ALBERT

Description: Two dimensional (2D) nanocrystals of noble metals (e.g., Au, Ag, Pt) often have unique structural and environmental properties which make them useful for applications in electronics, optics, sensors and biomedicines. In recent years, there has been a focus on discovering the fundamental mechanisms which govern the synthesis of the diverse geometries of these 2D metal nanocrystals (e.g., shapes, thickness, and lateral sizes). This has resulted in being able to better control the properties of these 2D structures for specific applications. In this review, a brief historical survey of the intrinsic anisotropic properties and quantum size effects of 2D noble metal nanocrystals is given and then a summary of synthetic approaches to control their shapes and sizes is presented. The unique properties and fascinating applications of these nanocrystals are also discussed.

Description: Foamable high melt strength polypropylene (HMSPP) was prepared by grafting styrene (St) onto polypropylene (PP) and simultaneously introducing polydimethylsiloxane (PDMS) through a one-step melt extrusion process. The effect of PDMS viscosity on the foaming behavior of HMSPP was systematically investigated using supercritical CO 2 as the foaming agent. The results show that the addition of PDMS has little effect on the grafting reaction of St and HMSPP exhibits enhanced elastic response and obvious strain hardening effect. Though the CO 2 solubility of HMSPP with PDMS (PDMS-HMSPP) is lower than that of HMSPP without PDMS, especially for PDMS with low viscosity, the PDMS-HMSPP foams exhibit narrow cell size distribution and high cell density. The fracture morphology of PDMS-HMSPP shows that PDMS with low viscosity disperses more easily and uniformly in HMSPP matrix, leading to form small domains during the extrusion process. These small domains act as bubble nucleation sites and thus may be responsible for the improved foaming performance of HMSPP.

Description: The secretion of osteocalcin (OCN) is an excellent differentiation marker for the osteogenic differentiation. This study investigated the secretion of OCN during the osteogenic differentiation of DFCs. During the differentiation of DFCs the extracellular concentrations of OCN were higher in standard cell culture medium than in osteogenic differentiation medium. However, after 4 weeks in the osteogenic differentiation medium the extracellular OCN concentration decreased strongly, whereas the concentration remains high in the control medium. At this point in time DFCs formed connective tissue like structures with mineralized clusters and OCN. Real-time RT-PCR analyses and western-blot analyses proved that OCN was expressed in both cell culture media. However, the expression of the mRNA was inhibited in the osteogenic differentiation medium. These results suggest that DFCs secrete constitutively OCN into the cell culture medium and that the osteogenic differentiation medium suppresses the gene expression of OCN. Moreover, OCN imbeds into the extracellular matrix after the formation of connective tissue like structures, and the soluble OCN in the cell culture medium disappears. Hence, extracellular OCN in the cell culture medium is not a marker for the osteogenic differentiation of DFCs.

Description: Cardanol is a biobased raw material derived from cashew nut shell liquid. In order to extend its utility, new derivatives and additional applications are useful. In this work cardanol was first epoxidized, and a novel aniline derivative prepared from it under mild reaction conditions with the help of an ionic liquid catalyst. The reaction chemistry was studied by using nuclear magnetic resonance. The resulting aminohydrin adduct showed antioxidant property and should also be a useful synthon for further reactions. As an example, the aminohydrin was shown to undergo a condensation reaction with formaldehyde to form a prepolymer, which could be further reacted to form thermosetting resins.

Description: Silver nanoparticles (AgNPs) have been synthesized in the presence of polyacrylate through the reduction of silver nitrate by sodium borohydride in aqueous solution. The AgNO3 and polyacrylate carboxylate group concentrations were kept constant at 2.0 × 10 –4 and 1.0 × 10 –2 mol·L –1 , respectively, while the ratio of [NaBH 4 ]/[AgNO 3 ] was varied from 1 to 100. The ultraviolet-visible plasmon resonance spectra of these solutions were found to vary with time prior to stabilizing after 27 d, consistent with changes of AgNP size and distribution within the polyacrylate ensemble occurring. These observations, together with transmission electron microscopic results, show this rearrangement to be greatest among the samples at the lower ratios of [NaBH 4 ]/[AgNO 3 ] used in the preparation, whereas those at the higher ratios showed a more even distribution of smaller AgNP. All ten of the AgNP samples, upon a one thousand-fold dilution, catalyze the reduction of 4-nitrophenol to 4-aminophenol in the temperature range 283.2–303.2 K with a substantial induction time being observed at the lower temperatures.

Description: Lowland Anoa has become endangered due to hunting and human activity. Protection and breeding of endangered species in a controlled environment is the best way of conservation. However, it is not possible to adopt this approach for all endangered species because of the cost involved and the ever-increasing number of critically endangered species. In consideration of these limitations to the conventional conservation methods, we established a primary cell culture of endangered buffalo (Lowland Anoa, Bubalus quarlesi ), for the preservation of this biological resource. In addition, we introduced human derived, mutant cyclin dependent kinase 4 (CDK4), Cyclin D, and telomerase reverse transcriptase (TERT) into the primary cells. The successful introduction of these three genes was confirmed by western blot with specific antibodies, and enzymatic activity. We also showed that the expression of mutant CDK4, Cyclin D, and TERT allows us to efficiently establish an immortalized cell line, with an intact chromosome pattern, from Lowland Anoa. To the best of our knowledge, this study is the first investigation that established an immortalized cell line of an endangered wild animal species.

Description: Elevated bone loss induced by osteoclasts is a critical and most commonly observed pathological complication during osteolytic diseases such as osteoporosis. Hence, attenuation of osteoclast formation or function is a classical therapeutic approach to regulate bone loss. In this study, we found that ferulic acid (FA), a natural compound potently inhibited osteoclast formation in human CD14+ peripheral blood monocytes ex vivo with an IC 50 of 39 µM. Moreover, due to impaired differentiation of osteoclast progenitors, actin ring formation and bone resorption activity were also perturbed. Investigation of underlying molecular mechanisms revealed that FA inhibited the RANKL-induced expression of dendritic cell-specific transmembrane protein (DC-STAMP), a critical regulator of osteoclast fusion. In addition, expression of matrix metalloproteinase-9 (MMP-9) and cathepsin K, the key osteoclast specific lysosomal proteases involved in bone matrix resorption were severely aggravated by FA. A significant reduction in mature osteoclast numbers was detected in the presence of FA accompanied by increased caspase-3 activity and DNA-fragmentation, a characteristic hallmark of apoptosis. Collectively, these results suggested that FA inhibited osteoclast fusion by suppressing the expression of DC-STAMP and induced apoptosis in mature osteoclasts by the caspase-3 pathway.

Description: To ensure cell-based assays are performed properly, both cell concentration and viability have to be determined so that the data can be normalized to generate meaningful and comparable results. Cell-based assays performed in immuno-oncology, toxicology, or bioprocessing research often require measuring of multiple samples and conditions, thus the current automated cell counter that uses single disposable counting slides is not practical for high-throughput screening assays. In the recent years, a plate-based image cytometry system has been developed for high-throughput biomolecular screening assays. In this work, we demonstrate a high-throughput AO/PI-based cell concentration and viability method using the Celigo image cytometer. First, we validate the method by comparing directly to Cellometer automated cell counter. Next, cell concentration dynamic range, viability dynamic range, and consistency are determined. The high-throughput AO/PI method described here allows for 96-well to 384-well plate samples to be analyzed in less than 7 min, which greatly reduces the time required for the single sample-based automated cell counter. In addition, this method can improve the efficiency for high-throughput screening assays, where multiple cell counts and viability measurements are needed prior to performing assays such as flow cytometry, ELISA, or simply plating cells for cell culture.

Description: Oral cancer is the eleventh most prevalent cancer worldwide. The most prevalent oral cancer is oral squamous cell carcinoma (OSCC). Damnacanthal (DAM) and nordamnacanthal (NDAM), the anthraquinone compounds, are isolated from the root of Morinda citrifolia L. (Noni), which has been used for the treatment of several chronic diseases including cancer. The objectives of this study were to evaluate the cytotoxicity, cell death mode, cell cycle, and the molecular mechanism of apoptosis induced by DAM and NDAM on OSCC. The cytotoxic effects of these compounds against OSCC cell lines were determined by MTT assay. The cell death mode was analysed by DNA laddering and FITC-annexin V/PI flow cytometric assays. In addition, the mechanism of apoptosis induced by DAM and NDAM was detected using mitochondrial membrane potential, Cytochrome c, and caspases assays. Finally, the effect of DAM and NDAM on cell cycle phase distribution of OSCC cells was detected by flow cytometry. In the present study, DAM and NDAM showed cytotoxicity towards OSCC cell lines and the maximum growth inhibition for both compounds was observed in H400 cells with IC 50 value of 1.9 and 6.8 μg/ml, respectively, after 72 h treatment. The results also demonstrated the inhibition of H400 OSCC cells proliferation, internucleosomal cleavage of DNA, activation of intrinsic apoptosis pathway, and cell cycle arrest caused by DAM and NDAM. Therefore, these findings suggest that DAM and NDAM can be potentially used as antitumor agents for oral cancer therapy.

Description: While drug resistance appears to be an inevitable problem of an increasing number of anticancer drugs in monotherapy, combination drug therapy has become a prosperous method to reduce the administered total drug dosages as well as overcome the drug resistance of carcinoma cells. Curcumin, considered to possess multifaceted roles in cancer treatment according to its multiple anti-neoplastic mechanisms as a depressor of chemoresistance, can significantly facilitate its anti-cancer functions and improve therapeutic effects via combination usage with a variety of other drugs with different reaction mechanisms. To explore this possibility, four anti-cancer chemotherapeutic agents that all possess a certain degree of drug resistance problems, including three tyrosine kinase inhibitors (erlotinib, sunitinib and sorafenib) that are acting on different cell pathways and a typical anticancer drug doxorubicin, were combined with curcumin individually to examine the synergistic anti-tumor effect both in vitro and in vivo . Results revealed that sunitinib combined with curcumin at the molar ratio of 0.46 yielded the most potent synergistic effect in vitro , and was therefore chosen for further animal evaluation. To further enhance the anticancer effect, bovine serum albumin (BSA) nanoparticles were utilized as a carrier to deliver the selected drug combination in situ . Preliminary in vivo findings confirmed our hypothesis of being able to maintain a similar injected drug ratio for prolonged time periods in tested animals by our approach, thereby maximizing the therapeutic potency yet minimizing the toxicity of these drugs. This work could open up a new avenue on combination drug therapy and realization the clinical utility of such drugs.

Description: With the aim to utilize human mesenchymal stem cells (hMSCs) grown in large scale for regenerative medicine, effects of agitation rate on aggregation during beads-to-beads subcultivation of microcarrier culture of hMSCs were studied. hMSCs could attach and grew on surface-type microcarriers of Cytodex 1, whereas almost no cell elongation and growth were observed on porous type microcarriers of Cytopores. The percentages of aggregated Cytodex 1 microcarriers at an agitation rate of 60 and 90 rpm were lower than that at 30 rpm, which was the lowest agitation rate necessary for the suspension of Cytodex 1 microcarriers, and the cells grew fastest at 60 rpm. hMSC could be subcultivated on Cytodex 1 by the beads-to-beads method at both 30 and 60 rpm without trypsinization. However, agitation at 60 rpm resulted in a markedly lower percentage of aggregated microcarriers not only before but also after subcultivation. The percentages of CD90- and CD166-positive cells among cells grown on Cytodex 1 at 60 rpm (91.5 and 87.6 %) were comparable to those of cells grown in the pre-culture on dishes. In conclusion, hMSCs could be subcultivated on Cytodex 1 by beads-to-beads method maintaining the expressions of the cell surface antigens CD90 and CD166, while adjusting agitation rate could decrease the microcarrier aggregation.

Description: The health benefits of Mediterranean diet has long been reported and attributed to the consumption of virgin olive oil (VOO). Here, we evaluated the neuroprotective effect of VOO against Alzheimer’s disease by determining its effect on β-amyloid (Aβ)-induced cytotoxicity and oxidative stress, and explored the possibility that its hydroxycinnamic acids (Hc acids) content contribute significantly to this effect. SH-SY5Y cells treated with or without Aβ and with VOO or Hc acids (mixture of p- coumaric acid, ferulic acid, vanillic acid, and caffeic acid) were subjected to MTT assay and the results showed that both samples alleviated Aβ-induced cytotoxicity. Furthermore, both VOO and Hc acids decreased the reactive oxygen species level. Using western blot to determine the effect of these samples on Aβ-induced activation of pERK1/2, p38, and JNK MAPKs, results revealed that both VOO and Hc acids inhibited the activation of pERK1/2 and p-p38 MAPK, but not JNK. Moreover, VOO upregulated the glycolytic enzymes genes hexokinase ( HK1), and phosphofructokinase ( PFKM ) expression which means that VOO enhanced the energy metabolism of the neurotypic cells, and therefore suggests another mechanism by which VOO could provide protection against Aβ-induced cytotoxicity. The findings in this study suggest that VOO has a neuroprotective effect, attributable to its hydroxycinnamic acids component, against Aβ-induced cytotoxicity and oxidative stress through the inhibition of the activation of MAPKs ERK and p38 and by enhancing the energy metabolism of the neurotypic cells.

Description: Endothelial cell activation, injury and dysfunction have been regarded as one of the initial key events in the pathogenesis of atherosclerosis. Lipopolysaccharide (LPS), an important mediator of inflammation, can cause endothelial cell damage and apoptosis. Naringin (Nar), one major flavanone glycoside from citrus fruits, shows various pharmacological actions, but the effect of Nar on LPS-induced damage in human umbilical vein endothelial cells (HUVECs) remains unknown. The present results showed that Nar significantly improved the survival rate of HUVECs, and decreased reactive oxygen species and intracellular Ca 2+ levels caused by LPS compared with model group. In addition, Nar obviously decreased cytochrome c release from mitochondria into cytosol. Moreover, Nar significantly down-regulated the protein or mRNA levels of IL-1, IL-6, TNF-α, VCAM-1, ICAM-1, NF-κB, AP-1, cleaved-3,-7,-9, p53, Bak and Bax, and up-regulated the expressions of Bcl-xl, Bcl-2 to suppress inflammation and apoptosis. Furthermore, Nar obviously inhibited phosphorylation levels of JNK, ERK and p38 MAPK. In conclusion, Nar exhibited potent effects against LPS-induced damage in HUVECs through the modulation of oxidative stress, inflammation, apoptosis and MAPK pathways, which should be developed as a potent candidate for the treatment of atherosclerosis in the future.

Description: Animal cells in suspension experience shear stress in different situations such as in vivo due to hemodynamics, or in vitro due to agitation in large-scale bioreactors. Shear stress is known to affect cell physiology, including binding and uptake of extracellular cargo. In adherent cells the effects of exposure to shear stress on particle binding kinetics and uptake have been studied. There are however no reports on the effect of shear stress on extracellular cargo delivery to suspension cells. In this study, we have evaluated the effect of shear stress on transfection of CHO-S cells using Lipofectamine 2000 in a simple flow apparatus. Our results show decreased cell growth and transfection efficiency upon lipoplex assisted transfection of CHO-S while being subjected to shear stress. This effect is not seen to the same extent when cells are exposed to shear stress in absence of the lipoplex complex and subsequently transfected, or if the lipoplex is subjected to shear stress and subsequently used to transfect the cells. It is also not seen to the same extent when cells are exposed to shear stress in presence of liposome alone, suggesting that the observed effect is dependent on interaction of the lipoplex with cells in the presence of shear stress. These results suggest that studies involving liposomal DNA delivery in presence of shear stress such as large scale transient protein expression should account for the effect of shear during lipoplex assisted DNA delivery.

Description: Modeling structural and thermodynamic properties of nucleic acids has long been a challenge in the development of force fields. Polarizable force fields are a new generation of potential functions to take charge redistribution and induced dipole into account, and have been proved to be reliable to model small molecules, polypeptides and proteins, but their use on nucleic acids is still rather limited. In this article, the interactions between nucleic acids and a small molecule or ion were modeled by AMOEBAbio09, a modern polarizable force field, and conventional non-polarizable AMBER99sb and CHARMM36 force fields. The resulting intermolecular interaction energies were compared with those calculated by ab initio quantum mechanics methods. Although the test is not sufficient to prove the reliability of the polarizable force field, the results at least validate its capability in modeling energetics of static configurations, which is one basic component in force field parameterization.

Description: A sol-gel technique has been developed for the synthesis of a magnetite-silica-titania (Fe 3 O 4 -SiO 2 -TiO 2 ) tertiary nanocomposite with improved photocatalytic properties based on the use of inexpensive titania and silica precursors. The exceptional photocatalytic activity of the resulting materials was demonstrated by using them to photocatalyze the degradation of methylene blue solution. The best formulation achieved 98% methylene blue degradation. An interesting feature of the present work was the ability to magnetically separate and reuse the catalyst. The efficiency of the catalyst remained high during two reuses. The synthesized nanomaterials were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, ultra-violet-visible spectroscopy, diffuse reflectance spectroscopy, and thermogravimetric analysis. XRD analysis revealed the formation of multicrystalline systems of cubic magnetite and anatase titania crystals. SEM and TEM characterization revealed well-developed and homo-geneously dispersed particles of size less than 15 nm. FTIR spectra confirmed the chemical interaction of titania and silica. It was further noticed that the optical properties of the prepared materials were dependent on the relative contents of their constituent metal oxides.

Description: Amphiphilic block copolymers (ABCs) assemble into a spherical nanoscopic supramolecular core/shell nanostructure termed a polymeric micelle that has been widely researched as an injectable nanocarrier for poorly water-soluble anticancer agents. The aim of this review article is to update progress in the field of drug delivery towards clinical trials, highlighting advances in polymeric micelles used for drug solubilization, reduced off-target toxicity and tumor targeting by the enhanced permeability and retention (EPR) effect. Polymeric micelles vary in stability in blood and drug release rate, and accordingly play different but key roles in drug delivery. For intravenous (IV) infusion, polymeric micelles that disassemble in blood and rapidly release poorly water-soluble anticancer agent such as paclitaxel have been used for drug solubilization, safety and the distinct possibility of toxicity reduction relative to existing solubilizing agents, e.g., Cremophor EL. Stable polymeric micelles are long-circulating in blood and reduce distribution to non-target tissue, lowering off-target toxicity. Further, they participate in the EPR effect in murine tumor models. In summary, polymeric micelles act as injectable nanocarriers for poorly water-soluble anticancer agents, achieving reduced toxicity and targeting tumors by the EPR effect.

Description: The genotoxicity of copper oxychloride was investigated in human lymphocytes using chromosome aberration (CA) and micronucleus (MN) tests and the randomly amplified polymorphic DNA-polymerase chain reaction technique. The lymphocytes were treated with 3, 6, and 12 µg/mL of copper oxychloride for 24 and 48 h. Copper oxychloride increased CA and abnormal cells in a dose-dependent manner. The frequency of MN and micronucleated binuclear cells also increased at all concentrations and treatment periods. However, copper oxychloride cytotoxicity, observed through lower mitotic and nuclear division index, was significantly lower only at the higher concentrations (6 and 12 µg/mL). Copper oxychloride increased the polymorphic bands and decreased genomic template stability. In conclusion, in this study it was confirmed that copper oxychloride has genotoxic potential for human lymphocytes in vitro. Additionally, caution is advised for its use as a fungicide, because it may increase the risk of exposure through the food chain.

Description: Endothelial cells are constantly exposed to blood flow and the resulting frictional force, the wall shear stress, varies in magnitude and direction with time, depending on vasculature geometry. Previous studies have shown that the structure and function of endothelial cells, and ultimately of the vessel wall, are deeply affected by the nature of wall shear stress waveforms. To investigate the in vitro effects of these stimuli, we developed a compact, programmable, real-time operated system based on cone-and-plate geometry, that can be used within a standard cell incubator. To verify the capability to replicate realistic shear stress waveforms, we calculated both analytically and numerically to what extent the system is able to correctly deliver the stimuli defined by the user at plate level. Our results indicate that for radii greater than 25 mm, the shear stress is almost uniform and directly proportional to cone rotation velocity. We further established that using a threshold of 10 Hz of wall shear stress waveform frequency components, oscillating flow conditions can be reproduced on cell monolayer surface. Finally, we verified the capability of the system to perform long-term flow exposure experiments ensuring sterility and cell culture viability on human umbilical vein endothelial cells exposed to unidirectional and oscillating shear stress. In conclusion, the system we developed is a highly dynamic, easy to handle, and able to generate pulsatile and unsteady oscillating wall shear stress waveforms. This system can be used to investigate the effects of realistic stimulations on endothelial cells, similar to those exerted in vivo by blood flow.

Description: The platelet is a component of blood that functions to initiate blood clotting. Abnormal platelet count and function can lead to a life-threatening condition caused by excessive bleeding. At present, platelet supply for transfusion can be obtained only from platelet donation. However, platelets cannot be stored for longer than 7 days, meaning that routine isolation is required to maintain platelet supply for transfusion. To mitigate for potential platelet shortages, several strategies have been proposed to generate platelets ex vivo. By employing both of natural and artificial approaches, several researchers have successfully generated biomaterials with characteristics similar to human-derived platelets. Their reports indicated that the biomaterials could mimic the aggregation of human-isolated platelets, further suggesting the possibility to substitute or complement human-isolated platelets. The current review summarizes the progress in ex vivo platelet production and gives a prospect for the possible approaches to achieving a feasible platelet factory, based on the Good Manufacturing Practice standards.

Description: The aim of this study was to establish a tree shrew metabolic syndrome model and demonstrate the utility of MSCs in treating metabolic syndrome. We used tree shrew umbilical cord mesenchymal stem cell (TS-UC-MSC) transplantation for the treatment of metabolic syndrome to demonstrate the clinical application of these stem cells and to provide a theoretical basis and reference methods for this treatment. Tree shrew metabolic syndrome model showed significant insulin resistance, high blood sugar, lipid metabolism disorders, and hypertension, consistent with the diagnostic criteria. TS-UC-MSC transplantation at 16 weeks significantly reduced blood sugar and lipid levels, improved insulin resistance and the regulation of insulin secretion, and reduced the expression levels of the pro-inflammatory cytokines IL-1 and IL-6 ( P  〈 0.05). The transplanted TS-UC-MSCs targeted the liver, kidney and pancreas; reduced liver cell degeneration, necrosis, and inflammatory exudation; mitigated bleeding congestion and inflammatory cell infiltration in the kidney; and reduced islet cell degeneration and necrosis. We successfully developed a tree shrew metabolic syndrome model and showed that MSC migrate in diseased organs and can attenuate metabolic syndrome severity in a tree shrew model.

Description: Enterolactone (ENL) is formed by the conversion of dietary precursors like strawberry lignans via the gut microbiota. Urinary concentrations of lignan metabolites are reported to be significantly associated with a lower risk of Type 2 diabetes (T2D). In the present study, antidiabetic effect of ENL and its modes of action were studied in vitro and in vivo employing a rat skeletal muscle-derived cell line, L6 myocytes in culture, and T2D model db/db mice. ENL dose-dependently increased glucose uptake in L6 myotubes under insulin absent condition. This increase by ENL was canceled by compound C, an inhibitor of 5′-adenosine monophosphate-activated protein kinase (APMK). Activation (=phosphorylation) of AMPK and translocation of glucose transporter 4 (GLUT4) to plasma membrane in L6 myotubes were demonstrated by Western blotting analyses. Promotion by ENL of GLUT4 translocation to plasma membrane was also visually demonstrated by immunocytochemistry in L6 myoblasts that were transfected with glut4 cDNA-coding vector. T2D model db/db mice were fed the basal 20 % casein diet (20C) or 20C supplemented with ENL (0.001 or 0.01 %) for 6 weeks. Fasting blood glucose (FBG) levels were measured every week and intraperitoneal glucose tolerance test (IPGTT) was conducted. ENL at a higher dose (0.01 % in 20C) suppressed the increases in FBG levels. ENL was also demonstrated to improve the index of insulin resistance (HOMA-IR) and glucose intolerance by IPGTT in db/db mice. From these results, ENL is suggested to be an antidiabetic chemical entity converted from dietary lignans by gut microbiota.

Description: In the recent years, the possibility of utilizing extracellular vesicles for drug delivery purposes has been investigated in various models, suggesting that these vesicles may have such potential. In addition to the choice of donor cell type for vesicle production, a major obstacle still exists with respect of loading the extracellular vesicles efficiently with the drug of choice. One of the proposed solutions to this problem has been drug loading by electroporation, where small pores are created in the membrane of the extracellular vesicles, hereby allowing for free diffusion of the drug compound into the interior of the vesicle. We investigated the utility of adipose-derived stem cells (ASCs) as an efficient exosome donor cell type with a particular focus on the treatment of glioblastoma multiforme (GBM). In addition, we evaluated electroporation-induced effects on the ASC exosomes with respect to their endogenous potential of stimulating GBM proliferation, and morphological changes to single and multiple ASC exosomes. We found that electroporation does not change the endogenous stimulatory capacity of ASC exosomes on GBM cell proliferation, but mediates adverse morphological changes including aggregation of the exosomes. In order to address this issue, we have successfully optimized the use of a trehalose-containing buffer system as a way of maintaining the structural integrity of the exosomes.

Description: Monoclonal antibodies and antibody fragments are used for diverse diagnostic and therapeutic applications. We have investigated the secretory production of Fab fragments from insect cells cotransfected with plasmid vectors carrying heavy- and light-chain genes. In the present study, to promote the formation of the disulfide bond between the heavy and light chains, some positively charged amino acid residues were introduced near the cysteine residue for the disulfide bond at the C-terminus of C L , while some negatively charged amino acid residues were added near the cysteine residue for the disulfide bond at the C-terminus of C H1 . This electrostatic steering led to an increase in Fab secretions from insect cells.

Description: Brown bears ( Ursus arctos ) exhibit hyperphagia each fall and can become obese in preparation for hibernation. They do this without displaying the physiological problems typically seen in obese humans, such as Type 2 diabetes and heart disease. The study of brown bear hibernation biology could therefore aid in the development of novel methods for combating metabolic diseases. To this end, we isolated mesenchymal stem cells from subcutaneous fat biopsies, and culture methods were developed to differentiate these into the adipogenic lineage. Biopsies were taken from 8 captive male (N = 6) and female (N = 2) brown bears, ages 2–12 years. Plastic adherent, fibroblast-like cells were proliferated and subsequently cryopreserved or differentiated. Differentiation conditions were optimized with respect to fetal bovine serum content and time spent in differentiation medium. Cultures were characterized through immunostaining, RT-qPCR, and Oil red O staining to quantify lipid accumulation. Adiponectin, leptin, and glycerol medium concentrations were also determined over the course of differentiation. The culturing protocol succeeded in generating hormone-sensitive lipase-expressing, lipid-producing white-type adipocytes (UCP1 negative). Serum concentration and time of exposure to differentiation medium were both positively related to lipid production. Cells cultured to low passage numbers retained similar lipid production and expression of lipid markers PLIN2 and FABP4. Ultimately, the protocols described here may be useful to biologists in the field investigating the health of wild bear populations and could potentially increase our understanding of metabolic disorders in humans.

Description: The need to have a better and safer culture condition for expansion of human mesenchymal stem cells (MSCs) is crucial particularly to prevent infection and immune rejection. This is normally associated with the use of animal-based serum in the culture media for cell expansion. The aim of this study is to investigate alternative culture conditions which may provide better and safer environment for cell growth. In the present study, human adipose-derived stem cells (ASCs) at passage 3 were subjected to treatment in 4 conditions: (1) 21 % O 2 with fetal bovine serum (FBS), (2) 21 % O 2 without FBS, (3) 2 % O 2 with FBS and (4) 2 % O 2 without FBS followed by subsequent analysis of their phenotype, viability and functionality. We observed that ASCs cultured in all conditions present no significant phenotypic changes. It was found that ASCs cultured in 2 % O 2 without serum showed an increase in viability and growth to a certain extent when compared to those cultured in 21 % O 2 without serum. However, ASCs cultured in 2 % O 2 without serum displayed a relatively low adipogenic and osteogenic potential. On the other hand, interestingly, there was a positive enhancement in chondrogenic differentiation of ASCs cultured in 21 % O 2 without serum. Our findings suggest that different culture conditions may be suitable for different indications. In summary, ASCs cultured in serum-free condition can still survive, proliferate and undergo subsequent adipogenic, osteogenic and chondrogenic differentiation. Therefore, FBS is feasible to be excluded for culture of ASCs, which avoids clinical complications.

Description: The goal of this study was to determine the effects of PGZ and MK886 on the mRNA expression of PPARα and other associated genes in MDA-MB-231 cells, and the biological mechanisms induced by both drugs were also assessed. The levels of PPARα mRNA expression in PGZ-treated and MK886-treated MDA-MB-231 cells were determined using real-time PCR; the growth inhibitory effects of PGZ and MK886 were determined using the trypan blue exclusion assay; the induction of apoptosis by PGZ and MK886 was determined using DNA fragmentation assay and real-time PCR; and the invasion of PGZ-treated and MK886-treated MDA-MB-231 cells was determined using the wound healing and transwell migration assays. In addition, we correlated the expression of PPARα mRNA with other genes, including PPARγ, FGF4 and 5LOX, in drug-treated MDA-MB-231 cells. Our results demonstrated that the treatment of MDA-MB-231 cells with PGZ increased the expression of PPARα/γ mRNA and that this expression could be inhibited by treatment with MK886. Both drugs reduced the viability of MDA-MB-231 cells independently of PPARα/γ mRNA expression but did not induce apoptosis. The wound caused by invasion was not healed by PGZ-treated MDA-MB-231 cells, but it was healed by MK886-treated cancer cells, indicating that the reduction of invasion in PGZ-treated MDA-MB-231 cells was eliminated by treatment with MK886, and this finding was validated by the transwell migration assay. This phenomenon might also be associated with the expression of PPARα/γ, FGF4 and 5LOX mRNA in the treated cancer cells. This study provides useful information regarding the mRNA expression levels of PPARα and other related genes in MDA-MB-231 cells. These genes could be attractive targets for reducing the invasion of breast cancer.

Description: In vitro studies about biomaterials biological properties are essential screening tests. Yet cell cultures encounter difficulties related to cell retention on material surface or to the observation of both faces of permeable materials. The objective of the present study was to develop a reliable in vitro method to study cell behavior on rigid and flexible/permeable biomaterials elaborating two specific insert-based systems (IBS-R and IBS-F respectively). IBS-R was designed as a specific cylindrical polytetrafluoroethylene (PTFE) system to evaluate attachment, proliferation and morphology of human gingival fibroblasts (HGFs) on grade V titanium and lithium disilicate glass-ceramic discs characteristics of dental prostheses. The number of cells, their covering on discs and their morphology were determined from MTS assays and microscopic fluorescent images after 24, 48 and 72 h. IBS-F was developed as a two components system to study HGFs behavior on guided bone regeneration polyester membranes. The viability and the membrane barrier effect were evaluated by metabolic MTS assays and by scanning electron microscopy. IBS-R and IBS-F were shown to promote (1) easy and rapid handling; (2) cell retention on biomaterial surface; (3) accurate evaluation of the cellular proliferation, spreading and viability; (4) use of non-toxic material. Moreover IBS-F allowed the study of the cell migration through degradable membranes, with an access to both faces of the biomaterial and to the bottom of culture wells for medium changing.

Description: Three-dimensional (3D) spheroids of mesenchymal stromal cells (MSC) have been demonstrated to improve a wide range of MSC features, such as multilineage potential, secretion of therapeutic factors, and resistance against hypoxic condition. Accordingly, they represent a promising tool in regenerative medicine for several biological and clinical applications. Many approaches have been proposed to generate MSC spheroids. They usually require specific generation systems, such as rotatory bioreactors or low-attachment plates, and each approach has its own disadvantages. Furthermore, an over-time analysis of morphological homogeneity and architectural stability of the spheroids generated is rarely provided. In this work we adapted the “pellet culture” method to obtain homogenous spheroids of MSC and maintain them in vitro for long term studies. We analysed their outer and inner structure over a 2-month period to provide morphological and architectural information regarding the spheroids generated. Quantitative and qualitative data were obtained using brightfield and confocal microscope imaging coupled to a computational analysis to estimate volume, sphericity, and jagging degree. In addition, histological evaluation was performed to more thoroughly assess the cellular composition and the internal architecture of the 3D spheroids. The results provided show that MSC spheroids generated with the proposed approach are homogeneous and stable, from both morphological and architectural points of view, for a period of at least 15 days, approximately between day 15 and day 30 after their generation. Accordingly, the approach proposed serves as a rapid, cost-effective, and efficient method to generate and maintain MSC spheroids using common entry-level laboratory equipment only.

Description: A palladium catalyst supported on 2-aminopyridine functionalized cellulose was synthesized and fully characterized by inductively coupled plasma atomic emission spectroscopy, transmission electron microscope, Fourier transform infrared spectroscopy, thermogravimetric analysis and X-ray photoelectron spectrometry. This catalyst can be applied in the Suzuki cross-coupling reaction of aryl halides with arylboronic acids in 50% ethanol to afford biaryls in good yields, and easily recycled by simple filtration after reaction without the loss of metal Pd.

Description: Dental pulp stem cells (DPSCs) are a type of mesenchymal stem cells, which have the self-renewal and multi-lineage differentiation potential, including chondrocytes, adipocytes, neural cells and osteoblasts. So they play a significant role in pulp repair and bone regeneration. Oncostatin M (OSM), one of the IL-6 family cytokines, inhibits adipogenic differentiation and stimulates osteogenic differentiation of human bone marrow mesenchymal stem cells. However, the effect of OSM on DPSCs is unclear. We found that OSM induced osteogenic differentiation of DPSCs, promoting matrix mineralization as measured by Alizarin Red S staining. OSM also increased expression of osteogenesis-associated gene products Alkaline phosphatase, Bone morphogenetic protein 2 (BMP2), Runt-related transcription factor 2 and Osteocalcin (OCN) as assessed by immunoblotting. We also found that OSM activated the Signal Transducer And Activator Of Transcription 3 (STAT3) pathway during the osteogenic differentiation of DPSCs. Blocking the osteogenic differentiation by silencing of STAT3 can significantly inhibit OSM-induced osteogenic differentiation of DPSCs and the expression of related genes, furthermore matrix mineralization was also suppressed. In summary, OSM promotes osteoblastic differentiation of DPSCs and osteogenesis-related genes expression through the JAK3/STAT3 signaling pathway which may be useful for the autologous transplantation of DPSCs.

Description: Hypertrophic growth is a response of the heart to increased mechanical load or physiological stress. Thereby, cardiomyocytes grow in length and/or width to maintain cardiac pump function. Major signaling pathways involved in cardiomyocyte growth and remodeling have been identified during recent years including calcineurin–NFAT and PI3K–Akt signaling. Modulation of these pathways is of certain interest for therapeutic treatment of cardiac hypertrophy. However, quantification and characterization of hypertrophy in response to different stimuli or modulators is difficult. This study aims to test different read-out systems for detection and quantification of differences in hypertrophic growth in response to prohypertrophic stimuli. Real-time impedance measurements allowed the detection of distinct differences in hypertrophic growth in response to endothelin, norepinephrine, phenylephrine or BIO, which were not observable by other methods such as flow cytometry. Endothelin treatment induced a rapid and strong peak in the impedance signal concomitant with a massive reorientation of the actin cytoskeleton. Changes in expression of hypertrophy-associated genes were detected and stabilization of β-catenin was identified as a common response to all hypertrophic stimuli used in this study. Hypertrophic growth was blocked by the PI3K/mTOR inhibitor PI-103.

Description: Great interests have arisen over the last decade in the development of hierarchically porous materials. The hierarchical structure enables materials to have maximum structural functions owing to enhanced accessibility and mass transport properties, leading to improved performances in various applications. Hierarchical porous materials are in high demand for applications in catalysis, adsorption, separation, energy and biochemistry. In the present review, recent advances in synthesis routes to hierarchically porous materials are reviewed together with their catalytic contributions.

Description: Indoxyl sulfate (IS) is a digestive intermediate product that is a known indicator of chronic kidney disease. Its toxicity has also been suggested to accelerate chronic kidney disease. Recently, mesenchymal stem cells (MSCs) have been confirmed as a potential treatment in kidney regeneration. To determine the universal alteration in gene expression, we combined high-throughput microarray technology and in vitro culture of adipose-derived mesenchymal stem cells at different doses of IS (20, 40, 60 mg/l). We found that indoxyl sulfate has a remarkable interconnection with stem cell and calcium/calmodulin-dependent kinase pathways. In vitro results showed that indoxyl sulfate exerts anti-proliferation and anti-migration effects on ADMSCs. In addition, IS effects lead to increase in apoptotic cells and cells arrested at the G1 phase. Moreover, MEF2-A, MEF2-D and CACNA1S expression significantly decreased after indoxyl sulfate treatment. It can be speculated that following treatment with indoxyl sulfate, the function of ADMSCs is decreased and ADMSCs’ ability to support renal tubule regeneration in chronic kidney disease patients may be lower.

Description: The effect of thermal pretreatment on the active sites and catalytic performances of PtSn/SiO 2 catalyst in acetic acid (AcOH) hydrogenation was investigated in this article. The catalysts were characterized by N 2 physical adsorption, X-ray diffraction, transmission electron microscopy, pyridine Fourier-transform infrared spectra, and H 2 -O 2 titration on its physicochemical properties. The results showed that Pt species were formed primarily in crystalline structure and no PtSn x alloy was observed. Meanwhile, with the increment of thermal pretreatment temperature, Pt dispersion showed a decreasing trend due to the aggregation of Pt particles. Simultaneously, the amount of Lewis acid sites was remarkably influenced by such thermal pretreatment owning to the consequent physicochemical property variation of Sn species. Interestingly, the catalytic activity showed the similar variation trend with that of Lewis acid sites, confirming the important roles of Lewis acid sites in AcOH hydrogenation. Moreover, a balancing effect between exposed Pt and Lewis acid sites was obtained, resulting in the superior catalytic performance in AcOH hydrogenation.

Description: Sequences of 5S ribosomal RNA (rRNA) are extensively used in fish cytogenomic studies, once they have a flexible organization at the chromosomal level, showing inter- and intra-specific variation in number and position in karyotypes. Sequences from the genome of Imparfinis schubarti (Heptapteridae) were isolated, aiming to understand the organization of 5S rDNA families in the fish genome. The isolation of 5S rDNA from the genome of I. schubarti was carried out by reassociation kinetics (C 0 t) and PCR amplification. The obtained sequences were cloned for the construction of a micro-library. The obtained clones were sequenced and hybridized in I. schubarti and Microglanis cottoides (Pseudopimelodidae) for chromosome mapping. An analysis of the sequence alignments with other fish groups was accomplished. Both methods were effective when using 5S rDNA for hybridization in I. schubarti genome. However, the C 0 t method enabled the use of a complete 5S rRNA gene, which was also successful in the hybridization of M. cottoides. Nevertheless, this gene was obtained only partially by PCR. The hybridization results and sequence analyses showed that intact 5S regions are more appropriate for the probe operation, due to conserved structure and motifs. This study contributes to a better understanding of the organization of multigene families in catfish’s genomes.

Description: Brazilian flora biodiversity has been widely investigated to identify effective and safe phytotherapeutic compounds. Among the investigated plant species, the Byrsonima genus exhibits promising biological activities. This study aimed at evaluating the cytotoxicity of B. correifolia , B. verbascifolia , B. fagifolia and B. intermedia extracts using different assays in two cell lines (primary gastric and HepG2 cells). The different extract concentrations effects on cell viability were assayed using the MTT, aquabluer, neutral red and LDH assays. Non-cytotoxic concentrations were selected to generate cell proliferation curves and to assess cell cycle kinetics by flow cytometry. Byrsonima extracts differentially affected cell viability depending on the metabolic cellular state and the biological parameter evaluated. B. fagifolia and B. intermedia extracts exhibited lower cytotoxic effects than B. correifolia and B. verbascifolia in all assays. The results obtained with LDH and flow cytometry assays were more reliable, suggesting that they can be useful in the screening for herbal medicine and to further characterize these extracts as phytotherapeutic compounds.

Description: In this study, 24 marine macroalgae collected from the coastline of Izmir Gulf were examined for their antioxidant activities and their effects on cell proliferation. Crude extracts were obtained from samples with cold methanol extraction. Antioxidant activity was evaluated as 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging activity and total phenolic content (TPC); growth inhibitory effects of the extracts were determined by using WST-8. Amongst the species, Polysiphonia denuata (Rhodophyta) and Cystoseira species ( Phaeophyceae ) have been noticed by their high DPPH radical scavenging activities and TPCs. As expected, there was a strong correlation between these tests. Dictyota dichotoma ( Phaeophyceae ) showed the highest anti-cancer activity on MCF-7 cells with an IC 50 of 17.2 ng mL −1 . Flow cytometry analyses demonstrated that D. dichotoma methanolic extract strongly induced apoptosis. This extract exhibited moderate viability inhibition on MCF10A cells (IC 50 : 49.3 ng mL −1 ), suggesting a potential use of the extracts or its compounds for cancer therapy. There was no correlation between anti-cancer potential and antioxidant content of the extracts.

Description: Acid ceramidases are enzymes with a vital role in metabolizing ceramide to sphingosine-1-phosphate that is an antiproliferative metabolite in the ceramide pathway. Inhibition of exogenous ceramides with ceramidase inhibitors lead to augmented ceramide levels in cells and in turn lead to cell cycle arrest and apoptosis. Our study aimed at targeting ceramide metabolic pathway to induce apoptosis in human breast cancer cell line (MCF7) and we examined the antiproliferative and apoptotic activities of ceranib-2, an inhibitor of human ceramidase, on this cell line as well ultrastructural and mophological changes. Methods used for our examinations in this study were the colorimetric MTT assay, Annexin V/Propidium iodide and JC-1 staining, transmission electron microscopy and confocal microscopy. Ceranib-2 effectively inhibited the viability of MCF7 cells in 24 h in a dose dependent manner leading to apoptosis via the mitochondrial pathway by reducing the potential of mitochondrial membrane. Additionally, significant changes on cell morphology and ultrastructure were observed on MCF7 cells exposed to ceranib-2 indicating apoptotic cell death. Collectively, our data demonstrate that ceranib-2 exerts a great potential to be an antineoplastic compound and that the mechanism of its action rely on its apoptosis inducing ability.

Description: Interferon regulatory factor 8 (Irf8) is a transcription factor that negatively regulates osteoclast differentiation and Irf8 global knockout ( Irf8 −/− ) mice have been shown to have reduced bone volume resulting from increased osteoclast numbers. However, detailed analysis of the functions of Irf8 in osteoclast precursors with a monocyte/macrophage linage is difficult, because the population and properties of hematopoietic cells in Irf8 −/− mice are severely altered. Therefore, to clearly elucidate the functions of Irf8 during osteoclastogenesis, we established myeloid cell-specific Irf8 conditional knockout ( Irf8 fl/fl ;Lyz2 cre/ + ) mice. We found that trabecular bone volume in the Irf8 fl/fl ;Lyz2 cre/ + mice was not significantly affected, while exposure to M-CSF and RANKL significantly increased TRAP activity in vitro in osteoclasts that underwent osteoclastogenesis from bone marrow-derived macrophages (BMMs) induced from bone marrow cells (BMCs) of those mice by addition of M-CSF. Our results also showed that expression of Irf8 mRNA and protein in BMMs obtained from Irf8 fl/fl ;Lyz2 cre/ + mice and cultured with M-CSF was reduced. These findings predicted that Lyz2/Lyz2-cre expression is induced when BMCs differentiate into BMMs in cultures with M-CSF. In osteoclast differentiation cultures, Lyz2 was gradually increased by M-CSF during the first 3 days of culture, then rapidly decreased by the addition of RANKL with M-CSF during the next 3 days. Furthermore, BMCs differentiated into osteoclasts while maintaining a low level of Lyz2 expression when cultured simultaneously with both M-CSF and RANKL from the initiation of culture. These findings suggest that Lyz2-cre expression is induced along with differentiation to BMMs by BMCs obtained from Irf8 fl/fl ;Lyz2 cre/ + mice and cultured with M-CSF. In addition, Irf8 was down-regulated by activation of the cre/loxP recombination system in BMMs and osteoclastogenesis was accelerated. Based on our results, we propose the existence in vivo of a new lineage of osteoclast precursors among BMCs, which differentiate into osteoclasts without up-regulation of Lyz2 expression.

Description: As a critical quality attribute, glycosylation represents an important consideration when analyzing the success of a glycoprotein production process. Though critical, glycosylation is not the only measure of culture success; other factors, including culture size, maintenance, and productivity, are also critical. A new metric was developed to address both product quality, as measured through glycosylation, and product quantity, as measured through product concentration. A monoclonal antibody Chinese hamster ovary cell culture model system was used to assess this metric across various media formulations. In a model test system, the metric discriminated that some media supplements had a net positive impact on productivity and glycosylation, while others had a net negative impact on productivity and glycosylation.

Description: DNA methylation in mammals is an epigenetic marker and necessary for normal embryogenesis. The global genomic demethylation of 5-methylcytosine occurs during the first cell cycle following fertilization. Activation-induced cytidine deaminase (AID), which is well-known for the function in antibody diversification, has been implicated to play a role in active demethylation, but its role in cell reprogramming and its crosstalk with other DNA demethylation mechanism need to be clarified. In this study, the dynamic epigenetic regulation of cell pluripotency and embryo development by AID in bovine preimplantation embryos was investigated. The analysis of an AID overexpressing transgenic cell line showed that AID overexpression did not change the global genomic methylation but did change the methylation status of the promoters of the OCT4, NANOG and SOX2 genes, thereby causing changes in their expression. The siRNA-mediated AID knockdown in early embryonic development indicated that AID interference did not affect oocyte maturation or the following embryo development after in vitro fertilization but influenced the DNA methylation status of OCT4 and NANOG . To clarify the role of AID in preimplantation embryos, SCNT embryos were obtained using AID -overexpressing cells as nuclear donors. Compared to the control group, the cleavage and blastocyst rates were both significantly improved in the AID -overexpression group. The expression of OCT4 and NANOG was increased in the SCNT embryos, whereas the methylation levels of their promoters were reduced. In conclusion, this study demonstrated that AID selectively catalyzes DNA demethylation of pluripotency genes to play a role in regulation their expression, improves bovine SCNT embryo development by increased expression levels.

Description: Traditionally, muscle cell lines are cultured on glass coverslips and differentiated to investigate myoblast fusion and differentiation. Efficient differentiation of myoblasts produces a dense network of myotubes with the correct organisation for contraction. Here we have tested the ability of artificially generated, precisely controlled peptide surfaces to enhance the efficiency of myoblast differentiation. We focused on specific short peptides from α-laminin-2 (IKVSV, VQLRNGFPYFSY and GLLFYMARINHA) as well as residues 15–155 from FGF1. We tested if these peptides in isolation, and/or in combination promoted muscle differentiation in culture, by promoting fusion and/or by improving sarcomere organisation. The majority of these peptides promoted fusion and differentiation in two different mouse myogenic cell lines and in primary human myoblasts. The additive effects of all four peptides gave the best results for both mouse cell lines tested, while primary human cell cultures differentiated equally well on most peptide surfaces tested. These data show that a mixture of short biomimetic peptides can reliably promote differentiation in mouse and human myoblasts.

Description: Eukaryotic elongation factor 2 (eEF2) plays an important role in eukaryotic polypeptide chain elongation. Adenosine diphosphate (ADP)-ribosylation is a post-translational modification reaction that catalyzes the transfer of ADP-ribose group to eEF2 and this causes the inhibition of protein synthesis. Indeed, in the absence of diptheria toxin, endogenous ADP-ribosylation can occur. eEF2 is phosphorylated by eEF2 kinase which prevents binding to ribosomes thus inhibiting its activity. Increase in endogenous ADP-ribosylation level approximately 70–75 % was observed in IL-1 β treated HUVECs. Moreover, a 70 % rise of phosphorylation of eEF2 was measured. Alteration of endogenous ADP-ribosylation of eEF2 activity was related with cellular mono-ADP-ribosyltransferases (ADPrT). Increment of endogenous ADP-ribosylation on eEF2 did not seem to occur as a direct effect of IL-1 β ; it arises from the activation of ADPrT. This 2.5 fold increase was abolished by ADPrT inhibitors. Due to these post-translational modifications, global protein synthesis is inhibited. After dephosphorylation of phospho-eEF2, around 20 % increase in protein synthesis was observed. In conclusion, systemic IL-1 β has an important role in the regulation of global protein synthesis.

Description: In vitro studies might be an interesting screening method for targeted in vivo studies in the field of immunonutrition and help to reduce and refine animal studies. As the role of amino acids for immune function of cats has not been evaluated in detail so far, the present study aimed at investigating the effects of eight different amino acids (arginine, leucine, isoleucine, valine, glutamine, lysine, threonine and tryptophan) in six concentrations each (0, 0.25, 0.5, 1, 2 and 8x the cat blood level) on cytokine secretion and proliferative activity of feline T cells (MYA-1) in vitro. The results demonstrated that high doses of arginine increased IL-4, IL-10 and TNF-α secretion of T cells, while increasing concentrations of lysine increased IL-10 secretion and proliferative activity of the T cells. High doses of leucine enhanced GM-CSF and IL-10 secretion, while concentrations of threonine in the cell culture media greater than blood concentration also increased GM-CSF and additionally TNF-α secretion of the cells. The effects of glutamine and isoleucine on T cell function were only small. In conclusion, the present in vitro study could evaluate the immunomodulating potential of specific amino acids for feline T cell function. High doses of arginine, lysine, leucine and threonine had a significant impact on cytokine secretion and proliferative activity of the T cells. Targeted in vivo studies should investigate the clinical relevance of dietary supplementation of those amino acids in healthy and diseased cats as a next step.

Description: Hyperuricemia is characterized by the high uric acid (UA) level in serum (or plasma) and has been considered to be an important risk factor for gout. In the present study, we have attempted to construct an assay system for UA production in vitro employing cultured AML12 hepatocytes. UA levels in balanced salt solution (BSS) in the presence of UA precursor nucleosides, adenosine, inosine, guanosine and xanthine, at 12.5, 25, and 100 µM were significantly higher than BSS alone and their effects were dose-dependent, while all the UA precursors did not significantly increase intracellular UA levels. Hence, UA levels in BSS were thereafter adopted as an index of UA production. UA production from nucleosides was significantly higher than that from nucleotides (GMP, IMP and AMP). UA production from guanosine and inosine in combination (GI mixture) as well as nucleosides increased time-dependently and almost linearly up to 2 h. Selecting GI mixture, effects of allopurinol, a widely used anti-hyperuricemic agent, and quercetin, a well-known polyphenol in onion and strawberry, on UA production were examined. Both allopurinol and quercetin dose-dependently (0.1, 0.3 and 1 μM for allopurinol and 10, 30, and 100 μM for quercetin) and significantly reduced UA production in the hepatocytes. They also significantly reduced hyperuricemia induced by intraperitoneal injection of UA precursor purine bodies to mice at a single oral dose of 10 (allopurinol) or 200 (quercetin) mg/kg body weight. This assay system for UA production in cultured hepatocytes is considered to be useful to search for novel anti-hyperuricemic compounds in foods and natural resources with possibility to have human health benefits.

Description: Chinese hamster ovary (CHO) cell lines are widely used for therapeutic protein production. When a transgene is integrated into the genome of a CHO cell, the expression level is highly dependent on the site of integration because of positional effects such as gene silencing. To overcome negative positional effects and establish stable CHO cell lines with high productivity, several regulatory DNA elements are used in vector construction. Previously, we established the CHO DR1000L-4N cell line, a stable and high copy number Dhfr gene-amplified cell line. It was hypothesized that the chromosomal location of the exogenous gene-amplified region in the CHO DR1000L-4N genome contains regulatory motifs for stable protein production. Therefore, we isolated DNA regulatory motifs from the CHO DR1000L-4N cell line and determined whether these motifs act as an insulator. Our results suggest that stable expression of a transgene can be promoted by the CHO genome sequence, and it would be a powerful tool for therapeutic protein manufacturing.

Description: The oxadiazole moiety is known for its anticancer activity through its antiangiogenic and mitostatic potential. Taking this as a cue, the present study was designed to investigate the anti-cancer potential of selected oxadiazole derivatives. Twelve 1,3,4-oxadiazole derivatives (AMK OX-1 to AMK OX-12) were synthesized and were tested for IC 50 values through brine shrimp lethality assay and MTT assay on HeLa and A549 cell lines. Four compounds, AMK OX-8, 9, 11 and 12 showed potential cytotoxicity activity with low IC 50 value. These compounds produced considerable cytotoxic effect on Hep-2 and A549 cancer cell lines. However, they were found to be comparatively safer to normal cell lines, viz., V-79 cell lines than to the tested cancer cell lines, such as HeLa, A 549, and Hep2 cell lines. The mechanism of cytotoxicity was evaluated through nuclear staining and DNA ladder assay. Although DNA ladder assay showed DNA fragmentation (apoptotic phenomenon) in Hep-2 cells treated with only AMK OX-12, the staining procedures using acridine orange, ethidium bromide and propidium iodide showed apoptotic bodies in cells treated with AMK OX-8, 9 and 12 also. In JCI staining on isolated mitochondria of Hep2 cells, AMK OX-8, 9-11 and 12 displayed increasing fluorescence intensity with time which confirmed involvement of mitochondrial pathway and intrinsic pathway of apoptosis. All four compounds were found to be safe in acute oral toxicity study in Swiss albino mice. These derivatives were effective in reducing tumor size and weight in the in vivo DLA-induced solid tumor model. They were found to be significantly effective in reducing tumor volume and tumor weight.

Description: Nuclear Warfare and nuclear leakage can result in a large number of patients with radiation-induced bone marrow damage. Based on the fact that hematopoietic stem cells and hematopoietic growth factors are characterized as a novel strategy for therapy, the aim of this study was to explore a safe and routine stem cell/cytokine therapeutic strategy. Allogeneic multiplacenta derived hematopoietic and mesenchymal stem cells/cytokines were intraperitoneally injected into a moderate dose of total body irradiation-induced mouse bone marrow damage model a single time. Then, the mouse posttransplantation survival time, peripheral blood hemoglobin count, bone marrow architecture, and donor cell engraftment were assessed. Each mouse that received placenta-derived stem cells exhibited positive donor hematopoietic and mesenchymal stem cell engraftment both in the bone marrow and peripheral blood after transplantation. The peripheral blood hemoglobin count and survival time were greater in the group with the combined treatment of multiplacenta-derived stem cells and cytokines, compared with model-only controls (both P  〈 0.001). The blood smear mesenchymal/hematopoietic stem cell count was significantly higher in the combined treatment group than in the mice treated only with placenta-derived cells (28.08 ± 5.824 vs. 20.40 ± 5.989, P  〈 0.001; 7.74 ± 2.153 vs. 4.23 ± 1.608, P  〈 0.001, respectively). However, there was no marked change on the bone marrow pathology of any of the experimental mice after the transplantation. These results indicate that for radiation-induced bone marrow damage treatment, multiplacenta-derived stem cells and cytokines can increase the life span of model mice and delay but not abrogate the disease progression. Intraperitoneally transplanted stem cells can survive and engraft into the host body through the blood circulation. Improvement of peripheral blood hemoglobin levels, but not the bone marrow architecture response, probably explains the increase in survival time observed in this study.

Description: Cyanobacteria can produce useful renewable fuels and high-value chemicals using sunlight and atmospheric carbon dioxide by photosynthesis. Genetic manipulation has increased the variety of chemicals that cyanobacteria can produce. However, their uniquely abundant NADPH-pool, in other words insufficient supply of NADH, tends to limit their production yields in case of utilizing NADH-dependent enzyme, which is quite common in heterotrophic microbes. To overcome this cofactor imbalance and enhance cyanobacterial fuel and chemical production, various approaches for cofactor engineering have been employed. In this review, we focus on three approaches: (1) utilization of NADPHdependent enzymes, (2) increasing NADH production, and (3) changing cofactor specificity of NADH-dependent enzymes from NADH to NADPH.

Description: Pyrroloquinoline quinone (PQQ) plays a significant role as a redox cofactor in combination with dehydrogenases in bacteria. These dehydrogenases play key roles in the oxidation of important substrates for the biotechnology industry, such as vitamin C production. While biosynthesis of PQQ genes has been widely studied, PQQ-transport mechanisms remain unclear. Herein, we used both two-dimensional fluorescence-difference gel electrophoresis tandem mass spectrometry and RNA sequencing to investigate the effects of pqqB overexpression in an industrial strain of Gluconobacter oxydans WSH-003. We have identified 73 differentially expressed proteins and 99 differentially expressed genes, a majority of which are related to oxidation-reduction and transport processes by gene ontology analysis. We also described several putative candidate effectors that responded to increased PQQ levels resulting from pqqB overexpression. Furthermore, quantitative PCR was used to verify five putative PQQ-transport genes among different PQQ producing strains, and the results showed that ompW , B932_1930 and B932_2186 were upregulated in all conditions. Then the three genes were over-expressed in G. oxydans WSH-003 and PQQ production were detected. The results showed that extracellular PQQ of B932_1930 (a transporter) and B932_2186 (an ABC transporter permease) overexpression strains were enhanced by 1.77-fold and 1.67-fold, respectively. The results suggest that the proteins encoded by PqqB, B932_1930 and B932_2186 might enhance the PQQ secretion process.

Description: Oil bleed is a serious problem in elastomeric thermal silicone conductive pads. The components of the oil bleed and the effect of the silicone chemical parameters on the amount of oil bleed have been determined. The main components of oil bleeds are the uncrosslinked silicones in the cured resins, which include the unreacted silicone materials and the macromolecular substances produced by the hydrosilylation reaction. Cured resins with a high crosslinking density and a high molecular weight of vinyl silicone residues had a lower amount of oil bleed. In addition, a low Si-H content also reduced the amount of oil bleed.

Description: Leaching selectivity during metal recovery from complex electronic waste using a hydrochemical process is always one of the generic issues. It was recently improved by using ammonia-based leaching process, specifically for electronic waste enriched with copper. This research proposes electrodeposition as the subsequent approach to effectively recover copper from the solutions after selective leaching of the electronic waste and focuses on recognising the electrochemical features of copper recovery. The electrochemical reactions were investigated by considering the effects of copper concentration, scan rate and ammonium salts. The diffusion coefficient, charge transfer coefficient and heterogeneous reaction constant of the electrodeposition process were evaluated in accordance with different solution conditions. The results have shown that electrochemical recovery of copper from ammoniabased solution under the conditions of selective electronic waste treatment is charge transfer controlled and provide bases to correlate the kinetic parameters with further optimisation of the selective recovery of metals from electronic waste.

Description: Careful selection of housekeeping genes (HKG) is prerequisite to yield sound qPCR results. HKG expression varies in response to hypoxia but the effect of manipulations of serum availability, a common experimental procedure, remains unknown. Also, no data on HKG expression stability across colon adenocarcinoma lines that would aid selection of normalizers suitable for studies involving several lines are available. Thus, we evaluated the effect of serum availability on the expression of commonly used HKG ( ACTB , B2M , GAPDH , GUSB , HPRT1 , IPO8 , MRPL19 , PGK1 , PPIA , RPLP0 , RPS23 , SDHA , TBP , UBC , and YWHAZ ) in seven colon adenocarcinoma cell lines (Caco-2, DLD-1, HCT116, HT29, Lovo, SW480, and SW620). Sets of stably expressed line-specific and pan-line HKG were validated against absolutely quantified CDKN1A , TP53 , and MDK transcripts. Both serum availability and line type affected HKG expression. UBC was fourfold down-regulated and HPRT1 1.75-fold up-regulated in re-fed HT29 cultures. Line-to-line variability in HKG expression was more pronounced than that caused by altering serum availability and could be found even between isogenic cell lines. PPIA , RPLP0 , YWHAZ , and IPO8 were repeatedly highly ranked while ACTB , B2M , UBC , and PGK1 were ranked poorly. Normalization against PPIA / RPLP0 / SDHA was found optimal for studies involving various colon adenocarcinoma cell lines subjected to manipulations of serum availability. We found HKG expression to vary, more pronouncedly by line type than growth conditions with significant differences also between isogenic cell lines. Although using line-specific normalizers remains optimal, a set of pan-line HKG that yields good estimation of relative expression of target genes was proposed.

Description: Adipose derived mesenchymal stromal/stem cells (ASCs) are a heterogeneous population characterized by (a) their ability to adhere to plastic; (b) immunophenotypic expression of certain cell surface markers, while lacking others; and (c) the capacity to differentiate into lineages of mesodermal origin including osteocytes, chondrocytes and adipocytes. The long-term goal is to utilize these cells for clinical translation into cell-based therapies. However, preclinical safety and efficacy need to be demonstrated in animal models. ASCs can also be utilized as biological vehicles for vector-based gene delivery systems, since they are believed to home to sites of inflammation and infection in vivo. These factors motivated the development of a labelling system for ASCs using lentiviral vector-based green fluorescent protein (GFP) transduction. Human ASCs were transduced with GFP-expressing lentiviral vectors. A titration study determined the viral titer required to transduce the maximum number of ASCs. The effect of the transduced GFP lentiviral vector on ASC immunophenotypic expression of surface markers as well as their ability to differentiate into osteocytes and adipocytes were assessed in vitro. A transduction efficiency in ASC cultures of approximately 80 % was observed with an MOI of ~118. No significant immunophenotypic differences were observed between transduced and non-transduced cells and both cell types successfully differentiated into adipocytes and osteocytes in vitro. We obtained 〉80 % transduction of ASCs using GFP lentiviral vectors. Transduced ASCs maintained plastic adherence, demonstrated ASC immunophenotype and the ability to differentiate into cells of the mesodermal lineage. This GFP-ASC transduction technique offers a potential tracking system for future pre-clinical studies.

Description: Hepatocytes differentiated from induced pluripotent stem cells and adult stem cells could be utilized as a tool for the study of liver diseases, screening for drug metabolism and hepatotoxicity. Thus further investigation of the method to efficiently generate hepatocytes is in great need. Bone Mesenchymal Stem Cells (BMSCs) were collected from rat femurs and tibias. FOXA2 and HNF1α genes were constructed into a lentiviral vector and introduced into BMSCs by a lentivirus-mediated overexpression system. Three weeks after the induction, the expressions of FOXA2 and HNF1α, and liver specific genes were analyzed, and hepatocyte-function related assays were performed. Overexpression of both FOXA2 and HNF1α induced the BMSCs to differentiate into hepatocyte-like cells (HLCs). Hepatocyte-specific gene and protein were detected by RT-PCR, Western Blot and Immunofluorescence. These HLCs also exerted some typical hepatocyte functions such as glycogen storage, indocyanine green absorption and lipid accumulation. The combination of FOXA2 and HNF1α can effectively induce BMSCs to differentiate into HLCs. This is a novel and efficient method to prepare HLCs within a short timeline.

Description: Heterogeneous catalysis with core-shell structures has been a large area of focus for many years. This paper reviews the most recent work and research in coreshell catalysts utilizing noble metals, specifically gold, as the core within a metal oxide shell. The advantage of the core-shell structure lies in its capacity to retain catalytic activity under thermal and mechanical stress, which is a pivotal consideration when synthesizing any catalyst. This framework is particularly useful for gold nanoparticles in protecting them from sintering so that they retain their size, structure, and most importantly their catalytic efficiency. The different methods of synthesizing such a structure have been compiled into three categories: seed-mediated growth, post selective oxidation treatment, and one-pot chemical synthesis. The selective oxidation of carbon monoxide and reduction of nitrogen containing compounds, such as nitrophenol and nitrostyrene, have been studied over the past few years to evaluate the functionality and stability of the core-shell catalysts. Different factors that could influence the catalyst’s performance are the size, structure, choice of metal oxide shell and noble metal core and thereby the interfacial synergy and lattice mismatch between the core and shell. In addition, the morphology of the shell also plays a critical role, including its porosity, density, and thickness. This review covers the synthesis and characterization of gold-metal oxide core-shell structures, as well as how they are utilized as catalysts for carbon monoxide (CO) oxidation and selective reduction of nitrogen-containing compounds.

Description: Amyloid peptides are renowned to be related to neurodegenerative diseases, however, a fruitful avenue is to employ them as high-performance nanomaterials. These materials benefit from the intrinsic outstanding mechanical robustness of the amyloid backbone made of β-strands. In this work, we exploited amyloid-like fibrils as functional material to attach pristine L-cysteine aggregates (cystine oligomers) and gold nanoparticles, without the need of templating compounds. This work will open new avenues on functional materials design and their realisation.

Description: The repair of meniscus in the avascular zone remains a great challenge, largely owing to their limited healing capacity. Stem cells based tissue engineering provides a promising treatment option for damaged meniscus because of their multiple differentiation potential. We hypothesized that meniscus-derived stromal cells (MMSCs) may be present in meniscal tissue, and if their pluripotency and character can be established, they may play a role in meniscal healing. To test our hypothesis, we isolated MMSCs, bone marrow-derived stromal cells (BMSCs) and fibrochondrocytes from rabbits. In order to avoid bone marrow mesenchymal stromal cell contamination, the parameniscal tissues and vascular zone of meniscus were removed. The characters of these three types of cells were identified by evaluating morphology, colony formation, proliferation, immunocytochemistry and multi-differentiation. Moreover, a wound in the center of rabbit meniscus was created and used to analyze the effect of BMSCs and MMSCs on wounded meniscus healing. BMSCs & MMSCs expressed the stem cell markers SSEA-4, Nanog, nucleostemin and STRO-1, while fibrochondrocytes expressed none of these markers. Morphologically, MMSCs displayed smaller cell bodies and larger nuclei than ordinary fibrochondrocytes. Moreover, it was certified that MMSCs and BMSCs were all able to differentiate into adipocytes, osteocytes, and chondrocytes in vitro. However, more cartilage formation was found in wounded meniscus filled with MMSCs than that filled with BMSCs. We showed that rabbit menisci harbor the unique cell population MMSCs that has universal stem cell characteristics and posses a tendency to differentiate into chondrocytes. Future research should investigate the mechanobiology of MMSCs and explore the possibility of using MMSCs to more effectively repair or regenerate injured meniscus.

Description: In this study, in order to investigate the anticancer mechanism of Calvatia gigantea extract, edible mushroom species, which belong to Lycoperdaceae family, changes of CCND1, CCND2, CDK4, p21, Akt, Bax, Bcl-2, p53, caspase-3 and caspase-9 were evaluated in A549 lung cancer cells. Cytotoxic effect of C. gigantea extract was evaluated by using XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5 carboxanilide). The C. gigantea extract was treated in a time and dose dependent manner within the range 25 μg/ml–2 mg/ml to determine the IC 50 dose. IC 50 dose for C. gigantea extract was detected as 500 μg/ml for 72 h. According to expression results, while CCND1, CCND2, CDK4, Akt and Bcl-2 expression clearly decreased, Bax, p53, caspase - 3 and caspase - 9 expression clearly increased in the dose group cells (A549 cells treated with 500 μg/ml dose of C. gigantea extract for 72 h). However, there was no change in p21 expression. C. gigantea extract induced cell cycle arrest and apoptosis by decreasing the CCND1, CCND2, CDK4, Akt and Bcl-2 expression and by increasing Bax, p53, caspase-3 and caspase-9 expression in A549 cells. Mushrooms are eukaryotic organisms heavily used because of their supposedly anticancer effect. Many mushroom species have been used for medical purposes, as a result of also having many effects such as antibiotic, antiviral and anticancer effects. It is thought that the C. gigantea extract may be a significant agent for treatment of lung cancer as a single agent or in combination with other drugs.

Description: Hepatic stellate cells (HSCs) are key players in liver fibrosis and regeneration via collagen degradation and synthesis. These phenomena involve inflammatory cytokines released from non-parenchymal liver cells such as Kupffer cells. Although the effects of individual cytokines on many cell types have been investigated in various conditions, such as inflammation and tissue fibrosis, investigating the effect of combined cytokines would further our understanding of the regulatory mechanisms in tissue fibrosis. Here, we report the effect of multiple cytokine combinations on primary HSCs. We first examined the effect of individual cytokines and then the simultaneous exposure of different cytokines, including interleukin-6 (IL-6), IL-1 alpha (IL-1α), platelet-derived growth factor (PDGF), tumour necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β), on matrix metalloproteinase-1 (MMP1) gene expression in primary HSCs. We observed that the combination of all five cytokines induced higher levels of MMP1 gene expression. Of these cytokines, TNF-α and IL-1α were found to be the key cytokines for not only inducing MMP1 expression, but also increasing α-smooth muscle actin gene expression. In conclusion, the combined treatment of TNF-α and IL-1α on HSCs had an enhanced effect on the expression of the fibrotic genes, MMP1 and α-smooth muscle actin, so appears to be an important regulator for tissue regeneration. This finding suggests that stimulation with combined anti-fibrotic cytokines is a potential approach in the development of a novel therapy for the recovery of liver fibrosis.

Description: Aqueous solutions of methyldiethanolamine (MDEA) and piperazine (PZ) are commonly used solvent nowadays. In this work a thermodynamic analysis with the Electrolyte-NRTL model has been performed for systems composed of acidic gases and MDEA + PZ aqueous solution. ASPEN Plus® has been used for thermodynamic modeling. Values of binary interaction parameters for liquid phase activity coefficients have been estimated from regressions of experimental data. Moreover, the influence of the interactions between ion pairs and MDEA or PZ molecular species has been analyzed. The final aim is to obtain a reliable tool for design and simulation of absorption and stripping columns, fundamentals also in order to carry out energy saving studies.

Description: Epithelial cells from oral mucosa (EOM) are responsible for important functions, like the primary protection of oral mucosa against external aggressions building a mechanical barrier against microorganisms, mechanical damage, toxic material, thermal regulation and secretion of different classes of inflammatory mediators. EOM could be an interesting tool for cellular and molecular biology research. Usually, EOM are collected by a painful and invasive process. In this study, we propose an alternative method to cultivate EOM collected by non-invasive scraping method of oral mucosa. Papanicolaou staining showed mainly two kinds of epithelial cell population after EOM scraping. As result of the five culture methods tested here, our results revealed that the EOM were successfully cultured on a murine feeder layer. In addition, EOM could be frozen and thawed, without morphology changes and loss of viability. Our findings suggest that EOM can be considered as a good cell source for many purposes, such as genetic studies, diagnosis and cell therapy.

Description: A largely increased number of GGGGCC repeats located in the non-coding region of C9orf72 gene have been identified as the leading cause of two related neurological disorders, familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We examined G-quadruplex forming ability of GGGGCCrepeat containing oligonucleotides with four guanine tracts chosen as the smallest possible model to form a unimolecular G-quadruplex. These oligonucleotides are readily to folded into G-quadruplexes in the presence of K + ions. However, the formation of multiple structures makes structural analysis challenging and time consuming. We observed that flanking sequences on 5'- and 3'-ends as well as mutations of loop residues have a profound effect on folding. Sequence d[(G 4 C 2 ) 3 G 4 ] was chosen for further scrutiny and optimization of nuclear magnetic resonance (NMR) spectroscopic properties with dG to 8Br-dG substitutions at specific positions in the sequence under different folding conditions. Expectedly, folding into desired predominant topology is facilitated when substituted residue adopted a syn conformation in the naturally-occurring structure. Single dG to 8Br-dG substitution at position 21 and fine tuning of folding conditions facilitate folding of d[(G 4 C 2 ) 3 GG Br GG] into (mostly) a single G-quadruplex, and thus enable determination of its high-resolution structure by high-field NMR.

Description: Small pore zeolites, containing 8-rings as the largest, are widely employed as catalysts in the process of methanol-to-olefins (MTO). Reactants and products diffuse with constraints through 8-rings and this is one of the reaction bottlenecks related to zeolite micropore topology. Small pore zeolites and silicon-aluminophosphates (SAPOs) containing cavities, where olefins are mainly formed through the hydrocarbon pool (HP) mechanism, are frequently tested for MTO. Shape selectivity of transition states within the side-chain methylation will be reviewed as this is one of the controlling steps of the MTO process, with particular attention to the role of hexamethylbenzene (HMB) and heptamethylbenzenium cation (HeptaMB+), which are the most tipically detected reaction intermediates, common to the paring and side-chain routes within the HP mechanism. The relative stability of these and other species will be reviewed in terms of confinement effects in different cage-based zeolites. The role of the different alkylating agents, methanol, dimethyl ether (DME), and surface methoxy species (SMS) will also be reviewed from the computational viewpoint.

Description: Rheumatoid arthritis is characterized by inflammation of the synovial membrane, which can result in joint destruction within the first few years after onset. Lipopolysaccharides (LPS) are glycolipids found in abundance on the outer membranes of all Gram-negative bacteria and incite a vigorous inflammatory response. We studied the potential involvement of mitogen-activated protein kinase serine/threonine kinases in LPS-induced growth of HS synovial cells. Various concentrations of LPS were applied to cultured HS cells and growth rate, as well as changes in the phosphorylation states of extracellular regulated kinase 1/2, Jun N-terminal kinase (JNK), and p38 were determined. As results, growth of LPS-treated HS cells was inhibited primarily by phosphorylated JNK and this phosphorylation was mediated by the LPS receptor. Our results suggest that LPS inhibits growth of HS cells primarily through the JNK pathway.

Description: The enteric nervous system (ENS) is a complex network of neurons in the gut, regulating many local, vital functions of the gastro-intestinal tract. The ENS is also part of the bidirectional gut-brain axis. The murine immorto fetal enteric neuronal (IM-FEN) cell line was chosen as a model to study enteric neurons. This cell line can be differentiated into cells with a neuronal phenotype, although they do not produce action potentials in vitro. It was concluded that the differentiation process in our laboratory was successful, based on positive staining for neuronal proteins. Proliferating IM-FEN cells have an unstable growth rate in our laboratory. An indicator of growth rate was calculated, and this indicator was found to be related to seeding density and number of days in culture, and was unrelated to person culturing, previous overconfluency or passage number. The indicator of growth rate was also unrelated to successful use of differentiated cells in follow-up experiments. We recommend the following conditions for optimal culture of IM-FEN cells. Keep cells in culture until 80 % confluent before passaging, seed cells at a density of 0.0133 million cells per cm 2 , and anticipate on unstable growth rates and the risk for overconfluency.

Description: Nowadays a lot of low-grade heat is wasted from the industry through the off- and flue-gasses with different compositions. These gases provide the sensitive heat with utilisation potential and latent heat with the components for condensation. In this paper, process integration methodology has been applied to the partly condensed streams. A hot composite curve that represents the gas mixture cooling according to equation of state for real gases was drawn to account the gas-liquid equilibrium. According to the pinch analysis methodology, the pinch point was specified and optimal minimal temperature difference was determined. The location of the point where gas and liquid phases can be split for better recuperation of heat energy within heat exchangers is estimated using the developed methodology. The industrial case study of tobacco drying process off-gasses is analysed for heat recovery. The mathematical model was developed by using MathCad software to minimise the total annualised cost using compact plate heat exchangers for waste heat utilisation. The obtained payback period for the required investments is less than six months. The presented method was validated by comparison with industrial test data.

Description: A review of recent research related to microporous polymeric membranes formed via thermally induced phase separation (TIPS) and the morphologies of these membranes is presented. A summary of polymers and suitable diluents that can be used to prepare these microporous membranes via TIPS are summarized. The effects of different kinds of polymer materials, diluent types, cooling conditions, extractants and additive agents on the morphology and performance of TIPS membranes are also discussed. Finally new developments in TIPS technology are summarized.

Description: Natural products and their derivatives represent a rich source for the discovery and development of new cancer therapeutic drugs. Bioactive components derived from natural sources including marine compounds have been shown to be effective agents in the clinic or in preclinical settings. In the present review, we present a story of discovery, synthesis and evaluation of three synthetic tricyclic pyrroloquinone (TPQ) alkaloid analogs as cancer therapeutic agents. Chemical synthesis of these compounds (BA-TPQ, TBA-TPQ, and TCBA-TPQ) has been accomplished and the mechanisms of action (MOA) and structure-activity relationships (SAR) have been investigated. In the past, the complexity of chemical synthesis and the lack of well-defined MOA have dampened the enthusiasm for the development of some makaluvamines. Recent discovery of novel molecular targets for these alkaloids (unrelated to inhibition of Topoisomerase II) warrant further consideration as clinical candidates in the future. In addition to the establishment of novel synthetic approaches and demonstration of in vitro and in vivo anticancer activities, we have successfully demonstrated that these makaluvamines attack several key molecular targets, including the MDM2-p53 pathway, providing ample opportunities of modulating the compound structure based on SAR and the use of such compounds in combination therapy in the future.

Description: In vitro cell culture models used to study epithelia and epithelial diseases would benefit from the recognition that organs and tissues function in a three-dimensional (3D) environment. This context is necessary for the development of cultures that more realistically resemble in vivo tissues/organs. Our aim was to establish and characterize biologically meaningful 3D models of epithelium. We engineered 3D epithelia cultures using a kidney epithelia cell line (MDCK) and spherical polymer scaffolds. These kidney epithelia were characterized by live microscopy, immunohistochemistry and transmission electron microscopy. Strikingly, the epithelial cells displayed increased physiological relevance; they were extensively polarized and developed a more differentiated phenotype. Using such a growth system allows for direct transmission and fluorescence imaging with few restrictions using wide-field, confocal and Light Sheet Fluorescence Microscopy. We also assessed the wider relevance of this 3D culturing technique with several epithelial cell lines. Finally, we established that these 3D micro-tissues can be used for infection as well as biochemical assays and to study important cellular processes such as epithelial mesenchymal transmission. This new biomimetic model could provide a broadly applicable 3D culture system to study epithelia and epithelia related disorders.

Description: This paper presents a novel synthesis method for designing integrated processes for oil-in-water (O/W) emulsions treatment. General superstructure involving alternative separation technologies is developed and modelled as a mixed integer linear programming (MILP) model for maximum annual profit. Separation processes in the superstructure are divided into three main sections of which the pretreatment and final treatment are limited to the selection of one alternative (or bypass) only, while within the intermediate section various combinations of different technologies in series can be selected. Integrated processes composed of selected separation techniques for given ranges of input chemical oxygen demand (COD) can be proposed by applying parametric analyses within the superstructure approach. This approach has been applied to an existing industrial case study for deriving optimal combinations of technologies for treating diverse oil-inwater emulsions within the range of input COD values between 1000 mg·L -1 and 145000 mg·L -1 . The optimal solution represents a flexible and profitable process for reducing the COD values below maximal allowable limits for discharging effluent into surface water.

Description: Despite the surgical and other insertional interventions, the complete recuperation of myocardial disorders is still elusive due to the insufficiency of functioning myocardiocytes. Thus, the use of stem cells to regenerate the affected region of heart becomes a prime important. In line with this human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have gained considerable interest due to their potential use for mesodermal cell based replacement therapy and tissue engineering. Since MSCs are harvested from various organs and anatomical locations of same organism, thus the cardiac regenerative potential of human cardiac-derived MSCs (hC-MSCs) and human umbilical cord Wharton’s Jelly derived MSC (hUC-MSCs) were tested concurrently. At in vitro culture, both hUC-MSCs and hC-MSCs assumed spindle shape morphology with expression of typical MSC markers namely CD105, CD73, CD90 and CD44. Although, hUC-MSCs and hC-MSCs are identical in term of morphology and immunophenotype, yet hUC-MSCs harbored a higher cell growth as compared to the hC-MSCs. The inherent cardiac regenerative potential of both cells were further investigated with mRNA expression of ion channels. The RT-PCR results demonstrated that both MSCs were expressing a notable level of delayed rectifier-like K + current ( I KDR ) ion channel, yet the relative expression level was considerably varied between hUC-MSCs and hC-MSCs that Kv1.1(39 ± 0.6 vs 31 ± 0.8), Kv2.1 (6 ± 0.2 vs 21 ± 0.12), Kv1.5 (7.4 ± 0.1 vs 6.8 ± 0.06) and Kv7.3 (27 ± 0.8 vs 13.8 ± 0.6). Similarly, the Ca2 + -activated K + current ( I KCa ) channel encoding gene, transient outward K + current ( I to ) and TTX-sensitive transient inward sodium current ( I Na.TTX ) encoding gene (Kv4.2, Kv4.3 and hNE-Na) expressions were detected in both groups as well. Despite the morphological and phenotypical similarity, the present study also confirms the existence of multiple functional ion channel currents I KDR , I KCa , I to , and I Na.TTX in undifferentiated hUC-MSCs as of hC-MSCs. Thus, the hUC-MSCs can be exploited as a potential candidate for future cardiac regeneration.

Description: Limitations of current treatments for skin loss caused by major injuries leads to the use of skin substitutes. It is assumed that secretion of wound healing mediators by these skin substitutes plays a role in treating skin loss. In our previous study, single layer keratinocytes (SK), single layer fibroblast (SF) and bilayer (BL; containing keratinocytes and fibroblasts layers) skin substitutes were fabricated using fibrin that had shown potential to heal wounds in preclinical studies. This study aimed to quantify the secretion of wound healing mediators, and compare between single and bi-layer skin substitutes. Skin samples were digested to harvest fibroblasts and keratinocytes, and expanded to obtain sufficient cells for the construction of skin substitutes. Acellular fibrin (AF) construct was used as control. Substitutes i.e. AF, SK, SF and BL were cultured for 2 days, and culture supernatant was collected to analyze secretion of wound healing mediators via multiplex ELISA. Among 19 wound healing mediators tested, BL substitute secreted significantly higher amounts of CXCL1 and GCSF compared to SF and AF substitute but this was not significant with respect to SK substitute. The BL substitute also secreted significantly higher amounts of CXCL5 and IL-6 compared to other substitutes. In contrast, the SK substitute secreted significantly higher amounts of VCAM-1 compared to other substitutes. However, all three skin substitutes also secreted CCL2, CCL5, CCL11, GM-CSF, IL8, IL-1α, TNF-α, ICAM-1, FGF-β, TGF-β, HGF, VEGF-α and PDGF-BB factors, but no significant difference was seen. Secretion of these mediators after transplantation may play a significant role in promoting wound healing process for the treatment of skin loss.

Description: Although there is a considerable demand for cell culture protocols from invertebrates for both basic and applied research, few attempts have been made to culture neural cells of crustaceans. We describe an in vitro method that permits the proliferation, growth and characterization of neural cells from the visual system of an adult decapod crustacean. We explain the coating of the culture plates with different adhesive substrates, and the adaptation of the medium to maintain viable neural cells for up to 7 days. Scanning electron microscopy allowed us to monitor the conditioned culture medium to assess cell morphology and cell damage. We quantified cells in the different substrates and performed statistical analyses. Of the most commonly used substrates, poly- l -ornithine was found to be the best for maintaining neural cells for 7 days. We characterized glial cells and neurons, and observed cell proliferation using immunocytochemical reactions with specific markers. This protocol was designed to aid in conducting investigations of adult crustacean neural cells in culture. We believe that an advantage of this method is the potential for adaptation to neural cells from other arthropods and even other groups of invertebrates.

Description: Water induced decomposition of Cu 3 (BTC) 2 (BTC = benzene-1,3,5-tricarboxylate) metal-organic framework (MOF) was studied using dynamic water vapour adsorption. Small-angle X-ray scattering, Fourier transform infrared spectroscopy and differential scanning calorimetry analyses revealed that the underlying mechanism of Cu 3 (BTC) 2 MOF decomposition under humid streams is the interpenetration of water molecules into Cu- BTC coordination to displace organic linkers (BTC) from Cu centres.

Description: Ribozymes are widespread, and catalyze some extremely important reactions in the cell. Mechanistically most fall into one of two classes, using either metal ions or general acid-base catalysis. The nucleolytic ribozymes fall into the latter class, mostly using nucleobases. A sub-set of these use a combination of guanine base plus adenine acid to catalyze the cleavage reaction. New ribozymes are still being discovered at regular intervals and we can speculate on the potential existence of ribozymes that catalyze chemistry beyond phosphoryl transfer reactions, perhaps using small-molecule coenzymes.

Description: 15-Lipoxygenase-1 (15-Lox-1) as a member of fatty acid dioxygenases family has received considerable attention as an effector of cancer cell growth. The relevance of sodium butyrate on 15-Lox-1 pathway has not been determined in breast cancer. This study is aimed to investigate the possible involvement of 15-Lox-1 in the regulation of breast cancer cell growth by sodium butyrate. MTT assay was used to assess the cytotoxicity effect and Annexin-V-FITC staining was applied for detection of apoptosis using flow cytometry. The involvement of 15-Lox-1 was examined using 15-Lox-1 specific inhibitor and enzyme gene expression level and activity was further analyzed by Real-time PCR and measurement of 13(S)-HODE. The results revealed that sodium butyrate increased the expression of 15-Lox-1 and production of 13(S)HODE. 15-Lox-1 was also involved in the sodium butyrate-induced breast cancer cell cytotoxicity and apoptosis. This study provided more evidences on the positive effectiveness of 15-Lox-1/13(S)-HODE on controlling growth of breast cancer cells.

Description: In this study, highly purified hair follicle stem cells from Arbas Cashmere goat (gHFSCs) were isolated using enzyme digestion and adhesion to type IV collagen. The biological characteristics of the gHFSCs were identified by morphological observation, growth curve, markers assay and differentiation in vitro. The gHFSCs were in small cell size with typical cobblestone morphology, good adhesion and high refractive index. Immunocytochemistry staining showed the cells were expressing Krt15, Krt19, CD34, Itgβ1 and Krt14. Cell growth curve indicated that cultured gHFSCs had strong proliferation ability. Krt14 and CD34 were high expressed at the mRNA level, respectively, 39.68 and 24.37 times of the Cashmere goat keratinocytes, and krt15 expression was 5.62 times and itgβ1 expression was 1.81 times higher ( p  〈 0.01). Western blot detected the expression of all the above markers. After osteogenic induction, the cells were positive for Von Kossa staining and expressed Osteocalcin. Sulfated proteoglycans in cartilaginous matrices were positively stained by Alcian blue after chondrogenic induction and COL2A1 was expressed. In myogenic induction, Hoechst 33342 staining evidenced cytoplasm fusion and positive expression of MyoG was detected by immunocytochemistry.

Description: Autographa californica nuclearpoly hedrosis virus (AcMNPV) is one of the most important baculoviridae. However, the application of AcMNPV as a biocontrol agent has been limited. Previously, we engineered Buthus martensii Karsch insect toxin ( Bm K IT) gene into the genome of AcMNPV. The bioassay data indicated that the recombinant baculovirus AcMNPV- Bm K IT significantly enhanced the anti-insect efficacy of the virus. The actin cytoskeleton is the major component beneath the surface of eukaryotic cells. In this report, the effects of AcMNPV- Bm K IT on the formation of early cables of actin and nuclear filamentous-actin (F-actin) were studied. The results indicated that these baculovirus induced rearrangement of the actin cytoskeleton of host cells during infection and actin might participate in the transportation of baculovirus from cytoplasm to the nuclei. AcMNPV- Bm K IT delayed the formation of early cables of actin and nuclear F-actin and accelerated the clearance of actin in the nuclei.

Description: In mammals, interferon-inducible transmembrane proteins (IFITMs) prevent infections by various enveloped viruses. The expression of IFITMs in chicken was herein examined in the adult and embryonic organs using a quantitative reverse-transcription-polymerase chain reaction. The results obtained revealed that IFITM3 was expressed at a higher level than IFITM1, 2 and 5, in both embryonic and adult organs. However, the expression levels of IFITMs in embryonic organs were less than 5 % of those in adult lungs. Among the embryonic tissues examined, primordial germ cells (PGCs) at day 2.5 expressed relatively higher levels of IFITM3. IFITM3 expression levels were 1.5-fold higher in the chicken cell line DF-1 than in PGCs. The knockdown of IFITM3 in DF-1 cells by siRNA increased the infectivity of a vesicular stomatitis virus G protein-pseudotyped lentiviral vector, suggesting that lower levels of IFITM3 are still sufficient to restrict this viral vector.

Description: Magnetic field has been widely used in clinical diagnostics or for clinical treatment and is an important biomedical technology. Glioblastoma multiforme U87 and U251 are models of a fast growing malignant cancer. We focused on cellular level drafting of these cell lines as a time-dependent effect indicator of static magnetic fields (2000 ± 600 Gauss) by using their fast-growing properties. Cell viability showed a significant decrease ( p  〈 0.01). The results coincided with the occurrence of apoptotic signals or protein expression of cyclin B1 and cyclin dependent kinase 1 in a non-apoptotic manner. Cdk1 was decreased in proportion to ankyrin G and cyclin B1 (Chi-square test, p  = 0.0366). Our findings suggest that static magnetic stimulation creates a specific cyto-proliferative pattern, rather than producing randomized growth impairment.

Description: Solid-liquid equilibrium data of cefquinome sulfate is important to develop industrial crystallization processes for cefquinome sulfate. The solubilities of cefquinome sulfate in five pure solvents (methanol, ethanol, ethylene glycol, acetic acid and water) from 277.15 to 305.15 K and in a binary acetone-water solvent from 278.15 to 293.15 K were measured at atmospheric pressure. The pure-solvent solubility data was correlated to the modified Apelblat and Van’t Hoff equations whereas the mixed-solvent system data was correlated to the modified Apelblat, Van’t Hoff, CNIBS/R-K and Jouyban- Acree models. It was found that the solubilities of cefquinome sulfate in all tested solvents decreased with the increasing of temperature. In addition, the thermodynamic properties of the dissolution processes, including standard Gibbs free energy, enthalpy and entropy changes, were calculated using the Van’t Hoff equation. It was found that the dissolution of cefquinome sulfate is exothermic.

Description: Ni/SiO 2 -ZrO 2 catalysts with Ni loadings of 1 to 13 wt-% were prepared, characterized by elemental analysis, X-ray diffraction, N 2 sorption, temperature programmed oxidation, temperature programmed reduction, and tested for their activity and stability in the dry reforming of methane with carbon dioxide at 850 °C, gas hourly space velocity of 6000 and 1800 h –1 and atmospheric pressure. The SiO 2 -ZrO 2 support as obtained through a simple and efficient sol-gel synthesis is highly porous ( A BET = 90 m 2 ∙g –1 , d P = 4.4 nm) with a homogeneously distributed Si-content of 3 wt-%. No loss of Si or formation of monoclinic ZrO 2 , even after steaming at 850 °C for 160 h, was detectable. The catalyst with 5 wt-% Ni loading in its fully reduced state is stable over 15 h on-stream in the dry reforming reaction. If the catalyst was not fully reduced, a reduction during the early stages of dry reforming is accompanied by the deposition of up to 44 mg∙g –1 carbon as shown by experiments in a magnetic suspension balance. Rapid coking occurs for increased residence times and times-on-stream starting at 50 h. The Ni loading of 5 wt-% on SiO 2 -ZrO 2 was shown to provide an optimal balance between activity and coking tendency.

Description: The productivity of cell culture-derived vaccines grown in anchorage-dependent animal cells is limited by bioreactor surface area. One way to increase the available surface area is by growing cells as monolayers on small spheres called microcarriers, which are approximately 100–250 μm in diameter. In order for microcarrier-based cell culture to be a success, it is important to understand the kinetics of cell growth on the microcarriers. Micro-flow imaging (MFI) is a simple and powerful technique that captures images and analyzes samples as they are drawn through a precision flow cell. In addition to providing size distribution and defect frequency data to compare microcarrier lots, MFI was used to generate hundreds of images to determine cell coverage and confluency on microcarriers. Same-day manual classification of these images provided upstream cell culture teams with actionable data that informed in-process decision making (e.g. time of infection). Additionally, an automated cell coverage algorithm was developed to increase the speed and throughput of the analyses.

Description: Dendritic cells (DCs) are potent antigen presenting cells (APCs). They are also specialized in the induction of cytotoxic T lymphocyte mediated responses against extracellular antigens, including tumour-specific antigens, by presenting peptide-Major Histocompatibility Complex (MHC) I complexes to naïve CD8 + T cells in lymphoid tissues, a process called cross-presentation. Emerging evidence suggests that the efficiency of cross-presentation can be influenced by a unique set of microRNAs (miRNAs). Some are differentially expressed in the course of morphological and functional development of DCs while tumorigenic miRNAs (onco-miRs) can be delivered to and inserted into DCs via exosomes. The latter reprogram the miRNA repertoire of DCs, transforming them from effective APCs to negative modulators of immunity, ultimately aiding cancers to evade host immunity. On the other hand, endogenous microRNAs can influence cross-presentation either positively or negatively. In this review, we discuss the possible mechanisms by which specific miRNAs influence cross-presentation as well as the viability of manipulating the expression of miRNAs that regulate DC cross-presentation as a potential cancer immunotherapy intervention.

Description: Methylglyoxal (MG), a reactive sugar-derived metabolite, exerts harmful effects by inducing oxidative stress, which aggravates a series of diabetic complications, including osteoporosis. The present study was performed to examine the effects of luteolin, a dietary polyphenolic flavonoid, on MG-induced cytotoxicity in MC3T3-E1 osteoblastic cells. Pretreatment of MC3T3-E1 osteoblastic cells with luteolin prevented MG-induced cell death and production of tumor necrosis factor-alpha, intracellular reactive oxygen species, mitochondrial superoxide, and cardiolipin peroxidation. In addition, luteolin increased the levels of glutathione and nuclear factor erythroid 2-related factor 2 (Nrf2) and decreased the inhibition of heme oxygenase-1 activity by MG. Pretreatment with luteolin prior to MG exposure reduced MG-induced mitochondrial dysfunction and increased the peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) and nitric oxide levels, suggesting that luteolin may induce mitochondrial biogenesis. Taken together, these observations indicated that luteolin has potential as a preventive agent against the development of diabetic osteopathy related to MG-induced oxidative stress in diabetes.

Description: This study evaluated the ability of human osteoprotegerin gene-modified autologous periodontal ligament cells (PDLCs) in combination with cell transplantation to promote periodontal regeneration in beagle dogs. Adenovirus Ad5-hOPG-EGFP-transfected PDLCs and BME-10X collagen membranes were fabricated and used for periodontal repair. Buccal periodontal defects (mesiodistal width × depth: 5 × 5 mm) were created on the second, third, and fourth mandibular premolars in six normal beagle dogs, and the defects were histologically and histomorphometrically assessed for periodontal regeneration in the following four groups: (1) hOPG-PDLCs + BME-10X, (2) mock-PDLCs + BME-10X, (3) PDLCs + BME-10X, and (4) BME-10X. The radiographic and histological results suggested that hOPG-PDLCs significantly promoted periodontal defect repair. This study demonstrates the potential of hOPG-modified PDLCs for periodontal tissue regeneration.

Description: Two-dimensional self-assembly of melem at pH-controlled aqueous solution-Au(111) interfaces has been investigated by electrochemical scanning tunneling microscopy. In the solutions with pH〉p K b1 of melem, two ordered self-assembled structures (honeycomb and close-packed structures) and one disordered fibrillar structure were observed as a function of the surface coverage of melem controlled by the electrode potential. In contrast, in the acidic solution with pH〈p K b1 of melem, only the self-assembled honeycomb network was observed in a relatively wide potential range probably due to the presence of monoprotonated melem cations. Dots attributed to counteranions were frequently observed in the pores of the honeycomb network. The lack of close-packed and fibrillar structures at low pH (〈p K b1 ) is attributed to ionic repulsion of melemium cations.

Description: During the last decade biomaterial sciences and tissue engineering have become new scientific fields supplying rising demand of regenerative therapy. Tissue engineering requires consolidation of a broad knowledge of cell biology and modern biotechnology investigating biocompatibility of materials and their application for the reconstruction of damaged organs and tissues. Stem cell-based tissue regeneration started from the direct cell transplantation into damaged tissues or blood vessels. However, it is difficult to track transplanted cells and keep them in one particular place of diseased organ. Recently, new technologies such as cultivation of stem cell on the scaffolds and subsequently their implantation into injured tissue have been extensively developed. Successful tissue regeneration requires scaffolds with particular mechanical stability or biodegradability, appropriate size, surface roughness and porosity to provide a suitable microenvironment for the sufficient cell–cell interaction, cell migration, proliferation and differentiation. Further functioning of implanted cells highly depends on the scaffold pore sizes that play an essential role in nutrient and oxygen diffusion and waste removal. In addition, pore sizes strongly influence cell adhesion, cell–cell interaction and cell transmigration across the membrane depending on the various purposes of tissue regeneration. Therefore, this review will highlight contemporary tendencies in application of non-degradable scaffolds and stem cells in regenerative medicine with a particular focus on the pore sizes significantly affecting final recover of diseased organs.

Description: Approximately half of the transplantable pancreatic islet tissue is lost during isolation, including the digestion and purification steps. Modifying the isolation method could increase the yield. This would enable the one donor-one recipient concept and improve the therapeutic effects of islet transplantation. This study aims to improve islet transplantation by increasing the yield of islets from the pancreas, both the number of islets and their size. Therefore, we used a sericin-containing isolating solution. Rat pancreatic islets were isolated by collagenase digestion and hand picking. We refer to islets isolated with or without sericin in the isolation solution as the sericin and control group, respectively. Volume yield, endocrine function, and islet morphology were compared between the groups. Histological distribution of sericin was evaluated by immunofluorescence staining to examine its mechanism of action in pancreatic islets. The pancreatic islet yield in the sericin group was significantly higher than that in the control group. The endocrine function of islets in the sericin group was comparable to that of islets isolated by conventional methods. Sericin adhered to the surface of isolated pancreatic islets and colocalized with E-cadherin, a cell membrane protein, which might explain the cytoprotective effects of sericin. The islet morphology tended to be better preserved in the sericin group. Sericin could prevent cytoarchitectural damage during the isolation and purification process, resulting in increased pancreatic islet yield. This suggests that sericin could contribute to islet therapy by enhancing the stability of islets.

Description: Cholesterol oxidation products (oxycholesterols) are produced from cholesterol by automatic and/or enzymatic oxidation of the steroidal backbone and side-chain. Oxycholesterols are present in plasma and serum, suggesting that oxycholesterols are related to the development and progression of various diseases. However, limited information is available about the absolute amounts of oxycholesterols in organs and tissues, and the physiological significance of oxycholesterols in the body. In the present study, we quantified the levels of 13 oxycholesterols in white adipose tissue (WAT) of mice and then evaluated correlations between each oxycholesterol level and WAT weight. The sum of the levels of 13 oxycholesterols in WAT (white adipose tissue) was 15.9 ± 3.4 μg/g of WAT weight and approximately 1 % of cholesterol level. Among oxycholesterols, the levels of 27-hydroxycholesterol (27-OH), an endogenous oxycholesterol produced by enzymatic oxidation, and the relative WAT weights were significantly negatively correlated. Next, we evaluated the effects of 27-OH on lipogenesis and adipogenesis in 3T3-L1 cells. TO901317 (TO), a potent and selective agonist for LXRα, significantly increased intracellular TAG contents, while 27-OH significantly reduced the contents to half when compared with control (DMSO) and completely abolished the effect of TO. In addition, 27-OH significantly reduced the mRNA levels of lipogenic (LXRα and FAS) and adipogenic genes (PPARγ and aP2) during adipocyte maturation of 3T3-L1 cells. In conclusion, our results indicate that 27-OH suppresses lipid accumulation by down-regulating lipogenic and adipogenic gene expression in 3T3-L1 cells.

Description: Generation of multiple cell types from embryonic stem (ES) cells and induced pluripotent stem cells is crucial to provide materials for regenerative medicine. EGAM1N has been found in preimplantation mouse embryos and mouse ES cells as a functionally unclassified homeoprotein. Recently, we reported that expression of EGAM1N suppressed the in vitro differentiation of ES cells into progenitor cells that arise in early embryogenesis. To clarify the effect of EGAM1N on terminal differentiation, embryoid bodies (EBs) were prepared from ES cells expressing exogenous Egam1n . In EBs expressing Egam1n , cardiomyogenesis was inhibited by impairing the expression of crucial transcription factors Brachyury T and Nkx2.5 in the generation of mesoderm and cardiomyocytes, respectively. Expression levels of Mef2c , another crucial gene for cardiomyogenesis, were unaffected. Conversely, the expression levels of Gata6 and Plat , markers for the primitive endoderm lineage, and Cdx2 , a marker for the trophectoderm lineage, were increased. These results suggested that certain cell populations in EBs expressing Egam1n preferentially differentiated to such cell lineages. Our results suggest that EGAM1N not only affects the generation of progenitor cells during early embryogenesis, but also the progression of terminal differentiation, such as cardiomyogenesis, in mouse ES cells.

Description: A series of Mn-promoted 15 wt-% Ni/Al 2 O 3 catalysts were prepared by an incipient wetness impregnation method. The effect of the Mn content on the activity of the Ni/Al 2 O 3 catalysts for CO 2 methanation and the comethanation of CO and CO 2 in a fixed-bed reactor was investigated. The catalysts were characterized by N2 physisorption, hydrogen temperature-programmed reduction and desorption, carbon dioxide temperature-programmed desorption, X-ray diffraction and highresolution transmission electron microscopy. The presence of Mn increased the number of CO 2 adsorption sites and inhibited Ni particle agglomeration due to improved Ni dispersion and weakened interactions between the nickel species and the support. The Mn-promoted 15 wt-% Ni/Al 2 O 3 catalysts had improved CO 2 methanation activity especially at low temperatures (250 to 400 °C). The Mn content was varied from 0.86% to 2.54% and the best CO 2 conversion was achieved with the 1.71Mn-Ni/Al 2 O 3 catalyst. The co-methanation tests on the 1.71Mn-Ni/Al 2 O 3 catalyst indicated that adding Mn markedly enhanced the CO 2 methanation activity especially at low temperatures but it had little influence on the CO methanation performance. CO 2 methanation was more sensitive to the reaction temperature and the space velocity than the CO methanation in the co-methanation process.

Description: We have previously shown that cultured adipocytes have the ability to biosynthesize prostaglandin (PG) I 2 called alternatively as prostacyclin during the maturation phase by the positive regulation of gene expression of PGI synthase and the prostanoid IP receptor. To clarify how prostacyclin regulates adipogenesis, we investigated the effects of prostacyclin and the specific agonists or antagonists for the IP receptor on the storage of fats during the maturation phase of cultured adipocytes. Exogenous PGI 2 and the related selective agonists for the IP receptor including MRE-269 and treprostinil rescued the storage of fats attenuated by aspirin, a cyclooxygenase inhibitor. On the other hand, selective antagonists for IP such as CAY10441 and CAY10449 were effective to suppress the accumulation of fats as GW9662, a specific antagonist for peroxisome proliferator-activated receptor (PPAR)γ. Thus, pro-adipogenic action of prostacyclin can be explained by the action mediated through the IP receptor expressed at the maturation stage of adipocytes. Cultured adipocytes incubated with each of PGI 2 and MRE-269 together with troglitazone, an activator for PPARγ, exhibited additively higher stimulation of fats storage than with either compound alone. The combined effect of MRE-269 and troglitazone was almost abolished by co-incubation with GW9662, but not with CAY10441. Increasing concentrations of troglitazone were found to reverse the inhibitory effect of CAY10441 in a dose-dependent manner while those of MRE-269 failed to rescue adipogenesis suppressed by GW9662, indicating the critical role of the PPARγ activation as a downstream factor for the stimulated adipogenesis through the IP receptor. Treatment of cultured adipocytes with cell permeable stable cAMP analogues or forskolin as a cAMP elevating agent partly restored the inhibitory effect of aspirin. However, excess levels of cAMP stimulated by forskolin attenuated adipogenesis. Supplementation with H-89, a cell permeable inhibitor for protein kinase A (PKA), had no effect on the promoting action of PGI 2 or MRE-269 along with aspirin on the storage of fats, suggesting that the promotion of adipogenesis mediated by the IP receptor does not require the PKA activity.

Description: Unmonitored use of plant extractions alone or in combination with drugs may cause important health problems and toxic effects. Limonium (Plumbaginaceae) plants are known as antibacterial, anticancer and antivirus agent. But it is possible that this genus may have toxic effects. This study evaluated the mutagenic and cytotoxic effects of Limonium globuliferum (Boiss. et Heldr.) O. Kuntze (Plumbaginaceae) acetone/methanol (2:1), and methanol extracts of root, stem, and leaf. Different parts of this species were used in order to compare the mutagenic and cytotoxic effects of these parts. Ames test was carried out with S. typhimurium TA98, and TA100 strains. Strains were incubated at 37 °C for 72 h. MDBK cell line was used in MTT test. 10,000, 1000, 100, 10, 1 and 0.1 µg/plate concentrations of plant extracts were used in Ames test. 50, 25, 12.5, 6.25 and 3.125 µg/ml concentrations of root, stem and leaf acetone/methanol (2:1) and methanol extracts were used in MTT test. Ames test results indicated that only methanol leaf extract (10,000 µg/plate) had mutagenic activity. L. globuliferum root methanol extracts (3.125 and 6.25 µg/ml) increased the proliferation rates. Root acetone/methanol (2:1) extracts were found highly cytotoxic in all treatments. The results indicated that leaf extracts had lower cytotoxic effects than root and stem extracts. High concentrations of L. globuliferum stem and leaf methanol extracts showed cytotoxic activity in all treatment periods while low concentrations of the stem methanol extracts increased the proliferation rates.

Description: The embryonic cardiomyocyte cell line H9C2 is commonly used in numerous in vitro studies, including cardiotoxicity analyses of new drugs. So far no results were published for studies on cell parameters variability during the cell line ageing process. For this reason the aim of the study was to evaluate the effect of a number of H9C2 rat embryonic cardiomyocytes passages on repeatability of study results for selected cytotoxicity parameters, with doxorubicin as a model toxic agent. The cultures were passaged twenty-five times. Cells from passage 1, 5, 10, 15, 20 and 25 were treated with doxorubicin for 24 h. Then drug cytotoxicity was evaluated with the MTT test and additionally the nuclear factor erythroid 2–related factor (Nrf2) gene expression was examined. The analysis of oxidative stress intensity and cell morphology was also assessed. The microscopic appearance of cells indicates that untreated cardiomyocytes morphology changes as well as sensitivity to toxic effects increases with the number of passages. Also an increase in oxidative stress in cells occurs with further passaging of cardiomyocytes. Statistical significance of differences in conducted tests results depended on doxorubicin concentration but in many cases the H9C2 line was found to be a reliable in vitro model only for the first five passages. For this reason it is important to take into consideration that further culturing of cardiomyocytes may not ensure repeatability of study results due to the culture ageing.

Description: The atmospheric pressure plasma jet (APPJ) was used to enhance the sensitivity of industrially important polyaniline (PANI) for detection of organic vapors from amides. The gas sensing mechanism of PANI is operating on the basis of reversible protonation or deprotonation, whereas the driving force to improve the sensitivity after plasma modifications is unknown. Herein we manage to solve this problem and investigate the sensing mechanism of atmospheric plasma treated PANI for vapor detection of amides using urea as a model. The results from various analytical techniques indicate that the plausible mechanism responsible for the improved sensitivity after plasma treatment is operating through a cyclic transition state formed between the functional groups introduced by plasma treatment and urea. This transition state improved the sensitivity of PANI towards 15 ppm of urea by a factor of 2.4 times compared to the non-treated PANI. This plasma treated PANI is promising for the improvement of the sensitivity and selectivity towards other toxic and carcinogenic amide analytes for gas sensing applications such as improving material processing and controlling food quality.