. PFHxA modulated the TC microenvironment at three levels: cell viability, pro-tumorigenic chemokines, and EMT-genes. The results provide further evidence of the pro-tumorigenic effect of PFOA. On the other hand, a marginal effect was observed for C6O4 on pro-tumorigenic chemokines.

Effect of human H3N2 influenza virus reassortment on influenza incidence and severity during the 2017–18 influenza season in the USA: a retrospective observational genomic analysis

During the 2017-18 influenza season in the USA, there was a high incidence of influenza illness and mortality. However, no apparent antigenic change was identified in the dominant H3N2 viruses, and the severity of the season could not be solely attributed to a vaccine mismatch. We aimed to investigate whether the altered virus properties resulting from gene reassortment were underlying causes of the increased case number and disease severity associated with the 2017-18 influenza season. Samples included were collected from patients with influenza who were prospectively recruited during the 2016-17 and 2017-18 influenza seasons at the Johns Hopkins Hospital Emergency Departments in Baltimore, MD, USA, as well as from archived samples from Johns Hopkins Health System sites. Among 647 recruited patients with influenza A virus infection, 411 patients with whole-genome sequences were available in the Johns Hopkins Center of Excellence for Influenza Research and Surveillance network during the 2016-17 and 2017-18 seasons. Phylogenetic trees were constructed based on viral whole-genome sequences. Representative viral isolates of the two seasons were characterised in immortalised cell lines and human nasal epithelial cell cultures, and patients' demographic data and clinical outcomes were analysed. Unique H3N2 reassortment events were observed, resulting in two predominant strains in the 2017-18 season: HA clade 3C.2a2 and clade 3C.3a, which had novel gene segment constellations containing gene segments from HA clade 3C.2a1 viruses. The reassortant re3C.2a2 viruses replicated with faster kinetics and to a higher peak titre compared with the parental 3C.2a2 and 3C.2a1 viruses (48 h vs 72 h). Furthermore, patients infected with reassortant 3C.2a2 viruses had higher Influenza Severity Scores than patients infected with the parental 3C.2a2 viruses (median 3·00 [IQR 1·00-4·00] vs 1·50 [1·00-2·00]; p=0·018). Our findings suggest that the increased severity of the 2017-18 influenza season was due in part to twointrasubtypes, cocirculating H3N2 reassortant viruses with fitness advantages over the parental viruses. This information could help inform future vaccine development and public health policies. The Center of Excellence for Influenza Research and Response in the US, National Science and Technology Council, and Chang Gung Memorial Hospital in Taiwan.

A proposed in vitro cytotoxicity test battery to detect and predict hepatotoxicity via multiple mechanisms and pathways: a minireview.

The 21st-century toxicity testing program recommends the use of cytotoxicity data from human cells in culture for rapid in vitro screening focusing on biological pathways of potential toxicants to predict in vivo toxicity. Liver is the major organ for both endogenous and exogenous chemical metabolism of xenobiotics. Therefore, this review was undertaken to evaluate side by side five different currently used commercial cytotoxicity assay kits for purpose of rapid predictive screening of potential hepatotoxicants. The test compounds for this review were selected from the NIH LiverTox and FDA Liver Toxicity Knowledge Base (LTKB) databases. Human liver HepG2, HepaRG, and rat liver Clone 9 cell cultures were used as the in vitro liver models. Five commercial assay kits representing different biomarkers or pathways were selected for this review. These kits are Vita-Orange Cell Viability Assay Kit (Sigma-Aldrich), CellTiter-Glo Cell Viability Assay Kit (Promega), CytoTox-ONE Homogeneous Membrane Integrity Assay Kit (Promega), DNA Quantitation Fluorescence Assay Kit (Sigma-Aldrich), and Neutral Red Based In Vitro Toxicology Assay Kit (Sigma-Aldrich). This review found that these kits can all be used for rapid predictive cytotoxicity screening of potential hepatotoxicants in human liver HepG2 and rat liver Clone 9 cells in culture as in vitro liver models without compromising quality and accuracy of endpoint measurements as well as the length of toxicity screening time. Unraveling the structure-activity relationship of potential hepatotoxins would help to classify their hepatotoxic effects. Therefore, in addition to the current regulatory hepatotoxicity testing strategies, development and regulatory approval of hepatotoxins need to be discussed in order to identify potential gaps in the safety assessment. The overall results of our study support the hypothesis that a battery of rapid, simple, and reliable assays is an excellent tool for predicting in vivo effects of suspected liver toxins. The human liver HepaRG cells do not appear to be an ideal in vitro liver model for this purpose.

СТЕКЛОКЕРАМИЧЕСКИЕ ПОКРЫТИЯ НА ДЕНТАЛЬНЫХ ИМПЛАНТАТАХ С БАРЬЕРНЫМИ СВОЙСТВАМИ

Pateks — the technology of applying a biocompatible glass-ceramic coating based on silicon, carbon and nitrogen on dental implants by PECVD is developed by research and production company Plasmacentre and Pavlov University (St. Petersburg, Russia). Purpose of the research — to evaluate the success of optimization of the surface of doped titanium endosseous dental implants which may be achieved with our glass-ceramic barrier coating technology using physicochemical, cytological and spectrophotometric researches. The main task is to carry out a comparative assessment of the studies’ results between dental implants with and without Pateks glass-ceramic coating (doped with titanium alloy). Materials and methods. Titanium implants (grade 5) from BioMed were used in this research. Some of them were coated with Pateks according to our technology. A human lung fibroblast cells culture was used for the cytological study. Spectrophotometric studies were carried out on atomic absorption spectrophotometer and a atomic absorption spectrometer KVANT-Z.ETA (Russia). Findings. Pateks-coated implants demonstrate advantage in comparison with non-coated ones in certain physicochemical parameters such as decrease of defects’ area in the initial surface of titanium implants which is formed on the previous stages of surface shaping and processing, providing higher surface hydrophilicity, increasing of dielectric characteristics and higher pH value. Pateks coating is safe for fibroblast monolayers and does not inhibit respiratory processes in them. Applying of Pateks glass-ceramic coating with a thickness of 0.5 μm reduces the content of aluminum and vanadium ions in the model medium by almost 2 times and pro- vides a barrier that helps to reduce the negative biological effect of these ions on peri-implant tissues.

Senescence of human pancreatic beta cells enhances functional maturation through chromatin reorganization and promotes interferon responsiveness.

Senescent cells can influence the function of tissues in which they reside, and their propensity for disease. A portion of adult human pancreatic beta cells express the senescence marker p16, yet it is unclear whether they are in a senescent state, and how this affects insulin secretion. We analyzed single-cell transcriptome datasets of adult human beta cells, and found that p16-positive cells express senescence gene signatures, as well as elevated levels of beta-cell maturation genes, consistent with enhanced functionality. Senescent human beta-like cells in culture undergo chromatin reorganization that leads to activation of enhancers regulating functional maturation genes and acquisition of glucose-stimulated insulin secretion capacity. Strikingly, Interferon-stimulated genes are elevated in senescent human beta cells, but genes encoding senescence-associated secretory phenotype (SASP) cytokines are not. Senescent beta cells in culture and in human tissue show elevated levels of cytoplasmic DNA, contributing to their increased interferon responsiveness. Human beta-cell senescence thus involves chromatin-driven upregulation of a functional-maturation program, and increased responsiveness of interferon-stimulated genes, changes that could increase both insulin secretion and immune reactivity.

Cross-species spill-over potential of the H9N2 bat influenza A virus

In 2017, a novel influenza A virus (IAV) was isolated from an Egyptian fruit bat. In contrast to other bat influenza viruses, the virus was related to avian A(H9N2) viruses and was probably the result of a bird-to-bat transmission event. To determine the cross-species spill-over potential, we biologically characterize features of A/bat/Egypt/381OP/2017(H9N2). The virus has a pH inactivation profile and neuraminidase activity similar to those of human-adapted IAVs. Despite the virus having an avian virus–like preference for α2,3 sialic acid receptors, it is unable to replicate in male mallard ducks; however, it readily infects ex-vivo human respiratory cell cultures and replicates in the lungs of female mice. A/bat/Egypt/381OP/2017 replicates in the upper respiratory tract of experimentally-infected male ferrets featuring direct-contact and airborne transmission. These data suggest that the bat A(H9N2) virus has features associated with increased risk to humans without a shift to a preference for α2,6 sialic acid receptors.

Tumour-associated myeloid cells expressing IL-10R2/IL-22R1 as a potential biomarker for diagnosis and recurrence of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a poor survival rate, largely due to the lack of early diagnosis. Although myeloid cells are crucial in the tumour microenvironment, whether their specific subset can be a biomarker of PDAC progression is unclear. We analysed IL-22 receptor expression in PDAC and peripheral blood. Additionally, we analysed gene expression profiles of IL-10R2+/IL-22R1+ myeloid cells and the presence of these cells using single-cell RNA sequencing and murine orthotropic PDAC models, respectively, followed by examining the immunosuppressive function of IL-10R2+/IL-22R1+ myeloid cells. Finally, the correlation between IL-10R2 expression and PDAC progression was evaluated. IL-10R2+/IL-22R1+ myeloid cells were present in PDAC and peripheral blood. Blood IL-10R2+ myeloid cells displayed a gene expression signature associated with tumour-educated circulating monocytes. IL-10R2+/IL-22R1+ myeloid cells from human myeloid cell culture inhibited T cell proliferation. By mouse models for PDAC, we found a positive correlation between pancreatic tumour growth and increased blood IL-10R2+/IL-22R1+ myeloid cells. IL-10R2+/IL-22R1+ myeloid cells from an early phase of the PDAC model suppressed T cell proliferation and cytotoxicity. IL-10R2+ myeloid cells indicated tumour recurrence 130 days sooner than CA19-9 in post-pancreatectomy patients. IL-10R2+/IL-22R1+ myeloid cells in the peripheral blood might be an early marker of PDAC prognosis.

Generalized Multifunctional Coating Strategies Based on Polyphenol-Amine-Inspired Chemistry and Layer-by-Layer Deposition for Blood Contact Catheters.

Blood-contacting catheters play a pivotal role in contemporary medical treatments, particularly in the management of cardiovascular diseases. However, these catheters exhibit inappropriate wettability and lack antimicrobial characteristics, which often lead to catheter-related infections and thrombosis. Therefore, there is an urgent need for blood contact catheters with antimicrobial and anticoagulant properties. In this study, we employed tannic acid (TA) and 3-aminopropyltriethoxysilane (APTES) to create a stable hydrophilic coating under mild conditions. Heparin (Hep) and poly(lysine) (PL) were then modified on the TA-APTES coating surface using the layer-by-layer (LBL) technique to create a superhydrophilic TA/APTES/(LBL)4 coating on silicone rubber (SR) catheters. Leveraging the superhydrophilic nature of this coating, it can be effectively applied to blood-contacting catheters to impart antibacterial, antiprotein adsorption, and anticoagulant properties. Due to Hep's anticoagulant attributes, the activated partial thromboplastin time and thrombin time tests conducted on SR/TA-APTES/(LBL)4 catheters revealed remarkable extensions of 276 and 103%, respectively, when compared to uncoated commercial SR catheters. Furthermore, the synergistic interaction between PL and TA serves to enhance the resistance of SR/TA-APTES/(LBL)4 catheters against bacterial adherence, reducing it by up to 99.9% compared to uncoated commercial SR catheters. Remarkably, the SR/TA-APTES/(LBL)4 catheter exhibits good biocompatibility with human umbilical vein endothelial cells in culture, positioning it as a promising solution to address the current challenges associated with blood-contact catheters.