Search results for: recombinant protein expression

Authors: Tiancheng Lan

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E10A is a kind of replication-defective adenovirus which carries the human endostatin gene to inhibit the growth of tumors. Kringle 5(K5) has almost the same function as angiostatin to also inhibit the growth of tumors since they are all the byproduct of the proteolytic cleavage of plasminogen. Tumor size increasing can be suppressed because both of the endostatin and K5 can restrain the angiogenesis process. Therefore, in order to improve the treatment effect on tumor, 2A peptide is used to construct a fusion gene carrying both E10A and K5. Using 2A peptide is an ideal strategy when a fusion gene is expressed because it can avoid many problems during the expression of more than one kind of protein. The overlap extension PCR is also used to connect 2A peptide with E10A and K5. The final construction of fusion gene E10A-2A-K5 can provide a possible new method of the anti-angiogenesis treatment with a better expression performance.

Keywords: E10A, Kringle 5, 2A peptide, overlap extension PCR

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Authors: Hatairuk Tungkasen, Somrudee Phetchrid, Suwapat Jaidee, Supinya Yoomak, Chantana Kankamol, Mayuree Pumipaiboon, Mayuva Areekijseree

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Ovaries of reproductive female pigs were obtained from local slaughterhouses in Nakorn Pathom Province, Thailand. Follicular fluid of medium follicle (5-6 diameters) and large follicles (7-8 mm and 10 mm in diameter) were aspirated and collected by sterile technique and analyzed protein pattern. The follicular fluid protein bands were found by SDS-PAGE which has no protein band in difference compared to standard protein band. So we chose protein band molecular weight 50, 62-65, 75-80, 90, 120-160, and >220 kDa were analyzed by LC/MS/MS. The result was found immunoglobulin gamma chain, keratin, transferrin, heat shock protein, and plasminogen precursor, ceruloplasmin, and hemopexin, and protease, respectively. All proteins play important roles in promotion and regulation on growth and development of reproductive cells. The result of this study found many proteins which were useful and important for in vitro oocyte maturation and embryonic development of cell technology in animals. The further study will be use porcine follicular fluid protein of medium and large follicles as feeder cells in in vitro condition to promote oocyte and embryo maturation.

Keywords: follicular fluid protein, LC/MS/MS, porcine oocyte, SDS-PAGE

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Authors: Ceyda Okudu, Secil Eroglu, Khandakar A. S. M. Saadat, Sibel O. Balci

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Ring Finger Protein 2 (RNF2) is a member of polycomb repressive complex 1 (PRC1), which is one of the epigenetic regulators in the genome. When RNF2 combines with other PRC1 members, it mediates the mono-ubiquitination of Histon2A (H2A). In breast cancer, RNF2 is commonly overexpressed, and also it promotes metastasis and invasion in other aggressive tumors like melanoma, prostate, and hepatocarcinoma. The role of RNF2 in the metastasis and invasion of breast cancer has not yet been elucidated. Our aim is to observe the role of RNF2 in metastasis and invasion in this study by miRNA mediated RNF2 gene silencing in breast cancer cell lines. We selected miRNAs, targeting to RNF2 by searching online databases. miR-17-5p, miR20a-5p, and miR-106b-5p were transfected to breast cancer cell lines (MCF-7, MDA-MB-231, SK-BR-3, and ZR-75-1), and also we used normal breast epithelial cell line (hTERT-HME1) to compare RNF2 gene expression level. After 48-72 hours post-transfection, mRNAs were isolated from the cells, and gene expressions were measured by RT-qPCR after from cDNA syntheses. We observed that RNF2 was highly expressed in SK-BR-3 and MDA-MB-231 cell lines opposite to MCF-7 and ZR-75-1 cell lines. RNF2 was downregulated 5, 5 and 7 fold by miR17-5p, miR20a-5p and miR106b-5p respectively in MCF-7. However, in SK-BR-3 and ZR-75-1 cell lines, miRNAs did not affect significantly RNF2 gene expression level. miR20a-5p decreased RNF2 3 fold and miR17-5p and miR106b-5p did not affect MDA-MB-231. After gene expression analysis, we performed metastasis and invasion assay in MCF-7 cells. For metastasis, we used both wound healing assay and Transwell Cell Migration Assay, and we used Transwell Cell Invasion Assay for invasion. The data of this assay showed that miR17-5p and miR20a-5p decreased both invasion and metastasis level, but miR106b-5p has no effect. We would like to conclude that RNF2 can be targeted by miR17-5p, miR20a-5p and miR106b-5p in MCF-7 cells and also RNF2, which is one of the upregulated genes in aggressive tumor, can be decreased by using these miRNAs. In future, we would like to confirm these results at the protein level and also whether these miRNAs are direct target of RNF2 or not.

Keywords: breast cancer, epigenetic, microRNAs, RNF2

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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: S. Junaid S. Qazi, Denice C. Bay, Raymond Chew, Raymond J. Turner

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EmrE, a member of the small multidrug resistance protein family in bacteria is considered to be the archetypical member of its family. It confers host resistance to a wide variety of quaternary cation compounds (QCCs) driven by proton motive force. Generally, purification yield is a challenge in all membrane proteins because of the difficulties in their expression, isolation and solubilization. EmrE is extremely hydrophobic which make the purification yield challenging. We have purified EmrE protein using two different approaches: organic solvent membrane extraction and hexahistidine (his6) tagged Ni-affinity chromatographic methods. We have characterized changes present between ligand affinity of untagged and his6-tagged EmrE proteins in similar membrane mimetic environments using biophysical experimental techniques. Purified proteins were solubilized in a buffer containing n-dodecyl-β-D-maltopyranoside (DDM) and the conformations in the proteins were explored in the presence of four QCCs, methyl viologen (MV), ethidium bromide (EB), cetylpyridinium chloride (CTP) and tetraphenyl phosphonium (TPP). SDS-Tricine PAGE and dynamic light scattering (DLS) analysis revealed that the addition of QCCs did not induce higher multimeric forms of either proteins at all QCC:EmrE molar ratios examined under the solubilization conditions applied. QCC binding curves obtained from the Trp fluorescence quenching spectra, gave the values of dissociation constant (Kd) and maximum specific one-site binding (Bmax). Lower Bmax values to QCCs for his6-tagged EmrE shows that the binding sites remained unoccupied. This lower saturation suggests that the his6-tagged versions provide a conformation that prevents saturated binding. Our data demonstrate that tagging an integral membrane protein can significantly influence the protein.

Keywords: small multidrug resistance (SMR) protein, EmrE, integral membrane protein folding, quaternary ammonium compounds (QAC), quaternary cation compounds (QCC), nickel affinity chromatography, hexahistidine (His6) tag

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Authors: Hanan Azzam1, Nashwa Abousamra1, Amany Mansour1, Yaser Abd El-dayem2, , Solafa Elsharawy1

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Background: Inherited thrombophilias are a heterogenous group of conditions which have been implicated in a variety of pregnancy complications. More recently, deficiency of protein Z (PZ) has been liked to pregnancy complications, including preterm delivery. Aim: We designed this study to evaluate the association of inherited thrombophilias including [Protein C (PC), Protein S (PS), Anti thrombin III (ATIII) deficiency and activated protein C (APC) resistance] and protein Z deficiency with a variety of pregnancy complications. Patients and Methods: 60 women with different pregnancy complications, including 20 patients with preeclampsia, 20 patients with intrauterine growth resistance (IUGR), and 20 patients with intrauterine fetal death (IUFD), in addition to 30 healthy pregnant women were recruited for the present study. PC and free PS antigen, ATIII activity, modified functional APC-resistance, and PZ levels were determined. Results: There was no significant association between inherited thrombophilias and complicated pregnancies as regards PC deficiency (p=1.0), AT III and PS deficiency (p=0.312), and APC-resistance (P=0.083). PZ was significantly associated with complicated pregnancies (p=0.012). Patients with protein Z levels below 1.5 µg/ml were considered deficient. Accordingly, we demonstrated protein Z deficiency in 30% of complicated pregnancies (RR 6.0, 95% CI 1.29-27.90;p=0.022), 20% of preeclampsia (RR 3.5, 95% CI 0.57 – 21.28; P = 0.174), 40% of IUGR (RR 9.3 95% CI 1.72-50.61; P = 0.010) and 30% of IUFD (RR 6, 95% CI 1.07 – 33.64; P = 0.042). Conclusions: These findings indicate the absence of association of inherited thrombophilias, including PC, PS, AT III deficiency, and APC resistance with pregnancy complications. However, PZ deficiency is associated with increased risk of pregnancy complications, especially intrauterine growth restriction and intrauterine fetal death.

Keywords: protein C, protein S, thrombophelia, pregnancy, protein Z

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Authors: Felicia Segeth, Florian G. Klein, Lea Berger, Andreas Kolk, Per S. Holm

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The entry of oncolytic viruses (OVs) into clinical application opens groundbreaking changes in current and future treatment regimens. However, despite their potent anti-cancer activity in vitro, clinical studies revealed limitations of OVs as monotherapy. The same applies to CDK 4/6 inhibitors (CDK4/6i) targeting cell cycle as well as bromodomain and extra-terminal domain inhibitors (BETi) targeting gene expression. In this study, the anti-tumoral effect of XVir-N-31, an YB-1 dependent oncolytic adenovirus, was evaluated in combination with Ribociclib, a CDK4/6i, and JQ1, a BETi. The head and neck squamous cell carcinoma (HNSCC) cell lines Fadu, SAS, and Cal-33 were used. DNA replication and gene expression of XVir-N-31 was measured by RT-qPCR, protein expression by western blotting, and cell lysis by SRB assays. Treatment with CDK4/6i and BETi increased viral gene expression, viral DNA replication, and viral particle formation. The data show that the combination of oncolytic adenovirus XVir-N-31 with CDK4/6i & BETi acts highly synergistic in cancer cell lysis. Furthermore, additional molecular analyses on this subject demonstrate that the positive transcription elongation factor P-TEFb plays a decisive role in this regard, indicating an influence of the combinational therapy on gene transcription control. The combination of CDK4/6i & BETi and XVir-N-31 is an attractive strategy to achieve substantial cancer cell killing and is highly suitable for clinical testing.

Keywords: adenovirus, BET, CDK4/6, HNSCC, P-TEFb, YB-1

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Authors: Xianming Chen, Yu Lei, Meinan Wang

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The dikaryotic basidiomycete fungus, Puccinia striiformis, causes stripe rust, one of the most important diseases of wheat and barley worldwide. The pathogen is largely reproduced asexually, and asexual recombination has been hypothesized to be one of the mechanisms for the pathogen variations. To test the hypothesis and understand the genetic process of asexual recombination, somatic recombinant isolates were obtained under controlled conditions by inoculating susceptible host plants with a mixture of equal quantity of urediniospores of isolates with different virulence patterns and selecting through a series of inoculation on host plants with different genes for resistance to one of the parental isolates. The potential recombinant isolates were phenotypically characterized by virulence testing on the set of 18 wheat lines used to differentiate races of the wheat stripe rust pathogen, P. striiformis f. sp. tritici (Pst), for the combinations of Pst isolates; or on both sets of the wheat differentials and 12 barley differentials for identifying races of the barley stripe rust pathogen, P. striiformis f. sp. hordei (Psh) for combinations of a Pst isolate and a Psh isolate. The progeny and parental isolates were also genotypically characterized with 51 simple sequence repeat and 90 single-nucleotide polymorphism markers. From nine combinations of parental isolates, 68 potential recombinant isolates were obtained, of which 33 (48.5%) had similar virulence patterns to one of the parental isolates, and 35 (51.5%) had virulence patterns distinct from either of the parental isolates. Of the 35 isolates of distinct virulence patterns, 11 were identified as races that had been previously detected from natural collections and 24 were identified as new races. The molecular marker data confirmed 66 of the 68 isolates as recombinants. The percentages of parental marker alleles ranged from 0.9% to 98.9% and were significantly different from equal proportions in the recombinant isolates. Except for a couple of combinations, the greater or less contribution was not specific to any particular parental isolates as the same parental isolates contributed more to some of the progeny isolates but less to the other progeny isolates in the same combination. The unequal contributions by parental isolates appear to be a general role in somatic recombination for the stripe rust fungus, which may be used to distinguish asexual recombination from sexual recombination in studying the evolutionary mechanisms of the highly variable fungal pathogen.

Keywords: molecular markers, Puccinia striiformis, somatic recombination, stripe rust

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Authors: Alena Semeradtova, Marcel Stofik, Lucie Mareckova, Petr Maly, Ondrej Stanek, Jan Maly

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Recently, novel strategies based on so-called molecular evolution were shown to be effective for the production of various peptide ligand libraries with high affinities to molecular targets of interest comparable or even better than monoclonal antibodies. The major advantage of these peptide scaffolds is mainly their prevailing low molecular weight and simple structure. This study describes a new high-affinity binding molecules based immunesensor using a simple optical system for human serum albumin (HSA) detection as a model molecule. We present a comparison of two variants of recombinant binders based on albumin binding domain of the protein G (ABD) performed on micropatterned glass chip. Binding domains may be tailored to any specific target of interest by molecular evolution. Micropatterened glass chips were prepared using UV-photolithography on chromium sputtered glasses. Glass surface was modified by (3-aminopropyl)trietoxysilane and biotin-PEG-acid using EDC/NHS chemistry. Two variants of high-affinity binding molecules were used to detect target molecule. Firstly, a variant is based on ABD domain fused with TolA chain. This molecule is in vivo biotinylated and each molecule contains one molecule of biotin and one ABD domain. Secondly, the variant is ABD domain based on streptavidin molecule and contains four gaps for biotin and four ABD domains. These high-affinity molecules were immobilized to the chip surface via biotin-streptavidin chemistry. To eliminate nonspecific binding 1% bovine serum albumin (BSA) or 6% fetal bovine serum (FBS) were used in every step. For both variants range of measured concentrations of fluorescently labelled HSA was 0 – 30 µg/ml. As a control, we performed a simultaneous assay without high-affinity binding molecules. Fluorescent signal was measured using inverse fluorescent microscope Olympus IX 70 with COOL LED pE 4000 as a light source, related filters, and camera Retiga 2000R as a detector. The fluorescent signal from non-modified areas was substracted from the signal of the fluorescent areas. Results were presented in graphs showing the dependence of measured grayscale value on the log-scale of HSA concentration. For the TolA variant the limit of detection (LOD) of the optical immunosensor proposed in this study is calculated to be 0,20 µg/ml for HSA detection in 1% BSA and 0,24 µg/ml in 6% FBS. In the case of streptavidin-based molecule, it was 0,04 µg/ml and 0,07 µg/ml respectively. The dynamical range of the immunosensor was possible to estimate just in the case of TolA variant and it was calculated to be 0,49 – 3,75 µg/ml and 0,73-1,88 µg/ml respectively. In the case of the streptavidin-based the variant we didn´t reach the surface saturation even with the 480 ug/ml concentration and the upper value of dynamical range was not estimated. Lower value was calculated to be 0,14 µg/ml and 0,17 µg/ml respectively. Based on the obtained results, it´s clear that both variants are useful for creating the bio-recognizing layer on immunosensors. For this particular system, it is obvious that the variant based on streptavidin molecule is more useful for biosensing on glass planar surfaces. Immunosensors based on this variant would exhibit better limit of detection and wide dynamical range.

Keywords: high affinity binding molecules, human serum albumin, optical immunosensor, protein G, UV-photolitography

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Authors: Abdelkrim Khadir, Sina Kavalakatt, Preethi Cherian, Ali Tiss

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Soluble epoxide hydrolase (sEH) is an emerging therapeutic target in several chronic states that have inflammation as a common underlying cause such as immunometabolic diseases. Indeed, sEH is known to play a pro-inflammatory role by metabolizing anti-inflammatory, epoxyeicosatrienoic acids (EETs) to pro-inflammatory diols. Recently, it was shown sEH to be linked to diet and microbiota interaction in rat models of obesity. Nevertheless, the functional contribution of sEH and its anti-inflammatory substrates EETs in obesity remain poorly understood. In the current study, we compared the expression pattern of sEH between lean and obese nondiabetic human subjects using subcutaneous adipose tissue (SAT) and peripheral blood mononuclear cells (PBMCs). Using RT-PCR, western blot and immunofluorescence confocal microscopy, we show here that the level of sEH mRNA and protein to be significantly increased in obese subjects with concomitant increase in endoplasmic reticulum (ER) stress components (GRP78 and ATF6α) and inflammatory markers (TNF-α, IL-6) when compared to lean controls. The observation that sEH was overexpressed in obese subjects’ prompt us to investigate whether physical exercise could reduce its expression. In this study, we report here 3-months supervised physical exercise significantly attenuated the expression of sEH in both the SAT and PBMCs, with a parallel decrease in the expression of ER stress markers along with attenuated inflammatory response. On the other hand, homocysteine, a sulfur containing amino acid deriving from the essential amino acid methionine was shown to be directly associated with insulin resistance. When 3T3-L1 preadipocytes cells were treated with homocysteine our results show increased sEH levels along with ER stress markers. Collectively, our data suggest that sEH upregulation is strongly linked to ER stress in adiposity and that physical exercise modulates its expression. This gives further evidence that exercise might be useful as a strategy for managing obesity and preventing its associated complications.

Keywords: obesity, adipose tissue, epoxide hydrolase, ER stress

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Authors: Adel M. Zakri, Mohammed A. Al-Saleh, Judith. K. Brown, Ali M. Idris

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Betasatellites are small circular single stranded DNA molecules found associated with begomoviruses on field symptomatic plants. Their genome size is about half that of the helper begomovirus, ranging between 1.3 and 1.4 kb. The helper begomoviruses are usually members of the family Geminiviridae. Okra leaves showing vein thickening were collected from okra plants growing in Jazan, Saudi Arabia. Total DNA was extracted from leaves and used as a template to amplify circular DNA using rolling circle amplification (RCA) technology. Products were digested with PstI to linearize the helper viral genome(s), and associated DNA satellite(s), yielding a 2.8kbp and 1.4kbp fragment, respectively. The linearized fragments were cloned into the pGEM-5Zf (+) vector and subjected to DNA sequencing. The 2.8 kb fragment was identified as Cotton leaf curl Gezira virus genome, at 2780bp, an isolate closely related to strains reported previously from Saudi Arabia. A clone obtained from the 1.4 kb fragments he 1.4kb was blasted to GeneBank database found to be a betasatellite. The genome of betasatellite was 1357-bp in size. It was found to be a recombinant containing one fragment (877-bp) that shared 91% nt identity with Cotton leaf curl Gezira betasatellite [KM279620], and a smaller fragment [133--bp) that shared 86% nt identity with Tomato leaf curl Sudan virus [JX483708]. This satellite is thus a recombinant between a malvaceous-infecting satellite and a solanaceous-infecting begomovirus.

Keywords: begomovirus, betasatellites, cotton leaf curl Gezira virus, okra plants

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Authors: Muhammad Yousaf Ali, Shahid Mansoor, Javeria Qazi, Imran Amin, Musarrat Shaheen

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Cotton leaf curl disease (CLCuD) is the major threat to the cotton crop and is transmitted by whitefly (Bemisia tabaci). Since multiple begomoviruses and associated satellites are involved in CLCuD, approaches based on the concept of broad-spectrum resistance are essential for effective disease control. Gro-El and G5 are two proteins from whitefly endosymbiont and M13 bacteriophage origin, respectively. Gro-El encapsulates the virus particle when it enters the whitefly and protects the virus from the immune system of the whitefly as well as prevents viral expression in it. This characteristic of Gro-El can be exploited to get resistance against viruses if expressed in plants. G5 is a single-stranded DNA binding protein, expression of which in transgenic plants will stop viral expression on its binding with ssDNA. The use of tissue-specific promoters is more efficient than constitutive promoters. Transgenics of Nicotiana benthamiana for Gro-El under constitutive promoter and Gro-El under phloem specific promoter were made. In comparison to non-transgenic plants, transgenic plants with Gro-El under NSP promoter showed promising results when challenged against cotton leaf curl Multan virus (CLCuMuV) along with cotton leaf curl Multan beta satellite (CLCuMB), cotton leaf curl Khokhran virus (CLCuKoV) along with cotton leaf curl Multan beta satellite (CLCuMB) and Pedilenthus leaf curl virus (PedLCV) along with Tobacco leaf curl beta satellite (TbLCB).

Keywords: cotton leaf curl disease, whitefly, endosymbionts, transgenic, resistance

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Authors: Alexander Hudson, Shaogang Gong

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Structure determination is key to understanding protein function at a molecular level. Whilst significant advances have been made in predicting structure and function from amino acid sequence, researchers must still rely on expensive, time-consuming analytical methods to visualise detailed protein conformation. In this study, we demonstrate that it is possible to make accurate (≥80%) predictions of protein class and architecture from structures determined at low (>3A) resolution, using a deep convolutional neural network trained on high-resolution (≤3A) structures represented as 2D matrices. Thus, we provide proof of concept for high-speed, low-cost protein structure classification at low resolution, and a basis for extension to prediction of function. We investigate the impact of the input representation on classification performance, showing that side-chain information may not be necessary for fine-grained structure predictions. Finally, we confirm that high resolution, low-resolution and NMR-determined structures inhabit a common feature space, and thus provide a theoretical foundation for boosting with single-image super-resolution.

Keywords: transfer learning, protein distance maps, protein structure classification, neural networks

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Authors: Diana Karina Baigts Allende, Mariana Gonzalez Diaz, Luis Antonio Chel Guerrero, Mukthar Sandoval Peraza

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Poverty and food insecurity are still incident problems in the developing countries, where population´s diet is based on cereals which are lack in protein content. Nevertheless, during last years the use of native plants has been studied as an alternative source of protein in order to improve the nutritional intake. Chaya crop also called Spinach tree, is a prehispanic plant native from Central America and South of Mexico (Mayan culture), which has been especially valued due to its high nutritional content particularly protein and some medicinal properties. The aim of this work was to study the effect of protein isolation processing from Chaya leaf harvest in Yucatan, Mexico on its structure quality in order: i) to valorize the Chaya crop and ii) to produce low-cost and high-quality protein. Chaya leaf was extruded, clarified and recovered using: a) acid precipitation by decreasing the pH value until reach the isoelectric point (3.5) and b) thermal coagulation, by heating the protein solution at 80 °C during 30 min. Solubilized protein was re-dissolved in water and spray dried. The presence of Fraction I protein, known as RuBisCO (Rubilose-1,5-biphosfate carboxylase/oxygenase) was confirmed by gel electrophoresis (SDS-PAGE) where molecular weight bands of 55 KDa and 12 KDa were observed. The infrared spectrum showed changes in protein structure due to the isolation method. The use of high temperatures (thermal coagulation) highly decreased protein solubility in comparison to isoelectric precipitated protein, the nutritional properties according to amino acid profile was also disturbed, showing minor amounts of overall essential amino acids from 435.9 to 367.8 mg/g. Chaya protein isolate obtained by acid precipitation showed higher protein quality according to essential amino acid score compared to FAO recommendations, which could represent an important sustainable source of protein for human consumption.

Keywords: chaya leaf, nutritional properties, protein isolate, protein structure

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Authors: Shima Mahmoudi, Babak Pourakbari, Setareh Mamishi, Mostafa Teymuri, Majid Marjani

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Although cytokine analysis has greatly contributed to the understanding of tuberculosis (TB) pathogenesis, data on cytokine profiles that might distinguish progression from latency of TB infection are scarce. Since PE/PPE proteins are known to induce strong humoral and cellular immune responses, the aim of this study was to evaluate the diagnostic potential of interleukin-2 (IL-2) as biomarker after specific antigen stimulation with PE35 and PPE68 for the discrimination of active and latent tuberculosis infection (LTBI). The production of IL-2 was measured in the antigen-stimulated whole-blood supernatants following stimulation with recombinant PE35 and PPE68. All the patients with active TB and LTBI had positive QuantiFERON-TB Gold in Tube test. The level of IL-2 following stimulation with recombinant PE35 and PPE68 were significantly higher in LTBI group than in patients with active TB infection or control group. The discrimination performance (assessed by the area under ROC curve) for IL-2 following stimulation with recombinant PE35 and PPE68 between LTBI and patients with active TB were 0.837 (95%CI: 0.72-0.97) and 0.75 (95%CI: 0.63-0.89), respectively. Applying the 12.4 pg/mL cut-off for IL-2 induced by PE35 in the present study population resulted in sensitivity of 78%, specificity of 78%, PPV of 78% and NPV of 100%. In addition, a sensitivity of 81%, specificity of 70%, PPV of 67% and 87% of NPV was reported based on the 4.4 pg/mL cut-off for IL-2 induced by PPE68. In conclusion, peptides of the antigen PE35 and PPE68, absent from commonly used BCG strains, stimulated strong IL-2- positive T cell responses in patients with LTBI. This study confirms IL-2 induced by PE35 and PPE68 as a sensitive and specific biomarker and highlights IL-2 as new promising adjunct markers for discriminating of LTBI and Active TB infection.

Keywords: IL-2, PE35, PPE68, tuberculosis

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Authors: Rohita Gupta, G. S. Brah, R. Verma, C. S. Mukhopadhayay

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Although chicken strains show differences in susceptibility to a number of diseases, the underlying immunological basis is yet to be elucidated. In the present study, heterophils were subjected to LPS stimulation and total RNA extraction, further differential gene expression was studied in broiler, layer and indigenous Aseel strain by Real Time RT-PCR at different time periods before and after induction. The expression of the 14 AvBDs and chTLR 1, 2, 3, 4, 5, 7, 15 and 21 was detectable in heterophils. The expression level of most of the AvBDs significantly increased (P<0.05) 3 hours post in vitro lipopolysaccharide challenge. Higher expression level and stronger activation of most AvBDs, NFkB-1 and IRF-3 in heterophils was observed with the stimulation of LPS in layer compared to broiler, and in Aseel compared to both layer and broiler. This investigation will allow more refined interpretation of immuno-genetic basis of the variable disease resistance/susceptibility in divergent stock of chicken including indigenous breed. Moreover, this study will be helpful in formulation of strategy for isolation of antimicrobial peptides from heterophils.

Keywords: differential expression, heterophils, cytokines, defensin, TLR

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Authors: Alexandre Barbosa de Almeida, Telma Woerle de Lima Soares

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Protein structure prediction is a challenging task in the bioinformatics field. The biological function of all proteins majorly relies on the shape of their three-dimensional conformational structure, but less than 1% of all known proteins in the world have their structure solved. This work proposes a deep learning model to address this problem, attempting to predict some aspects of the protein conformations. Throughout a process of multiobjective dominance, a recurrent neural network was trained to abstract the particular bias of each individual multiobjective algorithm, generating a heuristic that could be useful to predict some of the relevant aspects of the three-dimensional conformation process formation, known as protein folding.

Keywords: Ab initio heuristic modeling, multiobjective optimization, protein structure prediction, recurrent neural network

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Authors: Ayoola, Simeon Oluwatoyin, Omogoriola Hannah Omoloye

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The effects of 17α-Methyltestosterone Hormone on blood composition of the Sex Reversed Sarotherodon melanotheron were investigated. S. melanotheron fry were reared in six (6) plastic tanks for three (3) months, of which three (3) tanks served as treatment tanks while the other three (3) served as the control. The fry were fed with 17α-methyl testosterone enzyme, which functions as a sex reversal hormone. The fry were administered this hormone for 30 days, to ensure complete sex reversal. All the S. melanotheron fry were reared to table size for duration of three (3) months, after which, blood samples were taken from both the control and treatment fishes. The blood parameters showed no significant differences with the same values of White Blood Cell count (WBC) and Total plasma protein for the control and experimental fishes. A total protein value for sex reversed specimens was 3.99g/dL, while urea and creatinine values were 0.2g/dL. Alkaline Phosphatase, Aspartate transaminase and Alanine transaminase for the treatment specimen were 183nm/mg protein/min, 98nm/mg protein/min and 105nm/mg protein/min respectively. A total protein value for control specimens was 2.81g/dL, while urea and creatinine values were 0.2g/dL. Alkaline Phosphatase, Aspartate transaminase and Alanine transaminase for the control species were 174nm/mg protein/min, 93nm/mg protein/min and 106nm/mg protein/min respectively. The safety of MT on S. melanotheron is therefore proved since there is no adverse effect on the fish.

Keywords: 17α-Methyltestosterone, haematology, sex reversal, sarotherodon melanotheron

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Authors: Mohammad K. Parvez, Mohammed S. Al-Dosari

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Hepatitis E virus (HEV) is an emerging RNA virus that causes acute and chronic liver disease with a global mortality rate of about 2%. Despite milestone developments in understanding of HEV biology, there is still lack of a robust culture system or animal model. Therefore, in a novel approach, two recombinant-baculoviruses (vBac-ORF2 and vBac-ORF3) that could overexpress HEV ORF2 (structural/capsid) and ORF3 (nonstructural/regulatory) proteins, respectively were constructed. The established HEV-SAR55 (genotype 1) replicon that contained GFP gene, in place of ORF2/ORF3 sequences was in vitro transcribed, and GFP production in RNA transfected S10-3 cells was scored by FACS. Enhanced infectivity, if any, of nascent virions produced by exogenously-supplied ORF2 and viral RNA by co-expression of ORF3 was tested on naïve HepG2 cells. Co-transduction with vBac-ORF2/vBac-ORF3 (108 pfu/microL) produced high amounts of native ORF2/ORF3 in approximately 60% of S10-3 cells, determined by immunofluorescence microscopy and Western analysis. FACS analysis showed about 9% GFP positivity of S10-3 cells on day6 post-transfection (i.e, day5 post-transduction). Further, FACS scoring indicated that lysates from S10-3 cultures receiving the RNA plus vBac-ORF2 were capable of producing HEV particles with about 4% infectivity in HepG2 cells. However, lysates of cultures co-transduced with vBac-ORF3, were found to further enhance virion infectivity by approximately 17%. This supported a previously proposed role of ORF3 as a minor-structural protein in HEV virion assembly and infectivity. In conclusion, the present model for efficient genomic RNA packaging and production of infectious virions could be a valuable tool to study various aspects of HEV molecular biology, in vitro.

Keywords: chronic liver disease, hepatitis E virus, ORF2, ORF3, replicon

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Authors: Alise Senberga, Laila Dubova, Liene Strauta, Ina Alsina, Ieva Erdberga

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Legume symbiotic relationship with nitrogen fixating bacteria Rhizobim leguminosarum is an important factor used to improve the productivity of legumes, due to the fact that rhizobia can supply plant with the necessary amount of nitrogen. R. leguminosarum strains have shown different activity in fixing nitrogen. Depending on the chosen R. leguminosarum strain, host plant biochemical content can be altered. In this study we focused particularly on the changes in protein content in beans (using two different varieties) and peas (five different varieties) due to the use of several different R. leguminosarum strains (four strains for both beans and peas). Overall, the protein content increase was observed after seed inoculation with R. leguminosarum. Strain and plant cultivar interaction specification was observed. The effect of R. leguminosarum inoculation on the content of protein was dependent on the R. leguminosarum strain used. Plant cultivar also appeared to have a decisive role in protein content formation with the help of R. leguminosaru.

Keywords: legumes, protein content, rhizobia strains, soil

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Authors: Rui Liu, Pengyu Cui, Nan Jiang

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At present, there is much research on the application of centralized management and cross-integration application of basic geographic information. However, the idea of information integration and sharing between land, sea, and air navigation targets is not deeply applied into the research of navigation information service, especially in the information expression. Targeting at this problem, the paper carries out works about the expression pattern of navigation electronic map for ground vehicles under air and land united navigation mechanism. At first, with the support from multi-source information fusion of GIS vector data, RS data, GPS data, etc., an air and land united information expression pattern is designed aiming at specific navigation task of emergency rescue in the earthquake. And then, the characteristics and specifications of the united expression of air and land navigation information under the constraints of map load are summarized and transferred into expression rules in the rule bank. At last, the related navigation experiment is implemented to evaluate the effect of the expression pattern. The experiment selects evaluation factors of the navigation task accomplishment time and the navigation error rate as the main index, and make comparisons with the traditional single information expression pattern. To sum up, the research improved the theory of navigation electronic map and laid a certain foundation for the design and realization of united navigation system in the aspect of real-time navigation information delivery.

Keywords: navigation electronic map, united navigation, multi-element expression pattern, multi-source information fusion

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Authors: Ala Rakhshandeh, Zahra Ashkar, Solmaz Dehghani Dolatabadi, Hossein Bayat

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The present study has been conducted to investigate the role of family emotional climate and emotional expression style in the academic well-being of students with military parents. Children, including 280 female students of Shahriar police officers, were selected by random sampling method, and they have been investigated through Alfred B. Hillburn's family emotional climate questionnaire (1964), King and Ammon's emotional expression questionnaire (1990), and Pitrinen, Sweeney, and Falto's academic well-being questionnaire (2014). The data were analyzed using statistical methods of correlation coefficient and stepwise multiple regression under the SPSS23 program. The results reveal that the variables of family emotional climate and emotional expression can explain 36.4% of the variance in academic well-being. This finding reveals that with an increase of standard deviation on the scores of family emotional climate and emotional expression, 0.513 and 0.155 standard deviations are added to the scores of academic well-being, respectively. The emotional climate of the family has a superior distinctive role in predicting the educational well-being of female students. Thus, the emotional climate of the family and the style of emotional expression play a meaningful role in the academic well-being of students with the military parent.

Keywords: emotional climate, family, emotional expression style, academic well-being

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Authors: Denise Schrama, Claudia Raposo, Pedro Rodrigues

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Background: Food allergies are conducted by a hypersensitive response of the immune system. These allergies are a global concern for the public health. Consumption of fish is increasing worldwide as it is a healthy meat with high nutritional value. Unfortunately, fish can cause adverse immune-mediate reactions, affecting part of the population with higher incidence in children. β-parvalbumin, a small, highly conserved stable, calcium or magnesium binding muscle protein is the main fish allergen. In fish-allergic patients, cross-reactivity between different fish species exist due to recognition of highly identical protein regions. Enolases, aldolases, or fish gelatin are other identified fish allergens in some fish species. With no available cure for fish allergies, clinical management is only based on an avoidance diet aiming at the total exclusion of offending food. Methods: Mediterranean fish (S. aurata and D. labrax) were fed specifically designed diets, enriched in components that target the expression or inactivation of parvalbumin (creatine and EDTA, respectively). After 90 days fish were sampled and biological tissues were excised. Proteomics was used to access fish allergens characterization and expression in muscle while IgE assays to confirm the lower allergenic potential are conducted in patients with history of fish allergies. Fish welfare and quality of flesh were established with biochemical, texture and sensorial analysis. Results: Fish welfare shows no major impact between diets. In case of creatine supplementation in D. labrax proteomic analysis show a slight decrease in parvalbumin expression. No accumulation of this compound was found in muscle. For EDTA supplementation in S. aurata IgE assay show a slight decrease in allergenicity when using sera of fish allergic patients. Conclusion: Supplementation with these two compounds seems to change slightly the allergenicity of the two mean Mediterranean species.

Keywords: fish allergies, fish nutrition, proteomics, aquaculture

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Authors: Khaleel Saleh Al-Rababah

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Amyloid fibril forming regions, which are known as protein aggregates, in sequences of some protein families are associated with a number of diseases known as amyloidosis. Mutations play a role in forming fibrils by accelerating the fibril formation process. In this paper we want to extract diseases that caused by those mutations as a result of the impact of the mutations on structural and functional properties of the aggregated protein. We propose a text mining system, to automatically extract mutations, diseases and relations between mutations and diseases. We presented an algorithm based on finite state to cluster mutations found in the same sentence as a sentence could contain different mutation cause different diseases. Also, we presented a co reference algorithm that enables cross-link sentences.

Keywords: amyloid, amyloidosis, co reference, protein, text mining

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Authors: Vun Yee Thien, Kenneth Francis Rodrigues, Clemente Michael Vui Ling Wong, Wilson Thau Lym Yong

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Transcriptomes associated with the process of photosynthesis have offered insights into the mechanism of gene regulation in terrestrial plants; however, limited information is available as far as macroalgae are concerned. This investigation aims to decipher the underlying mechanisms associated with photosynthesis in the red alga, Kappaphycus alvarezii, by performing a differential expression analysis on a de novo assembled transcriptomes. Comparative analysis of gene expression was designed to examine the alteration of light qualities and its effect on physiological mechanisms in the red alga. High-throughput paired-end RNA-sequencing was applied to profile the transcriptome of K. alvarezii irradiated with different wavelengths of light (blue 492-455 nm, green 577-492 nm and red 780-622 nm) as compared to the full light spectrum, resulted in more than 60 million reads individually and assembled using Trinity and SOAPdenovo-Trans. The transcripts were annotated in the NCBI non-redundant (nr) protein, SwissProt, KEGG and COG databases with a cutoff E-value of 1e-5 and nearly 30% of transcripts were assigned to functional annotation by Blast searches. Differential expression analysis was performed using edgeR. The DEGs were designated to six categories: BL (blue light) regulated, GL (green light) regulated, RL (red light) regulated, BL or GL regulated, BL or RL regulated, GL or RL regulated, and either BL, GL or RL regulated. These DEGs were mapped to terms in KEGG database and compared with the whole transcriptome background to search for genes that regulated by light quality. The outcomes of this study will enhance our understanding of molecular mechanisms underlying light-induced responses in red algae.

Keywords: de novo transcriptome sequencing, differential gene expression, Kappaphycus alvareziired, red alga

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Authors: Jiang Xu, Daoping Wang, Yinghong Pan

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The jointing-booting stage is a critical period of both vegetative growth and reproductive growth in rice. Therefore, the proteomic analysis of the mutant Osbri1, whose corresponding gene OsBRI1 encodes the putative BRs receptor OsBRI1, at jointing-booting stage is very important for understanding the effects of BRs on vegetative and reproductive growth. In this study, the proteomes of leaves from an allelic mutant of the DWARF 61 (D61, OsBRI1) gene, Fn189 (dwarf54, d54) and its wild-type variety T65 (Taichung 65) at jointing-booting stage were analysed by using a Q Exactive plus orbitrap mass spectrometer, and more than 3,100 proteins were identified in each sample. Ontology analysis showed that these proteins distribute in various space of the cells, such as the chloroplast, mitochondrion, and nucleus, they functioned as structural components and/or catalytic enzymes and involved in many physiological processes. Moreover, quantitative analysis displayed that 266 proteins were differentially expressed in two samples, among them, 77 proteins decreased and 189 increased more than two times in Fn189 compared with T65, the proteins whose content decreased in Fn189 including b5-like Heme/Steroid binding domain containing protein, putative retrotransposon protein, putative glutaminyl-tRNA synthetase, and higher content proteins such as mTERF, putative Oligopeptidase homologue, zinc knuckle protein, and so on. A former study founded that the transcription level of a mTERF was up-regulated in the leaves of maize seedling after EBR treatment. In our experiments, it was interesting that one mTERF protein increased, but another mTERF decreased in leaves of Fn189 at jointing-booting stage, which suggested that BRs may have differential regulation mechanisms on the expression of various mTERF proteins. The relationship between other differential proteins with BRs is still unclear, and the effects of BRs on rice protein contents and its regulation mechanisms still need further research.

Keywords: bri1 mutant, jointing-booting stage, proteomic analysis, rice

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Authors: Anahita Izadyar

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Using a recombinant enzyme derived from corn and a simple modification, we are fabricating a facile, fast, and cost-beneficial novel biosensor to measure glucose. We are applying Plant Produced Mn Peroxidase (PPMP), glucose oxidase (GOx), polyaniline (PANI) as conductive polymer and gold nanoparticles (AuNPs) on Au electrode using electrochemical response to detect glucose. We applied the entrapment method of enzyme composition, which is generally used to immobilize conductive polymer and facilitate electron transfer from the enzyme oxidation-reduction center to the sample solution. In this work, the oxidation of glucose on the modified gold electrode was quantified with Linear Sweep Voltammetry(LSV). We expect that the modified biosensor has the potential for monitoring various biofluids.

Keywords: plant-produced manganese peroxidase, enzyme-based biosensors, glucose, modified gold nanoparticles electrode, polyaniline

Procedia PDF Downloads 198

Authors: Qiaoyue Kuang, Yang Li, Mizuki Endo, Takeaki Ozawa

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G protein-coupled receptors (GPCR) are the largest family of receptor proteins that detect molecules outside the cell and activate cellular responses. Of the GPCRs, dopamine receptors, which recognize extracellular dopamine, are essential to mammals due to their roles in numerous physiological events, including autonomic movement, hormonal regulation, emotions, and the reward system in the brain. To precisely understand the physiological roles of dopamine receptors, it is important to spatiotemporally control the signaling mediated by dopamine receptors, which is strongly dependent on their surface expression. Conventionally, chemical-induced interactions were applied to trigger the endocytosis of cell surface receptors. However, these methods were subjected to diffusion and therefore lacked temporal and special precision. To further understand the receptor-mediated signaling and to control the plasma membrane expression of receptors, an optogenetic tool called E-fragment was developed. The C-terminus of a light-sensitive photosensory protein cyptochrome2 (CRY2) was attached to β-Arrestin, and the E-fragment was generated by fusing the C-terminal peptide of vasopressin receptor (V2R) to CRY2’s binding partner protein CIB. The CRY2-CIB heterodimerization triggered by blue light stimulation brings β-Arrestin to the vicinity of membrane receptors and results in receptor endocytosis. In this study, the E-fragment system was applied to dopamine receptors 1 and 2 (DRD1 and DRD2) to control dopamine signaling. First, confocal fluorescence microscope observation qualitatively confirmed the light-induced endocytosis of E-fragment fused receptors. Second, NanoBiT bioluminescence assay verified quantitatively that the surface amount of E-fragment labeled receptors decreased after light treatment. Finally, GloSensor bioluminescence assay results suggested that the E-fragment-dependent receptor light-induced endocytosis decreased cAMP production in DRD1 signaling and attenuated the inhibition effect of DRD2 on cAMP production. The developed optogenetic tool was able to induce receptor endocytosis by external light, providing opportunities to further understand numerous physiological activities by controlling receptor-mediated signaling spatiotemporally.

Keywords: dopamine receptors, endocytosis, G protein-coupled receptors, optogenetics

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Authors: Rohita Gupta, G. S. Brah, R. Verma, C. S. Mukhopadhayay

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Although chicken strains show differences in susceptibility to a number of diseases, the underlying immunological basis is yet to be elucidated. In the present study, heterophils were subjected to LPS stimulation and total RNA extraction, further differential gene expression was studied in broiler, layer and indigenous Aseel strain by Real Time RT-PCR at different time periods before and after induction. The expression of the 14 AvBDs and chTLR 1, 2, 3, 4, 5, 7, 15 and 21 was detectable in heterophils. The expression level of most of the AvBDs significantly increased (P<0.05) 3 hours post in vitro lipopolysaccharide challenge. Higher expression level and stronger activation of most AvBDs, NFkB-1 and IRF-3 in heterophils was observed, with the stimulation of LPS in layer compared to broiler, and in Aseel compared to both layer and broiler. This investigation will allow more refined interpretation of immuno-genetic basis of the variable disease resistance/susceptibility in divergent stock of chicken including indigenous breed. Moreover this study will be helpful in formulation of strategy for isolation of antimicrobial peptides from heterophils.

Keywords: differential expression, heterophils, cytokines, defensin, TLR

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Authors: J. Vinay , Kusumbati Besra, Niharika Pattnaik, Shivaram Prasad Singh, Manjusha Dixit

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Gallbladder cancer (GBC) is rare but highly malignant cancer; its prevalence is more in certain geographical regions and ethnic groups, which include the Northern and Eastern states of India. Previous studies in India have reported genetic predisposition as one of the risk factors in GBC pathogenesis. Although the matrix metalloproteinase-14 (MMP14) is a well-known modulator of the tumor microenvironment and tumorigenesis and TCGA data also suggests its upregulation yet, its role in the genetic predisposition for GBC is completely unknown. We elucidated the role of MMP14 promoter variants as genetic risk factors and their implications in expression modulation. We screened MMP14 promoter variants association with GBC using Sanger’s sequencing in approximately 300 GBC and 300 control subjects and 26 GBC tissue samples of Indian ethnicity. The immunohistochemistry was used to check the MMP14 protein expression in GBC tissue samples. The role of promoter variants on expression levels was elucidated using a luciferase reporter assay. The variants rs1004030 (p-value = 0.0001) and rs1003349 (p-value = 0.0008) were significantly associated with gallbladder cancer. The luciferase assay in two different cell lines, HEK-293 (p = 0.0006) and TGBC1TKB (p = 0.0036) showed a significant increase in relative luciferase activity in the presence of risk alleles for both the single nucleotide polymorphisms (SNPs). Similarly, genotype-phenotype correlation in patients samples confirmed that the presence of risk alleles at rs1004030 and rs1003349 increased MMP14 expression. Overall, this study unravels the genetic association of MMP14 promoter variants with gallbladder cancer, which may contribute to pathogenesis by increasing its expression.

Keywords: gallbladder cancer, matrix metalloproteinase-14, single nucleotide polymorphism, case control study, genetic association study

Procedia PDF Downloads 179