Fluorescein Diacetate Assay Research Articles

Increased antibiotic resistance of Pseudomonas aeruginosa isolates from chronic rhinosinusitis patients grown in anaerobic conditions.

In chronic rhinosinusitis (CRS), the congestion and blockage of the nose can cause anaerobic conditions within the sinus cavities which may promote the expression of virulence and antibiotic resistance genes in invading pathogens. Pseudomonas aeruginosa is a facultative anaerobic bacteria and causes severe recalcitrant CRS. In this study, we aimed to evaluate the antimicrobial resistance of P. aeruginosa isolates of CRS patients in planktonic and biofilm form grown in aerobic and anaerobic conditions. P. aeruginosa clinical isolates of CRS patients (n = 25) were grown in planktonic and biofilm form in aerobic and anaerobic conditions. Minimum inhibitory concentrations (MIC) of planktonic forms and minimum biofilm eradication concentrations (MBEC) were determined. Additionally, metabolic activity by fluorescein diacetate assay, biofilm biomass by crystal violet assay and eDNA concentration were assessed in both conditions. P. aeruginosa planktonic cells grown in anaerobic condition exhibited increased gentamicin resistance (p < .01), whereas P. aeruginosa biofilms grown in anaerobic condition displayed significantly increased MBEC values for gentamicin (p < .0001) and levofloxacin (p < .001). The metabolic activity of anaerobic biofilms was significantly higher compared with aerobic biofilms (p < .0001). However, the biofilm biomass of isolates grown in aerobic conditions was higher than anaerobic conditions (p < .5). P. aeruginosa isolates from CRS patients grown in anaerobic conditions showed significantly increased resistance to antibiotics with an increased metabolic activity but decreased biofilm biomass. NA.

Cytotoxic Effects of Indonesian Betel Quid Components on Oral Keratinocytes and Fibroblasts

A betel quid (BQ) chewing habit has been strongly associated with the development of several oral mucosal diseases. In order to investigate whether individual components of BQ mixtures have distinct physio-pathological effects on oral mucosal cells, we examined the impact of areca nut (AN), Piper betle leaf (Leaf), Piper betle stem inflorescence (SI), areca husk (Husk) and the complete BQ mixture on the growth of oral keratinocytes (OKF-6) and primary oral fibroblasts (MMF-1). Based on their known chemical properties, we selected BQ samples from Banda Aceh (BA) and West Papua (WP) regions for our in vitro study. We used a fluorescein diacetate assay (FDA) to assess the cell viability of BQ components on OKF-6 and MMF-1 cells. The cytotoxic effect of WP-AN on the OKF-6 cell line was observed at a concentration of 100 μg/mL, resulting in a 50% reduction in cell viability (IC50) after a 2-day incubation. Similarly, BA-AN exhibited cytotoxic effect, although at a higher concentration (500 μg/mL). WP-SI also displayed cytotoxic effects at a concentration of 500 μg/mL following 2 days of incubation. In contrast, Leaf, BQ mixture and husk extracts did not show any cytotoxic effects even after 3 days of incubation. No cytotoxic effects were observed at any concentration of BQ components when exposed to MMF-1 cells. Regarding cell proliferation, MMF-1 cells exposed to BA-AN and WP-AN showed increased growth on day 1, followed by decreased growth on day 2, in a dose- and time-dependent manner. Overall, our study indicates that BQ components induce distinctive cytotoxic effects on stromal and epithelial cells from the oral cavity.

Fluorescein diacetate hydrolytic activity as a sensitive tool to quantify nitrogen/sulfur gene content in urban river sediments in China.

The relative abundance of functional genes used to quantify the abundance of functional genes in communities is controversial. Quantitative PCR (qPCR) technology offers a powerful tool for quantifying functional gene abundance. However, humic substances can inhibit qPCR in sediment/soil samples. Therefore, finding a convenient and effective quantitative analysis method for sediment/soil samples is necessary. The functional genes and physicochemical properties in sediments with different-level pollutions were analyzed in this study. Correlations between physicochemical properties and the relative abundance of functional genes were used to test whether relative abundance in gene prediction quantifies the abundance of functional genes. The abundance of functional genes could be corrected by multiplying the fluorescein diacetate (FDA) hydrolytic rates by the relative abundance of functional genes since the FDA assay has been widely used as a rapid and sensitive method for quantifying microbial activity in sediments. Redundancy analysis showed significant interrelations between the functional genes and the physicochemical properties of sediments. The relative abundance of functional genes is unreliable for quantifying the abundance of functional genes because of the weak correlation (R < 0.5, P < 0.05) between different pollutants and the relative abundance of functional genes. However, a significant positive correlation between concentrations of different pollutants and the activities of associated enzymes was obtained (R > 0.933, P < 0.05), which revealed that the abundance of functional genes could be reliably quantified by the relative abundance and FDA hydrolytic rate. This study proposed an alternative method besides qPCR to quantify the absolute abundance of functional genes, which overcomes the problem of humic interference in the quantitative analysis of sediment/soil samples.

Biogenic nanosilver bearing antimicrobial and antibiofilm activities and its potential for application in agriculture and industry.

Due to the increasing resistance of bacteria and fungi to antimicrobials, it is necessary to search for effective alternatives to prevent and treat pathogens causing diseases in humans, animals, and plants. In this context, the mycosynthesized silver nanoparticles (AgNPs) are considered as a potential tool to combat such pathogenic microorganisms. AgNPs were synthesized from Fusarium culmorum strain JTW1 and characterized by Transmission Electron Microscopy (TEM), X-ray diffraction (XRD), Fourier Transform Infrared (FTIR) spectroscopy, Nanoparticle Tracking Analysis (NTA), Dynamic Light Scattering (DLS) and Zeta potential measurement. The minimum inhibitory (MIC) and biocidal concentrations (MBC) were determined against 13 bacterial strains. Moreover, the combined effect of AgNPs with antibiotics (streptomycin, kanamycin, ampicillin, tetracycline) was also studied by determining the Fractional Inhibitory Concentration (FIC) index. The anti-biofilm activity was examined by crystal violet and fluorescein diacetate (FDA) assays. Furthermore, antifungal activity of AgNPs was evaluated against a panel of phytopathogenic fungi viz., Botrytis, Colletotrichum, Fusarium, Phoma, Sclerotinia, and an oomycete pathogen Phytophthora by agar well-diffusion and micro-broth dilution method to evaluate the minimal AgNPs concentrations that inhibit fungal spore germination. Fungi-mediated synthesis resulted in the formation of small (15.56 ± 9.22 nm), spherical and stable (zeta potential of - 38.43 mV) AgNPs with good crystallinity. The results of FTIR spectroscopy indicated the presence of various functional groups, namely hydroxyl, amino, and carboxyl ones, from the biomolecules on the surface of AgNPs. The AgNPs showed antimicrobial and antibiofilm formation activities against Gram-positive and Gram-negative bacteria. The values of MIC and MBC ranged between 16-64 and 32-512 μg mL-1, respectively. The enhanced effect of AgNPs in combination with antibiotics was confirmed against human pathogens. The highest synergistic effect (FIC = 0.0625) was demonstrated by the combination of AgNPs with streptomycin against two strains of Escherichia coli (ATCC 25922 and ATCC 8739), followed by Klebsiella pneumoniae and Pseudomonas aeruginosa (FIC = 0.125). Enhanced effects of AgNPs with ampicillin were also shown against Staphylococcus aureus ATCC 25923 (FIC = 0.125) and P. aeruginosa (FIC = 0.25), as well as kanamycin against S. aureus ATCC 6538 (FIC = 0.25). The crystal violet assay revealed that the lowest concentration of AgNPs (0.125 μg mL-1) reduced the development of biofilms of Listeria monocytogenes and Salmonella enterica, while the maximum resistance was shown by Salmonella infantis, its biofilm was reduced after exposure to a concentration of 512 μg mL-1. A high inhibitory effect on the activity of bacterial hydrolases was observed by the FDA assay. AgNPs at a concentration of 0.125 μg mL-1 reduced the hydrolytic activity of all biofilms formed by the tested pathogens, except E. coli ATCC 25922, P. aeruginosa, and Pectobacterium carotovorum (efficient concentration was 2-fold higher, at 0.25 μg mL-1), while the hydrolytic activity of E. coli ATCC 8739, Salmonella infantis and S. aureus ATCC 6538 was suppressed after treatment with AgNPs at concentrations of 0.5, 2 and 8 μg mL-1, respectively. Moreover, AgNPs inhibited fungal growth and spore germination of Botrytis cinerea, Phoma lingam, and Sclerotinia sclerotiorum. MIC and MFC values of AgNPs against spores of these fungal strains were determined at 64, 256, and 32 μg mL-1, and zones of growth inhibition were 4.93, 9.54, and 3.41 mm, respectively. Fusarium culmorum strain JTW1 was found to be an eco-friendly biological system for an easy, efficient and inexpensive synthesis of AgNPs. In our study, the mycosynthesised AgNPs demonstrated remarkable antimicrobial (antibacterial and antifungal) and antibiofilm activities against a wide range of human and plant pathogenic bacteria and fungi singly and in combination with antibiotics. These AgNPs could be applied in medicine, agriculture, and food industry to control such pathogens that cause numerous human diseases and crop losses. However, before using them extensive animal studies are required to evaluate the toxicity, if any.

Low-Intensity Pulsed Ultrasound Enhanced Neurite Guidance Growth through Netrin-1/DCC Signal Pathway in Primary Cultured Cortical Neurons of Rats.

Low-intensity pulsed ultrasound is found to be effective in axonal regeneration, while the role of ultrasound in axonal growth guidance is still unclear. This study was performed to explore the neuroprotective role of low-intensity pulsed ultrasound (US) both in vitro and in vivo. Primary cultured rat cortical neurons were subjected to 1.0 MHz ultrasound for 5 min every day at intensity of 0, 0.008, 0.12, and 0.21 W/cm2. Our results demonstrated that low-intensity pulsed ultrasound significantly increased neuronal cell viability and inhibited neuronal apoptosis in vitro as determined by fluorescein diacetate assay (FDA) and a TdT-mediated biotin-dUTP nicked-end labeling (TUNEL) assay. Moreover, low-intensity pulsed ultrasound at 0.12 W/cm2 significantly enhanced the axonal growth guidance by activation of netrin-1 and DCC (deleted in colorectal carcinoma) expression as determined by Western blots assay. More interestingly, we further found that low-intensity pulsed ultrasound treatment at 0.21 W/cm2 promoted the functional restoration of rat injured nerves in vivo, decreased hemorrhage, and reversed the injury process by activating positive netrin-1 expression as seen in the immunohistochemistry (IHC) assay. Thus, our study strongly demonstrated that low-intensity pulsed ultrasound activated netrin-1/DCC signaling and further mediated neurite outgrowth. It would be a new approach to nerve regeneration in the future.

Lanostane Triterpenoid Metabolites from a Penares sp. Marine Sponge Protect Neuro-2a Cells against Paraquat Neurotoxicity.

The results of an investigation of the protective effects of five lanostane triterpenoids: 3β-acetoxy-7β,8β-epoxy-5α-lanost-24-en-30,9α-olide (1), 3β-hydroxy-7β,8β-epoxy-5α-lanost-24-en- 30,9α-olide (2), 29-nor-penasterone (3), penasterone (4), and acetylpenasterol (5), from a marine sponge, Penares sp., against paraquat-induced neuroblastoma Neuro-2a cell damage, are described. The influence of all compounds on viability of the Neuro-2a cells treated with paraquat (PQ) was studied with MTT and fluorescein diacetate assays as well as propidium iodide straining. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of the compounds as well as their influence on reactive oxygen species (ROS) level and mitochondrial membrane potential in PQ-treated neuronal cells were analyzed. Finally, the effect of the compounds on intracellular level of heat shock protein 70 kDa (Hsp70) and neurite outgrowth in PQ-treated Neuro-2a cells were studied. Studied triterpenoids demonstrated protective effects against PQ-induced neurotoxicity associated with the ability to reduce ROS intracellular level and diminish mitochondrial dysfunction. Acetylpenasterol (5), as a more promising neuroprotective compound, significantly increased the viability of Neuro-2a cells incubated with PQ as well as decreased intracellular ROS level in these cells. Moreover, acetylpenasterol induced Hsp70 expression in PQ-treated cells. It was also shown to inhibit PQ-induced neurite loss and recovered the number of neurite-bearing cells. The relationship between neuroprotective activity of the investigated compounds 1–5 and their chemical structure was also discussed.

Long-term effects of organic manure and inorganic fertilization on biological soil quality indicators of soybean-wheat rotation in the Indian mid-Himalaya

A field experiment was accompanied to scrutinize the effect of long-term (21-year) mineral and organic manure fertilizer treatments on carbon mineralization, glomalin related soil protein and some microbial characteristics of arable soils in Indian mid-Himalaya. The experiment was initiated in 1995 includes six treatments: Control, N 120, NPK, FYM, N + FYM and NPK + FYM. Long-term chemical fertilization has been reported to negative effects on soil biological properties, however, the impact of combined use of organic and inorganic fertilization on soil biological properties and its relationship remains poorly understood. Results showed that integrated use of organic and inorganic (NPK + FYM) for 21 years significantly increased in carbon mineralization, easily extractable glomalin related soil protein (EEGRSP) and total glomalin related soil protein (TGRSP). Soil microbial indices (microbial quotient: qMIC, microbial metabolic quotient: qCO2 and metabolic potential: MP) were enhanced in the NPK + FYM treatment as compared to rest of the treatments. The activities of seven enzymes, viz. dehydrogenase (DHA), β-glucosidase (β-GA), invertase (IA), acid phosphatase (Acid-PA), alkaline phosphatase (Alkaline-PA), arylsulphatase (ASA) and urease activity (UA) were significantly influenced by the application of NPK + FYM as compared rest of treatments. Soil fertilization with NPK and FYM was significantly higher fluorescein diacetate assay (FDA) activity as compared to other treatments. Soil microbial properties had strong positive correlation with qMIC, microbial biomass carbon (MBC) and soil organic carbon (SOC), while qCO2 had negative correlation under both soil layers. Results showed that the 21-year fertilization experiment clearly indicated that NPK + FYM embodied the best management practice (BMPs) for soil biological sustainability and for sustainable food production.

Prevention of 7-Ketocholesterol-Induced Overproduction of Reactive Oxygen Species, Mitochondrial Dysfunction and Cell Death with Major Nutrients (Polyphenols, ω3 and ω9 Unsaturated Fatty Acids) of the Mediterranean Diet on N2a Neuronal Cells.

The brain, which is a cholesterol-rich organ, can be subject to oxidative stress in a variety of pathophysiological conditions, age-related diseases and some rare pathologies. This can lead to the formation of 7-ketocholesterol (7KC), a toxic derivative of cholesterol mainly produced by auto-oxidation. So, preventing the neuronal toxicity of 7KC is an important issue to avoid brain damage. As there are numerous data in favor of the prevention of neurodegeneration by the Mediterranean diet, this study aimed to evaluate the potential of a series of polyphenols (resveratrol, RSV; quercetin, QCT; and apigenin, API) as well as ω3 and ω9 unsaturated fatty acids (α-linolenic acid, ALA; eicosapentaenoic acid, EPA; docosahexaenoic acid, DHA, and oleic acid, OA) widely present in this diet, to prevent 7KC (50 µM)-induced dysfunction of N2a neuronal cells. When polyphenols and fatty acids were used at non-toxic concentrations (polyphenols: ≤6.25 µM; fatty acids: ≤25 µM) as defined by the fluorescein diacetate assay, they greatly reduce 7KC-induced toxicity. The cytoprotective effects observed with polyphenols and fatty acids were comparable to those of α-tocopherol (400 µM) used as a reference. These polyphenols and fatty acids attenuate the overproduction of reactive oxygen species and the 7KC-induced drop in mitochondrial transmembrane potential (ΔΨm) measured by flow cytometry after dihydroethidium and DiOC6(3) staining, respectively. Moreover, the studied polyphenols and fatty acids reduced plasma membrane permeability considered as a criterion for cell death measured by flow cytometry after propidium iodide staining. Our data show that polyphenols (RSV, QCT and API) as well as ω3 and ω9 unsaturated fatty acids (ALA, EPA, DHA and OA) are potent cytoprotective agents against 7KC-induced neurotoxicity in N2a cells. Their cytoprotective effects could partly explain the benefits of the Mediterranean diet on human health, particularly in the prevention of neurodegenerative diseases.

Cold Atmospheric Plasma Treatment of Chondrosarcoma Cells Affects Proliferation and Cell Membrane Permeability.

Chondrosarcoma is the second most common malign bone tumor in adults. Surgical resection of the tumor is recommended because of its resistance to clinical treatment such as chemotherapy and radiation therapy. Thus, the prognosis for patients mainly depends on sufficient surgical resection. Due to this, research on alternative therapies is needed. Cold atmospheric plasma (CAP) is an ionized gas that contains various reactive species. Previous studies have shown an anti-oncogenic potential of CAP on different cancer cell types. The current study examined the effects of treatment with CAP on two chondrosarcoma cell lines (CAL-78, SW1353). Through proliferation assay, the cell growth after CAP-treatment was determined. A strong antiproliferative effect for both cell lines was detected. By fluorescein diacetate (FDA) assay and ATP release assay, alterations in the cell membrane and associated translocation of low molecular weight particles through the cytoplasmic membrane were observed. In supernatant, the non-membrane-permeable FDA and endogenously synthesized ATP detected suggest an increased membrane permeability after CAP treatment. Similar results were shown by the dextran-uptake assay. Furthermore, fluorescence microscopic G-/F-actin assay was performed. G- and F-actin were selectively dyed, and the ratio was measured. The presented results indicate CAP-induced changes in cell membrane function and possible alterations in actin-cytoskeleton, which may contribute to the antiproliferative effects of CAP.

The Effect of Cold Atmospheric Plasma on the Membrane Permeability of Human Osteosarcoma Cells

Cold atmospheric plasma (CAP) has a variety of anticancer effects on different cancer cell types. In osteosarcoma (OS) cells, CAP reduces growth and motility, induces apoptosis, and alters secretion of cellular factors. The influence of CAP on membrane integrity of OS cells is unknown. Two different OS cell lines (U-2 OS and MNNG-HOS) were treated with CAP. Proliferation assays for cell growth after treatment was performed. Alterations in membrane permeability and the associated translocation of low molecular weight particles through the cytoplasmic membrane of OS cells after CAP treatment were shown in fluorescein diacetate (FDA) assays. FDA increasingly passed the membrane after CAP treatment and this effect depended on the duration of treatment. It was also shown that after CAP treatment, FDA was able to diffuse into the cells from the outside as well as out of the cell interior. These effects were observed when CAP-treated buffer was used and therefore no direct contact between cells and CAP occurred. The observations suggest that changes in membrane permeability and function may contribute to the antiproliferative effects of CAP.

Understanding Ciprofloxacin Failure in Pseudomonas aeruginosa Biofilm: Persister Cells Survive Matrix Disruption.

Biofilms are commonly recalcitrant to antibiotics, through incompletely elucidated mechanisms such as tolerance and persistence. We aimed at investigating how a Pseudomonas aeruginosa biofilm escapes ciprofloxacin treatment. P. aeruginosa PA14 in vitro mature biofilms were challenged with supra-MIC ciprofloxacin concentrations. Cell viability was quantified by fluorescein diacetate assay. Population dynamics were determined by counts of surviving culturable cells. Biofilms were analyzed using confocal laser scanning microscopy (CLSM), and the expression of genes involved in stringent response, toxin-antitoxin HigB/HigA, and type 3 secretion system (T3SS) was quantified by RT-qPCR in untreated and treated biofilms. Ciprofloxacin exposure resulted in an initial reduction of bacterial counts following a biphasic time-kill curve. After 24 h of treatment, the overall cell activity and the density of culturable cells significantly decreased as compared to untreated biofilm. No resistant mutant was isolated among the <1% surviving cells. Phenotypic adaptation toward persistence appeared to start after only 1 h of antibiotic exposure, by an overexpression of the genes involved in stringent response and in the toxin-antitoxin system, whereas the expression of genes encoding for the T3SS remained unchanged. After 4 h of ciprofloxacin exposure, stringent response genes returned to their basal level of expression. After a prolonged ciprofloxacin exposure, a deep alteration in the matrix structure that became thinner and lost mushroom-like aggregates was observed, in relation with reduced biovolumes of exopolysaccharides and extracellular DNA. These results support that ciprofloxacin might first induce the bacterial killing of most bacterial cells, but simultaneously activate stringent response mechanisms contributing to the switch of a subpopulation toward a persister phenotype. Once the persister phenotype is expressed, and despite an unexpected alteration of the biofilm matrix, ciprofloxacin fails to eradicate biofilm.

Optimization of culture medium and growth conditions of the plant growth-promoting bacterium Herbaspirillum seropedicae BR11417 for its use as an agricultural inoculant using response surface methodology (RSM)

Culture media compositions and bioprocess conditions were studied to improve the production of cell biomass and indolic phytohormones by Herbaspirillum seropedicae BR11471, a plant growth promoting bacterium, and different inoculant formulations were also produced and tested for their stability and shelf life. Response surface methodology (RSM) based on central composite rotation designs (CCRD) was used to find bioprocess variables that lead to an increase in bacterial biomass and yield of indolic compounds. The major components of DYGS medium were optimized in small-scale shaken cultivations, in two sets of CCRD. High performance liquid chromatography was used to determine nutrient consumption and to correlate it with cell biomass production, and the Salkowski method was used to quantify indoles. Hydrolytic activity in the formulations was quantified with the fluorescein diacetate assay. Glycerol (5.5 g L−1) and yeast extract (2.8 g L−1), as the main carbon and nitrogen sources, respectively, increased biomass production by 87.5% when compared to original DYGS medium, reaching 3.0 g L−1 of dry cell weight (DCW). In a 2.0 L bioreactor, the optimized medium was used to enhance process conditions for DCW and indole-3-acetic acid (IAA). Biomass production reached 3.4 g L−1 and was restrained at highest air flow levels. The conditions of 34-36 °C, 150 rpm and 4.0 L min−1 of air flow rate resulted in 11.97 mg L−1 of IAA, an increase of 370% over original DYGS at 30 °C. Peat can still be regarded as a good cell carrier for solid state inoculants, whilst the additives tested for liquid formulations are individually more efficient than the mixture. The production of inoculants containing H. seropedicae strain BR11471 can be efficiently improved with the use of the RSM approach i.e. it maximizes the production of biomass and indolic compounds, and reduces culture media components, both key factors for large-scale industrial production.

Afzelin suppresses proinflammatory responses inparticulate matter-exposed human keratinocytes.

Particulate matter (PM), a widespread airborne contaminant, is a complex mixture of solid and liquid particles suspended in the air. Recent studies have demonstrated that PM induces oxidative stress and inflammatory reactions, and may cause certain skin diseases. Afzelin is a flavonoid isolated from Thesium chinense Turcz, which has anti‑inflammatory, anticancer and antibacterial properties. Therefore, the present study aimed to investigate if afzelin affected inflammatory responses in human keratinocytes exposed to PM. HaCaT cells were treated with PM (25 µg/cm2) in the presence or absence of afzelin (200 µM). Here, standard reference material 1649b was used as PM. Cell viability was assessed using the water‑soluble tetrazolium salt‑1 assay. The generation of reactive oxygen species (ROS) was measured using the dichloro‑dihydro‑​fluorescein diacetate assay. Gene and protein expression were investigated using reverse transcription‑quantitative polymerase chain reaction and western blot analysis, respectively. Levels of secreted inflammatory cytokines were measured using ELISA. The results suggested that afzelin inhibited PM‑induced proinflammatory cytokine mRNA expression and protein secretion in HaCaT cells. In addition, afzelin suppressed PM‑induced intracellular ROS generation, and p38 mitogen‑activated protein kinase and transcription factor activator protein‑1 component c‑Fos and c‑Jun activation. The results indicated that afzelin exerts anti‑inflammatory and antioxidant effects in PM‑exposed HaCaT. Afzelin may have potential for preventing PM‑induced inflammatory skin diseases.

Effectiveness of expanded clay as a bacteria carrier for self-healing concrete

Cracking of concrete over time, is a natural phenomenon. Longer service life of concrete structures is desirable. Self-healing concrete using bacteria, which could form CaCO3 crystals for crack sealing, has promised benefits to reduce cost for concrete maintenance, because cracks could be autonomously repaired without human intervention. However, because of harsh concrete internal environment render the effectiveness depending on the bacteria viability within concrete. In this study, expanded clay (EC) was used as a carrier, to protect bacteria (Lysinibacillus boronitolerans YS11) from the harsh environment during the process. Existence of bacteria inside EC was observed using electron microscopy. When exposed to bacterial solution of 1.0 × 109 cells/mL, bacterial density within EC reached approximately 0.82 × 107 cells/g of dry EC. Extent of bacterial viability within EC, submerged to solution containing 1.0 × 108 cells/mL, was 53.6% of free bacteria solution containing 1.0 × 107 cells/mL, as measured with fluorescein diacetate assay. When rate of calcium carbonate formation was measured with Ca2+ disappearance, rates were comparable between bacteria within EC (submerged to bacterial solution containing 1.0 × 108 cells/mL) and free bacteria (1.0 × 107 cells/mL). This finding indicates that bacteria with EC is very active for generation of CaCO3 within EC. All experimental results suggest that EC may be an adequate bacteria carrier for self-healing concrete.

Receptor tyrosine kinase inhibitors cause dysfunction in adult rat cardiac fibroblasts in vitro

The anti-cancer receptor tyrosine kinase inhibitors include known cardiotoxins: a component of this toxicity may be mediated by effects on cardiac fibroblasts (CFs). We hypothesised that imatinib mesylate (imatinib) and sunitinib malate (sunitinib) cause significant dysfunction in adult CFs. Following in vitro treatments with imatinib or sunitinib, adult rat CF viability was assessed by fluorescein diacetate assay, proliferation measured by bromodeoxyuridine nuclear incorporation and changes to the expression of CF secretome components determined by real time quantitative RT-PCR. Imatinib and sunitinib significantly reduced cell viability over 48 h, with EC50 values of 11.0 μM (imatinib) and 4.5 μM (sunitinib) respectively. Imatinib reduced CF proliferation from 35.5 ± 3.2% in control to 23.0 ± 5.5% (3 μM; p < 0.001) and to 9.4 ± 2.5% (10 μM; p < 0.001), whereas sunitinib reduced proliferation to 22.9 ± 3.1% (1 μM; p < 0.001) and to 15 ± 1.0% (3 μM; p < 0.001). Further, 10 μM imatinib increased mRNA expression of TGFB1 7-fold, (p < 0.01), IL6 6-fold (p < 0.01), and IL1B 7-fold (p < 0.05) and reduced PDGFD 15-fold (p < 0.01); whereas sunitinib specifically reduced IL1B mRNA expression 17-fold (p < 0.01). Overall, these findings show tyrosine kinase inhibitors cause significant dysfunction in CFs. These data point to an important role for the PDGF pathway in governing CF functions, including survival and proliferation.

The bulk fluorescein diacetate assay (FDA) as a technique for evaluating biotic impacts of crude oil to coastal sediments

This study adapted the bulk fluorescein diacetate (FDA) assay to evaluate the impact of crude oil on biological activity levels in marine sediments. Sediment cores from the Elkhorn Slough, California were incubated with different levels of sweet crude oil (Deepwater Horizon, Macondo Prospect) in the Moss landing Marine Laboratories aquarium facility over a 30-day period. During this period, the amount of fluorescein released, concentration of oxygen in overlying waters, and flux of oxygen into the sediments were monitored. For all treatments a strong negative correlation between added oil and FDA production was observed (p < 0.05). The concentration of oxygen in the first 2 mm of sediment cores decreased linearly with the addition of crude oil and correlated positively with fluorescein released (p < 0.05). A linear increase in sediment oxygen flux was also observed upon crude oil addition. The miniaturized bulk FDA assay could be powerful in evaluating oil spill impacts in coastal systems.

Fluorescein Diacetate Hydrolysis Using the Whole Biofilm as a Sensitive Tool to Evaluate the Physiological State of Immobilized Bacterial Cells

Due to the increasing interest and the use of immobilized biocatalysts in bioremediation studies, there is a need for the development of an assay for quick and reliable measurements of their overall enzymatic activity. Fluorescein diacetate (FDA) hydrolysis is a widely used assay for measuring total enzymatic activity (TEA) in various environmental samples or in monoculture researches. However, standard FDA assays for TEA measurements in immobilized samples include performing an assay on cells detached from the carrier. This causes an error, because it is not possible to release all cells from the carrier without affecting their metabolic activity. In this study, we developed and optimized a procedure for TEA quantification in the whole biofilm formed on the carrier without disturbing it. The optimized method involves pre-incubation of immobilized carrier in phosphate buffer (pH 7.6) on the orbital shaker for 15 min, slow injection of FDA directly into the middle of the immobilized carrier, and incubation on the orbital shaker (130 rpm, 30 °C) for 1 h. Biofilm dry mass was obtained by comparing the dried weight of the immobilized carrier with that of the unimmobilized carrier. The improved protocol provides a simple, quick, and more reliable quantification of TEA during the development of immobilized biocatalysts compared to the original method.

Effect of Cytochalasin B Treatment on the Improvement of Survival Rate in Vitrified Pig Oocyte.

To improve survival rates of vitrified pig oocytes, the treatment of cytoskeletal stabilizer on an appropriate time is one of the possible approaches. However, the exact treatment timing and effect of cytoskeletal stabilizer such as cytochalasin B (CB) is not well known during oocyte vitrification procedures. Thus, the present study was conducted to determine optimal treatment timing of CB during vitrification and warming procedures. In experiment 1, the survival rates of the post-warming pig oocytes were analyzed by fluorescein diacetate (FDA) assays with 4 classifications. In results, post-warming oocytes showed significantly (p<0.05) decreased number of alive oocytes (31.8% vs. 86.4%) compared to fresh control. In detail, the significant difference (p<0.05) was found only in strong fluorescence (18.2% vs. 70.5%) not in intermediate fluorescence groups (13.6% vs. 15.9%). In experiment 2, CB was treated before (CB-Vitri) and after (Vitri-CB) vitrification. In results, group of Vitri-CB showed significantly (p<0.05) higher (91.6%) survival rates compared to group of CB-Vitri (83.7%), significantly (p<0.05) and comparable with group of Vitri Control (88.7%) by morphological inspection. In FDA assay results, group of Vitri-CB showed significantly (p<0.05) higher (44.2%) survival rates compared to groups of CB-Vitri (36.7%) and Vitri Control (35.1%). In conclusion, the increased survival rates of post-warming pig oocyte treated with Vitri-CB method are firstly described here. The main finding of present study is that the CB treatment during recovery could be helpful to refresh the post-warming pig oocyte resulting its improved survival rates.

Protective Effect of Moderate Exogenous Electric Field Stimulation on Activating Netrin-1/DCC Expression Against Mechanical Stretch-Induced Injury in Spinal Cord Neurons

Nerve cells detect and respond to electric field stimulation and extrinsic chemical guidance cues during development and regeneration; therefore, the development and optimization of an approach for functional neuronal regeneration are necessary for a nerve injury. In this study, we proposed using electric field stimulation to repair a nerve injury triggered by serious mechanical stretch loading. A device that provides continuous mechanical stretch and constant electric field stimulation was designed. Primary dissociated spinal cord neurons were stimulated by mechanical stretch (tensile strain 2.5-10%) at different times (1, 4, 8, and 12h) to set up a moderate nerve injury model. Stimulated samples were evaluated with respect to cell viability, density, and axonal elongation by the MTT and immunofluorescence assays. The results indicated that mechanical stretch (S, 5% tensile strain, 4h) caused moderate axonal injury, resulting in significant loss of cell viability and a decrease in cell density. However, injured spinal cord neurons became viable after electric field stimulation (E, 33mA/m2, 4h) in the fluorescein diacetate assay. In addition, neuronal viability, density, and elongation increased significantly after electric field stimulation compared with those of stretch-injured neurons. Moreover, electric field stimulation significantly activated the axonal guidance cues Netrin-1 and deleted in colorectal cancer (DCC) receptor expression compared with the stretch-injury group. These results indicate that electric stimulation activates synergistic guidance cues of expression to improve axonal growth relevant to nerve injuries. Our study provides new insight into neuronal regeneration.

Potential Impact of Cypermethrin on Selected Groups of Soil Microorganisms

Applicationof cypermethrin as insecticide during agricultural practices is a globalconcern due to its toxicity and potential accumulation of its metabolites inthe aquatic environment. A mesocosm plot of agricultural land (500 m2)was demarcated and deliberately contaminated with cypermethrin to study itsimpact on the autochthonous microorganisms. Potential effects of3-phenoxybenzoic acid (3-PBA), a known suicide metabolite of cypermethrin, onhydrolytic activities were also investigated by using fluorescein diacetate assay(FDA). A significant increase in the abundance of total heterotrophic bacteriafrom 3.55 x 106cfu g-1to 1.30 x 108cfu g-1in12 weeks as against the control was induced by the insecticide in the mesocosm.The number of starch hydrolysing bacteria increased after the secondinsecticide treatment from 6.25 x 106cfu g-1to 2.70 x 107cfu g-1in three weeks. However, there was no significantincrease in the microbial count of fungi, presumptive actinomycetes andnitrogen fixing bacteria during the twelve weeks of experiment. The hydrolyticactivity of soil microorganisms was apparently inhibited insoil treated with differentconcentration of 3-PBAthan in untreated soil. These results suggest that theapplication of a commercial cypermethrin formulation increased the abundance ofselected soil microorganisms while3-PBA reduced the soil microbial hydrolyticactivity