Unlocking the potential of higher-molecular-weight 5-HT7R ligands: Synthesis, affinity, and ADMET examination.

An increasing number of drugs introduced to the market and numerous repositories of compounds with confirmed activity have posed the need to revalidate the state-of-the-art rules that determine the ranges of properties the compounds should possess to become future drugs. In this study, we designed a series of two chemotypes of aryl-piperazine hydantoin ligands of 5-HT7R, an attractive target in search for innovative CNS drugs, with higher molecular weight (close to or over 500). Consequently, 14 new compounds were synthesised and screened for their receptor activity accompanied by extensive docking studies to evaluate the observed structure-activity/properties relationships. The ADMET characterisation in terms of the biological membrane permeability, metabolic stability, hepatotoxicity, cardiotoxicity, and protein plasma binding of the obtained compounds was carried out in vitro. The outcome of these studies constituted the basis for the comprehensive challenge of computational tools for ADMET properties prediction. All the compounds possessed high affinity to the 5-HT7R (Ki below 250 nM for all analysed structures) with good selectivity over 5-HT6R and varying affinity towards 5-HT2AR, 5-HT1AR and D2R. For the best compounds of this study, the expression profile of genes associated with neurodegeneration, anti-oxidant response and anti-inflammatory function was determined, and the survival of the cells (SH-SY5Y as an in vitro model of Alzheimer's disease) was evaluated. One 5-HT7R agent (32) was characterised by a very promising ADMET profile, i.e. good membrane permeability, low hepatotoxicity and cardiotoxicity, and high metabolic stability with the simultaneous high rate of plasma protein binding and high selectivity over other GPCRs considered, together with satisfying gene expression profile modulations and neural cell survival. Such encouraging properties make it a good candidate for further testing and optimisation as a potential agent in the treatment of CNS-related disorders.

Synergistic Effect of Venetoclax and Bendamustine in Early T-cell Precursor Acute Lymphoblastic Leukemia.

BACKGROUND/AIM: To date, therapeutic options for T-cell acute lymphoblastic leukemia (T-ALL) remain very limited. This study evaluated the efficacy of monotherapies and combination therapies including a selective BCL-2 inhibitor for T-ALL cell lines, namely Jurkat, CCRF-CEM, and Loucy. MATERIALS AND METHODS: Loucy is an early T-precursor ALL (ETP-ALL) cell line characterized by an immature phenotype, whereas Jurkat and CCRF-CEM are late T-cell progenitor ALL (LTP-ALL) cell lines. Monotherapy was conducted with venetoclax, cytarabine, bendamustine, or azacytidine, whereas combination therapy was performed with venetoclax plus cytarabine, venetoclax plus bendamustine, or venetoclax plus azacytidine. Cell viability assay was conducted after 48 h using Trypan blue and the 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). Statistical analysis for evaluating synergistic interactions between anticancer drugs was performed by using the SynergyFinder Plus and drc R package. RESULTS: Adding venetoclax to cytarabine, bendamustine, or azacitidine achieved an additive effect, with Loewe synergic scores ranging from -10 to 10 in Jurkat and CCRF-CEM. Conversely, the combination of venetoclax and cytarabine displayed an additive effect (Loewe synergic score: 8.45 and 5.82 with MTS and Trypan blue assays, respectively), whereas venetoclax plus bendamustine or azacitidine exhibited a synergistic effect (Loewe synergic score >10 with MTS assay) in Loucy. Remarkably, the Bliss/Loewe score revealed that the combination of venetoclax and bendamustine was the most synergistic, yielding a score of 13.832±0.55. CONCLUSION: The combination of venetoclax and bendamustine demonstrated the greatest synergistic effect in suppressing ETP-ALL cell proliferation. Further studies are warranted to determine the mechanisms for the synergism between venetoclax and bendamustine in high-risk T-ALL.

A Study on Thiol-Michael Addition to Semi-Synthetic Elastin-Hyaluronan Material for Electrospun Scaffolds.

Thiol-Michael addition is a chemical reaction extensively used for conjugating peptides to polysaccharides with applications as biomaterials. In the present study, for designing a bioactive element in electrospun scaffolds as wound dressing material, a chemical strategy for the semi-synthesis of a hyaluronan-elastin conjugate containing an amide linker (ELAHA) was developed in the presence of tris(2-carboxyethyl)phosphine hydrochloride (TCEP ⋅ HCl). The bioconjugate was electrospun with poly-D,L-lactide (PDLLA), obtaining scaffolds with appealing characteristics in terms of morphology and cell viability of dermal fibroblast cells. For comprehending the factors influencing the efficiency of the bioconjugation reaction, thiolated amino acids were also investigated as nucleophiles toward hyaluronan decorated with Michael acceptors in the presence of TCEP ⋅ HCl through the evaluation of byproducts formation.

Fe3O4 Magnetic Nanoparticles Obtained by the Novel Aerosol-Based Technique for Theranostic Applications.

This work is aimed at presenting a novel aerosol-based technique for the synthesis of magnetite nanoparticles (Fe3O4 NPs) and to assess the potential medical application of their dispersions after being coated with TEA-oleate. Refinement of the processing conditions led to the formation of monodispersed NPs with average sizes of ∼5-6 nm and narrow size distribution (FWHM of ∼3 nm). The NPs were coated with Triethanolammonium oleate (TEA-oleate) to stabilize them in water dispersion. This allowed obtaining the dispersion, which does not sediment for months, although TEM and DLS studies have shown the formation of small agglomerates of NPs. The different behaviors of cancer and normal cell lines in contact with NPs indicated the diverse mechanisms of their interactions with Fe3O4 NPs. Furthermore, the studies allowed assessment of the prospective theranostic application of magnetite NPs obtained using the aerosol-based technique, particularly magnetic hyperthermia and magnetic resonance imaging (MRI).

Chemical Composition and Biological Activity of Salvia officinalis L. Essential Oil.

In our study, we investigated the chemical composition and cytotoxic activity of essential oils isolated from Dalmatian sage (Salvia officinalis L.) collected along the Adriatic coast of Croatia. Scanning electron microscopy (SEM) was used to examine the morphology of the stem and leaf surfaces. Essential oil excretory glands were detected on both the leaves and stem surfaces. The essential oils were isolated by hydrodistillation, and their chemical composition was determined by gas chromatography and mass spectrometry (GC-MS). Sage essential oils were mixtures of terpene compounds, among which the most common were: α- and ß-thujone, camphor, and 1,8-cineol. Cytotoxic activity was tested using MTS assay on multiple cell lines: normal and immortalized fibroblasts (HF77FA and HDF-Tert), immortalized lung line (BEAS-2B), and breast adenocarcinoma (MDA-MB-231). The growth of treated cells was determined relative to control conditions without treatment. The immortalized lung line was the least resistant to the activity of the essential oils, whereas immortalized fibroblasts were the most resistant. Statistical analysis has connected the cytotoxic effect and chemical composition of the studied essential oils. To the best of our knowledge, this work is the first testing of the cytotoxic activity of S. officinalis EO's on the BEAS-2B, HF77FA, and HDF-Tert cell lines. The presented data on essential oil chemical composition and cytotoxic effect on 4 types of human cells supports pharmacotherapeutic potential this plant is known to have.

Design, Physical Characterizations, and Biocompatibility of Cationic Solid Lipid Nanoparticles in HCT-116 and 16-HBE Cells: A Preliminary Study.

In this study, pEGFP-LUC was used as a model plasmid and three distinct cationic lipids (dioleyloxy-propyl-trimethylammonium chloride [DOTMA], dioleoyl trimethylammonium propane [DOTAP], and cetylpyridinium chloride [CPC]) were tested along with PEG 5000, as a nonionic surfactant, to prepare glyceryl monostearate (GMS)-based cationic solid lipid nanoparticles (cSLNs). Both the type and quantity of surfactant had an impact on the physicochemical characteristics of the cSLNs. Thermal analysis of the greater part of the endothermic peaks of the cSLNs revealed they were noticeably different from the individual pure compounds based on their zeta potential (ZP ranging from +17 to +56 mV) and particle size (PS ranging from 185 to 244 nm). The addition of cationic surfactants was required to produce nanoparticles (NPs) with a positive surface charge. This suggested that the surfactants and extensive entanglement of the lipid matrix GMS provided support for the behavioral diversity of the cSLNs and their capacity to interface with the plasmid DNA. Additionally, hemolytic assays were used to show that the cSLNs were biocompatible with the human colon cancer HCT-116 and human bronchial epithelial 16-HBE cell lines. The DOTMA 6-based cSLN was selected as the lead cSLN for further ex vivo and in vivo investigations. Taken together, these new findings might provide some guidance in selecting surfactants to prepare extremely efficient and non-toxic cSLN-based therapeutic delivery systems (e.g., gene therapy).

In Vitro Biological Testing of Dental Materials.

Dental materials are specially fabricated materials designed for use in dentistry. A variety of materials may be used, including cements, impression, lining, and dental restorative materials. Some of these dental materials provide temporary dressings while others are more permanent and are in contact with host tissue for prolonged periods of time. Consequently, newly developed dental materials not only require mechanical, chemical, and physical testing but also require in vitro analysis to ensure their safety and biocompatibility. The current chapter provides background on dental material characterization and a protocol for its in vitro biological testing.

Cytotoxicity evaluation of poly(ethylene) oxide nanofibre in MCF-7 breast cancer cell line.

Biocompatible polymers have received significant interest from researchers for their potential in diagnostic applications. This type of polymer can perform with an appropriate host response or carrier for a specific purpose. The current study aims to fabricate and characterise poly(ethylene) oxide (PEO) nanofibres with different concentrations for cytotoxicity evaluation in human breast cancer cell lines (MCF-7) and to get an optimal PEO nanofibre concentration (permissible limit) as a suitable polymer matrix or carrier with potential use in diagnostic applications. The fabrication of PEO nanofibres was done using electrospinning and was characterised by structure and morphology, surface roughness, chemical bonding and release profiles. The functional effects of PEO nanofibres were evaluated with MTS assay and colony formation assay in MCF-7 cells. The results showed that viscosity plays a vital role in synthesising a polymer solution in electrospinning for producing beadless nanofibrous mats ranging from 4.7 Pa·s to 77.7 Pa·s. As the PEO concentration increases, the nanofibre diameter and thickness will increase, but the surface roughness will be decreased. The average fibre diameter for 5 wt% PEO, 6 wt% PEO and 7 wt% PEO nanofibres were 129 ± 70 nm, 185 ± 55 nm and 192 ± 53 nm, respectively. In addition, the fibre thickness for 4 wt% PEO, 5 wt% PEO, 6 wt% PEO and 7 wt% PEO nanofibres were 269 ± 3 µm, 664 ± 4 µm, 758 ± 7 µm and 1329 ± 44 µm, respectively. Contrarily, the surface roughness for 4 wt% PEO, 5 wt% PEO, 6 wt% PEO and 7 wt% PEO nanofibres were 55.6 ± 9 nm, 42.8 ± 6 nm, 42.7 ± 7 nm and 36.6 ± 1 nm, respectively. PEO nanofibres showed the same burst release pattern and rate due to the same molecular weight of PEO with a stable release rate profile after 15 min. It also demonstrates that the percentage of PEO nanofibre release increased with the increasing PEO concentration due to the fibre diameter and thickness. The findings showed that all PEO nanofibres formulations were non-toxic to MCF-7 cells. It is suggested that 5 wt% PEO nanofibre exhibited non-cytotoxic characteristics by maintaining the cell viability from dose 0-1000 µg/ml and did not induce the number of colonies. Therefore, 5 wt% PEO nanofibre is the optimal nanofibre concentration and was suggested as a suitable base polymer matrix or carrier with potential use for diagnostic purposes. The findings in this study have demonstrated the influence of cell growth and viability, including the effects of PEO nanofibre formulations on cancer progress characteristics to achieve a permissible PEO nanofibre concentration limit that can be a benchmark in medical applications, particularly diagnostic applications.

Development of biodegradable thin films for efficient, specific and controlled delivery of capecitabine.

Cancer is the leading cause of death worldwide. Capecitabine (CP) shows severe side effects because of early metabolism in stomach that affects the normal cells and organs, particularly liver and stomach. In this scope, we report the biocompatible, nontoxic polymeric thin films loaded with anti-cancer drug, CP for target specific, sublingual delivery of CP. Chitosan (CS) and polyvinyl alcohol (PVA) were used as biodegradable polymers alongwith glutaraldehyde (GLA) cross linker. CP-loaded thin films (TFCP1-TFCP5) were fabricated by solvent casting method. The results of Fourier transform infrared spectroscopy confirmed the presence of CP and polymers (CS and PVA) with GLA which binds through hydrogen bonding, and compatibility of drug with different excipients. Thermogravemetric analysis showed that the thin films are highly stable while differential scanning calorimeter thermograms confirmed the complete miscibility/entrapment of CP within PVA/CS thin film matrix. X-ray diffraction patterns revealed the molecular ineractions between CP and polymer matrix. High degree of swelling index of thin films at pH 7.4 was observed in comparison to pH 5.5. CP release studies in acetate (pH 5.5) and phosphate buffer (pH 7.4) showed that the thin films swell and result in drug diffusion faster in phosphate buffer through diffusion governed by Higuchi's model. Cytotoxicity results displayed that CPTFs killed MCF-7 and T47D (human breast adenocarcinoma) cells more effectively as compared to CP alone. The results of adhesion assay also showed that the PVA and CS both are safe and biocompatible. TFCP1 and TFCP3 thin films efficiently induced the apoptosis as compared to CP alone. The improved ability of TFCP1 and TFCP3 to induce cytotoxicity in MCF-7 cells reflects the potential of these thin films for targeted drug delivery. The CPTFs were stable for 4 months at 4 °C/60% ± 2%RH and 25 °C/70% ± 2%RH. In conclusion, the thin film formulations showed target specific controlled and burst release properties and thus could prove to be effective for human breast cancer treatment.

Methods in Screening Antiviral Drugs Against Enterovirus 71.

Enteroviruses 71 (EV71) is a single-stranded, neurotrophic RNA virus responsible for the numerous outbreaks of hand, foot, and mouth disease (HFMD) in the Asia-Pacific regions. HFMD primarily affects children to cause range of infection, from mild symptoms to acute flaccid paralysis, and hemorrhage. Despite increased incidence of EV71 epidemics globally and research against EV71 becoming prioritized, no antiviral agent against EV71 has yet been licensed and approved worldwide. In this chapter, detailed EV71 antiviral screening techniques are described, including plaque assay which determines viral titers through the use of a semisolid overlay, carboxymethyl cellulose to allow even viral spread and infection across the host cellular monolayers as well as a crystal violet, a distinct counterstain to visualize circular regions of infectious zones-plaques. qRT-PCR is used to quantify the viral genomic RNA in the infected samples and MTS cell viability assay to quantify the cell viability after infection or toxicity of the compound on the cells. Furthermore, various antiviral inhibition assays including prophylactic, post infection, and virucidal assays are demonstrated for estimation of the antiviral activity of potential antiviral drugs against EV71. These methods can be effectively utilized in virology laboratories for effective high-throughput screening of antiviral molecules against EV71 that can assist in the future development of antiviral drugs.

Evaluation of the in vivo fate of ultrapure alginate in a BALB/c mouse model.

The linear anionic polysaccharide alginate (ALG) has been comprehensively studied for biomedical applications, yet thus far the in vivo fate of this polymer has not been explored in detail. The current study therefore evaluates the biodistribution of ultrapure ALG (M/G ratio ≥ 0.67 with a measured Mw of 530 kg/mol and polydispersity index; PDI of 1.49) over a 14-day period in BALB/c mice. The biodistribution pattern over 2-days after sample administration using PET imaging with 64Cu-labelled ALG showed liver and spleen uptake. This was confirmed by the 14-day biodistribution profile of cyanine 5-labelled ALG from in vivo and ex vivo fluorescence imaging. Using MacGreen mice confirmed the uptake of the ALG by macrophages in the spleen at the 2-day time point. This extended biodistribution study confirmed the clearance of only a portion of the administered ALG biopolymer, but also uptake by macrophage populations in the spleen over a 14-day period.

Ultrafine-Grained Ti-31Mo-Type Composites with HA and Ag, Ta2O5 or CeO2 Addition for Implant Applications.

Ultrafine-grained Ti31Mo alloy and Ti31Mo5HA, Ti31Mo5HA-Ag (or Ta2O5, CeO2) composites with a grain size of approximately 2 µm were produced by the application of mechanical alloying and powder metallurgy. Additionally, the surface of the Ti31Mo alloy was modified. In the first stage, the specimens were immersed in 5M NaOH for 24 h at 60 °C. In the second stage, hydroxyapatite (HA) was deposited on the sample surface. The cathodic deposition at -5 V vs. open circuit potential (OCP) in the electrolyte containing 0.25M CaNa2-EDTA (di-calcium ethylenediaminetetraacetic acid), 0.25M K2HPO4 in 1M NaOH at 120 °C for 2 h was applied. The bulk Ti31Mo alloy is a single ß-type phase. In the alkali-modified surface titanium oxide, Ti3O is formed. After hydrothermal treatment, the surface layer mostly consists of the Ca10(PO4)6(OH)2 (81.23%) with about 19% content of CaHPO4·2H2O. Using optical profiler, roughness 2D surface topography parameters were estimated. The in vitro cytocompatibility of synthesized materials was studied. The cell lines of normal human osteoblasts (NHost) and human periodontal ligament fibroblasts (HPdLF) was conducted in the presence of tested biomaterials. Ultrafine-grained Ti-based composites altered with HA and Ag, Ta2O5 or CeO2 have superior biocompatibility than the microcrystalline Ti metal. NHost and HPdLF cells in the contact with the synthesized biomaterial showed stable proliferation activity. Biocompatibility tests carried out indicate that the ultrafine-grained Ti31Mo5HA composites with Ag, Ta2O5, or CeO2 could be a good candidate for implant applications.

Synthesis and in vitro antitumour activity of carboplatin analogues containing functional handles compatible for conjugation to drug delivery systems.

We describe herein the synthesis of a series of carboplatin derivatives with different functional groups at position 3 of the cyclobutane ring. This pharmacomodulation approach aims at facilitating the vectorisation of these analogues, via their subsequent conjugation to a drug delivery system. Five different derivatives bearing a hydroxy, keto, iodo, azido or amino function at position 3 were synthesised. One of these compounds was coupled to a bifunctional maleimide-containing linker. All compounds were tested in vitro for their cytotoxicity on four different cell lines including two platinum-resistant colorectal cancer cell line (SK-OV-3, HCT116, D3E2, D5B7) using an MTS assay. Overall, the tested compounds were up to six times more potent than carboplatin, especially on D5B7 human colorectal cancer cells. We demonstrated that these modifications led to potent analogues which are compatible with conjugation to a drug delivery system.

Synergistic Combinations of Curcumin, Sulforaphane, and Dihydrocaffeic Acid against Human Colon Cancer Cells.

Nutraceutical combinations that act synergistically could be a powerful solution against colon cancer, which is the second deadliest malignancy worldwide. In this study, curcumin (C), sulforaphane (S), and dihydrocaffeic acid (D, a chlorogenic acid metabolite) were evaluated, individually and in different combinations, over the viability of HT-29 and Caco-2 colon cancer cells, and compared against healthy fetal human colon (FHC) cells. The cytotoxic concentrations to kill 50%, 75%, and 90% of the cells (CC50, CC75, and CC90) were obtained, using the MTS assay. Synergistic, additive, and antagonistic effects were determined by using the combination index (CI) method. The 1:1 combination of S and D exerted synergistic effects against HT-29 at 90% cytotoxicity level (doses 90:90 µM), whereas CD(1:4) was synergistic at all cytotoxicity levels (9:36-34:136 µM) and CD(9:2) at 90% (108:24 µM) against Caco-2 cells. SD(1:1) was significantly more cytotoxic for cancer cells than healthy cells, while CD(1:4) and CD(9:2) were similarly or more cytotoxic for healthy cells. Therefore, the SD(1:1) combination was chosen as the best. A model explaining SD(1:1) synergy is proposed. SD(1:1) can be used as a basis to develop advanced food products for the prevention/co-treatment of colon cancer.

Cancellation effect is present in high-frequency reversible and irreversible electroporation.

It was recently suggested that applying high-frequency short biphasic pulses (HF-IRE) reduces pain and muscle contractions in electrochemotherapy and irreversible ablation treatments; however, higher amplitudes with HF-IRE pulses are required to achieve a similar effect as with monophasic pulses. HF-IRE pulses are in the range of a microseconds, thus, the so-called cancellation effect could be responsible for the need to apply pulses of higher amplitudes. In cancellation effect, the effect of first pulse is reduced by the second pulse of opposite polarity. We evaluated cancellation effect with high-frequency biphasic pulses on CHO-K1 in different electroporation buffers. We applied eight bursts of 1-10 µs long pulses with inter-phase delays of 0.5 µs - 10 ms and evaluated membrane permeability and cell survival. In permeability experiments, cancellation effect was not observed in low-conductivity buffer. Cancellation effect was, however, observed in treatments with high-frequency biphasic pulses looking at survival in all of the tested electroporation buffers. In general, cancellation effect depended on inter-phase delay as well as on pulse duration, i.e. longer pulses and longer interphase delay cause less pronounced cancellation effect. Cancellation effect could be partially explained by the assisted discharge and not by the hyperpolarization by the chloride channels.

Genotoxicity assessment of antiepileptic drugs (AEDs) in human embryonic stem cells.

Several antiepileptic drugs (AEDs) are administrated during pregnancy according to recent therapeutic protocols. Ten percent of pregnant women with epilepsy give birth to offspring with malformations and teratogenic defects. Since the mechanism of action of AEDs is not yet completely understood, therefore, it could be hypothesized that they may cause cyto- or genotoxicity in embryonic fetus cells. To investigate this hypothesis, the genotoxicity and cell survival of AEDs treated human embryonic stem cells (hESCs) were investigated by single-cell gel electrophoresis (Comet assay) and MTS assay, respectively. HESCs (Royan H6 cell line) were treated in-vitro with high therapeutic doses of Carbamazepine, Gabapentin, Lamotrigine, Levetiracetam or Topiramate as monotherapy or combination therapy of each drug with Folic acid. After hESCs pluripotency confirmation, the effect of AEDs on cellular DNA damage of hESCs was investigated. levetiracetam and topiramate were found to damage the DNA significantly compared to untreated cells. The amount of DNA damage produced by carbamazepine and lamotrigine was similar while for gabapentin, the amount of DNA migration was very low and produced less DNA damage than the others. A considerable reduction in DNA damages occurred in genotoxicity in the presence of Folic acid in comparison to AEDs monotherapies. According to our results, all mentioned AEDs caused DNA damage, while Levetiracetam and topiramate caused more extensive DNA damages than the others. Noticeably, the addition of Folic acid to the treated cells decreased the DNA damages considerably.

Synthesis of Ultrastable Gold Nanoparticles as a New Drug Delivery System.

Nanotechnologies are increasingly being developed for medical purposes. However, these nanomaterials require ultrastability for better control of their pharmacokinetics. The present study describes three types of ultrastable gold nanoparticles stabilized by thiolated polyethylene glycol groups remaining intact when subjected to some of the harshest conditions described thus far in the literature, such as autoclave sterilization, heat and freeze-drying cycles, salts exposure, and ultracentrifugation. Their stability is characterized by transmission electron microscopy, UV-visible spectroscopy, and dynamic light scattering. For comparison purposes, two conventional nanoparticle types were used to assess their colloidal stability under all conditions. The ability of ultrastable gold nanoparticles to encapsulate bimatoprost, a drug for glaucoma treatment, is demonstrated. MTS assays on human corneal epithelial cells is assessed without changing cell viability. The impact of ultrastable gold nanoparticles on wound healing dynamics is assessed on tissue engineered corneas. These results highlight the potential of ultrastable gold nanoparticles as a drug delivery system in ocular therapy.

Colorimetric assay for the in vitro evaluation of Saprolegnia biofilm inhibitors.

A quantitative and reproducible 96-well microtiter method that is easily adaptable for the screening of Saprolegnia biofilm inhibitors is described. As opposed to other methods previously developed for the screening of Saprolegnia inhibitors on spore germination or mycelial growth, this technique is of particular significance as it investigates potential inhibitors against surface-attached mycelial mats of Saprolegnia spp. (biofilm). In this study, we have investigated the effects of propionic acid (PPA) on reducing the viability of induced Saprolegnia biofilms using colorimetric MTS assay based on the reduction of tetrazolium salts. Viability of Saprolegnia hyphae in treated biofilms was reduced significantly following treatment with different PPA concentrations. The effect was enhanced after combining each of the tested PPA concentrations with 500 mg/L of boric acid (BA). However, the percentage of non-viable hyphae was still higher in 200 mg L-1 bronopol-treated biofilms (positive control) following 6- and 12-hr exposure. Similar results were observed using other recently described fluorescence-based assays for viability.

Oct3/4 is potentially useful for the suppression of the proliferation and motility of hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) cells are immature compared with healthy mature hepatocytes. Transcription factors serve a role in hepatocyte differentiation. The expression levels of transcription factors in HCC cell lines have been investigated to determine potential therapeutic targets. In the present study, the HLE, HLF, PLC/PRF/5, Huh-7, Hep3B, Huh-6 and HepG2 HCC cell lines were subjected to reverse-transcription polymerase chain reaction (RT-PCR) of transcription factors, including NANOG, Oct3/4, GATA binding protein 4 (GATA4), GATA6 and hematopoietically expressed homeobox (HHEX). In addition, these cell lines were analyzed using RT-quantitative PCR (RT-qPCR) of NANOG and Oct3/4. The 201B7 human induced pluripotent stem cells were evaluated as a model of pluripotent cells. The HLF cells were transfected with Oct3/4 small interfering RNA (siRNA) and used in an MTS colorimetric assay and a scratch assay. NANOG was not expressed in any of the cell lines. However, GATA4, GATA6 and HHEX were expressed in the majority of the HCC cell lines. In addition, NANOG and Oct3/4 were expressed in 201B7 cells. Oct3/4 was expressed in HLE, HLF and Hep3B cells; however, its expression levels were significantly reduced compared with those in 201B7 cells. RT-qPCR demonstrated that the expression of Oct3/4 siRNA suppressed the proliferation and motility of HLF cells. Oct3/4 siRNA may be a potentially effective therapy for the suppression of the proliferation and motility of HCC cells.

UPLC-MS/MS analysis of ochratoxin A metabolites produced by Caco-2 and HepG2 cells in a co-culture system.

Ochatoxin A (OTA) is one of the most important mycotoxins based on its toxicity. The oral route is the main gateway of entry of OTA into the human body, and specialized epithelial cells constitute the first barrier. The present study investigated the in vitro cytotoxic effect of OTA (5, 15 and 45 µM) and production of OTA metabolities in Caco-2 and HepG2 cells using a co-culture Transwell System to mimic the passage through the intestinal epithelium and hepatic metabolism. The results derived from MTS cell viability assays and transepithelial electrical resistance measurements showed that OTA was slightly cytotoxic at the lowest concentration at 3 h, but significant toxicity was observed at all concentrations at 24 h. OTA metabolites generated in this co-culture were ochratoxin B (OTB), OTA methyl ester, OTA ethyl ester and the OTA glutathione conjugate (OTA-GSH). OTA methyl ester was the major metabolite found in both Caco-2 and HepG2 cells after all treatments. Our results showed that OTA can cause cell damage through several mechanisms and that the OTA exposure time is more important that the dosage in in vitro studies. OTA methyl ester is proposed as an OTA exposure biomarker, although future studies should be conducted.