The microtubule regulator ringer functions downstream from the RNA repair/splicing pathway to promote axon regeneration.

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.

Metabolic engineering of the acid-tolerant yeast Pichia kudriavzevii for efficient L-malic acid production at low pH.

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.

Anti-Pulmonary Fibrosis Activities of Triterpenoids from Oenothera biennis.

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), 3ß,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-ß1-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.

Simultaneous Evaluation of Dissolution and Absorption Study of Compound Danshen Tablets and Capsules Based on Cellular Electrical Sensing Model.

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.

Synergistic cytotoxicity of binary combinations of inorganic and organic disinfection byproducts assessed by real-time cell analysis.

Chlorine, chlorine dioxide, and ozone are widely used as disinfectants in drinking water treatments. However, the combined use of different disinfectants can result in the formation of various organic and inorganic disinfection byproducts (DBPs). The toxic interactions, including synergism, addition, and antagonism, among the complex DBPs are still unclear. In this study, we established and verified a real-time cell analysis (RTCA) method for cytotoxicity measurement on Chinese hamster ovary (CHO) cell. Using this convenient and accurate method, we assessed the cytotoxicity of a series of binary combinations consisting of one of the 3 inorganic DBPs (chlorite, chlorate, and bromate) and one of the 32 regulated and emerging organic DBPs. The combination index (CI) of each combination was calculated and evaluated by isobolographic analysis to reflect the toxic interactions. The results confirmed the synergistic effect on cytotoxicity in the binary combinations consisting of chlorite and one of the 5 organic DBPs (2 iodinated DBPs (I-DBPs) and 3 brominated DBPs (Br-DBPs)), chlorate and one of the 4 organic DBPs (3 aromatic DBPs and dibromoacetonitrile), and bromate and one of the 3 organic DBPs (2 I-DBPs and dibromoacetic acid). The possible synergism mechanism of organic DBPs on the inorganic ones may be attributed to the influence of organic DBPs on cell membrane and cell antioxidant system. This study revealed the toxic interactions among organic and inorganic DBPs, and emphasized the latent adverse outcomes in the combined use of different disinfectants.

Formulation of Novel Liquid Crystal (LC) Formulations with Skin-Permeation-Enhancing Abilities of Plantago lanceolata (PL) Extract and Their Assessment on HaCaT Cells.

Exposure to reactive oxygen species can easily result in serious diseases, such as hyperproliferative skin disorders or skin cancer. Herbal extracts are widely used as antioxidant sources in different compositions. The importance of antioxidant therapy in inflammatory conditions has increased. Innovative formulations can be used to improve the effects of these phytopharmacons. The bioactive compounds of Plantago lanceolata (PL) possess different effects, such as anti-inflammatory, antioxidant, and bactericidal pharmacological effects. The objective of this study was to formulate novel liquid crystal (LC) compositions to protect Plantago lanceolata extract from hydrolysis and to improve its effect. Since safety is an important aspect of pharmaceutical formulations, the biological properties of applied excipients and blends were evaluated using assorted in vitro methods on HaCaT cells. According to the antecedent toxicity screening evaluation, three surfactants were selected (Gelucire 44/14, Labrasol, and Lauroglycol 90) for the formulation. The dissolution rate of PL from the PL-LC systems was evaluated using a Franz diffusion chamber apparatus. The antioxidant properties of the PL-LC systems were evaluated with 2,2-diphenyl-1-picrylhydrazyl (DPPH) and malondialdehyde (MDA) assessments. Our results suggest that these compositions use a nontraditional, rapid-permeation pathway for the delivery of drugs, as the applied penetration enhancers reversibly alter the barrier properties of the outer stratum corneum. These excipients can be safe and highly tolerable thus, they could improve the patient's experience and promote adherence.

Bersaldegenin-1,3,5-orthoacetate induces caspase-independent cell death, DNA damage and cell cycle arrest in human cervical cancer HeLa cells.

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.

Cytotoxic activity of standardized extracts, a fraction, and individual secondary metabolites from fenugreek seeds against SKOV-3, HeLa and MOLT-4 cell lines.

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.

Enhanced activation of human NK cells by drug-exposed hepatocytes.

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.

In Vitro Evaluation of Different Prebiotics on the Modulation of Gut Microbiota Composition and Function in Morbid Obese and Normal-Weight Subjects.

The gut microbiota remains relatively stable during adulthood; however, certain intrinsic and environmental factors can lead to microbiota dysbiosis. Its restoration towards a healthy condition using best-suited prebiotics requires previous development of in vitro models for evaluating their functionality. Herein, we carried out fecal cultures with microbiota from healthy normal-weight and morbid obese adults. Cultures were supplemented with different inulin-type fructans (1-kestose, Actilight, P95, Synergy1 and Inulin) and a galactooligosaccharide. Their impact on the gut microbiota was assessed by monitoring gas production and evaluating changes in the microbiota composition (qPCR and 16S rRNA gene profiling) and metabolic activity (gas chromatography). Additionally, the effect on the bifidobacterial species was assessed (ITS-sequencing). Moreover, the functionality of the microbiota before and after prebiotic-modulation was determined in an in vitro model of interaction with an intestinal cell line. In general, 1-kestose was the compound showing the largest effects. The modulation with prebiotics led to significant increases in the Bacteroides group and Faecalibacterium in obese subjects, whereas in normal-weight individuals, substantial rises in Bifidobacterium and Faecalibacterium were appreciated. Notably, the results obtained showed differences in the responses among the tested compounds but also among the studied human populations, indicating the need for developing population-specific products.

Biological activities of leaf extracts from selected Kalanchoe species and their relationship with bufadienolides content.

CONTEXT: Kalanchoe species (Crassulaceae) are widely used in traditional medicine as remedies in infectious diseases and cancer treatment. OBJECTIVE: Cytotoxic and antimicrobial activities of Kalanchoe daigremontiana Raym.-Hamet & H. Perrier, K. pinnata (Lam.) Pers., and K. blossfeldiana Poelln. extracts were determined. The relationship between biological activities and the extracts bufadienolides content was also investigated. MATERIALS AND METHODS: Fresh leaves of Kalanchoe species were macerated with 95% ethanol or water. The quantitative analysis of bufadienolides in the extracts was carried out with mass spectrometry. Cytotoxicity tests were performed on human cancer cell lines - HeLa, SKOV-3, MCF-7, and A375 by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and Real-Time Cell Analysis system. The microbiological study was done using a few bacteria strains (ß-hemolytic Streptococcus, Corynebacterium diphtheriae, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus hirae, Escherichia coli) and Candida albicans. RESULTS: The K. blossfeldiana ethanol extract and K. daigremontiana water extract exhibited the most potent cytotoxic activity (IC50 < 19 µg/mL for HeLa and SKOV-3 cells). The strongest antibacterial effects showed ethanol extract of K. blossfeldiana and K. pinnata (MIC values were 8.45, 8.45, 0.25 and <33.75 µg/mL for S. aureus, S. epidermidis, and E. hirae, respectively). The highest total amount of bufadienolides was in K. daigremontiana ethanol extract. In contrast, K. blossfeldiana ethanol extract did not show the presence of these compounds. CONCLUSIONS: Kalanchoe blossfeldiana ethanol extract is a potential candidate for cancer and bacterial infection treatment. Additionally, the biological effects of Kalanchoe extracts are not dependent on the presence and amount of bufadienolides in the plant extracts.

The cyclic phosphodiesterase CNP and RNA cyclase RtcA fine-tune noncanonical XBP1 splicing during ER stress.

The activity of X box-binding protein 1 (XBP1), a master transcriptional regulator of endoplasmic reticulum (ER) homeostasis and the unfolded protein response (UPR), is controlled by a two-step noncanonical splicing reaction in the cytoplasm. The first step of nuclease cleavage by inositol-requiring enzyme 1 (IRE1), a protein kinase/endoribonuclease, is conserved in all eukaryotic cells. The second step of RNA ligation differs biochemically among species. In yeast, tRNA ligase 1 (Trl1) and tRNA 2'-phosphotransferase 1 (Tpt1) act through a 5'-PO4/3'-OH pathway. In metazoans, RNA 2',3'-cyclic phosphate and 5'-OH ligase (RtcB) ligate XBP1 exons via a 3'-PO4/5'-OH reaction. Although RtcB has been identified as the primary RNA ligase, evidence suggests that yeast-like ligase components may also operate in mammals. In this study, using mouse and human cell lines along with in vitro splicing assays, we investigated whether these components contribute to XBP1 splicing during ER stress. We found that the mammalian 2'-phosphotransferase Trpt1 does not contribute to XBP1 splicing even in the absence of RtcB. Instead, we found that 2',3'-cyclic nucleotide phosphodiesterase (CNP) suppresses RtcB-mediated XBP1 splicing by hydrolyzing 2',3'-cyclic phosphate into 2'-phosphate on the cleaved exon termini. By contrast, RNA 3'-terminal cyclase (RtcA), which converts 2'-phosphate back to 2',3'-cyclic phosphate, facilitated XBP1 splicing by increasing the number of compatible RNA termini for RtcB. Taken together, our results provide evidence that CNP and RtcA fine-tune XBP1 output during ER stress.

Alpha-Hederin, the Active Saponin of Nigella sativa, as an Anticancer Agent Inducing Apoptosis in the SKOV-3 Cell Line.

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.

Application of Real-Time Cell Electronic Analysis System in Modern Pharmaceutical Evaluation and Analysis.

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.

Study on the Quality Evaluation of Compound Danshen Preparations Based on the xCELLigence Real-Time Cell-Based Assay and Pharmacodynamic Authentication.

Objective: To perform a preliminary study on the quality evaluation of compound Danshen preparations based on the xCELLigence Real-Time Cell-based Assay (RTCA) system and make a pharmacodynamics verification. Methods: The compound Danshen was discussed as a methodological example, and the bioactivity of the compound Danshen preparations were evaluated by real-time cell electronic analysis technology. Meanwhile, an in vivo experiment on an acute blood stasis rat model was performed in order to verify this novel evaluation through the curative effect of dissipating blood stasis. Results: We determined the cell index (CI) and IC50 of the compound Danshen preparations and produced time/dose-dependent cell response profiles (TCRPs). The quality of the three kinds of compound Danshen preparations was evaluated through the RTCA data. The trend of CI and TCRPs reflected the effect of drugs on the cell (promoting or inhibiting), and it was verified that the results correlated with the biological activity of the drugs using a pharmacodynamics experiment. Conclusion: The RTCA system can be used to evaluate the quality of compound Danshen Preparations, and it can provide a new idea and new method for quantitatively characterizing the biological activity of traditional Chinese medicines (TCMs).

A novel release kinetics evaluation of Chinese compound medicine: Application of the xCELLigence RTCA system to determine the release characteristics of Sedum sarmentosum compound sustained-release pellets.

Purpose: To establish a novel release kinetics evaluation method of Chinese compound medicine (Sedum Sarmentosum compound) with xCELLigence Real-Time Cell-based Assay (RTCA) system. Methods: Cell lines sensitive to Sedum Sarmentosum compound are screened, and cell index-time (CI-T) graphs and cell index release kinetics models are established based on the cell index (CI) monitored. The methodological studies of precision and repeatability were processed by the cell monitors system. The release profiles of the sustained-release Sedum Sarmentosum compound were determined. Consequently, the sustained-release property was characterized by the kinetic parameters based on the cell-index. Results: The accumulative release rate based on cell index of Sedum sarmentosum compound sustained-release preparation was determined and it had a good correlation with time, fitting better with First-order model, Higuchi model and Ritger-Peppas model, and fitting best with Weibull model. It indicated that the release rate is proportional with the diffusion coefficient. Conclusion: The new method of cell-index release kinetics may provide a quantitative description for the release of the multi active agents from Traditional Chinese Medicines. The application of xCELLigence RTCA system for evaluating the release kinetics of Chinese compound medicine is feasible.

Size Effect on the Cytotoxicity of Layered Black Phosphorus and Underlying Mechanisms.

A systematic cytotoxicity study of layered black phosphorus (BP) is urgently needed before moving forward to its potential biomedical applications. Herein, bulk BP crystals are synthesized and exfoliated into layered BP with different lateral size and thickness. The cytotoxicity of as-exfoliated layered BP is evaluated by a label-free real-time cell analysis technique, displaying a concentration-, size-, and cell type-dependent response. The IC50 values can vary by 40 and 30 times among the BP sizes and cell types, respectively. BP-1 with the largest lateral size and thickness has the highest cytotoxicity; whereas the smallest BP-3 only shows moderate toxicity. The sensitivity of three tested cell lines follows the sequence of 293T > NIH 3T3 > HCoEpiC. Two possible mechanisms for BP to induce cytotoxicity are proposed and verified: (1) the generation of intracellular reactive oxygen species (ROS) is detected by a ROS sensitive probe using the inverted fluorescence microscopy and flow cytometry; (2) the interaction of layered BP and model cell membrane is examined by quartz crystal microbalance with dissipation, illustrating the disruption of cell membrane integrity especially by the largest BP-1. This systematic study of BP's cytotoxicity will shed light on its future biomedical and environmental applications.

Impact of Seasonal Hypoxia on Activity and Community Structure of Chemolithoautotrophic Bacteria in a Coastal Sediment.

Seasonal hypoxia in coastal systems drastically changes the availability of electron acceptors in bottom water, which alters the sedimentary reoxidation of reduced compounds. However, the effect of seasonal hypoxia on the chemolithoautotrophic community that catalyzes these reoxidation reactions is rarely studied. Here, we examine the changes in activity and structure of the sedimentary chemolithoautotrophic bacterial community of a seasonally hypoxic saline basin under oxic (spring) and hypoxic (summer) conditions. Combined 16S rRNA gene amplicon sequencing and analysis of phospholipid-derived fatty acids indicated a major temporal shift in community structure. Aerobic sulfur-oxidizing Gammaproteobacteria (Thiotrichales) and Epsilonproteobacteria (Campylobacterales) were prevalent during spring, whereas Deltaproteobacteria (Desulfobacterales) related to sulfate-reducing bacteria prevailed during summer hypoxia. Chemolithoautotrophy rates in the surface sediment were three times higher in spring than in summer. The depth distribution of chemolithoautotrophy was linked to the distinct sulfur oxidation mechanisms identified through microsensor profiling, i.e., canonical sulfur oxidation, electrogenic sulfur oxidation by cable bacteria, and sulfide oxidation coupled to nitrate reduction by Beggiatoaceae The metabolic diversity of the sulfur-oxidizing bacterial community suggests a complex niche partitioning within the sediment, probably driven by the availability of reduced sulfur compounds (H2S, S0, and S2O32-) and electron acceptors (O2 and NO3-) regulated by seasonal hypoxia.IMPORTANCE Chemolithoautotrophic microbes in the seafloor are dependent on electron acceptors, like oxygen and nitrate, that diffuse from the overlying water. Seasonal hypoxia, however, drastically changes the availability of these electron acceptors in the bottom water; hence, one expects a strong impact of seasonal hypoxia on sedimentary chemolithoautotrophy. A multidisciplinary investigation of the sediments in a seasonally hypoxic coastal basin confirms this hypothesis. Our data show that bacterial community structure and chemolithoautotrophic activity varied with the seasonal depletion of oxygen. Unexpectedly, the dark carbon fixation was also dependent on the dominant microbial pathway of sulfur oxidation occurring in the sediment (i.e., canonical sulfur oxidation, electrogenic sulfur oxidation by cable bacteria, and sulfide oxidation coupled to nitrate reduction by Beggiatoaceae). These results suggest that a complex niche partitioning within the sulfur-oxidizing bacterial community additionally affects the chemolithoautotrophic community of seasonally hypoxic sediments.

Diversity of Total Bacterial Communities and Chemoautotrophic Populations in Sulfur-Rich Sediments of Shallow-Water Hydrothermal Vents off Kueishan Island, Taiwan.

Shallow-water hydrothermal vents (HTVs) are an ecologically important habitat with a geographic origin similar to that of deep-sea HTVs. Studies on shallow-water HTVs have not only facilitated understanding of the influences of vents on local ecosystems but also helped to extend the knowledge on deep-sea vents. In this study, the diversity of bacterial communities in the sediments of shallow-water HTVs off Kueishan Island, Taiwan, was investigated by examining the 16S ribosomal RNA gene as well as key functional genes involved in chemoautotrophic carbon fixation (aclB, cbbL and cbbM). In the vent area, Sulfurovum and Sulfurimonas of Epsilonproteobacteria appeared to dominate the benthic bacterial community. Results of aclB gene analysis also suggested involvement of these bacteria in carbon fixation using the reductive tricarboxylic acid (rTCA) cycle. Analysis of the cbbM gene showed that Alphaproteobacterial members such as the purple non-sulfur bacteria were the major chemoautotrophic bacteria involving in carbon fixation via the Calvin-Benson-Bassham (CBB) cycle. However, they only accounted for <2% of the total bacterial community in the vent area. These findings suggest that the rTCA cycle is the major chemoautotrophic carbon fixation pathway in sediments of the shallow-water HTVs off Kueishan Island.

Effect of antibiotics on biofilm inhibition and induction measured by real-time cell analysis.

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.