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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]

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.

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.

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.

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

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.

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.

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]

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]

Objective: We summarized the progress of the xCELLigence real-time cell analysis (RTCA) technology application in recent years for the sake of enriching and developing the application of RTCA in the field of Chinese medicine. Background: The RTCA system is an established electronic cellular biosensor. This system uses micro-electronic biosensor technology that is confirmed for real-time, label-free, dynamic and non-offensive monitoring of cell viability, migration, growth, spreading, and proliferation. Methods: We summarized the relevant experiments and literature of RTCA technology from the principles, characteristics, applications, especially from the latest application progress. Results and conclusion: RTCA is attracting more and more attention. Now it plays an important role in drug screening, toxicology, Chinese herbal medicine and so on. It has wide application prospects in the area of modern pharmaceutical evaluation and analysis.

Background: Real-time cellular analysis systems enable impedance-based label-free and dynamic monitoring of various cellular events such as proliferation. In this study, we describe the effects of initial cell seeding density on the anti-proliferative effects of transient gene silencing monitored via real-time cellular analysis. We monitored the real-time changes in proliferation of Huh7 hepatocellular carcinoma and A7r5 vascular smooth muscle cells with different initial seeding densities following transient receptor potential canonical 1 (TRPC1) silencing using xCELLigence system. Huh7 and A7r5 cells were seeded on E-plate 96 at 10,000, 5000, 1250 and 5000, 2500 cells well-1, respectively, following silencing vector transfection. The inhibitory effects of transient silencing on cell proliferation monitored every 30 min for 72 h. Results: TRPC1 silencing did not inhibit the proliferation rates of Huh7 cells at 10,000 cells well-1 seeding density. However, a significant anti-proliferative effect was observed at 1250 cells well-1 density at each time point throughout 72 h. Furthermore, significant inhibitory effects on A7r5 proliferation were observed at both 5000 and 2500 cells well-1 for 72 h. Conclusions: Data suggest that the effects of transient silencing on cell proliferation differ depending on the initial cell seeding density. While high seeding densities mask the significant changes in proliferation, the inhibitory effects of silencing become apparent at lower seeding densities as the entry into log phase is delayed. Using the optimal initial seeding density is crucial when studying the effects of transient gene silencing. In addition, the results suggest that TRPC1 may contribute to proliferation and phenotypic switching of vascular smooth muscle cells. [ABSTRACT FROM AUTHOR]

The importance of growth and maintenance of skeletal muscle is vital for long term health and quality of life. Appropriate nutrition with specific bioactivities relevant to the functionalities of tissues such as skeletal muscle, can assist in maintaining and promoting adaptive responses to biological and environmental stresses which prevent muscle atrophy and promote hypertrophy. The aim of this investigation was to develop a novel in vitro cell-based electric impedance assay to study myoblast to myotube formation on the real time cell analysis (RTCA) platform (xCELLigenceTM, ACEA) and to validate the system by testing myotube responses to hypertrophic stimuli. C2C12 myoblasts were proliferated until 70% confluent in Dulbecco's Modified Eagles Medium (DMEM) (10% FBS) and subsequently differentiated to myotubes over 8 days in DMEM [2% horse serum (HS)]. Changes in cell behaviour and adhesion properties were monitored by measuring impedance via interdigitated microelectrodes in the base of E-16 cell culture dishes. To establish the suitability of this assay to monitor nutrient regulation of muscle hypertrophy, leucine, a known potent regulator of MPS was then supplemented to the fully formed myotubes in physiologically relevant conditions - 0.20 mM, 0.40 mM, 0.6 mM, 0.8 mM and above 1.0 mM, 1.5 mM, 2.0 mM and impedance subsequently monitored. Parallel experiments highlighting alterations in myotube thickness, muscle protein synthesis (MPS) (mammalian target of rapamycin; mTOR) and differentiation (myogenin) were conducted to support RTCA bioassay findings. This in vitro bioassay can be used to monitor skeletal muscle behaviour and identify nutrient compounds with bioactivities promoting skeletal muscle hypertrophy, reducing muscle atrophy and thus inform the development of novel nutrient formulations for the maintenance of skeletal muscle. [ABSTRACT FROM AUTHOR]

The intestinal overgrowth of Clostridium difficile, often after disturbance of the gut microbiota by antibiotic treatment, leads to C. difficile infection (CDI) which manifestation ranges from mild diarrhea to life-threatening conditions. The increasing CDI incidence, not only in compromised subjects but also in traditionally considered low-risk populations, together with the frequent relapses of the disease, has attracted the interest for prevention/therapeutic options. Among these, probiotics, prebiotics, or synbiotics constitute a promising approach. In this study we determined the potential of selected Bifidobacterium strains for the inhibition of C. difficile growth and toxicity in different carbon sources. We conducted co-cultures of the toxigenic strain C. difficile LMG21717 with four Bifidobacterium strains (Bifidobacterium longum IPLA20022, Bifidobacterium breve IPLA20006, Bifidobacterium bifidum IPLA20015, and Bifidobacterium animalis subsp. lactis Bb12) in the presence of various prebiotic substrates (Inulin, Synergy, and Actilight) or glucose, and compared the results with those obtained for the corresponding mono-cultures. C. difficile and bifidobacteria levels were quantified by qPCR; the pH and the production of short chain fatty acids was also determined. Moreover, supernatants of the cultures were collected to evaluate their toxicity using a recently developed model. Results showed that co-culture with B. longum IPLA20022 and B. breve IPLA20006 in the presence of short-chain fructooligosaccharides, but not of Inulin, as carbon source significantly reduced the growth of the pathogen. With the sole exception of B. animalis Bb12, whose growth was enhanced, the presence of C. difficile did not show major effects upon the growth of the bifidobacteria. In accordance with the growth data, B. longum and B. breve were the strains showing higher reduction in the toxicity of the co-culture supernatants. [ABSTRACT FROM AUTHOR]

Clostridium difficile is an opportunistic pathogen inhabiting the human gut, often being the aetiological agent of infections after a microbiota dysbiosis following, for example, an antibiotic treatment. C. difficile infections (CDI) constitute a growing health problem with increasing rates of morbidity and mortality at groups of risk, such as elderly and hospitalized patients, but also in populations traditionally considered low-risk. This could be related to the occurrence of virulent strains which, among other factors, have high-level of resistance to fluoroquinolones, more efficient sporulation and markedly high toxin production. Several novel intervention strategies against CDI are currently under study, such as the use of probiotics to counteract the growth and/or toxigenic activity of C. difficile. In this work, we have analyzed the capability of twenty Bifidobacterium and Lactobacillus strains, from human intestinal origin, to counteract the toxic effect of C. difficile LMG21717 upon the human intestinal epithelial cell line HT29. For this purpose, we incubated the bacteria together with toxigenic supernatants obtained from C. difficile. After this co-incubation new supernatants were collected in order to quantify the remnant A and B toxins, as well as to determine their residual toxic effect upon HT29 monolayers. To this end, the real time cell analyser (RTCA) model, recently developed in our group to monitor C. difficile toxic effect, was used. Results obtained showed that strains of Bifidobacterium longum and B. breve were able to reduce the toxic effect of the pathogen upon HT29, the RTCA normalized cell-index values being inversely correlated with the amount of remnant toxin in the supernatant. The strain B. longum IPLA20022 showed the highest ability to counteract the cytotoxic effect of C. difficile acting directly against the toxin, also having the highest capability for removing the toxins from the clostridial toxigenic supernatant. Image analysis showed that this strain prevents HT29 cell rounding; this was achieved by preserving the F-actin microstructure and tight- junctions between adjacent cells, thus keeping the typical epithelium-like morphology. Besides, preliminary evidence showed that the viability of B. longum IPLA20022 is needed to exert the protective effect and that secreted factors seems to have anti-toxin activity. [ABSTRACT FROM AUTHOR]

Cell viability and cell migration capacities are critical parameters for cell culture-related studies. It is essential to monitor the dynamic changes of cell properties under various co-culture conditions to our better understanding of their behaviours and characteristics. The real time cell analyzer (RTCA, xCELLigence, Roche) is an impedance-based technology that can be used for label-free and real-time monitoring of cell properties, such as cell adherence, proliferation, migration and cytotoxicity. The practicality of this system has been proven in our recent cancer studies. In the present method, we intend to use co-cultures of pancreatic cancer cells (HP62) and mesenchymal stem cells to describe in detail, the procedures and benefits of RTCA. [ABSTRACT FROM AUTHOR]