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5-flourouracil (5-FU) is typically modulated with leucovorin (LEU) in clinical practice to improve clinical efficacy and patient survival rates. However, this combination has undesirable side effects and makes 5-FU more toxic. Hence, integrating a vesicular system (proniosomes) with another delivery vehicle may improve drug targeting, while resolving the aforementioned drawbacks. This study aimed to engineer 5-FU/LEU proniosomes for possible delivery to the colon. The modified slurry approach was used to create drug-loaded proniosomes (150 mg/9 g of carrier) using both water-soluble (dextrin (DEX) and lactose (LAC)) and insoluble (Neusilin FH2 (NEU)) carriers. The powdered formulations were filled into Eudragit S100 (10 %)-coated capsules or Eudragit FS 30D capsules for enteric- or colon-specific delivery. In vitro evaluations (flow properties, powder X-ray diffractometry (XRD) analysis, particle size analysis, entrapment efficiency, drug release, scanning electron microscopy (SEM), polydispersity index, Fourier transform infrared spectroscopy (FTIR), and stability studies) were performed on the formulations. An in vitro cytotoxicity test [real-time cell assay (RTCA)] against HCT-116 colon cancer cell lines was performed using the optimized formulation. In vitro evaluations showed that the nanoparticles had good physicochemical properties. RTCA studies showed sustained cell death with the formulations compared to the pure drug and placebo. The sequential drug release of the colon-targeted capsules containing 5-FU and LEU- loaded proniosomes showed negligible drug release in SGF (pH 1.2) and phosphate buffer solution (pH 6.8) (approximately 11 %) but profound drug release (>80 %) at pH 7.4. Drug-loaded proniosomes engineered for colon targeting (Eudragit S100 (10 %) capsules or Eudragit FS 30D capsules) showed good colon-specific targeting and favorable in vitro cytotoxicity profiles.

Promoting axon regeneration in the central and peripheral nervous system is of clinical importance in neural injury and neurodegenerative diseases. Both pro- and antiregeneration factors are being identified. We previously reported that the Rtca mediated RNA repair/splicing pathway restricts axon regeneration by inhibiting the nonconventional splicing of Xbp1 mRNA under cellular stress. However, the downstream effectors remain unknown. Here, through transcriptome profiling, we show that the tubulin polymerization-promoting protein (TPPP) ringmaker/ringer is dramatically increased in Rtca-deficient Drosophila sensory neurons, which is dependent on Xbp1. Ringer is expressed in sensory neurons before and after injury, and is cell-autonomously required for axon regeneration. While loss of ringer abolishes the regeneration enhancement in Rtca mutants, its overexpression is sufficient to promote regeneration both in the peripheral and central nervous system. Ringer maintains microtubule stability/dynamics with the microtubule-associated protein futsch/MAP1B, which is also required for axon regeneration. Furthermore, ringer lies downstream from and is negatively regulated by the microtubule-associated deacetylase HDAC6, which functions as a regeneration inhibitor. Taken together, our findings suggest that ringer acts as a hub for microtubule regulators that relays cellular status information, such as cellular stress, to the integrity of microtubules in order to instruct neuroregeneration.

Aspergillus nidulans has been more extensively characterized than other Aspergillus species considering its morphology, physiology, metabolic pathways, and genetic regulation. As it has a rapid growth rate accompanied by simple nutritional requirements and a high tolerance to extreme cultural conditions, A. nidulans is a promising microbial cell factory to biosynthesize various products in industry. However, it remains unclear for whether it is also a suitable host for synthesizing abundant L-malic acid. In this study, we developed a convenient and efficient double-gene-editing system in A. nidulans strain TN02A7 based on the CRISPR–Cas9 and Cre-loxP systems. Using this gene-editing system, we made a L-malic acid-producing strain, ZQ07, derived from TN02A7, by deleting or overexpressing five genes (encoding Pyc, pyruvate carboxylase; OahA, oxaloacetate acetylhydrolase; MdhC, malate dehydrogenase; DctA, C4-dicarboxylic acid transporter; and CexA, citric acid transporter). The L-malic acid yield in ZQ07 increased to approximately 9.6 times higher (up to 30.7 g/L titer) than that of the original unedited strain TN02A7, in which the production of L-malic acid was originally very low. The findings in this study not only demonstrate that A. nidulans could be used as a potential host for biosynthesizing organic acids, but also provide a highly efficient gene-editing strategy in filamentous fungi. [ABSTRACT FROM AUTHOR]

This study is dedicated to the development of multimodal anticancer agents. We have obtained ruthenium complexes conjugated with the steroid-type antitumor drug abiraterone acetate in order to take advantage of the dual antitumor properties of both ruthenium and abiraterone. The compounds exhibit good antiproliferative activity against cancer cells, with selectivity over primary fibroblasts. Real-time cell analysis revealed that compound dichlorido(η6-p-cymene)(abiraterone acetate)ruthenium(II) had pronounced antiproliferation activity compared to abiraterone acetate. Flow cytometric studies on the mechanism of cell death have revealed that the most active compound induces apoptosis more effectively than abiraterone acetate. Our findings demonstrate the potential of this novel dual-action compound as promising candidates for further development as anticancer agents. Design and synthesis of ruthenium-abiraterone conjugates as dual-action anticancer agents, combining the anticancer properties of ruthenium complexes with the activity of abiraterone acetate. [Display omitted] • Ruthenium complexes conjugated with the abiraterone acetate exhibit good antiproliferative activity with selectivity towards cancer cells. • Complex 1 showed a more pronounced antiproliferative effect in comparison with abiraterone acetate in real-time cell analysis. • Complex 1 causes significant apoptosis induction at an early stage. [ABSTRACT FROM AUTHOR]

Objective: Anti-cancer drug research plays an important role for chemotherapeutic treatments in various types of cancer. Pyrolidine derived compounds have been reported by many researchers to be a potent anti-cancer compound. It is aimed to investigate the effects of pyrolidine-derived compounds that are thought to be new drug candidates with antiproliferative activity on DLD-1 and CCD-18CO cell lines. Material and Methods: The antiproliferative activity of the pyrrolidine-derived compound was determined for 24 hours at different concentrations (25-100 µM) on DLD-1 (human colon cancer) and CCD-18CO (normal colon fibroblast) cell lines by comparing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5 Diphenyltetrazolium bromide) and RTCA (real-time cell analysis) assays. The significance of the differences between data sets in the MTT assay was analyzed statistically by ANOVA with SPSS 20.0 program for DLD-1 and CCD-18CO cell lines. Results: It has been determined that pyrrolidine-derived compounds reduce the number of DLD-1 cancer cells according to negative control with the MTT method and suppress the DLD-1 cell according to the RTCA assay results. Thus, the compounds have been shown to inhibit cell proliferation and have antiproliferative activity. Conclusion: Pyrrolidine-derived compounds will be the first step for antiproliferative activity studies in DLD-1 cancer cells and will guide the next studies.

Currently, the biological production of L-malic acid (L-MA) is mainly based on the fermentation of filamentous fungi at near-neutral pH, but this process requires large amounts of neutralizing agents, resulting in the generation of waste salts when free acid is obtained in the downstream process, and the environmental hazards associated with the waste salts limit the practical application of this process. To produce L-MA in a more environmentally friendly way, we metabolically engineered the acid-tolerant yeast Pichia kudriavzevii and achieved efficient production of L-MA through low pH fermentation. First, an initial L-MA-producing strain that relies on the reductive tricarboxylic acid (rTCA) pathway was constructed. Subsequently, the L-MA titer and yield were further increased by fine-tuning the flux between the pyruvate and oxaloacetate nodes. In addition, we found that the insufficient supply of NADH for cytoplasmic malate dehydrogenase (MDH) hindered the L-MA production at low pH, which was resolved by overexpressing the soluble pyridine nucleotide transhydrogenase SthA from E. coli. Transcriptomic and metabolomic data showed that overexpression of EcSthA contributed to the activation of the pentose phosphate pathway and provided additional reducing power for MDH by converting NADPH to NADH. Furthermore, overexpression of EcSthA was found to help reduce the accumulation of the by-product pyruvate but had no effect on the accumulation of succinate. In microaerobic batch fermentation in a 5-L fermenter, the best strain, MA009-10-URA3 produced 199.4 g/L L-MA with a yield of 0.94 g/g glucose (1.27 mol/mol), with a productivity of 1.86 g/L/h. The final pH of the fermentation broth was approximately 3.10, meaning that the amount of neutralizer used was reduced by more than 50% compared to the common fermentation processes using filamentous fungi. To our knowledge, this is the first report of the efficient bioproduction of L-MA at low pH and represents the highest yield of L-MA in yeasts reported to date. • Efficient L-MA production at low pH was achieved in the acid-tolerant yeast P. kudriavzevii. • The L-MA titer and yield were increased by fine-tuning the flux between the pyruvate and oxaloacetate nodes. • The additional supply of NADH was critical for L-MA production at low pH and reduced the by-product accumulation of pyruvate. [ABSTRACT FROM AUTHOR]

This study explores the dissolution mechanism and absorption process of compound Danshen tablets (CDTs) and compound Danshen capsules (CDCs) in vitro. Taking the cell index as the evaluation index of dissolution and absorption of multi-component solid preparations of CDTs and CDCs, it breaks through the idea of traditional research. We used real-time cell-based assay (RTCA) to provide a new idea and method for the consistency evaluation of traditional Chinese medicine (TCM) compound preparations. The drug dissolution and absorption simulation system (DDASS) was established to obtain the dissolution and absorption samples of compound Danshen solid preparations at different time points. The cell index (CI) of the sample to H9C2 cells was detected by RTCA technology, and the dissolution and absorption percentage were calculated based on this index to obtain the dissolution and absorption kinetics model. Meanwhile, one batch of tablets and one batch of capsules (batch numbers ZKC1816 and 202101001) were selected to conduct the overall animal pharmacodynamic experiment to verify the feasibility of drug effect evaluation with cell index as an indicator. The best fitting model of dissolution curves of each batch of CDTs and CDCs is the Weibull model. There was a good correlation (r > 0.86) between the dissolution-absorption-pharmacodynamic curve. Based on RTCA technology, we have established the comprehensive evaluation method for cell biology of compound Danshen solid preparations in line with the overall concept of TCM and a synchronous evaluation system of dissolution and absorption in vitro of new TCM compound solid preparations. [ABSTRACT FROM AUTHOR]

The formulation of niosomes is influenced by a number of variables, and these variables may eventually affect the formulation’s outcome. One of the elements that can influence the physico-chemical properties of niosomes is the method used in preparation of the formulation. In this study, we established if various methods of preparation have any impact on the prepared vesicles when loaded with 5-fluorouracil. Thereafter, a real-time cell assay (an in vitro cytotoxicity test) against HCT-116 colon cancer cell lines was done on an optimised batch. 5-fluorouracil loaded niosomes were prepared with either Tween 60 or Span 60 by four different methods - namely thin film hydration (TFH), reverse phase evaporation (RPE), evaporation/sonication (EVP/SON), and the ethanol injection method (EIM). In vitro evaluations were done on the formulations, and these included particle size analysis, entrapment efficiency, scanning electron microscopy (SEM), photomicrography, drug release, polydispersity index, and Fourier transform infrared spectroscopy (FTIR). The effects of the preparation method and type of non-ionic surfactants on encapsulation efficiency, particle size, and in vitro drug release of the niosomes at pH 7.4 were evaluated. An in vitro cytotoxicity test (real time cell assay (RTCA)) against HCT-116 cells was carried out using the optimised formulation. Results showed physically stable formulations. The TFH method produced the smallest particle sizes (187 nm and 482 nm), while the EVP/SON method produced the largest particle sizes (4476 nm and 9111 nm). The Tween-based niosomes prepared by TFH or RPE had higher drug entrapment. The FTIR studies of niosomal formulations showed broad peaks at wavenumbers above 3000 cm−1, indicating strong hydrogen bonds. The RTCA showed 5-fluorouracil-loaded niosomes caused more sustained cell death compared to the pure drug and blank niosomes. The methods of preparation affected the particle size, polydispersity index, entrapment efficiency, and the physical stability of the vesicles. The thin film hydration method was more robust in the entrapped 5-fluorouracil and showed lower particle sizes when compared to all the other methods. RTCA showed sustained cell death in real time.

Five new triterpenoids, oenotheralanosterols C-G (1–5), with seven known triterpenoidcompounds, namely 2α,3α,19α-trihydroxy-24-norurs4,12-dien-28-oic acid (6), 3β,23-dihydroxy-1-oxo-olean-12-en-28-oic acid (7), remangilone C (8), knoxivalic acid A (9), termichebulolide (10), rosasecotriterpene A (11), androsanortriterpene C (12), were extracted and separated from the dichloromethane part of Oenothera biennis L. The anti-pulmonary fibrosis activities of all the compounds against TGF-β1-induced damage tonormal human lung epithelial (BEAS-2B) cells were investigated in vitro. The results showed that compounds 1–2, 6, 8, and 11 exhibited significant anti-pulmonary fibrosis activities, with EC50 values ranging from 4.7 μM to 9.9 μM. [ABSTRACT FROM AUTHOR]

Synthetic materials used in regenerative medicine, upon implantation, induce the development of an inflammatory reaction necessary for the effective regeneration of damaged bone tissue. Implant contact with tissues is accompanied by the deposition of blood proteins and interstitial fluid on its surface, contributing to the activation of the complement system, components of innate immunity, initiating coagulation hemostasis, leading to the formation of a fibrin clot. An extracellular matrix based on fibrin, collagen and elastin forms on the implant’s surface, which provides the basis for the formation of tissue structure through the adhesion of stem cells to the forming bone callus before the formation of bone regenerate. To prevent the development of postoperative pathological conditions caused by hypercoagulable syndrome, therapeutic strategies are used to use anticoagulants (heparin, warfarin). However, their use limits the normal formation of a fibrin clot in vivo. This can slow down the migration of mesenchymal stem cells (MSC) and disrupt the formation of callus, inhibiting the processes of osseointegration of the implant and bone healing. The study’s goal was to study the effect of heparin in a gradient of low and high concentrations on the migration activity and stem capacity of human MSCs under in vitro cultivation conditions. According to the results of flow cytometry, it was revealed that high concentrations of heparin (130, 260 IU/ml) in a 2D cultivation model contribute to an increase in the number of cells expressing surface markers CD73 and CD90, which indicates that MSCs retain high clonogenic potential. A 3D model of in vitro cultivation with the addition of heparin and osteosubstituting implants bearing a CF coating with a roughness index of Ra = 2.6-4.9 μm contributed to preserving the “stemness” character of MSCs through the expression of surface markers CD73 and CD90. According to the results obtained using the xCELLigence system, heparin at a later time (from 20-40 hours) increases the invasion of MSCs through micropores that simulate the state of the blood vessel walls. However, in the presence of HAP nanoparticles that mimic the remodeling processes of the mineral bone matrix and/or resorption of bone cement, the effect of heparin was less pronounced. The results can be used in the field of regenerative medicine associated with the introduction of MSCs. The data can serve as a prerequisite for developing new therapeutic strategies for surgical patients with a high risk of postoperative thrombosis after osteosynthesis.

Aspergillus nidulans has been more extensively characterized than other Aspergillus species considering its morphology, physiology, metabolic pathways, and genetic regulation. As it has a rapid growth rate accompanied by simple nutritional requirements and a high tolerance to extreme cultural conditions, A. nidulans is a promising microbial cell factory to biosynthesize various products in industry. However, it remains unclear for whether it is also a suitable host for synthesizing abundant L-malic acid. In this study, we developed a convenient and efficient double-gene-editing system in A. nidulans strain TN02A7 based on the CRISPR–Cas9 and Cre-loxP systems. Using this gene-editing system, we made a L-malic acid-producing strain, ZQ07, derived from TN02A7, by deleting or overexpressing five genes (encoding Pyc, pyruvate carboxylase; OahA, oxaloacetate acetylhydrolase; MdhC, malate dehydrogenase; DctA, C4-dicarboxylic acid transporter; and CexA, citric acid transporter). The L-malic acid yield in ZQ07 increased to approximately 9.6 times higher (up to 30.7 g/L titer) than that of the original unedited strain TN02A7, in which the production of L-malic acid was originally very low. The findings in this study not only demonstrate that A. nidulans could be used as a potential host for biosynthesizing organic acids, but also provide a highly efficient gene-editing strategy in filamentous fungi.

Safety certification of GNSS-aided inertial navigation systems (INS) in civil aircraft requires thorough testing to ensure proper operation, even in worst-case conditions. One error that must be considered is that of gravity compensation on accelerometer measurements. Prior to the work described in this paper, no stochastic models existed with the Gaussian bounding of the tails required to ensure integrity performance. This paper describes a method to determine efficient stochastic models of the error of current high-order gravity models such as EGM2008. The stochastic and high-order models are combined to achieve a high-fidelity model suitable for use in testing systems designed for low-approach operations such as RNP-AR. This paper also describes a method to determine efficient stochastic models for low-order gravity models such as the WGS-84 ellipsoidal model. Such models may be used in testing systems designed for operations with less stringent lateral requirements. [ABSTRACT FROM AUTHOR]

Simple Summary: Although artificial antiviral drugs can effectively inhibit the proliferation of viruses, these drugs not only have serious side effects, but also create drug-resistant virus strains and produce harmful drug residues. In comparison, natural antiviral compounds have many distinctive advantages. For example, natural compounds, in general, have a lower toxicity, lower price, and better antiviral effects. Nowadays, wide applications of natural antiviral compounds not only provide us with safe meat products, but also reduce the abuse of antibiotics, thus reducing the number of drug-resilient microbes and viruses. The purpose of this study was to demonstrate the antiviral activity of Ren's oligopeptides-1 against the pseudorabies virus (PRV). Ren's oligopeptides-1 is a plant-derived drug with an antiviral activity against herpesvirus. Ren's oligopeptides-1 contains ginger, garlic, onion, banana peel, and bitter melon. We discovered that the CC50 value of Ren's oligopeptides-1 was 15 mg/mL, while the EC50 value was 6 mg/mL. This indicated that the concentration of 6 mg/mL was optimal for inhibiting the replication of PRV. In this study, PRV was used to conduct in vitro experiments on PK15 cells to explore the antiviral effect of Ren's oligopeptides-1, which could become a promising feed additive. Newly synthesized Ren's oligopeptides-1 was found to have an antiviral effect in clinical trials, and the purpose of this study was to further demonstrate the antiviral activity of Ren's oligopeptides-1 against the PRV 152-GFP strain. We used the real-time cell analysis system (RTCA) to detect the cytotoxicity of different concentrations of Ren's oligopeptides-1. We then applied high content screening (HCS) to detect the antiviral activity of Ren's oligopeptides-1 against PRV. Meanwhile, the fluorescence signal of the virus was collected in real time and the expression levels of the related genes in the PK15 cells infected with PRV were detected using real-time PCR. At the mRNA level, we discovered that, at a concentration of 6 mg/mL, Ren's oligopeptides-1 reduced the expression of pseudorabies virus (PRV) genes such as IE180, UL18, UL54, and UL21 at a concentration of 6 mg/mL. We then determined that Ren's oligopeptides-1 has an EC50 value of 6 mg/mL, and at this level, no cytotoxicity was observed. [ABSTRACT FROM AUTHOR]

In many regulated industries there is an increasing pressure to provide timely and robust risk assessment data to support product launches. Real-time cell analysis (RTCA) is a tool that allows for the fast and relatively labour-free cytotoxic assessment of test compounds, compared to traditional methods. Here, we propose an application for the RTCA platform to provide a screening approach, to evaluate the cytotoxic potential of tobacco-free nicotine pouches, also termed modern oral product (MOP), to determine the contribution of differing nicotine strengths (4−11 mg) and a range of available flavour types from multiple markets, on overall product toxicity.Aqueous extracts were prepared for all products using 1 pouch in 20 mL cell culture media and applied to the cell system for 24 h. Test extract nicotine concentrations reflected the increases in product nicotine strength; however, these changes were not present in the same magnitude in the cytotoxicity data obtained from both primary human gingival fibroblasts (HGF) and an NCI-H292 human bronchial epithelial continuous cell line. Furthermore, across the range of flavours and product nicotine strengths tested, H292 cells whilst not the target organ for oral product use, accurately predicted the results seen in HGFs and could be considered a useful surrogate for fast screening studies. H292 cells are more easily cultured and for longer periods, offering a more compatible test system.In conclusion, the data demonstrate the utility of the RTCA platform for the quick assessment of a large range of product variants. Furthermore, for a cytotoxicity measure with this test product, the simple H292 cell line can predict outcomes in the more complex HGF and provide useful pre-clinical cytotoxicity screening data to inform the risk assessment of MOPs and the relative contribution of flavourings, nicotine and other components.

Context Trigonella foenum-graecum L. (Fabaceae) has many therapeutic properties and anticancer potential. Objective The cytotoxic activities of standardized extracts and a fraction from fenugreek seeds and their compounds (sapogenins, flavone C-glycosides, alkaloid trigonelline) against human cancer SKOV-3, HeLa and MOLT-4 cells were evaluated. Materials and methods Fenugreek seeds were extracted with 70% methanol (A) or water (B). Furthermore, the seeds were purified with petroleum ether and chloroform and next extracted with methanol to obtain fraction (C). The quantitative analysis of saponins and flavonoids in the extracts was done with HPLC methods. The extracts (5–120 µg/mL) and compounds (1–50 µg/mL) were tested on the cells by MTT assay and RTCA system. The effect of a fraction on ROS production, mitochondrial membrane potential and caspase-3/7 activity in HeLa and SKOV-3 cells was also evaluated by flow cytometry. Results The strongest cytotoxic activity on cancer cells showed the fraction C (IC50 was 3.91 ± 0.03 for HeLa, 3.97 ± 0.07 for SKOV-3, and 7.75 ± 0.37 for MOLT-4) with the highest content of steroidal saponins (163.18 ± 11.03 μg/mg) and flavone C-glycosides (820.18 ± 0.05 μg/mg). The fraction significantly increased ROS production (up to four times higher than in keratinocytes as control) and caspases activity in the cells. The examined flavonoids did not exhibit the cytotoxic activity in contrast to yamogenin, tigogenin, and diosgenin. Conclusions The obtained results complement the data on the cytotoxic activity of Foenugraeci Semen and synergistic effect of flavonoids and saponins complex contained in the plant.

Context Bufadienolide compounds occur in many plants and animal species and have strong cardiac and anti-inflammatory properties. The compounds have been recently investigated for cytotoxic and antitumor activity. Objective The cytotoxic effect of bersaldegenin-1,3,5-orthoacetate – a bufadienolide steroid occuring in plants from Kalanchoe genus (Crassulaceae), was evaluated with cervical cancer HeLa cells in vitro. Materials and methods The cytotoxic activity of the compound (at 0.1–20.0 μg/mL) on the cells was determined by Real-Time Cell Analysis (RTCA) system for 24 h. The estimation of cell cycle arrest, reactive oxygen species (ROS) production, reduction of mitochondrial membrane potential (MMP), and caspases-3/7/9 activity in the HeLa cells treated with the compound was done by flow cytometry and luminometric technique. DNA damage in the cells was estimated by immunofluorescence staining and the comet assay with etoposide as a positive control. Results The compound had strong effect on the cells (IC50 = 0.55 μg/mL) by the suppression of HeLa cells proliferation in G2/M phase of cell cycle and induction of cell death through double-stranded DNA damage and reactive oxygen species overproduction. Furthermore, we did not observe an increase in the activity of caspase-3/7/9 in the treated cells as well as a decrease in cellular mitochondrial membrane potential. Gene expression analysis revealed the overexpression of NF-Kappa-B inhibitors genes (>2-fold higher than control) in the treated cells. Conclusions Bersaldegenin-1,3,5-orthoacetate induces cell cycle arrest and caspase-independent cell death through double-stranded DNA damage. These results are an important step in further studies on cell death signalling pathways induced by bufadienolides.

Trigonella foenum-graecum L. (Fabaceae) has many therapeutic properties and anticancer potential. The cytotoxic activities of standardized extracts and a fraction from fenugreek seeds and their compounds (sapogenins, flavone C-glycosides, alkaloid trigonelline) against human cancer SKOV-3, HeLa and MOLT-4 cells were evaluated. Fenugreek seeds were extracted with 70% methanol (A) or water (B). Furthermore, the seeds were purified with petroleum ether and chloroform and next extracted with methanol to obtain fraction (C). The quantitative analysis of saponins and flavonoids in the extracts was done with HPLC methods. The extracts (5–120 µg/mL) and compounds (1–50 µg/mL) were tested on the cells by MTT assay and RTCA system. The effect of a fraction on ROS production, mitochondrial membrane potential and caspase-3/7 activity in HeLa and SKOV-3 cells was also evaluated by flow cytometry. The strongest cytotoxic activity on cancer cells showed the fraction C (IC50 was 3.91 ± 0.03 for HeLa, 3.97 ± 0.07 for SKOV-3, and 7.75 ± 0.37 for MOLT-4) with the highest content of steroidal saponins (163.18 ± 11.03 μg/mg) and flavone C-glycosides (820.18 ± 0.05 μg/mg). The fraction significantly increased ROS production (up to four times higher than in keratinocytes as control) and caspases activity in the cells. The examined flavonoids did not exhibit the cytotoxic activity in contrast to yamogenin, tigogenin, and diosgenin. The obtained results complement the data on the cytotoxic activity of Foenugraeci Semen and synergistic effect of flavonoids and saponins complex contained in the plant. [ABSTRACT FROM AUTHOR]

Bufadienolide compounds occur in many plants and animal species and have strong cardiac and anti-inflammatory properties. The compounds have been recently investigated for cytotoxic and antitumor activity. The cytotoxic effect of bersaldegenin-1,3,5-orthoacetate – a bufadienolide steroid occuring in plants from Kalanchoe genus (Crassulaceae), was evaluated with cervical cancer HeLa cells in vitro. The cytotoxic activity of the compound (at 0.1–20.0 μg/mL) on the cells was determined by Real-Time Cell Analysis (RTCA) system for 24 h. The estimation of cell cycle arrest, reactive oxygen species (ROS) production, reduction of mitochondrial membrane potential (MMP), and caspases-3/7/9 activity in the HeLa cells treated with the compound was done by flow cytometry and luminometric technique. DNA damage in the cells was estimated by immunofluorescence staining and the comet assay with etoposide as a positive control. The compound had strong effect on the cells (IC50 = 0.55 μg/mL) by the suppression of HeLa cells proliferation in G2/M phase of cell cycle and induction of cell death through double-stranded DNA damage and reactive oxygen species overproduction. Furthermore, we did not observe an increase in the activity of caspase-3/7/9 in the treated cells as well as a decrease in cellular mitochondrial membrane potential. Gene expression analysis revealed the overexpression of NF-Kappa-B inhibitors genes (>2-fold higher than control) in the treated cells. Bersaldegenin-1,3,5-orthoacetate induces cell cycle arrest and caspase-independent cell death through double-stranded DNA damage. These results are an important step in further studies on cell death signalling pathways induced by bufadienolides. [ABSTRACT FROM AUTHOR]

Real-time cell analysis (RTCA) is a technique based on impedance and microsensor electrodes. RTCA system allows label-free, real-time, and continuous monitoring of cell adhesion, morphology, and rate of cell proliferation. The system offers a wide range of applications, mainly in toxicological studies, new drug screening, and microbiology. Here, we describe the usefulness of the system in different applications and compare this technology with conventional endpoint assays based on tetrazolium salts. We present advantages and disadvantages of the system and endpoint methods and their limitations in cytotoxicity investigations.

Five new triterpenoids, oenotheralanosterols C-G (1–5), with seven known triterpenoidcompounds, namely 2α,3α,19α-trihydroxy-24-norurs4,12-dien-28-oic acid (6), 3β,23-dihydroxy-1-oxo-olean-12-en-28-oic acid (7), remangilone C (8), knoxivalic acid A (9), termichebulolide (10), rosasecotriterpene A (11), androsanortriterpene C (12), were extracted and separated from the dichloromethane part of Oenothera biennis L. The anti-pulmonary fibrosis activities of all the compounds against TGF-β1-induced damage tonormal human lung epithelial (BEAS-2B) cells were investigated in vitro. The results showed that compounds 1–2, 6, 8, and 11 exhibited significant anti-pulmonary fibrosis activities, with EC50 values ranging from 4.7 μM to 9.9 μM.

Newly synthesized Ren’s oligopeptides-1 was found to have an antiviral effect in clinical trials, and the purpose of this study was to further demonstrate the antiviral activity of Ren’s oligopeptides-1 against the PRV 152-GFP strain. We used the real-time cell analysis system (RTCA) to detect the cytotoxicity of different concentrations of Ren’s oligopeptides-1. We then applied high content screening (HCS) to detect the antiviral activity of Ren’s oligopeptides-1 against PRV. Meanwhile, the fluorescence signal of the virus was collected in real time and the expression levels of the related genes in the PK15 cells infected with PRV were detected using real-time PCR. At the mRNA level, we discovered that, at a concentration of 6 mg/mL, Ren’s oligopeptides-1 reduced the expression of pseudorabies virus (PRV) genes such as IE180, UL18, UL54, and UL21 at a concentration of 6 mg/mL. We then determined that Ren’s oligopeptides-1 has an EC50 value of 6 mg/mL, and at this level, no cytotoxicity was observed.

To compare the use of real-time cell analysis system (RTCA) and Cell Counting Kit-8 (CCK-8) in detecting the toxic effects of diesel exhaust particles (DEP) on human bronchial epithelial (HBE) cells, the HBE cells were exposed to two types of Standard Reference Materials (SRM 1650b and SRM 2975) with the exposure concentrations of (0, 3.5, 7, 14, 28 and 56 mg⋅L-1). After exposure for (6, 12, 24 and 48 h), the cytotoxicity of HBE cells induced by different DEP was detected. Then the advantages or shortcomings of RTCA and CCK-8 were analyzed by comparing the cell viability and normalized cell index (NCI) of different groups. And the cell apoptosis assay was used to detect the apoptosis rate between the different groups. Results show that: SRM 2975 was more toxic to HBE cells than SRM 1650b at the same exposure concentration and time. When RTCA was used to detect DEP-induced HBE cytotoxicity, the NCI value of low concentration DEP groups showed statistically significant difference (P <0.05). In comparison, the significant toxicity effects (P<0.05) were detected in relatively higher concentrations of DEP at the same exposure time by use of CCK-8 assay. And the cell apoptosis assay confirmed that the apoptosis rate of low concentration of DEP groups had already showed statistical difference compared with the control group (P< 0.05). It is indicated that compared with CCK-8, RTCA was more suitable for detecting cytotoxicity of DEP-induced adherent HBE cells. And CCK-8 assays could be a better choice for detecting DEP-induced cytotoxicity of suspended cells or adhesion/suspension cytotoxicity when combined with air-liquid interface exposure systems. [ABSTRACT FROM AUTHOR]

The initial bacterial adhesion phase is a pivotal and unstable step in the formation of biofilms. The initiation of biofilm formation is an unstable process caused by the reversible adhesion of bacteria, which is always time-consuming and yet to be elucidated. In this study, impedance-based real time cell analysis (RTCA) was employed to comprehensively investigate the initial bacterial adhesion process. Results showed that the time required for the unstable adhesion process was significantly (p

Recently, nano-scaled chirality has attained significant attraction from various research areas depending on its fundamental significance in the matter of living and nature. In this work, we designed and fabricated d-/l-Pe-Au nanoparticles (Chiral Penicillamine-modified Gold NPs), system for the treatment of Alzheimer's disease. RTCA (real-time cell analysis) assay was performed using rat PC12 (Pheochromocytoma) cells to investigate the capable cytotoxicity of d-/l-Pe-Au nanoparticles. At any point of given time, with increase in the concentration of d-Pe-Au nanoparticles, we observed a significant decrease in the cell index, which suggests that the cytotoxic effect on the PC12 cells depends on the concentration. We believe that the results obtained may be significant for other chiral nanoparticles in the treatment of the disease called Alzheimer's and also has an extensive utilization in the studies and applications based on NPs. [ABSTRACT FROM AUTHOR]

Drug-induced liver injury (DILI) represents one of the major causes why drugs have to be withdrawn from the market. In this study, we describe a new interaction between drug-exposed hepatocytes and natural killer (NK) cells. In a previous genome-wide expression analysis of primary human hepatocytes that had been exposed to clinically relevant concentrations of 148 drugs, we found that several activating ligands for NK cell receptors were regulated by various drugs (e.g., valproic acid, ketoconazole, promethazine, isoniazid). Especially expression of the activating NKG2D ligands (MICA, MICB and ULBPs) and the NKp30 ligand B7-H6 were upregulated in primary human hepatocytes upon exposure to many different drugs. Using the human hepatocyte cell lines Huh7 and HepG2, we confirmed that protein levels of activating NK cell ligands were elevated after drug exposure. Hepatocyte cell lines or primary human hepatocytes co-cultivated with NK cells caused enhanced NK cell activation after pretreatment with drugs at in vivo relevant concentrations compared to solvent controls. Enhanced NK cell activation was evident by increased cytotoxicity against hepatocytes and interferon (IFN)-γ production. NK cell activation could be blocked by specific antibodies against activating NK cell receptors. These data support the hypothesis that NK cells can modulate drug-induced liver injury by direct interaction with hepatocytes resulting in cytotoxicity and IFN-γ production. [ABSTRACT FROM AUTHOR]

In 2018, 3.8 million premature deaths were attributed to exposure to biomass burning nanoparticles from wood combustion. The objective of this study was to investigate and compare the toxic effect of wood-combustion-related biomass burning nanoparticles from three different combustion stages (i.e., flaming, smoldering, and pyrolysis) on alveolar lung cells, by studying cell proliferation, and structural and behavioral parameters. A549 lung epithelial cells were treated with 31, 62, 125, 250, and 500 µg/mL of water-soluble particulate pollutants from wood burning, and measured by means of real-time cell analysis, cell imaging, and phase imaging microscopy. At low concentrations (31 and 62 µg/mL), all three types of wood burning samples exhibited no toxicity. At 125 µg/mL, they caused decreased cell proliferation compared to the control. Exposure to higher concentrations (250 and 500 µg/mL) killed the cells. Cell physical parameters (area, optical volume, eccentricity, perimeter, and optical thickness) and behavioral parameters (migration, motility, and motility speed) did not change in response to exposure to wood burning materials up to a concentration of 125 µg/mL. Exposure to higher concentrations (250 and 500 µg/mL) changed cell perimeter, optical thickness for smoldering and flaming particles, and led to decreased migration, motility, and motility speed of cells. In conclusion, all three of the combustion water-soluble organic pollutants were identified as equally toxic by real-time cell analysis (RTCA) results. The parameters describing cell structure suggest that pyrolysis particles were slightly less toxic than others.

Xenotransplantation of pig organs receives substantial attention for being comparable to human’s. However, compatibility constraints involving hyper-acute rejection (HAR) still block clinical applications. Transgenesis of human complement regulatory proteins has been proposed to overcome xenorejection. Pigs expressing human-CD55 have been widely tested in experimental surgery. Still, no standardized method has been developed to determine tissue expression of human decay-accelerating factor (DAF), hCD55’s product, or to predict the ability to overpass HAR. Here we describe objective procedures addressing this need. Organs and tissues from five hCD55 transgenic pigs were collected and classified according to their xenotransplantation value. The ability to overcome HAR was assessed by classical complement pathway hemolysis assays. Quantitative PCR mRNA expression and Western blot protein level studies were performed. Real-time cytotoxicity assays (RTCA) on fibroblast cultures exposed to baboon and human sera informed on longer-term rejection dynamics. While greater hCD55/DAF expression correlated with better performance, the results obtained varied among specimens. Interestingly, the individual with highest mRNA and protein levels showed positive feedback for hCD55 transcript after challenge with human and baboon sera. Moreover, hCD55 expression correlated to DAF levels in the liver, lung and intestine, but not in the heart. Moreover, we found significant correlations among valuable and non-valuable tissues. In sum, the methodology proposed allows us to characterize the hCD55 transgene functioning and performance. Moreover, the correlations found could allow us to predict hCD55/DAF expression in surrogate tissues, thus eliminating the need for direct biopsies, resulting in preservation of organ integrity before xenotransplantation.

5-flourouracil (5-FU) is typically modulated with leucovorin (LEU) in clinical practice to improve clinical efficacy and patient survival rates. However, this combination has undesirable side effects and makes 5-FU more toxic. Hence, integrating a vesicular system (proniosomes) with another delivery vehicle may improve drug targeting, while resolving the aforementioned drawbacks. This study aimed to engineer 5-FU/LEU proniosomes for possible delivery to the colon. The modified slurry approach was used to create drug-loaded proniosomes (150 mg/9 g of carrier) using both water-soluble (dextrin (DEX) and lactose (LAC)) and insoluble (Neusilin FH 2 (NEU)) carriers. The powdered formulations were filled into Eudragit S100 (10 %)-coated capsules or Eudragit FS 30D capsules for enteric- or colon-specific delivery. In vitro evaluations (flow properties, powder X-ray diffractometry (XRD) analysis, particle size analysis, entrapment efficiency, drug release, scanning electron microscopy (SEM), polydispersity index, Fourier transform infrared spectroscopy (FTIR), and stability studies) were performed on the formulations. An in vitro cytotoxicity test [real-time cell assay (RTCA)] against HCT-116 colon cancer cell lines was performed using the optimized formulation. In vitro evaluations showed that the nanoparticles had good physicochemical properties. RTCA studies showed sustained cell death with the formulations compared to the pure drug and placebo. The sequential drug release of the colon-targeted capsules containing 5-FU and LEU- loaded proniosomes showed negligible drug release in SGF (pH 1.2) and phosphate buffer solution (pH 6.8) (approximately 11 %) but profound drug release (>80 %) at pH 7.4. Drug-loaded proniosomes engineered for colon targeting (Eudragit S100 (10 %) capsules or Eudragit FS 30D capsules) showed good colon-specific targeting and favorable in vitro cytotoxicity profiles., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

The microbial community mediated biogeochemical cycles play important role in global C-cycle and display a sensitive response to environmental changes. Limited information is available on microbial composition and functional diversity controlling biogeochemical cycles in the riverine environment. The Ganga River water and sediment samples were studied for environmental gene tags with reference to carbohydrate metabolism, photoheterotrophy and chemolithotrophy using high throughput shotgun metagenomic sequencing and functional annotation. The diversity of environmental gene tags specific microbial community was annotated against reference sequence database using Kaiju taxonomic classifier. The metagenomic analyses revealed that the river harbored a broad range of carbohydrate and energy metabolism genes. The in-depth investigation of metagenomic data revealed that the enzymes associated with reverse TCA cycle, Calvin-Benson cycle enzyme RuBisCO, starch and sucrose metabolism genes were highly abundant. The enzymes associated with sulfur metabolism such as EC:2.7.7.4 (sulfate to ammonium per sulfate), EC:1.8.1.2, EC:1.8.7.1 (sulfite to H 2 S) were prevalent in both the class of samples. The principal component analysis of the functional profiles revealed that the water and sediment samples were clustered distinctly suggesting that both the sites had variable abundance of functional genes and associated microbiota. The taxonomic classification showed abundance of Proteobacteria , Actinobacteria and Bacteroidetes phyla. Also, the metagenomic study showed the presence of purple sulfur bacteria viz. Thiodictyon , Nitrosococcus and purple non-sulfur bacteria viz. Bradyrhozobium and Rhodobacter. The study demonstrates that the Ganga River microbiome has prevalence of functional genes involved in carbohydrate anabolism and catabolism, and CO 2 fixation with great prospects in cellulose and sulfide degrading enzyme production and characterization. • River Ganga harbored broad range of carbohydrate and energy metabolism genes. • Enzymes involved in reverse TCA, Calvin-Benson cycle enzyme RuBisCO were prevalent. • Sulfur metabolism enzymes such as EC:2.7.7.4 and EC:1.8.1.2 were prevalent river. • Proteobacteria , Actinobacteria and Bacteroidetes were the highly abundant phyla. • Various purple sulfur and purple non sulfur bacteria were also detected. [ABSTRACT FROM AUTHOR]

In 2018, seven million people died prematurely due to exposure to pollution. Polycyclic aromatic hydrocarbons (PAHs) are a significant source of secondary organic aerosol (SOA) in urban areas. We investigated the toxic effects of by-products of naphthalene SOA on lung cells. These by-products were 1,4-naphthoquinone (1,4-NQ), 2-hydroxy-1,4-naphthoquinone (2-OH-NQ), phthalic acid (PA) and phthaldialdehyde (OPA). Two different assessment methodologies were used to monitor the toxic effects: real-time cell analysis (RTCA) and the Holomonitor, a quantitative phase contrast microscope. The chemicals were tested in concentrations of 12.5 to 100 µM for 1,4-NQ and 1 to 10 mM for 2-OH-NQ, PA and OPA. We found that 1,4-NQ is toxic to cells from 25 to 100 µM (EC50: 38.7 µM ± 5.2); 2-OH-NQ is toxic from 1 to 10mM (EC50: 5.3 mM ± 0.6); PA is toxic from 5 to 10 mM (EC50: 5.2 mM ± 0.3) and OPA is toxic from 2.5 to 10 mM (EC50: 4.2 mM ± 0.5). Only 1,4-NQ and OPA affected cell parameters (migration, motility, motility speed and optical volume). Furthermore, 1,4-NQ is the most toxic by-product of naphthalene, with an EC50 value that was one hundred times higher than those of the other compounds. RTCA and Holomonitor analysis showed a complementarity when studying the toxicity induced by chemicals.

Rift valley fever virus (RVFV) is a mosquito-borne virus endemic to sub-Saharan African countries, and the first sporadic outbreaks outside Africa were reported in the Asia-Pacific region. There are no approved therapeutic agents available for RVFV; however, finding an effective antiviral agent against RVFV is important. This study aimed to evaluate the antiviral, antioxidant and anti-inflammatory activity of medicinal plant extracts. Twenty medicinal plants were screened for their anti-RVFV activity using the cytopathic effect (CPE) reduction method. The cytotoxicity assessment of the extracts was done before antiviral screening using the MTT assay. Antioxidant and reactive oxygen/nitrogen species’ (ROS/RNS) inhibitory activity by the extracts was investigated using non-cell-based and cell-based assays. Out of twenty plant extracts tested, eight showed significant potency against RVFV indicated by a decrease in tissue culture infectious dose (TCID50) < 105. The cytotoxicity of extracts showed inhibitory concentrations values (IC50) > 200 µg/mL for most of the extracts. The antioxidant activity and anti-inflammatory results revealed that extracts scavenged free radicals exhibiting an IC50 range of 4.12–20.41 µg/mL and suppressed the production of pro-inflammatory mediators by 60–80% in Vero cells. This study demonstrated the ability of the extracts to lower RVFV viral load and their potency to reduce free radicals.

Cardiovascular disease incidence continues to rise and new treatment paradigms are warranted. We reported previously that activation of Angiotensin II receptor (encoded by the X-linked Agtr2 gene) by a new peptide agonist, NP-6A4, was more effective in protecting mouse cardiomyocyte HL-1 cells and human coronary artery vascular smooth muscle cells (hCAVSMCs) from acute nutrient deficiency than other drugs tested. To elucidate further the protective effects of NP-6A4 in human cells, we studied the effects of NP-6A4 treatment on functions of human coronary artery endothelial cells (hCAECs), and hCAVSMCs. In hCAVSMCs, NP-6A4 (1 μM) increased Agtr2 mRNA (sixfold, p < 0.05) after 12-h exposure, whereas in hCAECs, significant increase in Agtr2 mRNA (hCAECs: eightfold) was observed after prolonged exposure. Interestingly, NP-6A4 treatment (1 μM, 12 h) increased AT2R protein levels in all human cells tested. Pre-treatment with AT2R-antagonist PD123319 (20 μM) and anti-AT2R siRNA (1 μM) suppressed this effect. Thus, NP-6A4 activates a positive feedback loop for AT2R expression and signaling in hCAVSMCs and hCAECs. NP-6A4 (1–20 μM) increased cell index (CI) of hCAVSMCs as determined by real time cell analyzer (RTCA), indicating that high concentrations of NP-6A4 were not cytotoxic for hCAVSMCs, rather promoting better cell attachment and growth. Seahorse Extracellular Flux Assay revealed that NP-6A4 (1 μM) treatment for 7 days increased whole cell-based mitochondrial parameters of hCAVSMCs, specifically maximal respiration (p < 0.05), spare respiratory capacity (p < 0.05) and ATP production (p < 0.05). NP-6A4 (1 μM; 7 days) also suppressed Reactive Oxygen Species (ROS) in hCAVSMCs. Exposure to Doxorubicin (DOXO) (1 μM) increased ROS in hCAVSMCs and this effect was suppressed by NP-6A4 (1 μM). In hCAECs grown in complete medium, NP-6A4 (1 μM) and Ang II (1 μM) exerted similar changes in CI. Additionally, NP-6A4 (5 μM: 12 h) increased expression of eNOS (sixfold, p < 0.05) and generation of nitric oxide (1.3-fold, p < 0.05) in hCAECs and pre-treatment with PD123319 (20 μM) suppressed this effect partially (65%). Finally, NP-6A4 decreased phosphorylation of Jun-N-terminal kinase, implicated in apoptosis of ECs in atherosclerotic sites. Taken together, NP-6A4, through its ability to increase AT2R expression and signaling, exerts different cell-specific protective effects in human VSMCs and ECs.

The aim of this study is to investigate the effect of cardamonin on mRNA expression of apoptosisrelated genes and proliferation after treatment of ovarian epithelial cells, SKOV-3. Real time cellular assay and acridine orange/propidium iodide staining were used to determine cell proliferation rate and viability in the presence and absence of cardamonin, respectively. The apoptosis and necrosis were quantified by annexin V/ propidium iodide uptake. In addition, the mRNA expression levels in cells containing 25-75 µM cardamonin were determined using RT-PCR. The results indicated that the pre-treatment of cells with 25-150 µM cardamonin attenuated the cell proliferation rate and cell viability significantly in a dose-dependent manner. The early apoptotic cell number was increased significantly (p≤0, 01) by treatment of 50 µM cardamonin as compared the untreated cells after 72 h incubation. The analyses of mRNA expressions showed an increase in caspase-3 and Bax apoptotic genes after 25 µM cardamonin treatment versus untreated cells. However, the mRNA expression levels of Bcl-2, Bcl-xL, NF-κB and cyclin D1 were attenuated by cardamonin in cells. Taken together, cardamonin supressed the cells proliferation in a dose dependent manner. These effects could be explained by down regulation of mRNA expression of NF-κB and cyclin D1. In addition, cardamonin stimulated apoptosis by down regulation of anti-apoptotic genes and upregulation of caspase-3 and Bax in SKOV-3 cell line. [ABSTRACT FROM AUTHOR]

Abstract Several in vitro screening tests have been used for selecting probiotic strains; however they often show low predictive value and only a limited number of strains have demonstrated functionality in vivo. The most used in vitro tests represent a very simplified version of the gut environment, especially since they do not consider the accompanying microbiota. Therefore, there is a need to develop sensitive and discriminating in vitro models including the microbiota. Here we developed an in vitro model to discriminate among microbiotas/fecal waters from different population groups. To this end samples were obtained from seven healthy adults, five IBD-patients, ten full-term and ten preterm newborns. Fecal microbiotas were purified and their impact, as well as that of the fecal waters, on HT29 cells was continuously monitored for 22 h using a real-time cell analyzer (RTCA). The composition of the purified microbiotas was assessed by 16S rRNA gene profiling and qPCR and the levels of short chain fatty acids (SCFA) determined by gas chromatography. The microbiota fractions and SCFA concentrations obtained from IBD-patients, full-term and preterm babies, showed clear differences with regard to those of the control group (healthy adults). Moreover, the purified intestinal microbiotas and fecal waters also differed from the control group in the response induced on the HT29 cells assay developed. In short, we have developed a real-time, impedance-based in vitro model for assessing the functional response induced by purified microbiotas and fecal waters upon intestinal epithelial cells. The capability of the assay for discriminating the functional responses induced, by microbiotas or fecal waters from different human groups, promises to be of help on the search for compounds/strains to restore the functionality of the microbiota-host's interaction. Highlights • In vitro screening tests for selecting probiotics shown low predictive value. • There is a need for in vitro models including the microbiota. • We tested the ability of an epithelial-cells model to functionally discriminate microbiotas. • Microbiotas from different human groups induce different responses on the assay developed. • This assay may help on the search for strategies to restore the functionality of the microbiota. [ABSTRACT FROM AUTHOR]

Cardiovascular disease incidence continues to rise and new treatment paradigms are warranted. We reported previously that activation of Angiotensin II receptor (encoded by the X-linked Agtr2 gene) by a new peptide agonist, NP-6A4, was more effective in protecting mouse cardiomyocyte HL-1 cells and human coronary artery vascular smooth muscle cells (hCAVSMCs) from acute nutrient deficiency than other drugs tested. To elucidate further the protective effects of NP-6A4 in human cells, we studied the effects of NP-6A4 treatment on functions of human coronary artery endothelial cells (hCAECs), and hCAVSMCs. In hCAVSMCs, NP-6A4 (1 μM) increased Agtr2 mRNA (sixfold, p < 0.05) after 12-h exposure, whereas in hCAECs, significant increase in Agtr2 mRNA (hCAECs: eightfold) was observed after prolonged exposure. Interestingly, NP-6A4 treatment (1 μM, 12 h) increased AT2R protein levels in all human cells tested. Pre-treatment with AT2R-antagonist PD123319 (20 μM) and anti-AT2R siRNA (1 μM) suppressed this effect. Thus, NP-6A4 activates a positive feedback loop for AT2R expression and signaling in hCAVSMCs and hCAECs. NP-6A4 (1–20 μM) increased cell index (CI) of hCAVSMCs as determined by real time cell analyzer (RTCA), indicating that high concentrations of NP-6A4 were not cytotoxic for hCAVSMCs, rather promoting better cell attachment and growth. Seahorse Extracellular Flux Assay revealed that NP-6A4 (1 μM) treatment for 7 days increased whole cell-based mitochondrial parameters of hCAVSMCs, specifically maximal respiration (p < 0.05), spare respiratory capacity (p < 0.05) and ATP production (p < 0.05). NP-6A4 (1 μM; 7 days) also suppressed Reactive Oxygen Species (ROS) in hCAVSMCs. Exposure to Doxorubicin (DOXO) (1 μM) increased ROS in hCAVSMCs and this effect was suppressed by NP-6A4 (1 μM). In hCAECs grown in complete medium, NP-6A4 (1 μM) and Ang II (1 μM) exerted similar changes in CI. Additionally, NP-6A4 (5 μM: 12 h) increased expression of eNOS (sixfold, p < 0.05) and generation of nitric oxide (1.3-fold, p < 0.05) in hCAECs and pre-treatment with PD123319 (20 μM) suppressed this effect partially (65%). Finally, NP-6A4 decreased phosphorylation of Jun-N-terminal kinase, implicated in apoptosis of ECs in atherosclerotic sites. Taken together, NP-6A4, through its ability to increase AT2R expression and signaling, exerts different cell-specific protective effects in human VSMCs and ECs. [ABSTRACT FROM AUTHOR]

Chlorogenic acid (CGA) is a major polyphenol in primary human diet, displaying a wide range of biological activities such as anti-oxidant, anti-inflammatory and anti-cancer effects. Several studies reported the chemopreventive effects of CGA on different types of cancers including breast cancer, which is the most common cancer among women worldwide. In this comprehensive study, we determined the anti-proliferative and cytotoxic effects of pure CGA on the phenotype of various breast cancer cell lines (MCF-7, SKBR-3, MDA-MB-231, MDA-MB-468 and BT- 20) with well-defined molecular classification and characteristics, in a time and dose dependent manner by using iCELLigence real-time and label-free cell analysis technology. Cells were plated on iCELLigence system-specific E-plate L8 and treated with CGA for 72 hours at concentrations ranging from 250 µM to 8 mM. Obtained data were analyzed by RTCA data analysis software 1.0 and IC50 values of 952±32.5 µM for MCF-7, 940±21.2 µM for SKBR-3, 590.5±10.6 µM for MDA-MB-231, 882.5±12.0 µM for MDA-MB-468 and 1095±121.6 µM for BT-20 cell lines were calculated at the end of 72-hour assay. Based on our findings, CGA did not have cytotoxic activity on breast cancer cell lines, and IC50 values and growth curves displayed similar anti-proliferative patterns with slight variation. [ABSTRACT FROM AUTHOR]

A current focus of research is the development of new tools for removing bacterial biofilms in industrial settings. Bacteriophage-encoded proteins, such as endolysins, virion-associated peptidoglycan hydrolases, and exopolysaccharide depolymerases, have been shown to be efficient against these structures. However, the current screening techniques for the identification of antibiofilm properties of phage-derived proteins have important shortcomings. The aim of this work was to use the rapid, reproducible and accurate technology “real-time cell analyzer” for screening and comparing the antibiofilm ability of four phage-derived compounds, three lytic proteins (LysH5, CHAP-SH3b, and HydH5-SH3b) and one exopolysaccharide depolymerase (Dpo7) against Staphylococcus aureus biofilms, which have been associated with recurrent contamination of food products. The data generated after biofilm treatment allowed for the calculation of different antibiofilm parameters: (1) the minimum biofilm eradicating concentration that removes 50% of the biofilm (ranging from 3.5 ± 1.1 to 6.6 ± 0.5 μM), (2) the lowest concentration needed to observe an antibiofilm effect (∼1.5 μM for all the proteins), and (3) the specific antibiofilm activity and the percentage of biofilm removal that revealed LysH5 as the best antibiofilm compound. Overall, this technology might be used to quickly assess and compare by standardized parameters the disaggregating activity of phage antibiofilm proteins.

Ascidians are marine invertebrates that have been a source of numerous cytotoxic compounds. Of the first six marine-derived drugs that made anticancer clinical trials, three originated from ascidian specimens. In order to identify new anti-neoplastic compounds, an ascidian extract library (143 samples) was generated and screened in MDA-MB-231 breast cancer cells using a real-time cell analyzer (RTCA). This resulted in 143 time-dependent cell response profiles (TCRP), which are read-outs of changes to the growth rate, morphology, and adhesive characteristics of the cell culture. Twenty-one extracts affected the TCRP of MDA-MB-231 cells and were further investigated regarding toxicity and specificity, as well as their effects on cell morphology and cell cycle. The results of these studies were used to prioritize extracts for bioassay-guided fractionation, which led to the isolation of the previously identified marine natural product, eusynstyelamide B (1). This bis-indole alkaloid was shown to display an IC50 of 5 µM in MDA-MB-231 cells. Moreover, 1 caused a strong cell cycle arrest in G2/M and induced apoptosis after 72 h treatment, making this molecule an attractive candidate for further mechanism of action studies.

Alpha-hederin (α-HN), a pentacyclic triterpene saponin, has recently been identified as one of the active compounds of Nigella sativa, as a potential anticancer agent. However, no extensive studies on α-HN have been done as yet, as it was in the case of thymoquinone—the main ingredient of the N. sativa essential oil. To our knowledge, there are also no data available on how α-HN acts on the human cancer ovarian cell line SKOV-3. In this study we attempt to present the cytotoxic influence of α-HN on the SKOV-3 cell line by means of two methods: Real-Time xCELLigence and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The obtained IC50 values are 2.62 ± 0.04 μg/mL and 2.48 ± 0.32 μg/mL, respectively. An induction of apoptosis in SKOV-3 cells was confirmed by staining cellular nuclei with Hoechst 33342 dye and by flow cytometry analysis by binding annexin V to the cell membranes. We found that α-HN induces apoptosis in a dose-dependent manner. In the first stages of apoptosis, the mitochondrial membrane potential was found to decrease. Also, inactivation of anti-apoptotic protein Bcl-2 was observed, as well as the caspase-9 and then caspase-3/7 activation. In addition, the treatment of SKOV-3 cells with α-HN induced the cell cycle arrest of cancer cells in G0/G1 phase. The results of our investigations indicate that α-HN induces apoptosis in the SKOV-3 cell line and that the intrinsic mitochondrial pathway is involved in the programmed cancer cell death.

Nanometer-scale chirality has gained significant interest from different research fields due to its fundamental importance in nature and living matter. In this study, we design and synthesize chiral penicillamine-capped selenium nanoparticles ( l -/ d -Pen@Se NPs) that can act as a novel class of chiral amyloid-β (Aβ) inhibitors. In this work, d -Pen@Se NPs demonstrate higher inhibition efficiency, as well as ameliorate cognition and memory impairments. We used rat pheochromocytoma (PC12) cells to perform real-time cell analysis assay (RTCA) to probe the potential cytotoxicity of l -/ d -Pen@Se NPs. At any given time point, the cell index decreases as d -Pen@Se NPs concentration increases, demonstrating a concentration-dependent cytotoxic effect on PC12 cells. In addition, d -Pen@Se NPs also reduced Zn 2+ -induced intracellular Aβ 40 fibrillation, while l -Pen@Se NPs did not. The histological analysis demonstrates that mice treated with d -Pen@Se NPs did not exhibit signs of in vivo systemic toxicity in major organs. Our findings are highly encouraging in terms of providing substantial evidence of the safety of chiral d -Pen@Se NPs for biomedical application. We expect that these results will be relevant for other chiral NPs for treatment of Alzheimer’s disease and have broad implications in NP-based studies and applications. [ABSTRACT FROM AUTHOR]

A current focus of research is the development of new tools for removing bacterial biofilms in industrial settings. Bacteriophage-encoded proteins, such as endolysins, virion-associated peptidoglycan hydrolases, and exopolysaccharide depolymerases, have been shown to be efficient against these structures. However, the current screening techniques for the identification of antibiofilm properties of phage-derived proteins have important shortcomings. The aim of this work was to use the rapid, reproducible and accurate technology "real-time cell analyzer" for screening and comparing the antibiofilm ability of four phage-derived compounds, three lytic proteins (LysH5, CHAP-SH3b, and HydH5-SH3b) and one exopolysaccharide depolymerase (Dpo7) against Staphylococcus aureus biofilms, which have been associated with recurrent contamination of food products. The data generated after biofilm treatment allowed for the calculation of different antibiofilm parameters: (1) the minimum biofilm eradicating concentration that removes 50% of the biofilm (ranging from 3.5 ± 1.1 to 6.6 ± 0.5 μM), (2) the lowest concentration needed to observe an antibiofilm effect (~1.5 mM for all the proteins), and (3) the specific antibiofilm activity and the percentage of biofilm removal that revealed LysH5 as the best antibiofilm compound. Overall, this technology might be used to quickly assess and compare by standardized parameters the disaggregating activity of phage antibiofilm proteins. [ABSTRACT FROM AUTHOR]

Anaerobic thermophiles inhabit relic environments that resemble the early Earth. However, the lineage of these modern organisms co-evolved with our planet. Hence, these organisms carry both ancestral and acquired genes and serve as models to reconstruct early metabolism. Based on comparative genomic and proteomic analyses, we identified two distinct groups of genes in Thermovibrio ammonificans: the first codes for enzymes that do not require oxygen and use substrates of geothermal origin; the second appears to be a more recent acquisition, and may reflect adaptations to cope with the rise of oxygen on Earth. We propose that the ancestor of the Aquificae was originally a hydrogen oxidizing, sulfur reducing bacterium that used a hybrid carbon fixation pathway for CO2 fixation. With the gradual rise of oxygen in the atmosphere, more efficient terminal electron acceptors became available and this lineage acquired genes that increased its metabolic flexibility while retaining ancestral metabolic traits. [ABSTRACT FROM AUTHOR]

Aims The purpose of the current work was to study the pattern and dynamics of biofilm formation in clinical isolates of Staphylococcus aureus and Staphylococcus epidermidis in the presence of 10 antibiotics with different action mechanisms. Methods and Results By using impedance measurements in microtitre plates with gold electrodes we have assessed the antibiotic effect on bacterial biofilm growth in real time. The impedance measurements appear to combine both cellular growth and matrix production, representing a measurement of total biofilm mass. Several clinical and reference strains were tested, showing different slopes and cell index values which correlated with their capacity to form biofilms as assessed by attachment to standard microtitre well plates and safranin staining. Biofilms were heavily reduced in biofilm mutants or by protease treatment in protein-based biofilm matrixes. Antibiotic resistance patterns of biofilms, which were very different to those obtained by traditional methods like epsilon-tests on solid media, revealed features that would pass unnoticed by end-point methods. Conclusions Once the biofilm is formed, antibiotic efficacy dramatically reduced and sub-inhibitory concentrations of some antibiotics, such as linezolid and clarithromycin, stimulated biofilm growth, stressing the importance of studying antibiotic resistance under biofilm growth conditions in real time. Significance and Impact of the Study Real-time biofilm analysis provides a promising tool to evaluate antibiotic therapy in clinical biofilm-mediated infections. [ABSTRACT FROM AUTHOR]