Towards Understanding the Involvement of H+-ATPase in Programmed Cell Death of Psammosilene tunicoides after Oxalic Acid Application

Psammosilene tunicoides is a unique perennial medicinal plant species native to the Southwestern regions of China. Its wild population is rare and endangered due to over-excessive collection and extended growth (4–5 years). This research shows that H+-ATPase activity was a key factor for oxalate-inducing programmed cell death (PCD) of P. tunicoides suspension cells. Oxalic acid (OA) is an effective abiotic elicitor that enhances a plant cell’s resistance to environmental stress. However, the role of OA in this process remains to be mechanistically unveiled. The present study evaluated the role of OA-induced cell death using an inverted fluorescence microscope after staining with Evans blue, FDA, PI, and Rd123. OA-stimulated changes in K+ and Ca2+ trans-membrane flows using a patch-clamp method, together with OA modulation of H+-ATPase activity, were further examined. OA treatment increased cell death rate in a dosage-and duration-dependent manner. OA significantly decreased the mitochondria activity and damaged its electron transport chain. The OA treatment also decreased intracellular pH, while the FC increased the pH value. Simultaneously, NH4Cl caused intracellular acidification. The OA treatment independently resulted in 90% and the FC led to 25% cell death rates. Consistently, the combined treatments caused a 31% cell death rate. Furthermore, treatment with EGTA caused a similar change in intracellular pH value to the La3+ and OA application. Combined results suggest that OA-caused cell death could be attributed to intracellular acidification and the involvement of OA in the influx of extracellular Ca2+, thereby leading to membrane depolarization. Here we explore the resistance mechanism of P. tunicoides cells against various stresses endowed by OA treatment.

Use of Ultrasounds to Reduce the Count of Campylobacter coli in Water

The present study aimed to evaluate the effectiveness of low-frequency ultrasounds applied to eliminate Campylobacter spp. from water. The strains used in this research were isolated from water contaminated with sewage. Campylobacter coli alone was detected in the samples and used for further research. The reference strain C. coli ATCC 33559 was simultaneously tested. The isolate was exposed to ultrasounds at frequencies of 37 kHz and 80 kHz in a continuous operation device with ultrapure deionized water. After 5 min of sonication, the count of C. coli decreased by 5.78% (37 kHz) and 6.27% (80 kHz), whereas the temperature increased by 3°C (37 kHz), and 6°C (80 kHz). After 30 min of sonication, the death rates of bacterial cells were 40.15% (37 kHz) and 55.10% (80 kHz), whereas the temperature reached the maximum values of 36°C (37 kHz), and 39°C (80 kHz). Sonication at the frequency of 80 kHz reduced the bacterial count from 6.86 log CFU/ml to 3.08 log CFU/ml, whereas the frequency of 37 kHz reduced the bacterial count from 6.75 log CFU/ml to 4.04 log CFU/ml. Despite significant differences (p < 0.05) in the number of C. coli cells, the cell death rate remained at the same level.

Molecular Mechanism of Anti-Inflammatory Activities of a Novel Sulfated Galactofucan from Saccharina japonica

Seaweed of Saccharina japonica is the most abundantly cultured brown seaweed in the world, and has been consumed in the food industry due to its nutrition and the unique properties of its polysaccharides. In this study, fucoidan (LJNF3), purified from S. japonica, was found to be a novel sulfated galactofucan, with the monosaccharide of only fucose and galactose in a ratio of 79.22:20.78, and with an 11.36% content of sulfate groups. NMR spectroscopy showed that LJNF3 consists of (1→3)-α-l-fucopyranosyl-4-SO3 residues and (1→6)-β-d-galactopyranose units. The molecular mechanism of the anti-inflammatory effect in RAW264.7 demonstrated that LJNF3 reduced the production of nitric oxide (NO), and down-regulated the expression of MAPK (including p38, ENK and JNK) and NF-κB (including p65 and IKKα/IKKβ) signaling pathways. In a zebrafish experiment assay, LJNF3 showed a significantly protective effect, by reducing the cell death rate, inhibiting NO to 59.43%, and decreasing about 40% of reactive oxygen species. This study indicated that LJNF3, which only consisted of fucose and galactose, had the potential to be developed in the biomedical, food and cosmetic industries.

Which Contributes to Meniscal Repair, the Synovium or the Meniscus? An In Vivo Rabbit Model Study With the Freeze-Thaw Method

Background: During meniscal tissue repair, the origin of the reparative cells of damaged meniscal tissue remains unclear. Hypothesis: Comparison of the influence between meniscal and synovial tissues on meniscal repair by the in vivo freeze-thaw method would clarify the origin of meniscal reparative cells. Study Design: Controlled laboratory study. Methods: A total of 48 mature Japanese white rabbits were divided into 4 groups according to the tissue (meniscal or synovial) that received freeze-thaw treatment. The meniscus of each group had a 2 mm–diameter cylindrical defect filled with alginate gel. Macroscopic and histologic evaluations of the reparative tissues were performed at 1, 3, and 6 weeks postoperatively. Additional postoperative measurements included cell density, which was the number of meniscal cells in the cut area per cut area (mm2) of meniscus; cell density ratio, which was the cell density of the sample from each group per the average cell density of the intact meniscus; and cell death rate, which was the number of cells stained by propidium iodide per the number of cells stained by Hoechst 33342 of the meniscal tissue adjacent to the defect. Results: The macroscopic and histologic evaluations of the non–synovium freeze-thaw groups were significantly superior to those of the synovium freeze-thaw groups at 3 and 6 weeks postoperatively. Additionally, the meniscal cell density ratio and cell death rate in the freeze-thaw groups were significantly lower than those in the non–meniscal freeze-thaw groups at 3 and 6 weeks postoperatively. Conclusion: The freeze-thawed meniscus recovered few cells in its tissue even after 6 weeks. However, the defect was filled with fibrochondrocytes and proteoglycan when the synovium was intact. On the basis of these results, it is concluded that synovial cells are the primary contributors to meniscal injury repair. Clinical Relevance: In meniscal tissue engineering, there is no consensus on the best cell source for meniscal repair. Based on this study, increasing the synovial activity and contribution should be the main objective of meniscal tissue engineering. This study can establish the foundation for future meniscal tissue engineering.

Potential Phototoxicity of Indocyanine Green in Retinal Pigment Epithelial Cells after Angiography under Ambient Illumination

Indocyanine green (ICG) angiography is an indispensable inspection to diagnose and treat for chorioretinal diseases. In this study, we investigated the phototoxicity of ICG on RPE cells at the levels of residual ICG after angiography under ambient light. After incubation of ARPE-19 cells in a colorless medium containing 0 to 10 μg/mL ICG for 24 hours in the dark or under 2000 lx illumination from a fluorescent lamp, cell viability decreased and cell death rate increased in cultures with more than 5.0 μg/mL ICG under illumination. In culture with 10 μg/mL ICG under illumination, morphology of cells changed to be oval and TUNEL- and malondialdehyde-positive cells increased compared to other cultures with ICG in the dark or without ICG under illumination. Furthermore, the level of intracellular reactive oxygen species was also elevated. On the other hand, toxicity of ICG denatured by illumination was not observed. Blocking green to red light overlapping wavelengths of ICG absorbance exhibited decreased cell death rate. The present study indicated that ICG at the estimated intravenous concentrations after ICG angiography induces potential phototoxicity on human RPE cells via oxidative damage under continuous ambient illumination and that the cytotoxicity is reduced by blocking green to red light wavelengths.

Optimized vascular network by stereolithography for tissue engineered skin

This paper demonstrates the essential and efficient methods to design, and fabricate optimal vascular network for tissue engineering structures based on their physiological conditions. Comprehensive physiological requirements in both micro and macro scales were considered in developing the optimisation design for complex vascular vessels. The optimised design was then manufactured by stereolithography process using materials that are biocompatible, elastic and surface bio-coatable. The materials are self-developed photocurable resin consist of BPA-ethoxylated-diacrylate, lauryl acrylate and isobornylacrylate with Irgacure® 184, the photoinitiator. The optimised vascular vessel offers many advantages: 1) it provides the maximum nutrient supply; 2) it minimises the recirculation areas and 3) it allows the wall shear stress on the vessel in a healthy range. The stereolithography manufactured vascular vessels were then embedded in the hydrogel seeded with cells. The results of in vitro studies show that the optimised vascular network has the lowest cell death rate compared with a pure hydrogel scaffold and a hydrogel scaffold embedded within a single tube in day seven. Consequently, these design and manufacture routes were shown to be viable for exploring and developing a high range complex and specialised artificial vascular networks.

Calcitriol Reverses Induced Expression of Efflux Proteins and Potentiates Cytotoxic Activity of Gemcitabine in Capan-2 Pancreatic Cancer Cells

Purpose. Efflux and influx proteins play a major role in chemo-resistance by affecting the net cellular uptake of anti-cancer drugs. Hence, alteration of the efflux and influx protein expression may result in variations of chemotherapeutics uptake and consequently cell death rate. The present study investigated the effects of pre-treatment of capan-2 pancreatic cancer cells with calcitriol, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) or silibinin on the induction of three major efflux proteins and the main gemcitabine influx protein. The influence of the pre-treatments on the net cellular uptake of gemcitabine, total ATPase activity, and cell death rate were also evaluated. Methods. Capan-2 pancreatic cancer cells were pre-treated for 24 h with calcitriol, BHT, BHA, or silibinin, followed by gemcitabine treatment. The concentration of gemcitabine was quantified using ultra-performance liquid chromatography (UPLC). Real-time polymerase chain reaction (RT-PCR) was utilized in order to investigate the expression of the mRNAs. The expression of the proteins was assessed using western blotting. Measurement of the ATPase activity was conducted utilizing a colorimetric method and viability of the cells was determined using a luminescent cell viability assay. Results. Protein expression studies showed that BHT, silibinin, and BHA increased expression of the efflux proteins and decreased the overall uptake of gemcitabine, whereas calcitriol significantly inhibited expression of the efflux proteins and increased gemcitabine uptake. Expression of specific mRNAs correlated reasonably well with the levels of corresponding proteins. Additionally, the expression of efflux proteins and ATPase activity were well correlated, signifying that the induced efflux proteins are functionally active. Moreover, pre-treatment with calcitriol resulted in a significant increase in cell death with gemcitabine treatment, whereas, BHA significantly reduced the cell death rate. On the other hand, pre-treatment with BHT and silibinin had no significant effect on the cell death rate. Conclusions. Pre-treatment of the pancreatic cancer cells with calcitriol significantly increased gemcitabine cellular uptake and consequently decreased cell viability after treatment with gemcitabine, whereas BHA significantly reduced gemcitabine uptake and decreased cell death rate, which were at least partially attributed to the alteration of expression of efflux and influx proteins. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.

Carbon nanomaterials sensitize prostate cancer cells to docetaxel and mitomycin C via induction of apoptosis and inhibition of proliferation

We have previously shown that carbon nanofibers (CNFs) and carbon nanotubes (CNTs) can sensitize prostate cancer (PCa) cells to platinum-based chemotherapeutics. In order to further verify this concept and to avoid a bias, the present study investigates the chemosensitizing potential of CNFs and CNTs to the conventional chemotherapeutics docetaxel (DTX) and mitomycin C (MMC), which have different molecular structures and mechanisms of action than platinum-based chemotherapeutics. DU-145 PCa cells were treated with DTX and MMC alone or in combination with the carbon nanomaterials. The impact of the monotreatments and the combinatory treatments on cellular function was then systematically analyzed by using different experimental approaches (viability, short-term and long-term proliferation, cell death rate). DTX and MMC alone reduced the viability of PCa cells to 94% and 68%, respectively, whereas a combined treatment with CNFs led to less than 30% remaining viable cells. Up to 17- and 7-fold higher DTX and MMC concentrations were needed in order to evoke a similar inhibition of viability as mediated by the combinatory treatments. In contrast, the dose of platinum-based chemotherapeutics could only be reduced by up to 3-fold by combination with carbon nanomaterials. Furthermore, combinatory treatments with CNFs led mostly to an additive inhibition of short- and long-term proliferation compared to the individual treatments. Also, higher cell death rates were observed in combinatory treatments than in monotreatments, e.g., a combination of MMC and CNFs more than doubled the cell death rate mediated by apoptosis. Combinations with CNTs showed a similar, but less pronounced impact on cellular functions. In summary, carbon nanomaterials in combination with DTX and MMC evoked additive to partly synergistic anti-tumor effects. CNFs and CNTs possess the ability to sensitize cancer cells to a wide range of structurally diverse chemotherapeutics and thus represent an interesting option for the development of multimodal cancer therapies. Co-administration of chemotherapeutics with carbon nanomaterials could result in a reduction of the chemotherapeutic dosage and thus limit systemic side effects.