Ales Bezrouk

The cytotoxic effect of α-tomatine in MCF-7 human adenocarcinoma breast cancer cells depends on its interaction with cholesterol in incubation media and does not involve apoptosis induction

In recent years, α-tomatine has been studied for its anticancer activity. In the present study, we focused on the cytotoxic effect of α-tomatine in the MCF-7 human breast adenocarcinoma cell line, its mechanism of action, biotransformation and stability in the culture medium. We observed an inhibition of cell proliferation and viability at concentrations of 6 and 9 μM but then a recovery of cells occurred. The recovery was not caused by the biotransformation of α-tomatine in MCF-7 cells, but by a substantial decrease in the concentration of α-tomatine in the culture medium due to its binding with cholesterol. Regarding the mechanism of action of α-tomatine, we observed no DNA damage, no changes in the levels of the proteins p53 and p21WAF1/Cip1, and no apoptosis (neither activated caspase-8 and -9, nor sub-G1 peak, or morphological signs). We found a loss of ATP in α-tomatine-treated cells. These results support the conclusion that α-tomatine does not induce apoptosis in the MCF-7 cell line.

Effective Method of Purification of Betulin from Birch Bark: The Importance of Its Purity for Scientific and Medicinal Use.

A new and relatively simple method for purification of betulin from birch bark extract was developed in this study. Its five purification steps are based on the differential solubility of extract components in various solvents and their crystallization and/or precipitation, on their affinity for Ca(OH)2 in ethanol, and on the affinity of some impurities for silica gel in chloroform. In addition, all used solvents can be simply recycled. Betulin of more than 99% purity can be prepared by this method with minimal costs. Various observations including crystallization of betulin, changes in crystals during heating, and attempt of localization of betulin in outer birch bark are also described in this work. The original extract, fraction without betulinic acid and lupeol, amorphous fraction of pure betulin, final crystalline fraction of pure betulin and commercial betulin as a standard were employed to determine the antiproliferative/cytotoxic effect. We used WST-1 tetrazolium-based assays with triple negative breast cancer cell line BT-549. The decrease in cell survival showed clear relationship with the purity of the samples, being most pronounced using our final product of pure crystalline betulin. WST-1 proliferation/cytotoxicity test using triple negative breast cancer cell line BT-549 clearly showed the importance of purity of betulin for biological experiments and, apparently, for its medicinal use.

Utilizing Autologous Multipotent Mesenchymal Stromal Cells and β-Tricalcium Phosphate Scaffold in Human Bone Defects: A Prospective, Controlled Feasibility Trial

The purpose of this prospective controlled study was to compare healing quality following the implantation of ultraporous β-tricalcium phosphate, containing either expanded autologous mesenchymal stromal cells (trial group, 9 patients) or β-tricalcium phosphate alone (control group, 9 patients), into femoral defects during revision total hip arthroplasty. Both groups were assessed using the Harris Hip Score, radiography, and DEXA scanning at 6 weeks and 3, 6, and 12 months postoperatively. A significant difference in the bone defect healing was observed between both groups of patients (P < 0.05). In the trial group, trabecular remodeling was found in all nine patients and in the control group, in 1 patient only. Whereas, over the 12-month follow-up period, no significant difference was observed between both groups of patients in terms of the resorption of β-tricalcium phosphate, the significant differences were documented in the presence of radiolucency and bone trabeculation through the defect (P < 0.05). Using autologous mesenchymal stromal cells combined with a β-tricalcium phosphate scaffold is a feasible, safe, and effective approach for management of bone defects with compromised microenvironment. The clinical trial was registered at the EU Clinical Trials Register before patient recruitment has begun (EudraCT number 2012-005599-33).

Directed reprogramming of comprehensively characterized dental pulp stem cells extracted from natal tooth

The aim of this study was to extensively characterise natal dental pulp stem cells (nDPSC) and assess their efficiency to generate human induced pluripotent stem cells (hiPSC). A number of distinguishing features prompted us to choose nDPSC over normal adult DPSC, in that they differed in cell surface marker expression and initial doubling time. In addition, nDPSC expressed 17 out of 52 pluripotency genes we analysed, and the level of expression was comparable to human embryonic stem cells (hESC). Ours is the first group to report comprehensive characterization of nDPSC followed by directed reprogramming to a pluripotent stem cell state. nDPSC yielded hiPSC colonies upon transduction with Sendai virus expressing the pluripotency transcription factors POU5F1, SOX2, c-MYC and KLF4. nDPSC had higher reprogramming efficiency compared to human fibroblasts. nDPSC derived hiPSCs closely resembled hESC in terms of their morphology, expression of pluripotency markers and gene expression profiles. Furthermore, nDPSC derived hiPSCs differentiated into the three germ layers when cultured as embryoid bodies (EB) and by directed differentiation. Based on our findings, nDPSC present a unique marker expression profile compared with adult DPSC and possess higher reprogramming efficiency as compared with dermal fibroblasts thus proving to be more amenable for reprogramming.

Ionizing radiation increases primary cilia incidence and induces multiciliation in C2C12 myoblasts

Primary cilia act as physical‐chemical sensors and their functions include the perception of the extracellular milieu, regulation of organogenesis, and cell polarity. In general, these cells are monociliated and the single cilium possesses diverse receptors and channels which are involved in morphogenesis and growth signaling, and are, therefore, important for cell proliferation and differentiation. In this study, we used an in vitro model of C2C12 myoblasts to evaluate the effect of DNA damage induced by gamma ionizing radiation on primary cilia incidence. A significantly higher number of ciliated cells were observed after 1 day post‐irradiation with 2–20 Gy when compared with non‐irradiated cells. After 3 days post‐irradiation, the cilia incidence in cells had decreased slightly when treated with 2, 6, and 10 Gy, although an increase in incidence rate was observed in cells treated with 20 Gy. Multi‐ciliated cells were also detected in myoblasts irradiated with 10 and 20 Gy but not in non‐irradiated cells or after low irradiation (2–6 Gy). Irradiation also caused a dose‐dependent decrease in cell viability and proliferation and corresponding cell cycle arrest. Furthermore, an activation of caspases 3/7, 8, and 9 was observed after higher radiation (10 and 20 Gy) with increased apoptosis. Together, our results show that irradiation by gamma rays promotes myoblast ciliogenesis, with pronounced effects observed after 3 days post‐irradiation. We conclude that irradiation doses of 10 and 20 Gy are sufficient to induce cell death and are responsible for the formation of multiple cilia originating from multiple basal bodies.

Transepidermální absorpce polycyklických aromatických uhlovodíků

Polycyklické aromatické uhlovodíky (PAU) představují skupinu významných kontaminantů pracovního i životního prostředí. Při expozici PAU v pracovním prostředí se vedle inhalační expoziční cesty může významně uplatňovat i expozice dermální. Stávající experimentální údaje o intenzitě a rychlosti penetrace látek do systémové cirkulace jsou zatím omezené. Předkládaný článek se zabývá metodickou a interpretační problematikou transepidermální absorpce PAU in vitro. Byla sledována intenzita (Flux) a rychlost (Lag time) penetrace naftalenu, fenanthrenu, pyrenu a benzo[a]pyrenu přes epidermální membránu z prasečího boltce. Experiment byl prováděn ve vertikálních statických difuzních komůrkách dle Franze (n = 32) a jako rozpouštědlo byl použit isopropyl-myristát. Parametr Flux (nmol/cm2/hod) dosáhl hodnot 95,7 ± 45,5 u naftalenu, 19,5 ± 8,7 u fenanthrenu, 4,38 ± 1,98 u pyrenu a 0,21 ± 0,08 u benzo[a]pyrenu. Parametr Lag time (hod) hodnot 0,26 ± 0,17 u naftalenu, 2,12 ± 0,41 u fenanthrenu, 3,25 ± 0,50 u pyrenu a 11,2 ± 4,08 u benzo[a]pyrenu. Hodnota parametru Flux klesala s molární hmotností PAU, zatímco hodnota parametru Lag time s molární hmotností PAU stoupala. Množství PAU, které za daný časový úsek penetrovalo přes epidermální membránu, se pohybovalo mezi 0,24 % (benzo[a]pyren) až 0,84 % (fenanthren) aplikované dávky. Penetrace PAU přes epidermální membránu vykazovala, v porovnání s experimenty na plné kůži, nižší stupeň variability dat. Z výsledků vyplývá, že použití epidermální membrány by mohlo zpřesňovat jak odhad vnitřní dávky PAU po dermální expozici, tak i odhad souvisejícího zdravotního rizika v rámci konzervativního expozičního scénáře. Experimenty s epidermální membránou jsou však časově i experimentálně náročné, bez možnosti objektivní kontroly integrity epidermální membrány, což může vést k finanční náročnosti testování, ztrátám vzorků a v neposlední řadě i k nárůstu rozdílů hodnot dat, získaných v různých laboratořích. Klíčová slova: polycyklické aromatické uhlovodíky (PAU) – dermální absorpce, epidermální membrána, experimenty TRANSEPIDERMÁLNÍ ABSORPCE POLYCYKLICKÝCH AROMATICKÝCH UHLOVODÍKŮ

Comparative Study on the Application of Mesenchymal Stromal Cells Combined with Tricalcium Phosphate Scaffold into Femoral Bone Defects

This prospective study sought to evaluate the healing quality of implanted ultraporous β-tricalcium phosphate sown with expanded autologous mesenchymal stromal cells (MSCs) into femoral defects during revision hip arthroplasty. A total of 37 osseous defects in 37 patients were treated and evaluated concerning bone regeneration. Nineteen subjects received β-tricalcium phosphate graft material serving as a carrier of expanded autologous MSCs (the trial group A), nine subjects received β-tricalcium phosphate graft material only (the study group B) and nine subjects received cancellous allografts only (the control group C). Clinical and radiographic evaluations were scheduled at 6 weeks, 3, 6, and 12 months post-operatively, and performed at the most recent visit as well. All observed complications were recorded during follow-up to assess the use of an ultraporous β-tricalcium phosphate synthetic graft material combined with expanded MSCs in bone defect repair. The resulting data from participants with accomplished follow-up were processed and statistically evaluated with a Freeman–Halton modification of the Fischer’s exact test, a P < 0.05 value was considered to be significant. Whereas no significant difference was observed between the trial group A with β-tricalcium phosphate synthetic graft material serving as a carrier of expanded autologous MSCs and control group C with cancellous impaction allografting in terms of the bone defect healing, significant differences were documented between the study group B with β-tricalcium phosphate graft material only and control group C. Regarding adverse effects, six serious events were recorded during the clinical trial with no causal relationship to the cell product. β-tricalcium phosphate synthetic graft material serving as a carrier of expanded autologous MSCs appears safe and promotes the healing of bone defects in a jeopardized and/or impaired microenvironment. This clinical trial was registered at the EU Clinical Trials Register before patient recruitment (Registration number: EudraCT number 2012-005599-33; Date of registration: 2013-02-04).

SAMPA: A free software tool for skin and membrane permeation data analysis

Skin and membrane permeation experiments comprise an important step in the development of a transdermal or topical formulation or toxicological risk assessment. The standard method for analyzing these data relies on the linear part of a permeation profile. However, it is difficult to objectively determine when the profile becomes linear, or the experiment duration may be insufficient to reach a maximum or steady state. Here, we present a software tool for Skin And Membrane Permeation data Analysis, SAMPA, that is easy to use and overcomes several of these difficulties. The SAMPA method and software have been validated on in vitro and in vivo permeation data on human, pig and rat skin and model stratum corneum lipid membranes using compounds that range from highly lipophilic polycyclic aromatic hydrocarbons to highly hydrophilic antiviral drug, with and without two permeation enhancers. The SAMPA performance was compared with the standard method using a linear part of the permeation profile and a complex mathematical model. SAMPA is a user-friendly, open-source software tool for analyzing the data obtained from skin and membrane permeation experiments. It runs on a Microsoft Windows platform and is freely available as a Supporting file to this article.