Search results for: recombinant inbred lines

Authors: Niklas Ravn-Boess, Nainita Bhowmick, Takamitsu Hattori, Shohei Koide, Christopher Park, Dimitris Placantonakis

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Background: Glioblastoma (GBM) is the most common and deadly primary brain malignancy in adults. Tumor propagation, brain invasion, and resistance to therapy critically depend on GBM stem-like cells (GSCs); however, the mechanisms that regulate GSC self-renewal are incompletely understood. Given the aggressiveness and poor prognosis of GBM, it is imperative to find biomarkers that could also translate into novel drug targets. Along these lines, we have identified a cell surface antigen, CD97 (ADGRE5), an adhesion G protein-coupled receptor (GPCR), that is expressed on GBM cells but is absent from non-neoplastic brain tissue. CD97 has been shown to promote invasiveness, angiogenesis, and migration in several human cancers, but its frequency of expression and functional role in regulating GBM growth and survival, and its potential as a therapeutic target has not been investigated. Design: We assessed CD97 mRNA and protein expression in patient derived GBM samples and cell lines using publicly available RNA-sequencing datasets and flow cytometry, respectively. To assess CD97 function, we generated shRNA lentiviral constructs that target a sequence in the CD97 extracellular domain (ECD). A scrambled shRNA (scr) with no predicted targets in the genome was used as a control. We evaluated CD97 shRNA lentivirally transduced GBM cells for Ki67, Annexin V, and DAPI. We also tested CD97 KD cells for their ability to self-renew using clonogenic tumorsphere formation assays. Further, we utilized synthetic Abs (sAbs) generated against the ECD of CD97 to test for potential antitumor effects using patient-derived GBM cell lines. Results: CD97 mRNA expression was expressed at high levels in all GBM samples available in the TCGA cohort. We found high levels of surface CD97 protein expression in 6/6 patient-derived GBM cell cultures, but not human neural stem cells. Flow cytometry confirmed downregulation of CD97 in CD97 shRNA lentivirally transduced cells. CD97 KD induced a significant reduction in cell growth in 3 independent GBM cell lines representing mesenchymal and proneural subtypes, which was accompanied by reduced (~20%) Ki67 staining and increased (~30%) apoptosis. Incubation of GBM cells with sAbs (20 ug/ ml) against the ECD of CD97 for 3 days induced GSC differentiation, as determined by the expression of GFAP and Tubulin. Using three unique GBM patient derived cultures, we found that CD97 KD attenuated the ability of GBM cells to initiate sphere formation by over 300 fold, consistent with an impairment in GSC self-renewal. Conclusion: Loss of CD97 expression in patient-derived GBM cells markedly decreases proliferation, induces cell death, and reduces tumorsphere formation. sAbs against the ECD of CD97 reduce tumorsphere formation, recapitulating the phenotype of CD97 KD, suggesting that sAbs that inhibit CD97 function exhibit anti-tumor activity. Collectively, these findings indicate that CD97 is necessary for the proliferation and survival of human GBM cells and identify CD97 as a promising therapeutically targetable vulnerability in GBM.

Keywords: adhesion GPCR, CD97, GBM stem cell, glioblastoma

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Authors: Suchao Donpudsa, Suwattana Visetnan, Anchalee Tassanakajon, Vichien Rimphanitchayakit

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The Single Wap Domain (SWD) is a type III crustin antimicrobial peptide whose function is to defense the host animal against the bacterial infection by means of antimicrobial and antiproteinase activities. A study of LvSWD from Litopenaeus vannamei is reported herein about its activities and function against bacteria, particularly the Vibrio parahaemolyticus AHPND (VPAHPND) that causes acute hepatopancreatic necrosis disease. The over-expressed mature recombinant (r)LvSWD exhibits antimicrobial activity against both Gram-positive and Gram-negative bacteria, especially VPAHPND. With four times the MIC of rLvSWD, the treated post larval shrimp infected by VPAHPND is able to survive longer with the 50% survival rate as long as 78 h as compared to 36 h of the infected shrimp without rLvSWD. To a certain extent, we have demonstrated that the rLvSWD can be applied to protect the post larval shrimp.

Keywords: crustin, Litopenaeus vannamei, Vibrio parahaemolyticus AHPND, antimicrobial activity

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Authors: Deepak Kumar, Ravi Rajwanshi, Mohd. Aslam Yusuf, Nisha Kant Pandey, Preeti Singh, Mukesh Saxena, Neera Bhalla Sarin

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Global issues are leading to concerns over food security. These include climate change, urbanization, increase in population subsequently leading to greater energy and water demand. Futuristic approach for crop improvement involves gene pyramiding for agronomic traits that empower the plants to withstand multiple stresses. In an earlier study from the laboratory, the efficacy of overexpressing γ-tocopherol methyl transferase (γ-TMT) gene from the vitamin E biosynthetic pathway has been shown to result in six-fold increase of the most biologically active form, the α-tocopherol in Brassica juncea which resulted in alleviation of salt, heavy metal and osmoticum induced stress by the transgenic plants. The glyoxalase I (gly I) gene from the glyoxalase pathway has also been earlier shown by us to impart tolerance against multiple abioitc stresses by detoxification of the cytotoxic compound methylglyoxal in Brassica juncea. Recently, both the transgenes were pyramided in Brassica juncea lines through sexual crosses involving two stable Brassica juncea lines overexpressing γ-TMT and gly I genes respectively. The transgene integration was confirmed by PCR analysis and their mRNA expression was evident by RT-PCR analysis. Preliminary physiological investigations showed ~55% increased seed germination under 200 mM NaCl stress in the pyramided line and 81% higher seed germination under 200 mM mannitol stress as compared to the WT control plants. The pyramided lines also retained more chlorophyll content when the leaf discs were floated on NaCl (200, 400 and 600 mM) or mannitol (200, 400 and 600 mM) compared to the WT control plants. These plants had higher Relative Water Content and greater solute accumulation under stress compared to the parental plants having γ-TMT or the glyI gene respectively. The studies revealed the synergy of two components from different metabolic pathways in enhancing stress hardiness of the transgenic B. juncea plants. It was concluded that pyramiding of genes (γ-TMT and glyI) from diverse pathways can lead to enhanced tolerance to salt and mannitol stress (simulating drought conditions). This strategy can prove useful in enhancing the crop yields under various abiotic stresses.

Keywords: abiotic stress, brassica juncea, glyoxalase I, α-tocopherol

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Authors: Julia Kapr, Andrea Rossi, Haribaskar Ramachandran, Marius Pollet, Ilka Egger, Selina Dangeleit, Katharina Koch, Jean Krutmann, Ellen Fritsche

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It is widely acknowledged that animal models do not always represent human disease. Especially human brain development is difficult to model in animals due to a variety of structural and functional species-specificities. This causes significant discrepancies between predicted and apparent drug efficacies in clinical trials and their subsequent failure. Emerging alternatives based on 3D in vitro approaches, such as human brain spheres or organoids, may in the future reduce and ultimately replace animal models. Here, we present a human induced pluripotent stem cell (hiPSC)-based 3D neural in a vitro disease model for the Cockayne Syndrome B (CSB). CSB is a rare hereditary disease and is accompanied by severe neurologic defects, such as microcephaly, ataxia and intellectual disability, with currently no treatment options. Therefore, the aim of this study is to investigate the molecular and cellular defects found in neural hiPSC-derived CSB models. Understanding the underlying pathology of CSB enables the development of treatment options. The two CSB models used in this study comprise a patient-derived hiPSC line and its isogenic control as well as a CSB-deficient cell line based on a healthy hiPSC line (IMR90-4) background thereby excluding genetic background-related effects. Neurally induced and differentiated brain sphere cultures were characterized via RNA Sequencing, western blot (WB), immunocytochemistry (ICC) and multielectrode arrays (MEAs). CSB-deficiency leads to an altered gene expression of markers for autophagy, focal adhesion and neural network formation. Cell migration was significantly reduced and electrical activity was significantly increased in the disease cell lines. These data hint that the cellular pathologies is possibly underlying CSB. By induction of autophagy, the migration phenotype could be partially rescued, suggesting a crucial role of disturbed autophagy in defective neural migration of the disease lines. Altered autophagy may also lead to inefficient mitophagy. Accordingly, disease cell lines were shown to have a lower mitochondrial base activity and a higher susceptibility to mitochondrial stress induced by rotenone. Since mitochondria play an important role in neurotransmitter cycling, we suggest that defective mitochondria may lead to altered electrical activity in the disease cell lines. Failure to clear the defective mitochondria by mitophagy and thus missing initiation cues for new mitochondrial production could potentiate this problem. With our data, we aim at establishing a disease adverse outcome pathway (AOP), thereby adding to the in-depth understanding of this multi-faced disorder and subsequently contributing to alternative drug development.

Keywords: autophagy, disease modeling, in vitro, pluripotent stem cells

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Authors: Kheireddine El-Boubbou

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Drug delivery to target human acute myeloid leukemia (AML) using a nanoparticulate chemotherapeutic formulation that can deliver drugs selectively to AML cancer is hugely needed. In this work, we report the development of a nanoformulation made of polymeric-stabilized multifunctional magnetic iron oxide nanoparticles (PMNP) loaded with the anticancer drug Doxorubicin (Dox) as a promising drug carrier to treat AML. Dox@PMNP conjugates simultaneously exhibited high drug content, maximized fluorescence, and excellent release properties. Nanoparticulate uptake and cell death following addition of Dox@PMNPs were then evaluated in different types of human AML target cells, as well as on normal human cells. While the unloaded MNPs were not toxic to any of the cells, Dox@PMNPs were found to be highly toxic to the different AML cell lines, albeit at different inhibitory concentrations (IC₅₀ values), but showed very little toxicity towards the normal cells. In comparison, free Dox showed significant potency concurrently to all the cell lines, suggesting huge potentials for the use of Dox@PMNPs as selective AML anticancer cargos. Live confocal imaging, fluorescence and electron microscopy confirmed that Dox is indeed delivered to the nucleus in relatively short periods of time, causing apoptotic cell death. Importantly, this targeted payload may potentially enhance the effectiveness of the drug in AML patients and may further allow physicians to image leukemic cells exposed to Dox@PMNPs using MRI.

Keywords: magnetic nanoparticles, drug delivery, acute myeloid leukemia, iron oxide, cancer nanotherapy

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Authors: Amir Saber Gharamaleki, Beitollah Alipour, Zeinab Faghfoori, Ahmad YariKhosroushahi

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Colorectal cancer (CRC) is one of the most prevalent cancers and intestinal microbial community plays an important role in colorectal tumorigenesis. Probiotics have recently been assessed as effective anti-proliferative agents and thus this study was performed to examine whether CRC undergo apoptosis by treating with isolated Iranian native dairy yeast, Kluyveromyces marxianus YAS, secretion metabolites. The cytotoxicity assessments on cells (HT-29, Caco-2) were accomplished through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as well as qualitative DAPI (4',6-diamidino-2-phenylindole staining) and quantitative (flow cytometry assessments) evaluations of apoptosis. To evaluate the main mechanism of apoptosis, Real time PCR method was applied. Kluyveromyces marxianus YAS secretions (IC50) showed significant cytotoxicity against HT-29 and Caco-2 cancer cell lines (66.57 % and 66.34 % apoptosis) similar to 5-Fluorouracil (5-FU) while apoptosis only was developed in 27.57 % of KDR normal cells. The prophylactic effects of Kluyveromyces marxianus (PTCC 5195), as a reference yeast, was not similar to Kluyveromyces marxianus YAS indicating strain dependency of bioactivities on CRC disease prevention. Based on real time PCR results, the main cytotoxicity is related to apoptosis phenomenon and the core related mechanism is depended on the overexpression of BAX, CASP 9, CASP 8 and CASP 3 inducing apoptosis genes. However, several investigations should be conducted to precisely determine the effective compounds to be used as anticancer therapeutics in the future.

Keywords: anticancer, anti-proliferative, apoptosis, cytotoxicity, yeast

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Authors: Li Yuchen, Wu Qingxin, Jin Yuxing, Yang Qian

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Interleukin-11 (IL-11), a well-known anti-inflammatory factor, helps to protect against intestinal epithelium damage caused by physical or chemical factors. However, little is known about the role of IL-11 during viral infection. Herein, high mRNA and protein levels of IL-11 were found in epithelial cells and jejunum of piglets during porcine epidemic diarrhea virus (PEDV) infection, and IL-11 expression was positively correlated with the level of viral infection. Pretreatment with recombinant porcine IL-11 (pIL-11) suppressed PEDV replication in Vero E6 cells, while IL-11 knockdown promoted viral infection. Furthermore, pIL-11 inhibited viral infection by preventing PEDV-mediated apoptosis of cells through activating the IL-11/STAT3 signal pathway. Conversely, application of a STAT3 phosphorylation inhibitor significantly antagonized the anti-apoptosis function of pIL-11 and counteracted its inhibition of PEDV. Our data suggested that that IL-11 is a novel PEDV-inducible cytokine, and its production enhances the anti-apoptosis ability of epithelial cells against PEDV infection. The potential uses of IL-11 as a novel therapeutic against devastating viral diarrhea in piglets deserves more attention and study.

Keywords: Interleukin-11, Porcine epidemic diarrhea virus, STAT3, anti-apoptosis

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Authors: Hugo Badia

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This research answers which is the most suitable transit network structure to serve specific demand requirements in an increasing urban dispersion process. Two main approaches of network design are found in the literature. On the one hand, a traditional answer, widespread in our cities, that develops a high number of lines to connect most of origin-destination pairs by direct trips; an approach based on the idea that users averse to transfers. On the other hand, some authors advocate an alternative design characterized by simple networks where transfer is essential to complete most of trips. To answer which of them is the best option, we use a two-step methodology. First, by means of an analytical model, three basic network structures are compared: a radial scheme, starting point for the other two structures, a direct trip-based network, and a transfer-based one, which represent the two alternative transit network designs. The model optimizes the network configuration with regard to the total cost for each structure. For a scenario of dispersion, the best alternative is the structure with the minimum cost. This dispersion degree is defined in a simple way considering that only a central area attracts all trips. If this area is small, we have a high concentrated mobility pattern; if this area is too large, the city is highly decentralized. In this first step, we can determine the area of applicability for each structure in function to that urban dispersion degree. The analytical results show that a radial structure is suitable when the demand is so centralized, however, when this demand starts to scatter, new transit lines should be implemented to avoid transfers. If the urban dispersion advances, the introduction of more lines is no longer a good alternative, in this case, the best solution is a change of structure, from direct trips to a network based on transfers. The area of applicability of each network strategy is not constant, it depends on the characteristics of demand, city and transport technology. In the second step, we translate analytical results to a real case study by the relationship between the parameters of dispersion of the model and direct measures of dispersion in a real city. Two dimensions of the urban sprawl process are considered: concentration, defined by Gini coefficient, and centralization by area based centralization index. Once it is estimated the real dispersion degree, we are able to identify in which area of applicability the city is located. In summary, from a strategic point of view, we can obtain with this methodology which is the best network design approach for a city, comparing the theoretical results with the real dispersion degree.

Keywords: analytical network design model, network structure, public transport, urban dispersion

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Authors: Suzan Ghaddar, Aline Pinon, Manuel Gallardo-villagran, Mona Diab-assaf, Bruno Therrien, Bertrand Liagre

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Colorectal cancer (CRC) is the third most common cancer and exhibits a consistently rising incidence worldwide. Despite notable advancements in CRC treatment, frequent occurrences of side effects and the development of therapy resistance persistently challenge current approaches. Eventually, innovations in focal therapies remain imperative to enhance the patient’s overall quality of life. Photodynamic therapy (PDT) emerges as a promising treatment modality, clinically used for the treatment of various cancer types. It relies on the use of photosensitive molecules called photosensitizers (PS), which are photoactivated after accumulation in cancer cells, to induce the production of reactive oxygen species (ROS) that cause cancer cell death. Among commonly used metal-based drugs in cancer therapy, ruthenium (Ru) possesses favorable attributes that demonstrate its selectivity towards cancer cells and render it suitable for anti-cancer drug design. In vitro studies using distinct arene-Ru complexes, encapsulating porphin PS, are conducted on human HCT116 and HT-29 colorectal cancer cell lines. These studies encompass the evaluation of the antiproliferative effect, ROS production, apoptosis, cell cycle progression, molecular localization, and protein expression. Preliminary results indicated that these complexes exert significant photocytotoxicity on the studied colorectal cancer cell lines, representing them as promising and potential candidates for anti- cancer agents.

Keywords: colorectal cancer, photodynamic therapy, photosensitizers, arene-ruthenium complexes, apoptosis

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Authors: Yasmin M. Attia, Hanan S. El-Abhar, Mahmoud M. Al Marzabani, Samia A. Shouman

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Background: Tamoxifen (TAM) is the most commonly used hormone therapy for the treatment of early and metastatic breast cancer. Although it significantly decreases the tumor recurrence rate and provides an overall benefit, as much as 20–30% of women still relapse during or after long-term therapy. 3-Bromopyruvate (3-BP) is a promising agent with impressive antitumor effects in several models of animal tumors and cell lines. Aim: This study was designed to investigate the combined effect of (TAM) and (3-BP) in breast cancer cells and to explore their molecular interaction via assessment of apoptotic, angiogenic, and metastatic markers. Methods: In vitro cytotoxicity study was carried out for both compounds to determine the combination regimen producing a synergistic effect and mechanistic pathways were studied using RT-PCR and western techniques. Moreover, the anti-oncolytic and anti-angiogenic potentials were assessed in mice bearing solid Ehrlich carcinoma (SEC). Results: The combined treatment significantly increased the expressions and protein levels of caspase 7, 9, and 3 and decreased of angiogenic markers VEGF, HIF-1α, and HK2 compared to cells treated with either drug individually. However, there were no significant changes in MMP-2 and MMP-9 protein levels. Interestingly, the in vivo results supported the in vitro findings; there was a decrease in the tumor volume and VEFG using immunohistochemistry in the combination-treated groups compared to either TAM or 3-BP treated one. Conclusion: 3-BP synergizes the cytotoxic effect of TAM by increasing apoptosis and decreasing angiogenesis which makes this combination a promising regimen to be applied clinically.

Keywords: tamoxifen, 3-bromopyruvate, breast cancer, cytotoxicity, angiogenesis

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Authors: I. Witbooi, J. De Smidt, A. Oelofse

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Negative emotions that are common in depression are coupled with the activation of the corrugator supercilli and procerus muscles in the glabellar region of the face. This research investigated the impact of OnabotulinumtoxinA (BOTOX) in the improvement of emotional states in depressed subjects by relaxing the mentioned muscles. The aim of the study was to investigate the effectiveness of BOTOX treatment for glabellar frown lines as an adjunctive therapy for Major Depressive Disorder (MDD) and to improve the quality of life and self-esteem of the subjects. It is hypothesized that BOTOX treatment for glabellar frown lines reduces depressive symptoms significantly and therefore augment conventional antidepressant medication. Forty-five (45) subjects diagnosed with MDD were assigned to a treatment (n = 15), placebo (n = 15), and control (n = 15) group. The treatment group received BOTOX injection, while the placebo group received saline injection into the Procerus and Corrugator supercilli muscles with follow-up visits every 3 weeks (weeks 3, 6 and 12 respectively). The control group received neither BOTOX nor saline injections and were only interviewed again on the 12th week. To evaluate the effect of BOTOX treatment in the glabellar region on depressive symptoms, the Montgomery-Asberg Depression Rating (MADRS) scale and the Beck Depression Inventory (BDI) were used. The Quality of Life Enjoyment and Satisfaction Questionnaire-Short Form (Q-LES-Q-SF) and Rosenberg Self-Esteem Scale (RSES) were used in the assessment of self-esteem and quality of life. Participants were followed up for a 12 week period. The expected primary outcome measure is the response to treatment, and it is defined as a ≥ 50% reduction in MADRS score from baseline. Other outcome measures include a clinically significant decrease in BDI scores and the increase in quality of life and self-esteem respectively. Initial results show a clear trend towards such differences. Results showed trends towards expected differences. Patients in the Botox group had a mean MADRS score of 14.0 at 3 weeks compared to 20.3 of the placebo group. This trend was still visible at 6 weeks with the Botox and placebo group scoring an average of 10 vs. 18 respectively. The mean difference in MDRS scores from baseline to 3 weeks were 9.3 and 2.0 for the Botox and placebo group respectively. Similarly, the BDI scores were lower in the Botox group (17.25) compared to the placebo group (19.43). The two self-esteem questionnaires showed expected results at this stage with the RSES 19.1 in the Botox group compared to 18.6 in the placebo group. Similarly, the Botox patients had a higher score for the Q-LES-Q-SF of 49.2 compared to 46.1 for the placebo group. Conclusions: Initial results clearly demonstrated that the use of Botox had positive effects on both scores of depressions and that of self-esteem when compared to a placebo group.

Keywords: adjunctive therapy, depression, glabellar area, OnabotulinumtoxinA

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Authors: Letizia Maria Drammis, Maria Antonella Brandimonte

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The idea that people use space representations to think about time and number received support from several lines of research. However, how these representations develop in children and then shape space-time and space-number mappings is still a debated issue. In the present study, 40 children (20 pre-schoolers and 20 elementary-school children) performed 4 main tasks, which required the use of more concrete (non-symbolic) or more abstract (symbolic) space-time and space-number associations. In the non-symbolic conditions, children were required to order pictures of everyday-life events occurring in a specific temporal order (Temporal sequences) and of quantities varying in numerosity (Numerical sequences). In the symbolic conditions, they were asked to perform the typical time-to-position and number-to-position tasks by mapping time-related words and numbers onto lines. Results showed that children performed reliably better in the non-symbolic Time conditions than the symbolic Time conditions, independently of age, whereas only pre-schoolers performed worse in the Number-to-position task (symbolic) as compared to the Numerical sequence (non-symbolic) task. In addition, only older children mapped time-related words onto space following the typical left-right orientation, pre-schoolers’ performance being somewhat mixed. In contrast, mapping numbers onto space showed a clear left-right orientation, independently of age. Overall, these results indicate a cross-domain difference in the way younger and older children process time and number, with time-related tasks being more difficult than number-related tasks only when space-time tasks require symbolic representations.

Keywords: space-time associations, space-number associations, orientation, children

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Authors: Rakesh Kumar Meena, Tanushree Chatterjee

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Okra (Abelmoschus esculentas L. Moench) or lady’s finger is an important vegetable crop belonging to the Malvaceae family. Unfortunately, production and productivity of Okra are majorly affected by Yellow Vein mosaic virus (YVMV). The AO: 189 (resistant parent) X AO: 191(susceptible parent) used for the development of mapping population. The mapping population has 143 individuals (F₂:F₃). Population was characterized by physiological and pathological observations. Screening of 360 DNA markers was performed to survey for parental polymorphism between the contrasting parents’, i.e., AO: 189 and AO: 191. Out of 360; 84 polymorphic markers were used for genotyping of the mapping population. Total markers were distributed into four linkage groups (LG1, LG2, LG3, and LG4). LG3 covered the longest span (106.8cM) with maximum number of markers (27) while LG1 represented the smallest linkage group in terms of length (71.2cM). QTL identification using the composite interval mapping approach detected two prominent QTLs, QTL1 and QTL2 for resistance against YVMV disease. These QTLs were placed between the marker intervals of NBS-LRR72-Path02 and NBS-LRR06- NBS-LRR65 on linkage group 02 and linkage group 04 respectively. The LOD values of QTL1 and QTL2 were 5.7 and 6.8 which accounted for 19% and 27% of the total phenotypic variation, respectively. The findings of this study provide two linked markers which can be used as efficient diagnostic tools to distinguish between YVMV resistant and susceptible Okra cultivars/genotypes. Lines identified as highly resistant against YVMV infection can be used as donor lines for this trait. This will be instrumental in accelerating the trait improvement program in Okra and will substantially reduce the yield losses due to this viral disease.

Keywords: Okra, yellow vein mosaic virus, resistant, linkage map, QTLs

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Authors: Kaarour Abdelkrim, Ouardi Okkacha, Meskine Mohamed

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In this study, the υ_4 fundamental band of 12CD4 molecule has been studied by infrared spectroscopy with high resolution. Using XTDS and SPEVIEW software and the tensor formalism developed by ICB (laboratoire interdisciplinaire de Bourgogne) to several lines have been assigned and fitted with a standard deviation acceptable. This analysis allowed us to calculate several parameters of the molecule 12 CD4.

Keywords: XTDS, SPEVIEW, tetrahedral tensorial formalism, rovibrational band

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Authors: Mohsina Akhter

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Cancer has become a wide spread disease around the globe and takes many lives every year. Different treatments are being practiced but all have potential side effects with somewhat less specificity towards target sites. Pseudomonas aeruginosa is known to secrete a protein azurin with special anti-cancer function. It has unique cell penetrating peptide comprising of 18 amino acids that have ability to enter cancer cells specifically. Reported function of Azurin is to stabilize p53 inside the tumor cells and induces apoptosis through Bax mediated cytochrome c release from mitochondria. At laboratory scale, we have made recombinant azurin through cloning rpTZ57R/T-azu vector into E.coli strain DH-5α and subcloning rpET28-azu vector into E.coli BL21-CodonPlus (DE3). High expression was ensured with IPTG induction at different concentrations then optimized high expression level at 1mM concentration of IPTG for 5 hours. Purification has been done by using Ni+2 affinity chromatography. We have concluded that azurin can be a remarkable improvement in cancer therapeutics if it produces on a large scale. Azurin does not enter into the normal cells so it will prove a safe and secure treatment for patients and prevent them from hazardous anomalies.

Keywords: azurin, pseudomonas aeruginosa, cancer, therapeutics

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Authors: Attapon Kamlangdee, Brock Kingstad-Bakke, Tavis K. Anderson, Tony L. Goldberg, Jorge E. Osorio

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A critical failure in our preparedness for an influenza pandemic is the lack of a universal vaccine. Influenza virus strains diverge by 1 to 2% per year, and commercially available vaccines often do not elicit protection from one year to the next, necessitating frequent formulation changes. This represents a major challenge to the development of a cross-protective vaccine that can protect against circulating viral antigenic diversity. We have constructed a recombinant modified vaccinia virus Ankara (MVA) that expresses an H5N1 mosaic hemagglutinin (H5M) (MVA-H5M). This mosaic was generated in silico using 2,145 field-sourced H5N1 isolates. A single dose of MVA-H5M provided 100% protection in mice against clade 0, 1, and 2 avian influenza viruses and also protected against seasonal H1N1 virus (A/Puerto Rico/8/34). It also provided short-term (10 days) and long-term (6 months) protection post vaccination. Both neutralizing antibodies and antigen-specific CD4+and CD8+ T cells were still detected at 5 months post vaccination, suggesting that MVA-H5M provides long-lasting immunity.

Keywords: modified vaccinia Ankara, MVA, H5N1, hemagglutinin, influenza vaccine

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Authors: Saimir Heta, Ilma Robo, Dhimiter Papakozma, Eduart Kapaj, Vera Ostreni

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Introduction: The biocompatible materials applied to the implant surfaces are the target of recent literature studies. Methodologies: Modification of implant surfaces in different ways such as application of additional ions, surface microstructure change, surface or laser ultrasound alteration, or application of various substances such as recombinant proteins are among the most affected by articles published in the literature. The study is of review type with the main aim of finding the different ways that the mesenchymal cell reaction to these materials is, according to the literature, in the same percentage positive to the osteointegration process. Results: It is emphasized in the literature that implant success as a key evaluation key has more to implement implant treatment protocol ranging from dental health amenity and subsequent of the choice of implant type depending on the alveolar shape of the ridge level. Conclusions: Osteointegration is a procedure that should initially be physiologically independent of the type of implant pile material. With this physiological process, it can not "boast" for implant success or implantation depending on the brand of the selected implant, as the breadth of synthetic or natural materials that promote osteointegration is relatively large.

Keywords: mesenchymal cells, implants, review, biocompatible materials

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Authors: Omolola A. Tajelawi, Hari L. Garbharran

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Revenue leakages are one of the major challenges manufacturers face in production processes, as most of the input materials that should emanate as products from the lines are lost as waste. Rather than generating income from material input which is meant to end-up as products, losses are further incurred as costs in order to manage waste generated. In addition, due to the lack of a clear view of the flow of resources on the lines from input to output stage, acquiring information on the true cost of waste generated have become a challenge. This has therefore given birth to the conceptualization and implementation of waste minimization strategies by several manufacturing industries. This paper reviews the principles and applications of three environmental management accounting tools namely Activity-based Costing (ABC), Life-Cycle Assessment (LCA) and Material Flow Cost Accounting (MFCA) in the manufacturing industry and their effectiveness in curbing revenue leakages. The paper unveils the strengths and limitations of each of the tools; beaming a searchlight on the tool that could allow for optimal resource utilization, transparency in production process as well as improved cost efficiency. Findings from this review reveal that MFCA may offer superior advantages with regards to the provision of more detailed information (both in physical and monetary terms) on the flow of material inputs throughout the production process compared to the other environmental accounting tools. This paper therefore makes a case for the adoption of MFCA as a viable technique for the identification and reduction of waste in production processes, and also for effective decision making by production managers, financial advisors and other relevant stakeholders.

Keywords: MFCA, environmental management accounting, resource efficiency, waste reduction, revenue losses

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Authors: Yasir Ali, Farhatullah

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The present study was conducted to estimate the genetic variability, heritability and genetic advance in six parental lines and their 56 genotypes derived from five introgressed brassica populations on the basis of morphological and biochemical traits. The experiment was laid out in a randomized complete block design with two replications at The University of Agriculture Peshawar-Pakistan during growing season of 2015-2016. The ANOVA of all traits of F5:6 populations showed highly significant differences (P ≤ 0.01) for all morphological and biochemical traits. Among F5:6 populations, the genotype 2(526) was earlier in flowering (108.65 days), and genotype 14(485) was earlier in maturity (170 days). Tallest plants (182.5 cm), largest main raceme (91.5 cm) and maximum number of pods (80.5) on main raceme were recorded for genotype 17(34). Maximum primary branches plant-1(6.2) and longest pods (10.26 cm) were recorded for genotype 15, while genotype 16(171) had more seeds pod⁻¹ (22) and gave maximum yield plant-1 (30.22 g). The maximum 100-seed weight (0.60 g) was observed for genotype 10(506) while high protein content (22.61%) was recorded for genotype 4(99). Maximum oil content (54.08 %) and low linoleic acid (7.07 %) were produced by genotype (12(138) and low glucosinolate (59.01 µMg⁻¹) was recorded for genotype 21(113). The genotype 27(303) having high oleic acid content (51.73 %) and genotype 1(209) gave low erucic acid (35.97 %). Among the F5:6 populations moderate to high heritability observed for all morphological and biochemical traits coupled with high genetic advance. Cluster analysis grouped the 56 F5:6 populations along their parental lines into seven different groups. Each group was different from the other group on the basis of morphological and biochemical traits. Moreover all the F5:6 populations showed sufficient variability. Genotypes 10(506) and 16(171) were superior for high seed yield⁻¹, 100-seeds weight, and seed pod⁻¹ and are recommended for future breeding program.

Keywords: Brassicaceae, biochemical characterization, introgression, morphological characterization

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Authors: Soroosh Torabi, Brad Berron, Ren Xu, Christine Trinkle

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Microfluidics integrated with 3D cell culture is a powerful technology to mimic cellular environment, and can be used to study cell activities such as proliferation, migration and response to drugs. This technology has gained more attention in cancer studies over the past years, and many organ-on-a-chip systems have been developed to study cancer cell behaviors in an ex-vivo tumor microenvironment. However, there are still some barriers to adoption which include low throughput, complexity in 3D cell culture integration and limitations on non-optical analysis of cells. In this study, a user-friendly microfluidic multi-well plate was developed to mimic the in vivo tumor microenvironment. The microfluidic platform feeds multiple 3D cell culture sites at the same time which enhances the throughput of the system. The platform uses hydrophobic Cassie-Baxter surfaces created by microchannels to enable convenient loading of hydrogel/cell suspensions into the device, while providing barrier free placement of the hydrogel and cells adjacent to the fluidic path. The microchannels support convective flow and diffusion of nutrients to the cells and a removable lid is used to enable further chemical and physiological analysis on the cells. Different breast cancer cell lines were cultured in the device and then monitored to characterize nutrient delivery to the cells as well as cell invasion and proliferation. In addition, the drug response of breast cancer cell lines cultured in the device was compared to the response in xenograft models to the same drugs to analyze relevance of this platform for use in future drug-response studies.

Keywords: microfluidics, multi-well 3d cell culture, tumor microenvironment, tumor-on-a-chip

Procedia PDF Downloads 240

Authors: Ranjeeta Kumari, Makesh M, Gayatri Tripathi, K V Rajendran, Megha Bedekar

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A comparative study on DNA immunization with recombinant glyceraldehyde-3-phosphate dehydrogenase (GAPDH) construct of Edwardsiella tarda (pGPD group) and a bicistronic construct expressing GAPDH plus IFN-γ of Labeo rohita as adjuvant (pGPD+IFN group) was undertaken in Labeo rohita along with the control animals. Successful co-expression of two genes that is GAPDH and IFN-γ was confirmed in SSN-1 cells line by RT-qPCR and western blot. The protective immune response of host to DNA vaccine construct was determined by RPS and specific antibody production. Fishes immunized with plasmids via intramuscular injection (I/M) exhibited a considerable relative percentage survivability of 66.66% in pGPD+IFN immunized group and 53.34% in pGPD immunized group after challenge with E. tarda. Antibody response was also significantly high in pGPD+IFN group at all time points under study. This was analysed by competitive ELISA, using anti GAPDH monoclonal antibodies. The experiment revealed that the GAPDH gene of E. tarda is one of the ideal candidates for generating protective immune response in L. rohita. Further addition of Interferon gamma to DNA vaccine construct can enhance the immune response in host.

Keywords: DNA vaccine, Edwardsiella tarda, Labeo rohita, zoonosis, immune response

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Authors: Manal Kamel, Sara Maher

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Development of sensitive non-invasive tests for detection of SARS-CoV-2 antigens is imperative to manage the extent of infection throughout the population, yet, it is still challenging. Here, we designed and optimized a sandwich enzyme-linked immunosorbent assay (ELISA) for SARS-CoV-2 S1 antigen detection in saliva. Both saliva samples and nasopharyngeal swapswere collected from 170 PCR-confirmed positive and negative cases. Gold nanoparticles (AuNPs) were conjugated with S1protein receptor binding domain (RBD) nanobodies. Recombinant S1 monoclonal antibodies (S1mAb) as primery antibody and gold conjugated nanobodies as secondary antibody were employed in sandwich ELISA. Our developed system were optimized to achieve 87.5 % sensitivity and 100% specificity for saliva samples compared to 89 % and 100% for nasopharyngeal swaps, respectively. This means that saliva could be a suitable replacement for nasopharyngeal swaps No cross reaction was detected with other corona virus antigens. These results revealed that our developed ELISAcould be establishedas a new, reliable, sensitive, and non-invasive test for diagnosis of SARS-CoV-2 infection, using the easily collected saliva samples.

Keywords: COVID 19, diagnosis, ELISA, nanobodies

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Authors: Adriana Muñoz-Talavera, Ana Rosa Rincón-Sánchez, Abraham Escobedo-Moratilla, María Cristina Islas-Carbajal, Miguel Ángel Gómez-Lim

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The highest rates of diabetes incidence and prevalence worldwide will increase the number of diabetic patients requiring insulin or insulin analogues. Then, current production systems would not be sufficient to meet the future market demands. Therefore, developing efficient expression systems for insulin and insulin analogues are needed. In addition, insulin analogues with better pharmacokinetics and pharmacodynamics properties and without mitogenic potential will be required. SCI-57 (single chain insulin-57) is an insulin analogue having 10 times greater affinity to the insulin receptor, higher resistance to thermal degradation than insulin, native mitogenicity and biological effect. Plants as expression platforms have been used to produce recombinant proteins because of their advantages such as cost-effectiveness, posttranslational modifications, absence of human pathogens and high quality. Immunoglobulin production with a yield of 50% has been achieved by transient expression in Nicotiana benthamiana (Nb). The aim of this study is to produce SCI-57 by transient expression in Nb. Methodology: DNA sequence encoding SCI-57 was cloned in pICH31070. This construction was introduced into Agrobacterium tumefaciens by electroporation. The resulting strain was used to infiltrate leaves of Nb. In order to isolate SCI-57, leaves from transformed plants were incubated 3 hours with the extraction buffer therefore filtrated to remove solid material. The resultant protein solution was subjected to anion exchange chromatography on an FPLC system and ultrafiltration to purify SCI-57. Detection of SCI-57 was made by electrophoresis pattern (SDS-PAGE). Protein band was digested with trypsin and the peptides were analyzed by Liquid chromatography tandem-mass spectrometry (LC-MS/MS). A purified protein sample (20µM) was analyzed by ESI-Q-TOF-MS to obtain the ionization pattern and the exact molecular weight determination. Chromatography pattern and impurities detection were performed using RP-HPLC using recombinant insulin as standard. The identity of the SCI-57 was confirmed by anti-insulin ELISA. The total soluble protein concentration was quantified by Bradford assay. Results: The expression cassette was verified by restriction mapping (5393 bp fragment). The SDS-PAGE of crude leaf extract (CLE) of transformed plants, revealed a protein of about 6.4 kDa, non-present in CLE of untransformed plants. The LC-MS/MS results displayed one peptide with a high score that matches SCI-57 amino acid sequence in the sample, confirming the identity of SCI-57. From the purified SCI-57 sample (PSCI-57) the most intense charge state was 1069 m/z (+6) on the displayed ionization pattern corresponding to the molecular weight of SCI-57 (6412.6554 Da). The RP-HPLC of the PSCI-57 shows the presence of a peak with similar retention time (rt) and UV spectroscopic profile to the insulin standard (SCI-57 rt=12.96 and insulin rt=12.70 min). The collected SCI-57 peak had ELISA signal. The total protein amount in CLE from transformed plants was higher compared to untransformed plants. Conclusions: Our results suggest the feasibility to produce insulin analogue SCI-57 by transient expression in Nicotiana benthamiana. Further work is being undertaken to evaluate the biological activity by glucose uptake by insulin-sensitive and insulin-resistant murine and human cultured adipocytes.

Keywords: insulin analogue, mass spectrometry, Nicotiana benthamiana, transient expression

Procedia PDF Downloads 323

Authors: Scott A. Ochsner, Christopher M. Watkins, Apollo McOwiti, David L. Steffen Lauren B. Becnel, Neil J. McKenna

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Understanding signaling by nuclear receptors (NRs) requires an appreciation of their cognate ligand- and tissue-specific transcriptomes. While target gene regulation data are abundant in this field, they reside in hundreds of discrete publications in formats refractory to routine query and analysis and, accordingly, their full value to the NR signaling community has not been realized. One of the mandates of the Nuclear Receptor Signaling Atlas (NURSA) is to facilitate access of the community to existing public datasets. Pursuant to this mandate we are developing a freely-accessible community web resource, Transcriptomine, to bring together the sum total of available expression array and RNA-Seq data points generated by the field in a single location. Transcriptomine currently contains over 25,000,000 gene fold change datapoints from over 1200 contrasts relevant to over 100 NRs, ligands and coregulators in over 200 tissues and cell lines. Transcriptomine is designed to accommodate a spectrum of end users ranging from the bench researcher to those with advanced bioinformatic training. Visualization tools allow users to build custom charts to compare and contrast patterns of gene regulation across different tissues and in response to different ligands. Our resource affords an entirely new paradigm for leveraging gene expression data in the NR signaling field, empowering users to query gene fold changes across diverse regulatory molecules, tissues and cell lines, target genes, biological functions and disease associations, and that would otherwise be prohibitive in terms of time and effort. Transcriptomine will be regularly updated with gene lists from future genome-wide expression array and expression-sequencing datasets in the NR signaling field.

Keywords: target gene database, informatics, gene expression, transcriptomics

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Authors: Mohamad Najari, Gilbert Grondin, Marwan El-Rich

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Design trucks in standard codes are selected based on the amount of damage they cause on structures-specifically bridges- and roads to represent the real traffic loads. Some limited numbers of trucks are run on a bridge one at a time and the damage on the bridge is recorded for each truck. One design track is also run on the same bridge “n” times -“n” is the number of trucks used previously- to calculate the damage of the design truck on the same bridge. To make these damages equal a reduction factor is needed for that specific design truck in the codes. As the limited number of trucks cannot be the exact representative of real traffic through the life of the structure, these reduction factors are not accurately calculated and they should be modified accordingly. Started on July 2004, the vehicle load data were collected in six weigh in motion (WIM) sites owned by Alberta Transportation for eight consecutive years. This database includes more than 200 million trucks. Having these data gives the opportunity to compare the effect of any standard fatigue trucks weigh and the real traffic load on the fatigue life of the bridges which leads to a modification for the fatigue truck factor in the code. To calculate the damage for each truck, the truck is run on the bridge, moment history of the detail under study is recorded, stress range cycles are counted, and then damage is calculated using available S-N curves. A 2000 lines FORTRAN code has been developed to perform the analysis and calculate the damages of the trucks in the database for all eight fatigue categories according to Canadian Institute of Steel Construction (CSA S-16). Stress cycles are counted using rain flow counting method. The modification factors for design truck (CL-625) are calculated for two different bridge configurations and ten span lengths varying from 1 m to 200 m. The two considered bridge configurations are single-span bridge and four span bridge. This was found to be sufficient and representative for a simply supported span, positive moment in end spans of bridges with two or more spans, positive moment in interior spans of three or more spans, and the negative moment at an interior support of multi-span bridges. The moment history of the mid span is recorded for single-span bridge and, exterior positive moment, interior positive moment, and support negative moment are recorded for four span bridge. The influence lines are expressed by a polynomial expression obtained from a regression analysis of the influence lines obtained from SAP2000. It is found that for design truck (CL-625) fatigue truck factor is varying from 0.35 to 0.55 depending on span lengths and bridge configuration. The detail results will be presented in the upcoming papers. This code can be used for any design trucks available in standard codes.

Keywords: bridge, fatigue, fatigue design truck, rain flow analysis, FORTRAN

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Authors: T. S. Soliman, A. G. Galyas, E. V. Rusinova, S. A. Vshivkov

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The liquid crystals combining properties of a liquid and an anisotropic crystal substance play an important role in a science and engineering. Molecules of cellulose and its derivatives have rigid helical conformation, stabilized by intramolecular hydrogen bonds. Therefore the macromolecules of these polymers are capable to be ordered at dissolution and form liquid crystals of cholesteric type. Phase diagrams of solutions of some cellulose derivatives are known. However, little is known about the effect of a magnetic field on the viscosity of polymer solutions. The systems hydroxypropyl cellulose (HPC) – ethanol, HPC – ethylene glycol, HPC–DМАA, HPC–DMF, ethyl cellulose (EC)–ethanol, EC–DMF, were studied in the presence and absence of magnetic field. The solution viscosity was determined on a Rheotest RN 4.1 rheometer. The effect of a magnetic field on the solution properties was studied with the use of two magnets, which induces a magnetic-field-lines directed perpendicularly and parallel to the rotational axis of a rotor. Application of the magnetic field is shown to be accompanied by an increase in the additional assembly of macromolecules, as is evident from a gain in the radii of light scattering particles. In the presence of a magnetic field, the long chains of macromolecules are oriented in parallel with field lines. Such an orientation is associated with the molecular diamagnetic anisotropy of macromolecules. As a result, supramolecular particles are formed, especially in the vicinity of the region of liquid crystalline phase transition. The magnetic field leads to the increase in viscosity of solutions. The results were used to plot the concentration dependence of η/η0, where η and η0 are the viscosities of solutions in the presence and absence of a magnetic field, respectively. In this case, the values of viscosity corresponding to low shear rates were chosen because the concentration dependence of viscosity at low shear rates is typical for anisotropic systems. In the investigated composition range, the values of η/η0 are described by a curve with a maximum.

Keywords: rheology, liquid crystals, magnetic field, cellulose ethers

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Authors: Nalini Kaul, Barrie Drewitt, Elsie Kohoot

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Hyperpigmentation is the third most common pigmentary disorder where dermatologic treatment is sought. It affects all ages resulting in skin darkening because of melanin accumulation. An uneven skin tone because of either exposure to the sun (solar lentigos/age spots/sun spots or skin disruption following acne, or rashes (post-inflammatory hyperpigmentation -PIH) or hormonal changes (melasma) can lead to significant psychosocial impairment. Dyschromia is a result of various alterations in biochemical processes regulating melanogenesis. Treatments include the daily use of sunscreen with lightening, brightening, and exfoliating products. Depigmentation is achieved by various depigmenting agents: common examples are hydroquinone, arbutin, azelaic acid, aloesin, mulberry, licorice extracts, kojic acid, niacinamide, ellagic acid, arbutin, green tea, turmeric, soy, ascorbic acid, and tranexamic acid. These agents affect pigmentation by interfering with mechanisms before, during, and after melanin synthesis. While immediate correction is much sought after, patience and diligence are key. Our objective was to assess the effects of a facial product with pigmentation treatment and UV protection in 35 healthy F (35-65y), meeting the study criteria. Subjects with mild to moderate hyperpigmentation and fine lines with no use of skin-lightening products in the last six months or any dermatological procedures in the last twelve months before the study started were included. Efficacy parameters included expert clinical grading for hyperpigmentation, radiance, skin tone & smoothness, fine lines, and wrinkles bioinstrumentation (Corneometer®, Colorimeter®), digital photography and imaging (Visia-CR®), and self-assessment questionnaires. Safety included grading for erythema, edema, dryness & peeling and self-assessments for itching, stinging, tingling, and burning. Our results showed statistically significant improvement in clinical grading scores, bioinstrumentation, and digital photos for hyperpigmentation-brown spots, fine lines/wrinkles, skin tone, radiance, pores, skin smoothness, and overall appearance compared to baseline. The product was also well-tolerated and liked by subjects. Conclusion: Facial hyperpigmentation is of great concern, and treatment strategies are increasingly sought. Clinical trials with both subjective and objective assessments, imaging analyses, and self-perception are essential to distinguish evidence-based products. The multifunctional cosmetic product tested in this clinical study showed efficacy, tolerability, and subject satisfaction in reducing hyperpigmentation and global photoaging.

Keywords: hyperpigmentation; photoaging, clinical testing, expert visual evaluations, bio-instruments

Procedia PDF Downloads 50

Authors: Sadaf Ilyas

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Recombinant cytokines have been employed successfully as potential therapeutic agent. Some cytokine therapies are already used as a part of clinical practice, ranging from early exploratory trials to well established therapies that have already received approval. Interleukin 15 is a pleiotropic cytokine having multiple roles in peripheral innate and adaptive immune cell function. It regulates the activation, proliferation and maturation of NK cells, T-cells, monocytes/macrophages and granulocytes, and the interactions between them thus acting as a bridge between innate and adaptive immune responses. Unraveling the biology of IL-15 has revealed some interesting surprises that may point toward some of the first therapeutic applications for this cytokine. In this study, the human interleukin 15 gene was isolated, amplified and ligated to a TA vector which was then transfected to a bacterial host, E. coli Top10F’. The sequence of cloned gene was confirmed and it showed 100% homology with the reported sequence. The confirmed gene was then subcloned in pET Expression system to study the IPTG induced expression of IL-15 gene. Positive expression was obtained for number of clones that showed 15 kd band of IL-15 in SDS-PAGE analysis, indicating the successful strain development that can be studied further to assess the potential therapeutic intervention of this cytokine in relevance to human diseases.

Keywords: Interleukin 15, pET expression system, immune therapy, protein purification

Procedia PDF Downloads 385

Authors: Kah Yan How, Peh Fern Ong, Keang Peng Song

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Porphyromonas gingivalis is a black-pigmented, anaerobic Gram-negative bacterium that is important in the progression of chronic and severe periodontitis. This organism has an essential requirement for iron, which is usually obtained from hemin, using specific membrane receptors, proteases, and lipoproteins. In this study, we report the characterization of a novel 24 kDa hemin-binding protein, HmuX, in P. gingivalis W50. The hmuX gene is 651 bp long which encodes for a 217 amino acid protein. HmuX was found to be identical at the C-terminus to the previously reported HmuY protein, differing by an additional 74 amino acids at the N-terminus. Recombinant HmuX demonstrated hemin-binding ability by LDS- PAGE and TMBZ staining. Sequence analysis of HmuX revealed a putative lipoprotein attachment site, suggesting its possible role as a lipoprotein. HmuX was also localized to the outer cell surface by transmission electron microscopy. Northern analysis showed hmuX to be transcribed as a single gene and that hmuX mRNA was tightly regulated by the availability of extra-cellular hemin. P. gingivalis isogenic mutant deficient in hmuX gene exhibited significant growth retardation under hemin-limited conditions. Taken together, these results suggest that HmuX is a hemin-binding lipoprotein, important in hemin utilization for the growth of P. gingivalis.

Keywords: Porphyromonas gingivalis, periodontal diseases, HmuX, protein characterization

Procedia PDF Downloads 196

Authors: David A. Ullisch, Yantree D. Sankar-Thomas, Stefan Wilke, Thomas Selge, Matthias Pump, Thomas Leibold, Kai Schütte, Gilbert Gorr

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Plants have been considered as a source of natural substances for ages. Secondary metabolites from plants are utilized especially in medical applications but are more and more interesting as cosmetical ingredients and in the field of nutraceuticals. However, supply of compounds from natural harvest can be limited by numerous factors i.e. endangered species, low product content, climate impacts and cost intensive extraction. Especially in the pharmaceutical industry the ability to provide sufficient amounts of product and high quality are additional requirements which in some cases are difficult to fulfill by plant harvest. Whereas in many cases the complexity of secondary metabolites precludes chemical synthesis on a reasonable commercial basis, plant cells contain the biosynthetic pathway – a natural chemical factory – for a given compound. A promising approach for the sustainable production of natural products can be plant cell fermentation (PCF®). A thoroughly accomplished development process comprises the identification of a high producing cell line, optimization of growth and production conditions, the development of a robust and reliable production process and its scale-up. In order to address persistent, long lasting production, development of cryopreservation protocols and generation of working cell banks is another important requirement to be considered. So far the most prominent example using a PCF® process is the production of the anticancer compound paclitaxel. To demonstrate the power of plant suspension cultures here we present three case studies: 1) For more than 17 years Phyton produces paclitaxel at industrial scale i.e. up to 75,000 L in scale. With 60 g/kg dw this fully controlled process which is applied according to GMP results in outstanding high yields. 2) Thapsigargin is another anticancer compound which is currently isolated from seeds of Thapsia garganica. Thapsigargin is a powerful cytotoxin – a SERCA inhibitor – and the precursor for the derivative ADT, the key ingredient of the investigational prodrug Mipsagargin (G-202) which is in several clinical trials. Phyton successfully generated plant cell lines capable to express this compound. Here we present data about the screening for high producing cell lines. 3) The third case study covers ingenol-3-mebutate. This compound is found in the milky sap of the intact plants of the Euphorbiacae family at very low concentrations. Ingenol-3-mebutate is used in Picato® which is approved against actinic keratosis. Generation of cell lines expressing significant amounts of ingenol-3-mebutate is another example underlining the strength of plant cell culture. The authors gratefully acknowledge Inspyr Therapeutics for funding.

Keywords: Ingenol-3-mebutate, plant cell culture, sustainability, thapsigargin

Procedia PDF Downloads 221