24-h Incubation Research Articles

The Activity of Combination of Ethanol Extract of Artocarpus lacucha Buch.-Ham and Anredera cordifolia Steenis Leaves to Increase Wound Healing Process on NIH-3T3 Cell Line

AIM: This study aims to determine the effect of the combination of ethanol extract of Artocarpus lacucha Buch.-Ham and Anredera cordifolia Steenis leaves to increase cell proliferation and increase VEGFR-2 expression of NIH-3T3. METHODS: The samples used were Artocarpus lacucha Buch.-Ham and Anredera cordifilia Steenis leaves. The powder of simples was extracted using ethanol 80% with maceration method. The effect of extract combination on proliferation was evaluated using the MTT method. Wound healing assay was established by a cell migration method, and VEGFR-2 expression was determined using RT-PCR. RESULTS: The effect of combination of ethanol extract of Artocarpus lacucha leaves (EEALL) and ethanol extract of Anredera cordifolia leaves (EEACL) on cell proliferation after 24h, 48h and 72h incubation found as viable cells were showed (124.33 ± 0.32%; 128.52 ± 0.41%; 118.35 ± 0.22%). Percent of wound closed after 24 h and 48 h incubation are 64.88 ± 0.90% and 100.00 ± 0.00%, and expression of VEGFR-2 increased from 1 (control) to 1.58 ± 0.02. CONCLUSION: The results suggest that a combination of EEALL and EEACL (37.5 μg/mL−37.5 μg/mL) is effective in increasing cells proliferation and hence wound healing process.

Effects of Ionizing Radiation on Human Peripheral Blood Mononuclear Cells (PBMCs) in the Presence of Mentha-Pulegium Essential Oil: A Study on the Radioprotective Effect.

Background: The Mentha-Pulegium essential oil (MP-EO) contains different antioxidant compounds and reduces the indirect effects of dispersed ionizing radiation on biological systems.Objective: The current study aimed to assess a possible radio-protective effect of MP-EO on peripheral blood mononuclear cells (PBMCs).Material and Methods: In this experimental study, MP-EO was firstly prepared and PBMCs were then irradiated in various groups with doses of 25 and 200 cGy of X-rays in the presence of IC10 of MP-EO. After incubation times of 48h and 72h, the survival, apoptosis, and necrosis percentages of PBMCs were determined by MTT assay and flow cytometry analyses; the radio-protective effect of MP-EO was examined.Results: In the presence of 80 µg/ml (IC10) MP-EO, the mean survival percentage of irradiated PBMCs by radiation doses of 25 and 200 cGy was significantly increased after 48h of incubation compared with the control. At 72h of incubation, the mean survival percentage of irradiated PBMCs was significantly increased only at 25 cGy. The percentage of apoptosis and necrosis of PBMCs was significantly reduced in the presence of the MP-EO at both incubation times and radiation doses; therefore, the highest reduction was at 200 cGy and 48h incubation compared to the control.Conclusion: MP-EO as a natural, non-toxic, and cost-effective compound can exhibit a favorable in-vitro radio-protective effect by increasing the survival and decreasing the percentage of apoptosis and necrosis of irradiated PBMCs.

On-Site Deployment of an Air-Liquid-Interphase Device to Assess Health Hazard Potency of Airborne Workplace Contaminants: The Case of 3-D Printers.

Biomonitoring of workers is an approach of evaluating workers’ exposure to chemicals and particulate matter by measuring biomarkers of parent chemicals, their metabolites, and reaction products in workers’ biospecimens. Prerequisites for biological monitoring in the workplace include permission to enter the workplace, approval of the study plan from the IRB (Institutional Review Board), and obtaining consent from workers. Because of the complex legal process involved in biomonitoring, few studies have been conducted so far on biomonitoring of workers’ exposures to nanoparticles and other hazards from emerging materials and advanced nanotechnologies. We have developed a cell-based biomonitoring device that can evaluate acute cytotoxicity and various other effect biomakers, such as inflammation, at realistic workplace exposure. This device is based on air–liquid interphase (ALI) and can be used to evaluate cell toxicity and early effect biomarkers along adverse outcome pathways. Following exposure of A549 lung epithelial cells in ALI to workplace air for 1–2 h, the cells were processed to assess the induction of inflammatory and cell damage biomarkers. Initially, we estimated the deposition rate of nanoparticles in the transwell by exposing the cell-free ALI device to silver nanoparticle aerosols (AgNP 20–30 nm) for 2 h in the laboratory. Then A549 lung epithelial cells cultured on the transwell in the ALI device were exposed to AgNP nanoaerosols for 2 h and evaluated for cytotoxicity and induction of mRNAs of pro-inflammatory cytokines IL-1b, IL-6, and TNF-α. Then the cells in the ALI device were exposed to 3-D printer emissions at the workplace and evaluated for the same matched endpoints. The mRNA levels for IL-1b, IL-6, and TNF-α increased significantly at the end of 2-h exposure of A549 cells to the positive control AgNP aerosols. These mRNAs, as well as LDH and microprotein concentrations, increased even more after 24-h post-exposure incubation (p < 0.05). Cytotoxicity evaluation of 3-D printer emissions at 810 and 957 μg/m3, which was more than 80 times higher than the airborne total suspended particulate concentrations in the workplace air (9–12.5 μg/m3), suggested no significant acute cytotoxicity at the end of 2-h exposure to 3-D-printing emission, as well as at 24-h post-exposure incubation. Hyperspectral microscopic observation showed that 3-D printers emitted particles to be attached to A549 cells after 2-h exposure, and many particles were internalized by A549 cells after 24 h of post-exposure incubation. The mRNA expression of pro-inflammatory cytokine IL-1b and IL-6 increased significantly after 2-h exposure to 3-D printer emissions and after 24-h incubation (only IL-6). In contrast, the expression of TNF-α mRNA decreased significantly after 2 h of exposure to 3-D printers and decreased even more after 24-h post-exposure incubation. These results support the use of cell-based ALI devices for direct assessment of airborne hazards in the workplace, for probing toxicological properties of airborne contaminants using adverse molecular pathways, and for guiding study design for workplace biomonitoring. ALI devices can bridge conventional exposure assessment with cellular toxicity testing platforms for hazard and risk assessment.

Investigation of extracellular medium osmolality depending on zinc application and incubation time on A549 cancer cells.

Changes in the osmolality of the extracellular medium (ECM) affect cell volume and cellular processes such as cell migration and proliferation. Not only may high concentrations of zinc (Zn) lead to cell death by apoptosis, but Zn is also a physiological suppressor of apoptosis. The aim of our study was to examine whether Zn and regulation of extracellular osmolality had an effect on the lung cancer cell line (A549) and how to be changed in ECM according to elements and osmolality depending on incubation time and Zn application. Our study consisted of four groups: cell-free medium, ECM of cancer cell after 24h incubation (24hECM), ECM of cancer cell after 48h incubation (48hECM), and ECM of cancer cell after 48h incubation with ZnCl2 (48hECM + Zn). ECM osmolality was measured by using osmometer, and the levels of chromium (Cr), iron (Fe), and magnesium (Mg) elements were analyzed using ICP-OES device for all groups. According to the result of the analysis, a statistically significant difference was found when osmolality and element values of ECM of 24hECM and 48hECM groups were compared with the values of the 48hECM + Zn group. It was observed that there was a decrease in the levels of Cr, Fe, and Mg with Zn application and incubation period in ECM. The regulation of ECM osmolality is a promising method due to biophysical effects on cancer cells. In our study, we speculated that the understanding of the effects of Zn and osmolality with the relationship between ECM and cancer cell might lead to the discovery of biophysical approaches as a novel therapeutic strategy.

Influence of freezing and heating conditions on grape seed flavan-3-ol extractability, oxidation, and galloylation pattern

In cool-climate viticulture, the short growing season can influence grape seed maturation by reducing the apparent oxidation of flavan-3-ol monomers and associated increase in seed browning. A reduction in seed maturation increases the potential extraction of flavan-3-ol monomers into wine during maceration operations, heightening bitterness. Here, we carried out a 2 × 2 factorial experiment to test the ability of freezing and heating treatments to advance maturation (decrease flavan-3-ol, improve browning) of (Vitis vinifera L.) Pinot noir and Cabernet Sauvignon seeds over a 24-h incubation period. Only freezing significantly increased seed browning in both cultivars. Subsequent correlations with seed flavan-3-ol monomer concentrations suggest that freezing enhanced the oxidation of these compounds. Interestingly, natural ripening and freezing reduced galloylated flavan-3-ol monomers to a greater extent than non-galloylated ones. This study provides new information regarding the susceptibility of flavan-3-ol monomers to freezing and heating, and also suggests that freezing can advance the maturation the seeds of under-ripe red vinifera grapes.

Cytokine Response of CD4+ T-Lymphocytes with Red Rose (Rosa Rosaceae – Pierre de Ronsard) Extracts by in Vitro Evaluation

Background. Red rose extract is known to have anti-inflammatory and immune-modulation effects. In this study, the red rose extract was tested on CD4+T lymphocytes in vitro, and cytokine response was evaluated.&#x0D; Materials and Methods. The red rose (Rosa Rosaceae - Pierre de Ronsard) extract used in this study was prepared and stored at -20° C until use. CD4+T-cells were seeded in 96-well plates at 313,500 cells/well in 100μ l cell culture medium in duplicate. One-half of the wells were used for biomarker screening in the culture medium, and the other half was used for cytotoxicity assay. Twenty-four hours after plating, the cells were treated in duplicate with 100μ l of the red rose extract diluted at 0.5%, 0.1%, 0.05%, 0.01% and 0.005% (v/v) in the cell culture medium or with culture medium only as control for 72 hours. Some other wells were allocated for untreated cells, and cells treated with the rose extract at 0.005% for 48-h incubation time.&#x0D; Results. Several cytokines (GRO; IFN-γ; IL-1α, 6, 10; MCP-1; RANTES; TGF-β1; TIMP 1, 2; Ang1, Ang2; G-CSF; MMP-9; and VEGF R2) were elevated. Except for MMP-9, which had fold changes &gt; 2, other cytokines were minimally elevated at various concentrations and timing of rose extract treatment. None of the mentioned cytokines were less than 0.8-fold after treatment with the rose extract. Cytotoxicity assay revealed insignificant changes in the viability of T-cells.&#x0D; Conclusions. There was a mild elevation in few inflammatory markers by CD4+ T-lymphocytes after in vitro treatment with the red rose extract (Rosa Rosacea - Pierre De Ronsard). Further in vitro and in vivo studies are required to evaluate the benefits of the red rose extract in immune regulation.

Targeting of sialoadhesin-expressing macrophages through antibody-conjugated (polyethylene glycol) poly(lactic-co-glycolic acid) nanoparticles.

This research aims to evaluate different-sized nanoparticles consisting of (polyethylene glycol) (PEG) poly(lactic-co-glycolic acid) (PLGA), loaded with fluorescein isothiocyanate for nanoparticle uptake and intracellular fate in sialoadhesin-expressing macrophages, while being functionalized with anti-sialoadhesin antibody. Sialoadhesin is a macrophage-restricted receptor, expressed on certain populations of resident tissue macrophages, yet is also upregulated in some inflammatory conditions. The nanocarriers were characterized for nanoparticle size (84–319 nm), zeta potential, encapsulation efficiency, and in vitro dye release. Small (86 nm) antibody-functionalized PEG PLGA nanoparticles showed persisting benefit from sialoadhesin-targeting after 24 h compared to the control groups. For small (105 nm) PLGA nanoparticles, uptake rate was higher for antibody-conjugated nanoparticles, though the total amount of uptake was not enhanced after 24 h. For both plain and functionalized small-sized (PEG) PLGA nanoparticles, no co-localization between nanoparticles and (early/late) endosomes nor lysosomes could be observed after 1-, 4-, or 24-h incubation time. In conclusion, decorating (PEG) PLGA nanocarriers with anti-sialoadhesin antibodies positively impacts macrophage targeting, though it was found to be formulation-specific.

Differentiation Effect of Two Alkaloid Fractions from Vietnamese Lycopodiaceae on Mouse Neural Stem Cells

Various Lycopodium alkaloids have been studied for their various biological activities including anti-inflammatory, antioxidant, immunomodulatory, and neuroprotective activities. Moreover, these alkaloid compounds have high potential in the treatment of neuron degenerative disease. This study has been carried out to test the effect of Huperzia serrata (Thunb.) Trevis, and Lycopodium clavatum L alkaloid fractions on the mouse neural stem cells (NSCs). Firstly, the alkaloid fractions were used to verify its toxicity on NSCs. The multiple concentrations of alkaloid fractions from H. serrata (0.044; 0.088; 0.175; 0.35; 0.7; 1.4 mg/ml) and L. clavatum (0.031; 0.063; 0.125; 0.25; 0.50; 1.0; 2.0 mg/ml) have been used for the treatment of NSCs at period of 48h incubation. Results of the study suggested that the IC50 value of H. serrata and L. clavatum was 0.56 mg/ml and 0.50 mg/ml, respectively. Then, the NSCs were differentiated in the presence of 5 and 10 µg/ml of alkaloid fraction from H. serrata; 0.625 and 1.25 µg/ml of alkaloid fraction from L. clavatum for 6 days. Here, we observed the primary NSCs treated with alkaloid fraction extract from H. serrata showed the increased gene expression level of early neuron TUBB3 and neuron-specific cytoskeleton MAP2. On the other hand, the L. clavatum alkaloid fraction increased the expression of neural stem cell marker genes (Nestin and PAX6) and decreased neuron marker genes. In conclusion, these results established that alkaloid fraction from H. serrata promoted differentiation of the mouse NSCs to neuron cells, and L. clavatum extract had a capacity for stemness maintenance.

Enhanced Antioxidant Effects of the Anti-Inflammatory Compound Probucol When Released from Mesoporous Silica Particles.

Brain endothelial cells mediate the function and integrity of the blood brain barrier (BBB) by restricting its permeability and exposure to potential toxins. However, these cells are highly susceptible to cellular damage caused by oxidative stress and inflammation. Consequent disruption to the integrity of the BBB can lead to the pathogenesis of neurodegenerative diseases. Drug compounds with antioxidant and/or anti-inflammatory properties therefore have the potential to preserve the structure and function of the BBB. In this work, we demonstrate the enhanced antioxidative effects of the compound probucol when loaded within mesoporous silica particles (MSP) in vitro and in vivo zebrafish models. The dissolution kinetics were significantly enhanced when released from MSPs. An increased reduction in lipopolysaccharide (LPS)-induced reactive oxygen species (ROS), cyclooxygenase (COX) enzyme activity and prostaglandin E2 production was measured in human brain endothelial cells treated with probucol-loaded MSPs. Furthermore, the LPS-induced permeability across an endothelial cell monolayer by paracellular and transcytotic mechanisms was also reduced at lower concentrations compared to the antioxidant ascorbic acid. Zebrafish pre-treated with probucol-loaded MSPs reduced hydrogen peroxide-induced ROS to control levels after 24-h incubation, at significantly lower concentrations than ascorbic acid. We provide compelling evidence that the encapsulation of antioxidant and anti-inflammatory compounds within MSPs can enhance their release, enhance their antioxidant effects properties, and open new avenues for the accelerated suppression of neuroinflammation.

Effects of harvest date and growth stage on triticale forages in the southwest USA: kinetics of in vitro disappearance of fiber and dry matter.

Recently, there has been interest in including triticale (X Triticosecale Wittmack) within forage programs in the southwest USA. Our objectives were to evaluate in vitro disappearance kinetics of neutral detergent fiber (NDF) and dry matter (DM) for cultivars identified during 2019 as positively or negatively deviant from typical cultivars, based specifically on regressions of 48-h in vitro disappearance of NDF on growth stage (GRST). All NDF analyses included the use of heat-stable α-amylase and sodium sulfite, as well as correction for residual ash (asNDFom). Seven triticale cultivars were established on December 18, 2019 at the University of Arizona Maricopa Agricultural Center, located near Maricopa, AZ. Forage plots were arranged in a randomized complete block design with three complete blocks (replications), and then harvested on seven dates the following late-winter and spring (February 26, March 17, April 1, April 14, April 28, May 12, and May 26). Based on a linear model, GRST was highly variable among cultivars on March 17 (44 ± 10.6), April 1 (57 ± 12.1), April 14 (67 ± 8.9), and April 28 (79 ± 7.2) compared with other harvest dates (SD ≤ 1.7). For concentrations of asNDFom, all cultivars exhibited linear (P ≤ 0.042) and quadratic (P < 0.001) polynomial contrasts in response to harvest date, and all cultivars except Merlin Max (P ≥ 0.063) exhibited at least one additional cubic or quartic effect (P ≤ 0.015). A contributing factor to the unique response by Merlin Max was the numerically greater maximum canopy height (145 ± 9.8cm) compared with the mean of all cultivars (107 ± 17.7cm), which also was associated with greater percentages of stem, as well as reduced percentages of DM partitioned within the grain head. Regressions of asNDFom disappearance after 30- or 48-h incubations on GRST indicated this was an effective independent variable (R2 ≥ 0.927), and responses were most often linear in nature. Generally, relationships for DM disappearance were quadratic, ostensibly due to the complicating effect of grain fill, but GRST was again an effective predictor variable with R2 statistics ≥ 0.852 for 12 of 14 combinations of cultivar and incubation time. Predicted percentages of digestible DM attributed to asNDFom disappearance were ≥50.3% through the fully flowered stage of growth, but digestible contributions from nonfiber components following the onset of grain fill profoundly affected overall DM digestibility among cultivars harvested at later GRST.

Extract from Opuntia ficus-indica cladode delays the Aedes aegypti larval development by inducing an axenic midgut environment.

This study evaluated the effects of acute exposure of Aedes aegypti third instar (L3 ) larvae to the saline extract of Opuntia ficus-indica cladodes on the biological cycle and fertility of the emerging adults. For this, larvae were treated for 24 h with the extract at ¼ LC50 (lethal concentration to kill 50% of larvae), ½ LC50 or LC50 ; the development and reproduction of the emerged adults were evaluated after a recovery period of 9 days. The resistance of proteins in the extract to hydrolysis by L3 digestive enzymes and histomorphological alterations in the larval midgut were also investigated. The extract contained lectin, flavonoids, cinnamic derivatives, terpenes, steroids, and reducing sugars. It showed a LC50 of 3.71% for 48 h. The data indicated mean survival times similar in control and extract treatments. It was observed development delay in extract-treated groups, with a lower number of adults than in control. However, the females that emerged laid similar number of eggs in control and treatments. Histological evaluation revealed absence of bacterial and fungal microorganisms in the food content in midguts from larvae treated with cladode extract. Electrophoresis revealed that three polypeptides in the extract resisted to hydrolysis by L3 digestive proteases for 90 min. The lectin activity was not altered even after 24-h incubation with the enzymes. In conclusion, the extract from O. ficus-indica can delay the development of Ae. aegypti larvae, which may be linked to induction of an axenic environment at larval midgut and permanence of lectin activity even after proteolysis.

Evaluation of the antibacterial and antibiofilm activities of sodium benzoate against the soft rot disease of tomato in Iraq

Pectobacterium carotovorum is one of the most serious pectolytic bacterial pathogens causing significant pre- and postharvest soft rot diseases on a variety of agroeconomic fruits and vegetables worldwide. Application of chemical pesticides for controlling the postharvest diseases is still insufficient and unacceptable by consumers. The effect of sodium benzoate (C6H5COONa) (SBN) was evaluated against the growth of P. carotovorum utilizing the highly pathogenic strain BR03, confirmed molecularly using dnaA primers. The inhibition zone was the greatest when P. carotovorum was treated with 600 mM SBN both at pH 7.5 and 7.0. Flow cytometry (distinguishing live and dead bacterial cells) and the development of bacterial colonies on Petri plates confirmed the bactericidal action of SBN. In addition, these results validated that bacterial cells were killed when treated with 600 mM SBN. The biofilm formation was completely inhibited and destroyed following exposure to 600 mM SBN after a 24-h incubation period and the absorbance readings were reduced as the concentration of SBN increased. Disease severity on inoculated tomato fruits decreased in comparison with the control fruit at either 4 or 25 °C. SBN is safe for human health and commonly used in industries as a food preservative. SBN can be an effective alternative method for controlling soft rot diseases on tomato fruits.

In vitro anti-Toxocara vitulorum effect of silver nanoparticles.

Toxocara vitulorum is a common gastrointestinal nematode of buffaloes and cattle, primarily young calves. This parasitic infection is distributed worldwide, causing a huge economic loss due to reduced meat and milk production and animal mortality. Several studies have indicated that silver nanoparticles have an effective anthelmintic activity. Thus, this study aimed to evaluate the anthelmintic effects of different concentrations of silver nanoparticles on adult Toxocara vitulorum in vitro. Male and female adult worms were incubated for 48h in 50, 100, and 200mg/L silver nanoparticles synthesized using lemon juice. Oxidative stress markers, in addition to light and scanning electron microscopic studies of treated worms, were assessed following 48h incubation in 200mg/L silver nanoparticles. Light and scanning electron micrographs of treated worms revealed damage in the muscular layer, destruction of the cuticle, distortion in lips structure, and deformed excretory pore and sensory papillae. Also, oxidative stress markers recorded an increase in malondialdehyde and nitric oxide and decreased levels of glutathione reduced, glutathione S-transferase, and catalase after exposure to silver nanoparticles. In Conclusion, the current study demonstrated a substantial destructive effect of silver nanoparticles on adult Toxocara vitulorum, indicating its potential as an anthelmintic alternative to the more expensive drugs.

Inhibitory effect of 405-nm blue LED light on the growth of Candida albicans and Streptococcus mutans dual-species biofilms on denture base resin.

We investigated whether irradiation with 405-nm blue LED light could inhibit the growth of not only single- but dual-species biofilms formed by Candida albicans and Streptococcus mutans on denture base resin and cause the alteration in gene expression related to adhesion and biofilm formation. C. albicans and S. mutans single-/dual-species biofilms were formed on the denture base specimens. The biofilms were irradiated with 405-nm blue LED light (power density output: 280 mW/cm2) for 0 (control) and 40min. Dual-species biofilms were analyzed using CFU assay and fluorescence microscopy, and single-/dual-species biofilms were analyzed using alamarBlue assays and gene expression analysis. To assess the inhibitory effect of irradiation on dual-species biofilms, specimens after irradiation were aerobically incubated for 12h. After incubation, the inhibition of growth was assessed using CFU assays and fluorescence microscopy. Data were analyzed using the Mann-Whitney U or Student's t test (p < 0.05). Irradiation produced a significant inhibitory effect on biofilms. Fluorescence microscopy revealed that almost all C. albicans and S. mutans cells were killed by irradiation, and there was no notable difference in biofilm thickness immediately after irradiation and after irradiation and incubation for 12h. alamarBlue assays indicated the growth of the biofilms was inhibited for 12-13h. The expression of genes associated with adhesion and biofilm formation-als1 in C. albicans and ftf, gtfC, and gtfB in S. mutans-significantly reduced by irradiation. Irradiation with 405-nm blue LED light effectively inhibited the growth of C. albicans and S. mutans dual-species biofilms for 12h.

Cropping management in a livestock-pasture-crop integration modifies microbial communities, activity, and soil health score.

Understanding indicators of soil health is crucial for developing agricultural systems that are sustainable and climate resilient. Labile soil carbon (C), microbial properties, and nutrient status are all incorporated into the Haney Soil Health Tool with the goal of summarizing several indicators into one index. Monthly soil samples from an integrated crop-livestock system in Central Texas were collected from 2017 to 2019. Fields represented a range of management practices, including cover crops, no-till, rotational grazing, and a native prairie remnant. Soil samples were analyzed for total C, water-soluble C, macro- and micronutrient content and bioavailability, and phospholipid fatty acids (PLFAs). Microbial activity was determined via a 24-h CO2 incubation. Soil health score, C, and PLFAs were well correlated with each other. The greatest total PLFA (219.5nmol g-1 soil) and organic C (54.3g kg-1 soil) were found in the native prairie, and the lowest were found in the unfertilized continuous-corn system (60.5nmol PLFAs g-1 soil and 24.0g organic C kg-1 soil). Of all agroecosystems, the perennial grazing system (soil health score, 24.7) was most similar to the native prairie (soil health score, 27.4), having high soil C and a large microbial community. Of the row cropping systems, the no-till system approached the perennial systems better than the conventional till and unfertilized conventional till (soil health score, 11.1 vs. 8.0 and 5.3, respectively). This study highlights the value of perennial grass grazing in agroecosystems and appropriate best management practices. Expanding this analysis to other sites may provide additional insight.

Red-emission carbon dots as fluorescent "on-off-on" probe for highly sensitive and selective detection of Cu2+ and glutathione.

A highly sensitive and selective fluorescent "on-off-on" strategy is established for the synchronous detection of Cu2+ and glutathione in aqueous solution. Red carbon dots (R-CDs) were prepared by using precursors of 4,5-difluoro-1,2-phenylenediamine and citric acid via a one-step hydrothermal strategy. R-CDs show a relatively long fluorescence lifetime of 3.47ns under 455nm excitation and high absolute fluorescent quantum yield of 20.1% with an excitation wavelength of 550nm. R-CDs exhibit a marked pH-responsive fluorescence property with no significant perturbation from pH 4 to pH 13 even after five cycles. R-CDs with higher concentration of 750μg·mL-1 exhibit no significant cytotoxicity and good biocompatibility on HeLa cells and A549 cells after incubation for 48h. The fluorescence of R-CDs at 619nm (excited at 550nm) is quenched statically by Cu2+ and recovered by glutathione subsequently, resulting in a fluorescent "on-off-on" assay for the synchronous detection of Cu2+ and glutathione. Under optimal conditions, the linear response covers the Cu2+ concentration range of 1 to 50μM and the glutathione concentration range of 1 to 70μM. Detection limits of Cu2+ and glutathione are 0.16 and 0.41μM, respectively. This fluorescent probe is applied to the determination of Cu2+ and glutathione in authentic samples with satisfying results. Such an assay broadens the potential application of CDs in environmental areas and clinical therapy fields.

25-Hydroxyvitamin D potentializes extracellular cathelicidin release from human PBMC stimulated ex vivo with either bacterial (LPS) or viral (P: IC) mimetics.

Human cathelicidin refers to the cationic antimicrobial peptide hCAP18/LL-37. LL-37 is formed by cleavage of the propeptide hCAP18 coded by the CAMP gene. The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)D), has been shown to induce the CAMP gene expression through promoter activation. We previously failed to demonstrate in a clinical trial that supplementation of 25-hydroxyvitamin D (25(OH)D) improves LL-37 serum levels. The aim of this work was to evaluate the impact of 25(OH)D supplementation on intracellular expression of CAMP and secretion of LL-37 in an ex vivo model using the peripheral blood mononuclear cells (PBMC). PBMC collected from healthy donors and incubated with different concentrations of 25(OH)D (0 ng/ml: control (D0); 25 ng/ml: deficient (D25); 75 ng/ml: physiological (D75); 125 ng/ml: supraphysiological (D125)) were stimulated or not with lipopolysaccharide (LPS, 100 ng/ml) or synthetic double-stranded RNA Poly (I: C) (PIC, 10 µg/ml). The intracellular expressions of the CAMP gene and the hCAP18 peptide were measured respectively after 24-h and 48-h incubation periods. The concentration of LL-37 was determined in the culture medium after 48-h incubation. 25(OH)D significantly induced CAMP gene expression at 24 h with a maximum effect at a dose of D125 in either unstimulated (tenfold expression) or stimulated (LPS: 100-fold expression; PIC: 15-fold expression) conditions. Intracellular hCAP18 peptide was overexpressed at 48 h under unstimulated (1.5-fold, D125) and stimulated conditions, LPS (twofold, D125) and PIC (2.5-fold, D125). The secretion of LL-37 in the culture medium was significantly induced by 25(OH)D only in both stimulated (LPS and PIC) conditions in a dose-dependent manner. Our results demonstrate that 25(OH)D incubation increases intracellular expression of CAMP and hCAP18, but extracellular secretion of LL-37 antimicrobial peptide is increased by 25(OH)D only when PBMC from healthy donors were stimulated with bacterial or viral immune mimetic.

Cytotoxic and pro-apoptotic Effects of Spirulina Platensis Extract on PC-3 Prostate Adenocarcinoma Cell Line

Introduction: Prostate cancer is one of the most common cancers among men, with an increasing incidence and mortality rate. In the present study, cytotoxic and pro-apoptotic effects of spirulina platensis extract on PC-3 prostate adenocarcinoma cell line were investigated. Methods: In the present experimental study, the PC-3 prostatic cancer cells were treated in four experimental with 400, 200, 100 and 50 μg / ml extract of spirulina and incubated at 24 and 48 hours. Cytotoxicity was analyzed by MTS kit (3-(4, 5-Dimethylthiazol-2-yl)-5-(3-Carboxymethoxyphenyl)-2-(4-Sulfophenyl)-2H-Tetrazolium, Inner Salt) and apoptosis was analyzed by flow- cytometry using an Annexin V-FITC/PI kit according to the manufacturer protocol in both times. Statistical analysis was accomplished by ANOVA and Duncan tests using FlowJo and SPSS 16 software. Results: In the experimental groups treated with extract of spirulina, the viability of the cells showed a decrease compared to control group, while this decrease was more noticeable in the experimental group of 100 μg / ml at both incubation times (&lt;0.0071).Increased incidence of apoptosis was significantly higher in the experimental groups than the control group. However, this increase was significantly higher than the control group at concentrations of 200 μg / ml in 24h incubation time (Ƥ &lt; 0.0331) and 100 μg / ml of 48h incubation time (Ƥ &lt; 0.0502). Conclusion: Extract of Spirulina at specific concentrations reduced cell growth and induced apoptosis in PC-3 prostatic cancer cells. Evidence suggests that spirulina can be used as an anticancer drug for the treatment of prostate cancer.

Technical note: Design, development and validation of an automated gas monitoring equipment for measurement of the dynamics of microbial fermentation

The present technical note describes design, development and validation of an automated equipment for measurement of kinetics of gas production during fermentation in glass bottles. The overall repeatability and precision of the developed system was evaluated and compared with the manual gas measurement technique in respect to characterization of the fermentation kinetics of ruminant livestock feeds. Two incubations were carried out, during which the GP of six different feeds was measured with the automated system or manual technique. During a 48-hour incubation period, pressure data were collected at 15-minute intervals using automated equipment, yielding 192 head-space pressure measurements for each bottle. In manual measurement, incubations were performed with the nominal 60-mL serum bottle, and headspace pressure was read using a digital pressure gauge and then released at 2, 4, 6, 8, 12, 16, 24, 36, and 48 hours of incubation. The automated equipment recorded greater GP (+11.5%, over the 48-h incubation) than the manual measurement, and the repeatability and coefficient of repeatability values indicated that the GP data obtained with manual equipment were less repeatable. The automated equipment measures the fermentative GP kinetics with greater precision and repeatability than manual technique.•An automated batch GP equipment was designed, developed and validated, and a comparison was made with GP data obtained manually using a digital pressure gauge.•The automated equipment provided more reliable and repeatable data compared with manual measurement.•The automated equipment is available with lower cost and more functionality.

Effect of Breast Silicone Implant Topography on Bacterial Attachment and Growth: An In Vitro Study.

The mechanisms underlying capsular contracture remain unclear. Emerging evidence supports the inflammation hypothesis, according to which bacteria from an adherent biofilm cause chronic inflammation and collagen deposition on the implant and trigger capsular contracture. Our goal was to evaluate the effect of different types of breast implants on the growth of Staphylococcus aureus, S. epidermidis, and Pseudomonas aeruginosa, which are commonly found in biofilms in infection. Bacteria were grown in tryptic soy broth at 37°C for 2, 6, and 24 h and subsequently incubated for 24 h on 12 shell sections of smooth, nano-, and macrotextured breast implants. After incubation, the solutions were ultrasonicated and bacterial numbers were determined by serial dilution. S. aureus were fixed, washed with phosphate-buffered saline, dehydrated in ethanol, and coated with a platinum film to visualize the presence of biofilms by scanning electron microscopy. The numbers of S. aureus and S. epidermidis attached to the smooth and nanotextured surface implants were significantly lower than those on the macrotextured surface for all incubation times, whereas the number of P. aeruginosa was non-significantly lowest on the nanotextured surface after 24h incubation. Biofilms on smooth and nanotextured implant surfaces showed patchy patterns on scanning electron microscopy in contrast to the continuous pattern detected on macrotextured implants. Nanotextured breast implants may limit bacterial growth and thus prevent capsular contracture.