Hr Of Culture Research Articles

State of antioxidant systems and phenolic compounds’ production in Hypericum perforatum L. hairy roots

Hypericum perforatum hairy root clones (HR A–HR O) transformed with Agrobacterium rhizogenes A4 were evaluated for growth characteristics, phenolic compounds production, antioxidant/radical scavenging activity, antioxidant enzymes, and oxidative stress markers. The screening of growth characteristics revealed that HR clones were characterized with higher biomass accumulation, root elongation, and lateral branching in comparison to non-transformed roots. Significant increase of phenolics production in HR clones was related to phenylalanine ammonia lyase activity indicating an up-regulation of phenylpropanoid/flavonoid metabolism. Positive correlation of phenolics, flavonoids, and tannins with antioxidant assays indicated that these secondary metabolites significantly contributed to the antioxidant/radical scavenging properties of HR cultures. Regarding the enzymatic antioxidant state, an enhancement of superoxide dismutase activity in HR lines coincided with decrease in O 2 •− production rate, while ascorbate peroxidase and catalase greatly contributed to the reduction of H2O2 levels. The substantial accumulation of malondialdehyde in HR clones indicated an efficiency of antioxidant enzymes to reduce O 2 •− and H2O2 at levels that are not deleterious for membrane lipids. These results confirmed the involvement of an efficient antioxidant defense system in the response adjustment of H. perforatum HR cultures to transformation process. Two superior clones denoted as HR B and HR F were selected as fast-growing and high biomass yielding lines with up-regulated phenolic compounds’ production, antioxidant, and radical scavenging activity. Altogether, the best performing H. perforatum HR clones could be proposed as a prospective biotechnological system for scale-up production of antioxidant phenolic compounds.

THE APOPTOTIC EFFECT OF RESVERATROL ON HUMAN PAPILLOMA VIRUS POSITIVE CASKI CELL-LINE: A TIME AND DOSE DEPENDENCE STUDY.

Background & objectives: Resveratrol (trans-3,4,5’ –trihydroxystilbene) is an active compound in food, such as redgrapes, peanuts, and berries. A body of evidence shows that resveratrol is able to inhibit thegrowth of many cancers such as leukemia, breast cancer and primary brain tumors. The objective of the present study was to demonstrate the dose and time dependent apoptotic effect of Resveratrol on Human Caski cell line.
 Methods: We used cell viability assays like MTT and Trypan blue assays to demonstrate the inhibition of cancer cell proliferation in a dose and time dependent manner.
 Results: Our results have shown that Resveratrol inhibited proliferation of Caski cells in a concentration and time dependent manner in 48 hr cultures. No significant effect was observed in 24 hour cultures of cell viability assays.
 Interpretation & conclusions: The results indicated that Resveratrol and NAC inhibited cell growth in Caski cells as a function of dose and time.
 Keywords: Resveratrol , Cervical cancer, apoptosis

Increased endothelin-1-mediated vasoconstriction after organ culture in rat and pig ocular arteries can be suppressed with MEK/ERK1/2 inhibitors.

Even though retinal vascular changes following ischaemia have been poorly understood, the upregulation of vasoconstrictive endothelin-1 (ET-1) receptors (ETA /ETB ) following global cerebral ischaemia has been described. The aim of this study was to investigate whether or not the MEK/ERK1/2 pathway is involved in the observed upregulation and whether specific MEK/ERK1/2 inhibitors U0126 and trametinib can prevent it. The aim was also to localize ETA and ETB receptors using immunohistochemistry in both fresh rat ophthalmic arteries and after 24-hr organ culture and study the receptors functionally using myography. Pig retinal arteries also underwent 24-hr organ culture to validate similar responses across species and the retinal vasculature. Results showed that following organ culture there is a significant increase in ET-1-mediated vasoconstriction, in particular via the ETB receptor. Furthermore, immunohistochemistry revealed a clear increase in pERK in the smooth muscle cells of rat ophthalmic artery. U0126 and trametinib were successful in attenuating the functional vasoconstriction in both rat and pig, as well as restoring immunofluorescence of pERK to fresh levels and counteracting ETB expression in the smooth muscle cells of the rat ophthalmic artery. This is the first study to show that the MEK/ERK1/2 pathway in responsible for the increase in functional vasoconstriction via ET-1 receptor in rat ophthalmic and pig retinal arteries. Furthermore, this study is the first to suggest a way of inhibiting and preventing such an increase. With these results, we suggest a novel approach in retinal ischaemia therapy.

Spontaneous hepatocyte migration towards an endothelial cell tube network.

A crucial part of the engineering liver tissue is contribution of nonparenchymal cells and maintenance of a complex three-dimensional (3D) structure in vitro for their normal physiology and function. We generated 3D hepatic tissue using primary isolated rat hepatocytes and an endothelial cell tube network from human endothelial vein epithelial cells (HUVECs). To create the 3D hepatic tissue, coculture of primary hepatocytes and tube-structured HUVECs was performed on a Matrigel®. After the HUVECs formed the tube structures, primary isolated rat hepatocytes were inoculated onto the HUVEC tube-structured layer and cultured for 24hr. We investigated the cell migration, cellular interaction, and distributions of HUVEC tube structures and hepatocytes using multi cell-imaging incubator, confocal microscopy, and electron microscopy analyses. During the culture time, time-lapse imaging showed spontaneous migration of the hepatocytes in the gel, and after the 24-hr culture period, the vast majority of the hepatocytes had moved and adhered to the surface of the HUVEC tube structures. A confocal microscopy assay confirmed this unique 3D cellular interaction between hepatocytes and HUVEC tube structures. The hepatocytes were able to maintain their spherical shape, as well as HUVECs (tube-like form with tubular cavity). We speculate that coculturing of hepatocytes and endothelial cells replicates part of their normal physiology and may help induce migration in vitro and the growth of complex biological tissue structures.

P027 FCGRIIIA gene variant influences differential rituximab mediated NK cell response against different B-cell lymphoma targets

Aim Rituximab based therapy is the frontline treatment for aggressive B-cell malignancies (Diffuse large B cell lymphoma, DLBCL, Mantle Cell lymphoma, MCL and post-transplant lymphoproliferative disease, PTLD). NK cell mediated ADCC of target B-cells is one of the major modes of Rituximab action. The binding of Rituximab to FcγRIIIa during ADCC is influenced by FcγRIIIa (158 V/F; Valine to Phenylalanine) gene variant, where −158 V is the high affinity variant. However, the clinical relevance of FcγRIIIa gene variant in prediction of Rituximab response in contentious. Here, we analyzed if FcγRIIIa gene variant stratifies Rituximab mediated NK cell response against different B-cell lymphoma targets. Methods Peripheral blood mononuclear cells (MNCs) were obtained from 15 healthy volunteers carrying different FcγRIIIa genotypes (VV/VF n = 9; and FF, n = 6). Multiple representative cell lines of various B-cell lymphoma, and one representing EBV transformed B lymphoblast as a surrogate of PTLD, were selected due to their different phenotypic characteristics. K-562 cells were used as a positive control. MNCs were incubated with rituximab (5 μg/ml) coated different B-cell lymphoma cell lines in a 4hr culture. NK cell response (degranulation and IFN-γ production) was analyzed by multicolor flow cytometry. Results Rituximab mediated NK cell response against all B-cell lymphoma cells was consistently higher in individuals carrying FcγRIIIa 158 V allele (VV or VF genotypes) as compared to individuals carrying FF genotype. However, a considerable heterogeneity was observed in NK cell responses linked to FcγRIIIa 158 V allele against different lymphoma targets. Influence of FcγRIIIa gene variants was most evident against SUDHL-8, a GCB-DLBCL cell line (median % of functional NK cells was 3.2% and 1.1% in individuals with VV/VF and FF genotypes respectively, P = 0.007) and least evident against JeKo, an MCL cell line (5.8% and 4.2% in individuals with VV/VF and FF genotypes respectively, P = 0.27). Conclusions Presence of FcγRIIIa 158 V alleles led to stronger NK cell response to rituximab treated lymphoma cells, though there was a heterogeneity in responses to different lymphoma cell targets. Our findings have strong implications for prediction of Rituximab response in B-cell lymphoma patients.

Human DMBT1-Derived Cell-Penetrating Peptides for Intracellular siRNA Delivery.

Small interfering RNA (siRNA) is a promising molecule for gene therapy, but its therapeutic administration remains problematic. Among the recently proposed vectors, cell-penetrating peptides show great promise in in vivo trials for siRNA delivery. Human protein DMBT1 (deleted in malignant brain tumor 1) is a pattern recognition molecule that interacts with polyanions and recognizes and aggregates bacteria. Taking advantage of these properties, we investigated whether specific synthetic DMBT1-derived peptides could be used to formulate nanoparticles for siRNA administration. Using an electrophoretic mobility shift assay and UV spectra, we identified two DMBT1 peptides that could encapsulate the siRNA with a self- and co-assembly mechanism. The complexes were stable for at least 2 hr in the presence of either fetal bovine serum (FBS) or RNase A, with peptide-dependent time span protection. ζ-potential, circular dichroism, dynamic light scattering, and transmission electron microscopy revealed negatively charged nanoparticles with an average diameter of 10–800 nm, depending on the reaction conditions, and a spherical or rice-shaped morphology, depending on the peptide and β-helix conformation. We successfully transfected human MCF7 cells with fluorescein isothiocyanate (FITC)-DMBT1-peptide-Cy3-siRNA complexes. Finally, DMBT1 peptides encapsulating an siRNA targeting a fluorescent reporter gene showed efficient gene silencing in MCF7-recombinant cells. These results lay the foundation for a new research line to exploit DMBT1-peptide nanocomplexes for therapeutic siRNA delivery.

A review on the HR culture: India, US, Europe

The era of globalization of world economy has lead to the high complication, disturbance and competition in the market place. Human resource has become the indispensable part for the success of business. The competitive advantages of the organizations which need to be built sharpened and leveraged are the people. Agility, responsiveness, innovation or transcendence, efficiency and execution is the fundamental need of organizations for sustainable competitive advantage. It is here that organizations can be developed to their fullest capacity with the help of human resource. The work environment in which employees feel motivated and most satisfied when their needs and values are in consistency with those in their workplace is basically defined as the culture of that place. It is like a lifeguard on duty.

Lipopolysaccharides, but not Angiotensin ll, lnduces Direct Pro-lnflammatory Effects in Cultured Mouse Arteries and Human Endothelial and Vascular Smooth Muscle Cells.

Angiotensin II (Ang II) might induce pro-inflammatory effects directly in the vascular wall independently of its haemodynamic effects. The aim of our study was to investigate the putative direct pro-inflammatory and vasomotor effects of Ang II and compare to those of lipopolysaccharides (LPS) in mouse isolated mesenteric resistance-sized arteries (MRA) supported by experiments in cultured human primary endothelial and vascular smooth muscle cells. Results showed that 24-hr organ culture of mouse MRA with 10 nM Ang II had, unlike 100 ng/mL LPS, no effects on IL-6 or MCP-1 secretion, VCAM1 mRNA expression or endothelial function, while Ang II significantly decreased maximal vasomotor responses to phenylephrine. In support, 24-hr organ culture of mouse MRA significantly suppressed Agtr1a mRNA and augmented Tlr4 mRNA along with attenuated vasomotor responses to Ang II. Moreover, contrary to LPS and TNF-α, Ang II and [Sar1]-Ang II had no concentration- or time-dependent effects on IL-6 and MCP-1 secretion in human umbilical vein endothelial cells (HUVEC) and human aortic smooth muscle cells (HASMC). AGTR1 or AGTR2 mRNA expression was undetectable in HUVEC, whereas HASMC expressed only AGTR1 mRNA. In summary, contrary to previous studies and the observed effects of LPS, we could not demonstrate direct vascular pro-inflammatory effects of Ang II ex vivo or in vitro. As indicated by our results, down-regulation or desensitization of AT1 R during culture may explain our findings.

Maoto, a Traditional Japanese Herbal Medicine, Inhibits Uncoating of Influenza Virus

We previously reported in randomized controlled trials that maoto, a traditional herbal medicine, showed clinical and virological efficacy for seasonal influenza. In this study, a culturing system for influenza was used to test the effect of maoto. A549 cells in the culture were infected with influenza virus A (PR8) and followed after treatment with maoto; the virus titers in the culture supernatant, intracellular viral proteins, and viral RNA were determined. When infected cells were cultured with maoto for 24 hr, the virus titer and protein were significantly reduced compared with medium only. Other subtypes, A/H3N2, H1N1pdm, and B, were also inhibited by maoto. Proliferation of viral RNA in a 6 hr culture was inhibited by maoto in the early phase, especially in the first 30 min. Focusing on the entry step of the influenza virus, we found that endosomal pH, regulated by vacuolar-type H+ ATPase (V-ATPase) located in the membrane, was increased when treated with maoto. We also found that uncoating of influenza viruses was also inhibited by maoto, resulting in the increase of the number of virus particles in endosomes. These results strongly suggest that the inhibition of endosomal acidification by maoto results in blocking influenza virus entry to cytoplasm, probably through the inhibition of V-ATPase. The present study provides evidence that supports the clinical use of maoto for the treatment of influenza.

Standard Bank: The Agile Transformation

Josef Langerman, Head of IT Transformation for Standard Bank, reflected on the extraordinary transformation that his organization’s IT group had recently experienced. Over the past three years, Standard Bank’s IT group had changed from the relatively well accepted systems development lifecycle/waterfall model to a revolutionary large scale agile approach. The results had been gratifying. But it left a question unanswered. Now that things were starting to stabilize, what should be the next steps? The 154-year-old Standard Bank was the largest banking group in Africa, and the 5th largest company headquartered in South Africa. The bank offered a range of corporate, business and personal banking as well as financial services. Its 49,000 employees served over 15 million customers, in 20 countries across the continent of Africa, as well as other countries scattered around the globe. Standard Bank’s IT group, located within the company’s Johannesburg headquarters, had over 6000 employees. The group managed the bank’s technology infrastructure–including a network of nearly 10,000 ATMs, its applications development, testing, deployment, maintenance and operations. By 2014, the bank recognized that its IT performance was lagging industry benchmarks in productivity, turnaround time and employee satisfaction. Employing a “do it in-house” philosophy, it embarked on a major transformation. Abandoning traditional highly structured approaches to project management and development, it had adopted an agile philosophy that was most commonly seen in much smaller organizations and technology startups. The results had been impressive–productivity, cycle time and organizational health indicators had all risen dramatically. The group had also achieved substantial reductions in its budget. Even skeptics within the organization could not fail to be impressed. Now, however, Langerman wondered about the future. He had been cautioned by his group’s HR Culture Transformation Guide that rapid improvement could easily be followed by disillusionment. What could be done to keep the momentum going forward? Should the bank double down on the types of changes to culture, practice and training that had led to its success, or was it time to let things settle? And who should be guiding the change? Should the implementation continue entirely in-house, or should outside consultants–that were working in other areas of the bank–play a significant role? In the near future, he would need to present his recommendations to the group’s CIO.

Standard Bank: The Agile Transformation

South Africa’s largest bank has recently completed a transformation from traditional systems development to the scaled agile framework. The individual leading the transformation is now considering how to keep the momentum going and possible new directions. Josef Langerman, Head of IT Transformation for Standard Bank, reflected on the extraordinary transformation that his organization’s IT group had recently experienced. Over the past three years, Standard Bank’s IT group had changed from the relatively well accepted systems development lifecycle/waterfall model to a revolutionary large scale agile approach. The results had been gratifying. But it left a question unanswered. Now that things were starting to stabilize, what should be the next steps? The 154-year-old Standard Bank was the largest banking group in Africa, and the 5th largest company headquartered in South Africa. The bank offered a range of corporate, business and personal banking as well as financial services. Its 49,000 employees served over 15 million customers, in 20 countries across the continent of Africa, as well as other countries scattered around the globe. Standard Bank’s IT group, located within the company’s Johannesburg headquarters, had over 6000 employees. The group managed the bank’s technology infrastructure–including a network of nearly 10,000 ATMs, its applications development, testing, deployment, maintenance and operations. By 2014, the bank recognized that its IT performance was lagging industry benchmarks in productivity, turnaround time and employee satisfaction. Employing a “do it in-house” philosophy, it embarked on a major transformation. Abandoning traditional highly structured approaches to project management and development, it had adopted an agile philosophy that was most commonly seen in much smaller organizations and technology startups. The results had been impressive–productivity, cycle time and organizational health indicators had all risen dramatically. The group had also achieved substantial reductions in its budget. Even skeptics within the organization could not fail to be impressed. Now, however, Langerman wondered about the future. He had been cautioned by his group’s HR Culture Transformation Guide that rapid improvement could easily be followed by disillusionment. What could be done to keep the momentum going forward? Should the bank double down on the types of changes to culture, practice and training that had led to its success, or was it time to let things settle? And who should be guiding the change? Should the implementation continue entirely in-house, or should outside consultants–that were working in other areas of the bank–play a significant role? In the near future, he would need to present his recommendations to the group’s CIO.

Production of phenolic compounds, antioxidant and antimicrobial activities in hairy root and shoot cultures of Hypericum perforatum L.

Three hairy root clones of Hypericum perforatum (HR 2, HR 15 and HR 27) transformed with Agrobacterium rhizogenes A4M70GUS and their corresponding regenerated shoot culture clones (HRRS) were compared for differences in growth, production of phenolic compounds, antioxidant and antimicrobial activities. Transgenic clones were selected on the basis of morphological evaluation, genetic and molecular analyses. The clone HR 2 had the highest biomass accumulation, while HR 27 showed the highest shoot regeneration potential. The total phenolics and flavan-3-ols were enhanced in all tested transgenic cultures, while total flavonoids and hypericins were augmented in HRRS clones compared to non-transformed shoots. The HRRS clones produced substantial amounts of chlorogenic acid and 3-p-coumaroylquinic acid. Regarding the flavonoids, they produced significant contents of luteolin hexoside (HRRS 2), quercitrin and quercetin (HRRS 15) and isoquercetin (HRRS 27), while HR 2 and 15 accumulated 4-O-methylkaempferol-O-hexoside and quercetin 6-C-glucoside, respectively. The HR 15 was promising for the production of catechin and procyanidin derivatives and together with its HRRS clone exhibited a high potential for hyperforin and adhyperforin production. All identified naphtodianthrones were confirmed in HRRS 2 and 15 clones. Among xanthones, mangiferin was found as the major compound in HRRS, while trihydroxy-1-metoxy-C-prenyl xanthone was dominant in HR clones. Antimicrobial activity of transgenic cultures revealed that HRRS 15 strongly inhibited the growth of Bacillus cereus, Micrococcus flavus, Pseudomonas aeruginosa and Escherichia coli. Altogether, H. perforatum HR and HRRS cultures could be proposed as promising experimental systems for enhanced production of phenolic compounds with antioxidant and antibacterial properties.

High-throughput screening of clinically approved drugs that prime polyethylenimine transfection reveals modulation of mitochondria dysfunction response improves gene transfer efficiencies.

Nonviral gene delivery methods are advantageous over viral vectors in terms of safety, cost, and flexibility in design and application, but suffer from lower gene transfer efficiency. In addition to modifications to nucleic acid design and nonviral carriers, new tools are sought to enhance transfection. Priming is the pharmacological modulation of transfection efficiency and transgene expression, and has demonstrated transfection increase in several compounds, for example, chloroquine and glucocorticoids. To develop a library of transfection priming compounds, a high‐throughput screen was performed of the NIH Clinical Collection (NCC) to identify clinical compounds that prime polyethylenimine (PEI) transfection. HEK293T cells were treated with priming compounds, then transfected with enhanced green fluorescent protein (EGFP)‐encoding plasmid by PEI. After 48‐hr culture, primed and transfected cells were assayed for transfection, cell proliferation, and cell viability by fluorescence measurement of EGFP reporter, Hoechst 33342 nuclei stain, and resazurin metabolic assay. From the microscope image analysis and microplate measurements, transfection fold‐changes were determined, and compounds resulting in statistically significant transfection fold‐change were identified. NCC compounds were clustered using PubChem fingerprint similarity by Tanimoto coefficients in ChemmineTools. Fold‐changes for each compound were linked to drug clusters, from which drug classes that prime transfection were identified. Among the identified drugs classes that primed transfection increases were antioxidants, GABAA receptor modulators, and glucocorticoids. Resveratrol and piceid, stilbenoid antioxidants found in grapes, and zolpidem, a GABAA modulator, increased transfection nearly three‐fold. Literature indicate interaction of the identified transfection priming drug clusters with mitochondria, which may modulate mitochondrial dysfunction known to be associated with PEI transfection.

Transcriptome profiling reveals differential expression of interferon family induced by dengue virus 2 in human endothelial cells on tissue culture plastic and polyacrylamide hydrogel.

A cell model is critical for studying the molecular mechanisms of dengue virus 2 (DENV-2) invasions and cell bioactivity can be easily affected by the substrate matrix. Tissue culture plastic (TCP) and polyacrylamide hydrogel (PAMH) are two kinds of matrices widely used for cells. The effects of different matrices on the cultured cells with DENV-2 invasion remain unknown. To address the issue, the effects of TCP and PAMH were explored in primary human umbilical vein endothelial cells (HUVECs) with DENV-2 invasion. HUVECs were assigned into four groups: group A (cultured on TCP), group B (cultured on PAMH), group C (cultured on TCP with DENV-2 invasion), and group D (cultured on PAMH with DENV-2 invasion). Flow cytometry was performed on HUVECs after 48-hr culture. Gene expression patterns were analyzed by gene microarray. The levels of interleukin-29 (IL-29) were measured by real-time qRT-PCR and ELISA. There were no cell apoptosis induced by DENV-2 in HUVECs cultured on TCP and PAMH (P > 0.05). After DENV-2 invasion, the up-regulated genes involve in the activities of oligoadenylate synthetase (OAS), interferon-related cytokine, and growth factors so on. The up-regulated pathways involve in the responses to DENV-2 and innate immunity. IL-29 was induced in the HUVECs on PAMH when compared with the cells on TCP (P < 0.05). Thus, different matrices cause different immune responses, which should be considered in the cell models for exploring the molecular mechanisms of DENV-induced diseases.

Abstract 4216: Targeting NANOG: genes, proteins and response to viral RNAi in preclinical models

Abstract Purpose: Since the NANOG family controls stemness and neoplastic progression in human colorectal carcinoma (CRC), our postulate is that targeting the dominant NANOG and NANOGP8 members with viral vector delivered shRNA will inhibit CRC progression in preclinical models and that replicating viral vectors are more active than non-replicating vectors. Methods: Cells were human CRC lines (Clone A, CX-1 and LS174T) and mouse fibroblasts (3T3). shRNA specific to NANOG (shNG-1), NANOGP8 (shNp8-1) or a control (shNEG) were created in lentivirus (LV) and chimeric adenovirus 5/3 (Ad5/3). CRC and 3T3 were cultured in monolayer with serum-containing medium or in suspension in ultra low attachment plates without serum. Nanostring 770 gene cancer panel was analyzed in 72 hr cultures with NMF clustering and GSEA analysis. Annexin V expression and caspase-3, 8 or 9 activation in suspension culture assessed Apoptosis. LV or Ad5/3 shRNAs were added to spheroids in 3-D suspension cultures to test therapy in vitro. Intratumoral injection of LV or Ad5/3 shRNA into 5-10 mm sub cutis nodules in groups of 5-10 NOD/SCID mice measured activity in vivo. Results: LV shNp8-1 in both Clone A and CX-1 caused significant inhibition of TGF-beta, EGFR, NRF2, E2F3 and the Cyclin D1 pathways by NMF and GSEA analysis in monolayer culture. LV shNp8-1 inhibited growth of LS174T, Clone A and CX-1 by 50% in 3 days in 3-D suspension cultures without further decrease. Apoptosis was due to Caspase 9 activation. In contrast, Ad5/3-shNp8-1 decreased spheroid growth by up to 94% at 9 -14 days while Ad5/3 decreases spheroid growth by 80%. Ad5/3-shNp8-1 but nit Ad5/3 was active in co-cultures with 3T3 to a 2:1 CRC:3T3 ratio. Transduction level was ∼50% in LV treated cells but ∼90% in Ad5/3 treated cells. Intratumoral injection of LVshNG-1 or LVshNp8-1, decreased tumor size by 50% compared to LVshNEG or untreated CX-1 tumors for 11 days after which tumor growth rebounded to match controls. Ad5/3-shNp8-1 injection into 5-9 mm nodules inhibited CX-1 and LS14T growth by ∼50% until euthanized. Conclusions: Targeting NANOGP8 more than NANOG inhibits proliferation and activates apoptosis through multiple pathways. Nonreplicating LV delivered shRNA inhibited tumor growth in in vitro and in vivo preclinical models but replicating oncolytic Ad5/3 infects more cells and promotes shRNA efficacy. Replicating vectors targeting NANOG are important for further development. Citation Format: J. Milburn Jessup, Abid R. Mattoo, Nikolay Korokhov. Targeting NANOG: genes, proteins and response to viral RNAi in preclinical models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4216. doi:10.1158/1538-7445.AM2015-4216

프로바이오틱스 생산 exopolysaccharide에 의한 항염증 활성

The present study isolated seven different kinds of probiotics from various food sources and identified them with Bacillus sp. and Lactobacillus sp. by 16S rDNA sequencing. Their supernatants were prepared after a 24 hr culture, and their effects on nitric oxide (NO) production in mouse RAW 264.7 cells were investigated. Among the treated samples, the culture supernatants of two strains (Bacillus sp. FG-1 and Lactobacillus sp. FG-6) significantly decreased NO production in LPS-activated RAW 264.7 cells. Moreover, they dramatically reduced the expression of pro-inflammatory genes such as COX-2, iNOS, and TNF-α. To examine whether exopolysaccharide (EPS) is responsible for the anti-inflammatory effects of probiotics, EPS was purified from the culture supernatants of Bacillus sp. FG-1 and Lactobacillus sp. FG-6 strains. The EPS treatment produced by FG-1 and FG-6 strains decreased NO production in a dose-dependent manner in LPS-stimulated RAW 264.7 cells without affecting cell viability, while also reducing pro-inflammatory gene expression. Overall, these results suggest that EPS might be one of the key molecules responsible for the anti-inflammatory effects of probiotics.

Optimization of multi-epitopic HIV-1 recombinant protein expression in prokaryote system and conjugation to mouse DEC-205 monoclonal antibody: implication for in-vivo targeted delivery of dendritic cells.

Multi-epitopic protein vaccines and direction of vaccine delivery to dendritic cells (DCs) are promising approaches for enhancing immune responses against mutable pathogens. Escherichia coli is current host for expression of recombinant proteins, and it is important to optimize expression condition. The aim of this study was the optimization of multi-epitopic HIV-1 tat/pol/gag/env recombinant protein (HIVtop4) expression by E. coli and conjugation of purified protein to anti DEC-205 monoclonal antibody as candidate vaccine. In this study, expression was induced in BL21 (DE3) E. coli cells by optimization of induction condition, post induction incubation time, temperature and culture medium formula. Some culture mediums were used for cell culture, and isopropyl-beta-D-thiogalactopyranoside was used for induction of expression. Protein was purified by Ni-NTA column chromatography and confirmed against anti-His antibody in western-blotting. To exploit DCs properties for immunization purposes, recombinant protein chemically coupled to αDEC-205 monoclonal antibody and confirmed against anti-His antibody in western-blotting. The optimum condition for expression was 1 mM IPTG during 4 hr cultures in 2XYT medium, and final protein produced in soluble form. Conjugation of purified protein to αDEC-205 antibody resulted in smears of protein: antibodies conjugate in different molecular weights. The best cultivation condition for production of HIVtop4 protein is induction by 1 mM IPTG during 4 hr in 2XYT medium. The final concentration of purified protein was 500 µg/ml.

Effect of growth factors and steroid hormones on heme oxygenase and cyclin D1 expression in primary astroglial cell cultures.

Astrocyte activity may be modulated by steroid hormones and GFs. This study investigates the interaction between glucocorticoids or estrogens and GFs on the expression of heme oxygenase-1 (HO-1) and cyclin D1 in astrocyte cultures at 14 days treated for 48 or 60 hr with dexamethasone (DEX) or 48 hr with 17β-estradiol (E2) alone or with GFs added only in the last 12 or 24 hr. Twelve- or twenty-four-hour epidermal growth factor (EGF) treatment significantly enhanced HO-1 expression in astrocyte cultures pretreated for 48 hr with DEX. A highly significant increase in HO-1 expression was obtained after the last-12-hr EGF treatment in 48-hr E2-pretreated astrocyte cultures; this enhancement was particularly significant in 48-hr E2-pretreated cultures as well as in the last-12-hr insulin-treated ones pretreated for 48 hr with E2. Sixty-hour DEX-alone pretreatment as well as the last-12-hr EGF treatment in 60-hr DEX-pretreated astrocyte cultures showed a significant increase of cyclin D1 expression. A significant decrease of cyclin D1 expression in the last-12-hr insulin-like growth factor-I (IGF-1)-treated cultures pretreated for 60 hr with DEX was observed. A highly significant enhancement in cyclin D1 expression in 14 days in vitro astrocyte cultures pretreated with E2 alone for 48 hr and treated in the last 12 hr with IGF-1 in 48-hr E2-pretreated cultures was found. Finally, the data highlight an interactive dialogue between the growth factors and glucocorticoids or estrogens during the maturation of astroglial cells in culture that may control the HO-1 and cyclin D1 expression as well as proliferating astroglial cells during the cell cycle.

Bone marrow stromal cell adhesion and morphology on micro- and sub-micropatterned titanium.

The objective of this study was to investigate the adhesion and morphology of bone marrow derived stromal cells (BMSCs) on bulk titanium (Ti) substrates with precisely-patterned surfaces consisting of groove-based gratings with groove widths ranging from 50 micro m down to 0.5 micro m (500 nm). Although it is well known that certain surface patterning enhances osteoblast (bone-forming cell) functions, past studies on cell-pattern interactions reported in the literature have heavily relied on surface patterning on materials with limited clinical relevance for orthopedic applications, such as polymeric substrates. The clinical need for improving osseointegration and juxtaposed bone formation around load-bearing Ti implants motivated this in vitro study. BMSCs were selected as model cells due to their important role in bone regeneration. The results showed significantly greater BMSC adhesion density and more favorable cell morphology on sub-micropatterned gratings when compared with larger micropatterned gratings and non-patterned control surfaces after both 24 hr and 72 hr cultures. We observed increasing cellular alignment and elongation with decreasing feature size. We also identified two distinctive cellular morphologies: Type I-Attached and spread cells that elongated along the pattern axes; and Type II-Superficially adhered round cells. Sub-micropatterned gratings demonstrated significantly greater Type I cell density than the non-patterned control, and lower Type II cell density than the larger micropatterned gratings. Collectively, these results suggest potential for rationally designing nano-scale surface topography on Ti implants to improve osseointegration.

Production and characterization of calcium phosphate cement incorporated with platelet concentrate

Calcium phosphate cements (CPCs) incorporated with platelet concentrate (PC) were prepared and characterized in an attempt to improve their bone regenerating performance. The characteristics of CPCs, such as paste injectability, initial setting time, and mechanical strength, were assessed. The generation of hydroxyapatite (HA) was observed with X-Ray diffraction (XRD) and scanning electron microscopy (SEM). The release of platelet-derived growth factor-BB (PDGF-BB) and basic fibroblast growth factor (bFGF) was measured using enzyme linked immunosorbent assay (ELISA). The culture of Saos-2 cells was performed to assay the cell compatibility of CPCs. The paste injectability was enhanced but the cement setting was delayed by the incorporation of PC. The diametral tensile and compressive strengths were deteriorated by incorporated PC. The generation of HA was not affected by PC incorporation. The cumulative concentrations of released PDGF-BB and bFGF increased constantly for about 9 days. The release of PDGF-BB and bFGF linearly increased with the amount of PC added in CPC. According to 12 hr culture, the adhered Saos-2 cells increased with the amount of incorporated PC.