Disturbance of firefly luciferase-based bioassays by different aluminum species

Luciferase-dependent assays, important for biochemical analyses of cytotoxicity and reporter genes, may be perturbed by compounds interfering with the luciferase reaction. We analyzed the impact of different aluminum (Al) species on a luciferase-based assay for determination of cellular adenosine triphosphate. Al⁰ nanoparticles (Al⁰–NPs) but not Al₂O₃–NPs decreased luminescence, correlated to high absorbance of Al⁰–NPs. By contrast, Al ions increased the luminescent signal. Data demonstrate that luciferase-dependent assays can be reciprocally disturbed by Al–NPs or Al ions in a specific manner, depending on the particular Al species. Careful interpretation of data from such experiments is essential in order to obtain conclusive results.     

The effects of mitochondrial complex I blockade on ATP and permeability in rat pulmonary microvascular endothelial cells in culture (PMVEC) are overcome by coenzyme Q1 (CoQ1)

In isolated rat lung perfused with a physiological saline solution (5.5 mM glucose), complex I inhibitors decrease lung tissue ATP and increase endothelial permeability (K_f), effects that are overcome using an amphipathic quinone (CoQ₁) [Free Radic. Biol. Med. 65:1455–1463; 2013]. To address the microvascular endothelial contribution to these intact lung responses, rat pulmonary microvascular endothelial cells in culture (PMVEC) were treated with the complex I inhibitor rotenone and ATP levels and cell monolayer permeability (PS) were measured. There were no detectable effects on ATP or permeability in experimental medium that, like the lung perfusate, contained 5.5 mM glucose. To unmask a potential mitochondrial contribution, the glucose concentration was lowered to 0.2 mM. Under these conditions, rotenone decreased ATP from 18.4±1.6 (mean±SEM) to 4.6±0.8 nmol/mg protein, depolarized the mitochondrial membrane potential (Δψ_m) from −129.0±3.7 (mean±SEM) to −92.8±5.5 mV, and decreased O₂ consumption from 2.0±0.1 (mean±SEM) to 0.3±0.1 nmol/min/mg protein. Rotenone also increased PMVEC monolayer permeability (reported as PS in nl/min) to FITC–dextran (~40 kDa) continually over a 6 h time course. When CoQ₁ was present with rotenone, normal ATP (17.4±1.4 nmol/mg protein), O₂ consumption (1.5±0.1 nmol/min/mg protein), Δψ_m (−125.2±3.3 mV), and permeability (PS) were maintained. Protective effects of CoQ₁ on rotenone-induced changes in ATP, O₂ consumption rate, Δψ_m, and permeability were blocked by dicumarol or antimycin A, inhibitors of the quinone-mediated cytosol–mitochondria electron shuttle [Free Radic. Biol. Med. 65:1455–1463; 2013]. Key rotenone effects without and with CoQ₁ were qualitatively reproduced using the alternative complex I inhibitor, piericidin A. We conclude that, as in the intact lung, PMVEC ATP supply is linked to the permeability response to complex I inhibitors. In contrast to the intact lung, the association in PMVEC was revealed only after decreasing the glucose concentration in the experimental medium from 5.5 to 0.2 mM.     

Induced pluripotent stem cell‐derived melanocyte precursor cells undergoing differentiation into melanocytes

Induced pluripotent stem cell (iPSC) technology offers a novel approach for conversion of human primary fibroblasts into melanocytes. During attempts to explore various protocols for differentiation of iPSCs into melanocytes, we found a distinct and self‐renewing cell lineage that could differentiate into melanocytes, named as melanocyte precursor cells (MPCs). The MPCs exhibited a morphology distinctive from that of melanocytes, in lacking either the melanosomal structure or the melanocyte‐specific marker genes MITF, TYR, and SOX10. In addition, gene expression studies in the MPCs showed high‐level expression of WNT5A, ROR2, which are non‐canonical WNT pathway markers, and its related receptor TGFβR2. In contrast, MPC differentiation into melanocytes was achieved by activating the canonical WNT pathway using the GSK3β inhibitor. Our data demonstrated the distinct characteristic of MPCs' ability to differentiate into melanocytes, and the underlying mechanism of interfacing between canonical WNT signaling pathway and non‐canonical WNT signaling pathway.     

The conditioned medium from osteo‐differentiating human mesenchymal stem cells affects the viability of triple negative MDA‐MB231 breast cancer cells

This study aimed to investigate the effect of conditioned media (CM) from osteo‐differentiating and adipo‐differentiating human mesenchymal stem cells (MSCs) isolated from lipoaspirates of healthy female donors on the viability of triple‐negative breast cancer cells MDA‐MB231. The CM of undifferentiated and differentiating MSCs were collected after 7, 14, 21 and 28 days of culture. The effects of MSC CM on cell proliferation were assessed using an 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay after 24 h. The effects of osteo‐differentiating cell CM on apoptotic promotion, cell cycle impairment, mitochondrial transmembrane potential dissipation, production of reactive oxygen species and autophagosome accumulation were analysed by flow cytometry and Western blot. MTT assay showed that only CM collected from osteo‐induced cells at day 28 (d28O‐CM) reduced tumour cell viability. Treatment with d28O‐CM restrained cell cycle progression through G₂ phase, elicited a caspase‐8‐driven apoptotic effect already after 5 h of culture, and down‐regulated autophagosome accumulation and beclin‐1 expression. The finding that factor(s) secreted by osteo‐differentiating MSCs shows properties of an apoptotic inducer and autophagy inhibitor on triple‐negative breast cancer cells may have an important applicative potential that deserves further investigation. Copyright © 2015 John Wiley & Sons, Ltd.     

超声乳化白内障手术后患者角膜内皮细胞损伤的研究

目的:探讨超声乳化白内障手术患者角膜内皮细胞的损伤情况。方法:收集我院确诊为白内障的患者121例,随机分配为微切口组与常规切口组。常规切口组采用3.0 mm切口超声乳化白内障手术方案,微切口组采用1.8 mm小切口超声乳化白内障手术。手术前、手术后1日、7日、1个月、3个月监测患者角膜内皮细胞密度、六角形细胞比例、角膜内皮细胞变异系数及中央角膜厚度。结果:与治疗前相比,两组患者手术后1日、7日、1个月、3个月的角膜内皮细胞密度、六角形细胞比例及角膜内皮细胞变异系数均较治疗前显著降低(P0.05);手术后1日、7日时,两组患者的中央角膜厚度均较手术前明显变薄,有统计学差异(P0.05);手术后1个月、3个月,两组患者的中央角膜厚度均呈降低趋势,最终与手术前相似。微切口组患者不同时点六角形细胞比例与同期常规切口组比较均显著升高,角膜内皮细胞变异系数与同期常规切口组比较均明显降低有统计学差异(P0.05)。两组患者手术前后不同时间点角膜内皮细胞密度、中央角膜厚度组间比较均无统计学差异(P0.05)。结论:超声乳化白内障手术后患者角膜内皮细胞损伤与手术切口有相关性,缩小手术面积的小切口手术使术后修复增快,安全有效,适宜临床推广。     

Role of adipose-derived stromal cells in pedicle skin flap survival in experimental animal models

The use of skin flaps in reconstructive surgery is the first-line surgical treatment for the reconstruction of skin defects and is essentially considered the starting point of plastic surgery. Despite their excellent usability, their application includes general surgical risks or possible complications, the primary and most common is necrosis of the flap. To improve flap survival, researchers have used different methods, including the use of adiposederived stem cells, with significant positive results. In our research we will report the use of adipose-derived stem cells in pedicle skin flap survival based on current literature on various experimental models in animals.     

Nanofiber-expanded stem cells mitigate liver fibrosis: Experimental study

ObjectivesThis study examines a pretreatment strategy to strengthen the hepatic lineage divergence of mesenchymal stem cells (MSCs).Design and methodsBMSCs were expanded in the presence or absence of nanofiber (NF) and treated with growth factors (GF) prior to transplantation. Thioacetamide (TA) was used for liver fibrosis induction and transplantation of NF-expanded BMSCs was compared biochemically and histologically to the cells expanded without NF scaffold.ResultsThe ultraweb NF caused better proliferation and characterization of MSCs. MSCs transplantation significantly improved liver functions, increased hepatic HGF and Bcl-2 levels, whereas decreased serum fibronectin, hepatic TNF-α and TGF-β1 levels. Hepatic HNF4α, FOXa2, CYP7a1 genes expression were enhanced while β-5-Tub and AFP genes expression were depressed. Histological study documented these results. Differentiated NF-MSCs showed pronounced enhancement of the aforementioned parameters as compared to differentiated MSCs in the absence of NF.Conclusionpretreatment with growth factors in the presence of NF augment homing, repopulation and hepatic differentiation abilities of MSCs and proves to be a promising approach for the treatment of liver fibrosis.     

New N-benzhydrylpiperazine/1,3,4-oxadiazoles conjugates inhibit the proliferation,migration, and induce apoptosis in HeLa cancer cells via oxidative stress–mediated mitochondrial pathway

N-benzhydrylpiperazine and 1,3,4-oxadiazoles are pharmacologically active scaffolds which exhibits significant inhibitory growth effects against various cancer cells, however, antiproliferation effects and the underlying mechanism for inducing apoptosis for aforementioned scaffolds addressing HeLa cancer cells remains uncertain. In this study, N-benzhydrylpiperazine clubbed with 1,3,4-oxadiazoles ( 4a–4h ) were synthesized, subsequently characterized using high resolution spectroscopic techniques and eventually evaluated for their antiproliferation potential by inducing apoptosis in HeLa cancer cells. The MTT assay screening results revealed that among all, compound 4d ( N-benzhydryl-4-((5-(4-aminophenyl)-1,3,4-oxadiazol-2-yl)methyl)piperazine) in particular, exhibited IC ₅₀ value of 28.13 ± 0.21 μg/mL and significantly inhibited the proliferation of HeLa cancer cells in concentration-dependent manner. The in vitro anticancer assays for treated HeLa cells resulted in alterations in the cell morphology, reduction in colony formation, and inhibition of cell migration in concentration-dependent treatment. Furthermore, G2/M phase arrest, variations in the nuclear morphology, degradation of chromosomal DNA confirmed the ongoing apoptosis in treated HeLa cells. Increase in the expression of cytochrome C and caspase-3 confirmed the involvement of intrinsic mitochondrial pathway regulating the cell death. Also, elevation in reactive oxygen species level and loss of mitochondrial membrane potential signified that compound 4d induced apoptosis in HeLa cells by generating the oxidative stress. Therefore, compound 4d may act as a potent chemotherapeutic agent against human cervical cancer.