Reconfiguration of Langmuir Monolayers of Thermo-Responsive Branched Ionic Polymers with LCST Transition.

Thermo-responsive ionic polymers have the ability to form adaptive and switchable morphologies, which may offer enhanced control in energy storage and catalytic applications. Current thermo-responsive polymers are composed of covalently attached thermo-responsive moieties, restricting their mobility and global dynamic response. Here, we report the synthesis and assembly at the water-air interface of symmetric and asymmetric amphiphilic thermo-responsive branched polymers with weakly ionically bound arms of amine-terminated poly(N-isopropylacrylamide) (PNIPAM) macro-cations. As we observed, symmetric branched polymers formed multimolecular nanosized micellar assemblies, whereas corresponding asymmetric polymers formed large, interconnected worm-like aggregates. Dramatic changes in localized and large-scale chemical composition confirmed the reversible adsorption and desorption of the mobile PNIPAM macro-cations below and above the low critical solution temperature (LCST) and their non-uniform redistribution within polymer monolayer. Increasing the temperature above LCST led to the formation of large interconnected micellar aggregates because of the micelle-centered aggregation of the hydrophobized PNIPAM macro-cationic terminal chains in the aqueous subphase. Overall, this work provides insights into the dynamic nature of the chemical composition of branched ionic polymers with weakly ionically bound thermo-responsive terminal chains and its effect on both morphology and local/surface chemistry of monolayers at LCST transition.

Weakly Ionically Bound Thermosensitive Hyperbranched Polymers.

We synthesized novel amphiphilic hyperbranched polymers (HBPs) with variable contents of weakly ionically tethered thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) macrocations in contrast to traditional covalent linking. Their assembling behavior was studied below and above the lower critical solution temperature (LCST). The HBPs underwent a morphological transition under changing temperature and ionic strength due to the LCST transition of PNIPAM and the reduction in the ionization degree of terminal ionic groups, respectively. We suggest that, in contrast to traditional branched polymers, ionically linked PNIPAM macrocations can reversibly disassociate from the sulfonate groups and form mobile coronas, endowing the dynamic micellar morphologies. In addition, assembly at the air-water interface confined PNIPAM macrocations and resulted in the formation of heterogeneous Langmuir-Blodgett (LB) monolayers with diverse surface morphologies for different peripheral compositions with circular domains formed in the condensed state. The HBPs with 25% PNIPAM showed larger and more stable circular domains that were partially preserved at high compression than those of HBPs with 50% PNIPAM. Moreover, the LB monolayers showed variable surface mechanical and surface charge distribution, which can be attributed to net dipole redistribution caused by the behavior of mobile PNIPAM macrocations and core sulfonate groups.

Transformations of Thermosensitive Hyperbranched Poly(ionic liquid)s Monolayers.

We synthesized amphiphilic hyperbranched poly(ionic liquid)s (HBPILs) with asymmetrical peripheral composition consisting of hydrophobic n-octadecylurethane arms and hydrophilic, ionically linked poly(N-isopropylacrylamide) (PNIPAM) macrocations and studied low critical solution temperature (LCST)-induced reorganizations at the air-water interface. We observed that the morphology of HBPIL Langmuir monolayers is controlled by the surface pressure with uniform well-defined disk-like domains formed in a liquid phase. These domains are merged and transformed to uniform monolayers with elevated ridge-like network structures representing coalesced interdomain boundaries in a solid phase because the branched architecture and asymmetrical chemical composition stabilize the disk-like morphology under high compression. Above LCST, elevated individual islands are formed because of the aggregation of the collapsed hydrophobized PNIPAM terminal macrocations in a solid phase. The presence of thermoresponsive PNIPAM macrocations initiates monolayer reorganization at LCST with transformation of surface mechanical contrast distribution. The heterogeneity of elastic response and adhesion distributions for HBPIL monolayers in the wet state changed from highly contrasted two-phase distribution below LCST to near-uniform mechanical response above LCST because of the hydrophilic to hydrophobic transformation of the PNIPAM phase.

Stable Overexpression of the Constitutive Androstane Receptor Reduces the Requirement for Culture with Dimethyl Sulfoxide for High Drug Metabolism in HepaRG Cells.

Dimethylsulfoxide (DMSO) induces cellular differentiation and expression of drug metabolic enzymes in the human liver cell line HepaRG; however, DMSO also induces cell death and interferes with cellular activities. The aim of this study was to examine whether overexpression of the constitutive androstane receptor (CAR, NR1I3), the nuclear receptor controlling various drug metabolism genes, would sufficiently promote differentiation and drug metabolism in HepaRG cells, optionally without using DMSO. By stable lentiviral overexpression of CAR, HepaRG cultures were less affected by DMSO in total protein content and obtained increased resistance to acetaminophen- and amiodarone-induced cell death. Transcript levels of CAR target genes were significantly increased in HepaRG-CAR cultures without DMSO, resulting in increased activities of cytochrome P450 (P450) enzymes and bilirubin conjugation to levels equal or surpassing those of HepaRG cells cultured with DMSO. Unexpectedly, CAR overexpression also increased the activities of non-CAR target P450s, as well as albumin production. In combination with DMSO treatment, CAR overexpression further increased transcript levels and activities of CAR targets. Induction of CYP1A2 and CYP2B6 remained unchanged, whereas CYP3A4 was reduced. Moreover, the metabolism of low-clearance compounds warfarin and prednisolone was increased. In conclusion, CAR overexpression creates a more physiologically relevant environment for studies on hepatic (drug) metabolism and differentiation in HepaRG cells without the utilization of DMSO. DMSO still may be applied to accomplish higher drug metabolism, required for sensitive assays, such as low-clearance studies and identification of (rare) metabolites, whereas reduced total protein content after DMSO culture is diminished by CAR overexpression.

HepaRG microencapsulated spheroids in DMSO-free culture: novel culturing approaches for enhanced xenobiotic and biosynthetic metabolism.

The need for models that recapitulate liver physiology is perceived for drug development, study of liver disease and bioartificial liver support. The bipotent cell line HepaRG constitutes an efficient surrogate of liver function, yet its differentiated status relies on high concentrations of DMSO, which may compromise the study of drug metabolism and limit the applicability of this hepatic model. Herein, we present a three-dimensional (3D) strategy for the differentiation of HepaRG based on alginate microencapsulation of cell spheroids and culture in dimethyl sulfoxide (DMSO)-free conditions. A ratio of 2.9:1 hepatocyte-like to biliary-like cells was obtained in the 3D culture, with an improvement of 35.9 % in the hepatocyte differentiation when compared with two-dimensional (2D) cultures. The expression of the hepatic identity genes HNF4α and PXR in 3D cultures was comparable to 2D differentiated cultures, while the expression of homeostatic-associated genes albumin and carbamoyl phosphate synthase 1 was higher in 3D. Moreover, the spheroids presented a polarized organization, exhibiting an interconnected bile canalicular network and excretory functionality, assessed by specific activity of MRP2. Importantly, despite variability in basal gene expression levels, the activity of the phase I enzymes cytochrome P450 family 3, subfamily A, polypeptide 4 and cytochrome P450 family 1, subfamily A, polypeptide 2 upon induction was comparable to differentiated 2D cultures and albumin production and ammonia detoxification were enhanced in 3D. The presented model is suitable for toxicological applications, as it allows high throughput analysis of multiple compounds in a DMSO-free setting. Due to the high xenobiotic metabolism and maintenance of biosynthetic functions, the applicability of this model might be broadened to understand liver physiology and for bioartificial liver applications.

Star-shaped molecules with polyhedral oligomeric silsesquioxane core and azobenzene dye arms.

We synthesized a series of hybrid nanomaterials combining organic dyes with polyhedral oligomeric silsesquioxanes (POSS) based on three different azobenzenes: monoazobenzene (4-phenylazophenol), bis-azobenzene (Disperse Yellow 7 and Fast Garnet derivative), and push-pull azobenzene (Disperse Red 1) via hydrosilylation coupling. The azo-functionalized POSS compounds possess high thermal stability, and their branched architecture resulted in effective suppression of molecular aggregation and allowed for direct imaging of individual dye-POSS structures with expected molecular dimensions. Stable, uniform, smooth, and ultrathin nanocomposite films with mixed silica-organic composition and relatively low refractive indices can be fabricated from all azo-POSS branched conjugates. Finally, the photoisomerization behavior of POSS-conjugated 4-phenylazophenol was investigated in solution as well as in ultrathin nanocomposite film. We found that conjugation to POSS core did not affect the kinetics of trans-cis photoisomerization and thermal cis-trans relaxation. Furthermore, rapid and reversible photoisomerization was observed in azo-POSS nanocomposite films. We suggest that the highly stable branched azo-POSS conjugates with high dye grafting density described here can be considered for nanometer-sized photoswitches, active layer material with optical-limiting properties, and a medium with photoinduced anisotropy for optical storage.

HIV drug resistance among patients experiencing antiretroviral therapy failure in Russia, 2019-2021.

Increasing HIV drug resistance is an important public health concern. The current study aimed to assess HIV drug resistance among people who live with HIV (PLWH) experiencing virological failure. Blood samples and epidemiological characteristics were collected in four Siberian regions from PLWH experiencing ART failure. Partial pol gene sequences were obtained for the study individuals. Drug resistance mutations (DRMs) were predicted using the Stanford HIVdb Program. The association of HIV DRM with epidemiological characteristics was estimated using logistic regression analysis. Further analysis was performed for children (0-14 y old) and adults (≥15 y old) separately. In total, 815 (89.4%) patients were included in the final dataset. Overall, 501 (61.5%) patients had DRM detected. NRTI DRM was more common in children, while NRTI+NNRTI DRM was more frequent in adults (P < 0.001). Krasnoyarsk region, male sex and high viral load were positively associated with the presence of DRM in adults, while higher CD4 cell count and PI/INSTI-based ART had a negative association. No association between epidemiological characteristics and DRM was identified in children. The remaining 38.5% of patients with virological failure had no DRM detected; those patients were likely to have insufficient ART adherence. Most (55.5%) patients had HIV CRF63_02A6, followed by sub-subtype A6 (39.2%). This study revealed poor ART adherence as a main factor driving ART failure among PLWH in the Siberian region. DRM was detected in over 60% of PLWH experiencing ART failure. The current results highlight an urgent need for the introduction of special programs focusing on ART adherence improvement.

Ultrathin Films of MXene Nanosheets Decorated by Ionic Branched Nanoparticles with Enhanced Energy Storage Stability.

Two-dimensional (2D) materials such as MXenes have shown great potential for energy storage applications due to their high surface area and high conductivity. However, their practical implementation is limited by their tendency to restack, similar to other 2D materials, leading to a decreased long-term performance. Here, we present a novel approach to addressing this issue by combining MXene (Ti3C2Tx) nanosheets with branched ionic nanoparticles from polyhedral oligomeric silsesquioxanes (POSS) using an amphiphilicity-driven assembly for the formation of composite monolayers of nanoparticle-decorated MXene nanosheets at the air-water interface. The amphiphilic hybrid MXene/POSS monolayers allow for the fabrication of organized multilayered films with ionic nanoparticles supporting the nanoscale gap between MXene nanosheets. For these composite multilayers, we observed a 400% enhancement in specific capacitance compared to pure drop-cast MXene films. Furthermore, dramatically enhanced electrochemical cycling stability for ultrathin-film electrodes (<400 nm in thickness) with a 91% capacitance retention over 10,000 cycles has been achieved. Our results suggest that this insertion of 0D ionic nanoparticles with complementary interactions in between 2D MXene nanosheets could be extended to other hybrid 0D-2D nanomaterials, providing a promising pathway for the development of hybrid electrode architectures with enhanced ionic transport for long-term energy cycling and storage, capacitive deionization, and ionic filtration.

Flexible Sustained Ionogels with Ionic Hyperbranched Polymers for Enhanced Ion-Conduction and Energy Storage.

Flexible and mechanically robust gel-like electrolytes offer enhanced energy storage capabilities, versatility, and safety in batteries and supercapacitors. However, the trade-off between ion conduction and mechanical robustness remains a challenge for these materials. Here, we suggest that the introduction of ionic hyperbranched polymers in structured sustained ionogels will lead to both enhanced ion conduction and mechanical performance because of the hyperbranched polymers' ionically conductive groups and the complementary interfacial interactions with ionic liquids. More specifically, we investigate the effect of hyperbranched polymers with carboxylate terminal groups and imidazolium counterions with various ionic group densities on the properties of ionogels composed of coassembled cellulose nanofibers (CNFs) and cellulose nanocrystals (CNCs) as sustainable open pore frame for ionic liquid immersion. The addition of hyperbranched polymers leads to the formation of highly interconnected openly porous, lightweight, and shape-persistent materials by harnessing hydrogen bonding between the polymers and the CNFs/CNCs "frame". Notably, these materials possess a 2-fold improvement in ionic conductivity combined with many-fold increase in Young's modulus, tensile strength, and toughness, making them comparable to common reinforced nanocomposite materials. Furthermore, the corresponding thin-film gel supercapacitors possess enhanced electrochemical cycling stability upon repeated bending with an 85% capacitance retention after 10 000 cycles, promising new insight in the development of simultaneously conductive and flexible gel electrolytes with sustained performance.

Effectiveness of bortezomib and temozolomide for eradication of recurrent human glioblastoma cells, resistant to radiation.

BACKGROUND: Glioblastoma multiforme (GBM) is a primary human brain tumor with the highest mortality rate. The prognosis for such patients is unfavorable, since the tumor is highly resistant to treatment, and the median survival of patients is 13 months. Chemotherapy might extend patients' life, but a tumor, that reappears after chemoradiotherapy, is resistant to temozolomide (TMZ). Using postgenome technologies in clinical practice might have a positive effect on the treatment of a recurrent GBM. METHODS: T98G cells of human GBM have been used. Radiation treatment was performed with Rokus-M gamma-therapeutic system, using 60Сo as a source of radionuclide emissions. High-performance liquid chromatography-mass spectrometry was used for proteome analysis. Mass spectrometry data were processed with MaxQuant (version 1.6.1.0) and Perseus (version 1.6.1) software, Max Planck Institute of Biochemistry (Germany). Biological processes, molecular functions, cells locations and protein pathways were annotated with a help of PubMed, PANTHER, Gene Ontology and KEGG and STRING v10 databases. Pharmaceutical testing was performed in vitro with a panel of traditional chemotherapeutic agents. RESULTS: GBM cells proliferation speed is inversely proportional to the irradiation dose and recedes when the dosage is increased, as expected. Synthesis of ERC1, NARG1L, PLCD3, ROCK2, SARNP, TMSB4X and YTHDF2 in GBM cells, treated with 60Gy of radiation, shows more than a fourfold increase, while the synthesis level of PSMA2, PSMA3, PSMA4, PSMB2, PSMB3, PSMB7, PSMC3, PSMD1, PSMD3 proteins increases significantly. Traditional chemotherapeutic agents are not very effective against cancer cells of the recurrent GBM. Combination of TMZ and CCNU with a proteasome inhibitor-bortezomib-significantly increases their ability to eradicate cells of a radioresistant GBM. CONCLUSIONS: Bortezomib and temozolomide effectively destroy cells of a radioresistant recurrent human glioblastoma; proteome mapping of the recurrent GBM cancer cells allows to identify new targets for therapy to improve the treatment results.

Proteins of the Wnt signaling pathway as targets for the regulation of CD133+ cancer stem cells in glioblastoma.

Glioblastoma multiforme (GBM) is one of the most aggressive types of brain tumor and is highly resistant to therapy. The median survival time for patients with GBM is 15 months. GBM resistance to treatment is associated with cancer stem cells (CSCs). CD133 membrane glycoprotein is the best­known marker of GBM CSCs. The Wnt signaling pathway plays an important role in the proliferation of all stem cells. To the best of our knowledge, the present study was the first to examine the expression levels of proteins associated with the Wnt signaling pathway in СD133+ CSCs of human GBM. Furthermore, potential targets that may regulate СD133+ CSCs in human GBM were investigated. The human GBM U­87MG cell line was cultured in neurobasal medium supplemented with B27, fibroblast growth factor, epidermal growth factor and no serum. Immunohistochemical characteristics of glioma spheres were investigated based on the expression of key markers of CSCs. CD133+ cells were extracted from glioma spheres by cell sorting and then lysed. High­performance liquid chromatography­mass spectrometry was used for proteome analysis. Lysates of CD133­ cells in GBM were used for comparison. The present study was the first to describe the conceptual proteome differences between GBM and CD133+ CSCs of the common pool. Major differences were identified in the glycolysis/gluconeogenesis, focal adhesion, tight junction and Wnt signaling pathways. This study aimed to analyze the crucial role that proteins of the Wnt signaling pathway play in stem cell proliferation. The identified proteins were analyzed for their association with the Wnt signaling pathway using the international open databases PubMed, Protein Analysis Through Evolutionary Relationships, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes and Search Tool for the Retrieval of Interacting Genes/Proteins. An increased expression of 12 proteins associated with the Wnt signaling pathway were identified in GBM CD133+ CSCs, which included catenin ß­1, disheveled associated activator of morphogenesis 1, RAC family small GTPase 2 and RAS homolog gene family member A, a number of which are also associated with adherens junctions. The Wnt signaling pathway is not upregulated in CSCs; however, the high expression levels of adenomatous polyposis coli, ß­catenin, C­terminal binding protein (CtBP) and RuvB­like AAA ATPase 1 (RUVBL1 or Pontin52) proteins suggest the possibility of alternative activation of specific genes in the nuclei of these cells. Calcyclin­binding protein, casein kinase II α, casein kinase II ß, CtBP1, CtBP2, CUL1 and RUVBL1 proteins may be used as targets for the pharmaceutical regulation of CSCs in complex GBM treatment.

In-Depth Proteome Analysis Highlights HepaRG Cells as a Versatile Cell System Surrogate for Primary Human Hepatocytes.

Of the hepatic cell lines developed for in vitro studies of hepatic functions as alternatives to primary human hepatocytes, many have lost major liver-like functions, but not HepaRG cells. The increasing use of the latter worldwide raises the need for establishing the reference functional status of early biobanked HepaRG cells. Using deep proteome and secretome analyses, the levels of master regulators of the hepatic phenotype and of the structural elements ensuring biliary polarity were found to be close to those in primary hepatocytes. HepaRG cells proved to be highly differentiated, with functional mitochondria, hepatokine secretion abilities, and an adequate response to insulin. Among differences between primary human hepatocytes and HepaRG cells, the factors that possibly support HepaRG transdifferentiation properties are discussed. The HepaRG cell system thus appears as a robust surrogate for primary hepatocytes, which is versatile enough to study not only xenobiotic detoxification, but also the control of hepatic energy metabolism, secretory function and disease-related mechanisms.

In Silico Modeling for the Prediction of Dose and Pathway-Related Adverse Effects in Humans From In Vitro Repeated-Dose Studies.

Long-term repeated-dose toxicity is mainly assessed in animals despite poor concordance of animal data with human toxicity. Nowadays advanced human in vitro systems, eg, metabolically competent HepaRG cells, are used for toxicity screening. Extrapolation of in vitro toxicity to in vivo effects is possible by reverse dosimetry using pharmacokinetic modeling. We assessed long-term repeated-dose toxicity of bosentan and valproic acid (VPA) in HepaRG cells under serum-free conditions. Upon 28-day exposure, the EC50 values for bosentan and VPA decreased by 21- and 33-fold, respectively. Using EC(10) as lowest threshold of toxicity in vitro, we estimated the oral equivalent doses for both test compounds using a simplified pharmacokinetic model for the extrapolation of in vitro toxicity to in vivo effect. The model predicts that bosentan is safe at the considered dose under the assumed conditions upon 4 weeks exposure. For VPA, hepatotoxicity is predicted for 4% and 47% of the virtual population at the maximum recommended daily dose after 3 and 4 weeks of exposure, respectively. We also investigated the changes in the central carbon metabolism of HepaRG cells exposed to orally bioavailable concentrations of both drugs. These concentrations are below the 28-day EC(10) and induce significant changes especially in glucose metabolism and urea production. These metabolic changes may have a pronounced impact in susceptible patients such as those with compromised liver function and urea cycle deficiency leading to idiosyncratic toxicity. We show that the combination of modeling based on in vitro repeated-dose data and metabolic changes allows the prediction of human relevant in vivo toxicity with mechanistic insights.

Selecting Cells for Bioartificial Liver Devices and the Importance of a 3D Culture Environment: A Functional Comparison between the HepaRG and C3A Cell Lines.

Recently, the first clinical trials on Bioartificial Livers (BALs) loaded with a proliferative human hepatocyte cell source have started. There are two cell lines that are currently in an advanced state of BAL development; HepaRG and HepG2/C3A. In this study we aimed to compare both cell lines on applicability in BALs and to identify possible strategies for further improvement. We tested both cell lines in monolayer- and BAL cultures on growth characteristics, hepatic differentiation, nitrogen-, carbohydrate-, amino acid- and xenobiotic metabolism. Interestingly, both cell lines adapted the hepatocyte phenotype more closely when cultured in BALs; e.g. monolayer cultures produced lactate, while BAL cultures showed diminished lactate production (C3A) or conversion to elimination (HepaRG), and urea cycle activity increased upon BAL culturing in both cell lines. HepaRG-BALs outperformed C3A-BALs on xenobiotic metabolism, ammonia elimination and lactate elimination, while protein synthesis was comparable. In BAL cultures of both cell lines ammonia elimination correlated positively with glutamine production and glutamate consumption, suggesting ammonia elimination was mainly driven by the balance between glutaminase and glutamine synthetase activity. Both cell lines lacked significant urea cycle activity and both required multiple culture weeks before reaching optimal differentiation in BALs. In conclusion, culturing in BALs enhanced hepatic functionality of both cell lines and from these, the HepaRG cells are the most promising proliferative cell source for BAL application.

Architecture, Assembly, and Emerging Applications of Branched Functional Polyelectrolytes and Poly(ionic liquid)s.

Branched polyelectrolytes with cylindrical brush, dendritic, hyperbranched, grafted, and star architectures bearing ionizable functional groups possess complex and unique assembly behavior in solution at surfaces and interfaces as compared to their linear counterparts. This review summarizes the recent developments in the introduction of various architectures and understanding of the assembly behavior of branched polyelectrolytes with a focus on functional polyelectrolytes and poly(ionic liquid)s with responsive properties. The branched polyelectrolytes and poly(ionic liquid)s interact electrostatically with small molecules, linear polyelectrolytes, or other branched polyelectrolytes to form assemblies of hybrid nanoparticles, multilayer thin films, responsive microcapsules, and ion-conductive membranes. The branched structures lead to unconventional assemblies and complex hierarchical structures with responsive properties as summarized in this review. Finally, we discuss prospectives for emerging applications of branched polyelectrolytes and poly(ionic liquid)s for energy harvesting and storage, controlled delivery, chemical microreactors, adaptive surfaces, and ion-exchange membranes.

Light-responsive plasmonic arrays consisting of silver nanocubes and a photoisomerizable matrix.

We report on the synthesis of novel branched organic-inorganic azo-polyhedral oligomeric silsesquioxane (POSS) conjugates (Azo-POSS) and their use as a stable active medium to induce reversible plasmonic modulations of embedded metal nanostructures. A dense monolayer of silver nanocubes was deposited on a quartz substrate using the Langmuir-Blodgett technique and subsequently coated with an ultrathin Azo-POSS layer. The reversible light-induced photoisomerization between the trans and cis states of the azobenzene-terminated branched POSS material results in significant changes in the refractive index (up to 0.17) at a wavelength of 380 nm. We observed that the pronounced and reversible change in the surrounding refractive index results in a corresponding hypsochromic plasmonic shift of 6 nm in the plasmonic band of the embedded silver nanocubes. The reversible tuning of the plasmonic modes of noble-metal nanostructures using a variable-refractive-index medium opens up the possibility of fabricating photoactive, hybrid, ultrathin coatings with robust, real-time, photoinitiated responses for prospective applications in photoactive materials that can be reversibly tuned by light illumination.

Synthesis of (5Z,8Z,11Z,14Z)-18- and 19-azidoeicosa-5,8,11,14-tetraenoic acids and their [5,6,8,9,11,12,14,15-3H8]-analogues through a common synthetic route.

Total synthesis of (5Z,8Z,11Z,14Z)-18- and 19-azidoeicosa-5,8,11,14-tetraenoic acids and their [5,6,8,9,11,12,14,15-3H8]-analogues via the corresponding p-toluenesulphonates is reported. This synthetic approach allows the preparation of radioactively labelled arachidonic acid derivatives following a common synthetic route. Activity assays indicated that 15-lipoxygenases may tolerate the azido group in the substrate binding pocket and thus, radioactively labelled azido compounds may be used as photo-affinity probes to investigate mechanistic features of eicosanoid biosynthesis.

3D organotypic cultures of human HepaRG cells: a tool for in vitro toxicity studies.

Drug-induced human hepatotoxicity is difficult to predict using the current in vitro systems. In this study, long-term 3D organotypic cultures of the human hepatoma HepaRG cell line were prepared using a high-throughput hanging drop method. The organotypic cultures were maintained for 3 weeks and assessed for (1) liver specific functions, including phase I enzyme and transporter activities, (2) expression of liver-specific proteins, and (3) responses to three drugs (acetaminophen, troglitazone, and rosiglitazone). Our results show that the organotypic cultures maintain high liver-specific functionality during 3 weeks of culture. The immunohistochemistry analyses illustrate that the organotypic cultures express liver-specific markers such as albumin, CYP3A4, CYP2E1, and MRP-2 throughout the cultivation period. Accordingly, the production rates of albumin and glucose, as well as CYP2E1 activity, were significantly higher in the 3D versus the 2D cultures. Toxicity studies show that the organotypic cultures are more sensitive to acetaminophen- and rosiglitazone-induced toxicity but less sensitive to troglitazone-induced toxicity than the 2D cultures. Furthermore, the EC50 value (2.7mM) for acetaminophen on the 3D cultures was similar to in vivo toxicity. In summary, the results from our study suggest that the 3D organotypic HepaRG culture is a promising in vitro tool for more accurate assessment of acute and also possibly for chronic drug-induced hepatotoxicity.

Bulk and surface assembly of branched amphiphilic polyhedral oligomer silsesquioxane compounds.

This study probes the behavior of two series of organic-functionalized core-shell silsesquioxane (POSS-M)p-(x/y) derivatives with various hydrophobic-hydrophilic terminal group compositions in the bulk state and within mono- and multilayered films at the air-water interface and on solid surface. POSS-M refers to mixed silsesquioxane cores, in contrast to the geometrically specific POSS compounds. It is composed of polyhedra, incompletely condensed polyhedra, ladder-type structures, linear structures, and all the possible combinations thereof and attracts great interest because of its facile preparation, low polydispersity, high yield, and low cost. The two series of (POSS-M)p-(x/y) molecules are different in hydrophobic-hydrophilic balance of their terminal groups, with x and y respectively referring to the molar percent of -OCONH-C(18)H(37) tails and -OH for p = 1 and the percent of -OCONH-C(18)H(37) tails and -OCO-C(6)H(4)COOH terminal groups for p = 2. In the bulk state, the presence of aromatic rings in (POSS-M)2-(x/y) series resulted in a lower symmetry crystal structure than the (POSS-M)1-(x/y) series. Moreover, the (POSS-M)p-(x/y) molecules that contain a sufficient amount of -OCONH-C(18)H(37) tails exhibit double endothermic transition, which attributed to the melting of alkyl chains followed by the melting of the unit cells of (POSS-M) cores. The surface morphologies for the various hydrophobic-hydrophilic combinations at surface pressure p = 0.5 mN/m are similar to that observed for the classical amphiphilic star polymers. However, at higher surface pressure (p > or = 5 mN/m), the POSS-M compounds with lower content of hydrophilic groups form a uniform monolayer.