Search results for: mismatch repair protein

Authors: Jong-Bae Lee, Seongsoo Lee

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This paper proposes an adaptive discharge time control method to balance cell voltages in alternating battery cell discharging method. In the alternating battery cell discharging method, battery cells are periodically discharged in turn. Recovery effect increases battery output voltage while the given battery cell rests without discharging, thus battery operation time of target system increases. However, voltage mismatch between cells leads two problems. First, voltage difference between cells induces inter-cell current with wasted power. Second, it degrades battery operation time, since system stops when any cell reaches to the minimum system operation voltage. To solve this problem, the proposed method adaptively controls cell discharge time to equalize both cell voltages. In the proposed method, battery operation time increases about 19%, while alternating battery cell discharging method shows about 7% improvement.

Keywords: battery, recovery effect, low-power, alternating battery cell discharging, adaptive discharge time control

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Authors: Leixuri Aguirre, Iñaki Milton-Laskibar, Elizabeth Hijona, Luis Bujanda, Agnes M. Rimando, Maria P. Portillo

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Introduction: In recent years great attention has been paid by scientific community to phenolic compounds as active biomolecules naturally present in foodstuffs due to their beneficial effects on health. Pterostilbene is a resveratrol dimethylether derivative which shows higher biodisponibility. Objective. To analyze the effects of two doses of pterostilbene on several markers of thermogenic capacity in a model of genetic obesity, which shows reduced thermogenesis. Methods: The experiment was conducted with thirty Zucker (fa/fa) rats that were distributed in 3 experimental groups, the control group and two groups orally administered with pterostilbene at 15 and 30 mg/kg body weight/day for 6 weeks. Gene expression of Ucp1, Pgc-1α, Cpt1b, Pparα, Nfr1, Tfam and Cox-2 were assessed by RT-PCR, protein expression of UCP1 and GLUT4 by western blot and enzyme activity of carnitine palmitoyl transferase 1b and citrate synthase by spectrophotometry in interscapular brown adipose tissue (iBAT). Statistical analysis was performed by using one way ANOVA and Newman-Keuls as post-hoc test. Results: Pterostilbene did not change gene expression of Pgc-1α. However, significant increases were found in the expression of Ucp1, Pparα, Nfr-1 and Cox-2. Protein expression of UCP1 and GLUT4 was increased in animals treated with pterostilbene, as well as the activities of CPT-1b and CS. These effects were observed with both doses of pterostilbene, without differences between them. Conclusions: These results show that pterostilbene increases thermogenic and oxidative capacity of brown adipose tissue in obese rats. Whether these effects effectively contribute to the anti-obesity properties of these compound needs further research. Acknowledgments: MINECO-FEDER (AGL2015-65719-R), Basque Government (IT-572-13), University of the Basque Country (ELDUNANOTEK UFI11/32), Institut of Health Carlos III (CIBERobn). Iñaki Milton is a fellowship from the Basque Government.

Keywords: brown adipose tissue, pterostilbene, thermogenesis, uncoupling protein 1

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Authors: Wizna, Helmi Muis, Hafil Abbas

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An experiment was conducted to improve the nutrient value of sago pith (Metroxylon sago Rottb) supplemented with Zn, Sulfur and urea through fermentation by using cellulolytic bacteria (Bacillus amyloliquefaciens) as inoculums. The experiment was determination of the optimum dose combination (dosage of Zn, S and urea) for sago pith fermentation based on nutrient quality and quantity of these fermented products. The study was conducted in experimental method, using the completely randomized design in factorial with 3 treatments consist of: factor A (Dose of urea: A1 = 2.0%, A2 = 3.0%), factor B (Dose of S: B1 = 0.2%, B2 = 0.4%) and factor C (Dose of Zn: C1 = 0.0025%, C2 = 0.005%). Results of study showed that optimum condition for fermentation process of sago pith with B. amyloliquefaciens caused a change of nutrient content was obtained at urea (3%), S (0,2%), and Zn (0,0025%). This fermentation process was able to increase amino acid average, reduce crude fiber content by 67% and increase crude protein by 433%, which made the nutritional value of the product based on dry matter was 18.22% crude protein, 12.42% crude fiber, 2525 Kcal/kg metabolic energy and 65.73% nitrogen retention.

Keywords: fermentation, sago pith, sulfur, Zn, urea, Bacillus amyloliquefaciens

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Authors: Apisaraporn Baicharoen, Prapasiri Pongprayoon

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Betaine aldehyde dehydrogenase 2 (BADH2) is an enzyme that inhibits the accumulation of 2-acetyl-1-pyrroline (2AP), a potent flavor compound in rice fragrance. BADH2 contains three domains (NAD-binding, substrate-binding, and oligomerization domains). It catalyzes the oxidation of amino aldehydes. The lack of BADH2 results in the formation of 2AP and consequently an increase in rice fragrance. To date, inadequate data on BADH2 structure and function are available. An insight into the nature of BADH2 can serve as one of key starting points for the production of high quality fragrant rice. In this study, we therefore constructed the homology model of BADH2 and employed 500-ns Molecular Dynamics simulations (MD) to primarily understand the structural and dynamic properties of BADH2. Initially, Ramachandran plot confirms the good quality of modeled protein structure. Principle Component Analysis (PCA) was also calculated to capture the protein dynamics. Among 3 domains, the results show that NAD binding site is found to be more flexible. Moreover, interactions from key amino acids (N162, E260, C294, and Y419) that are crucial for function are investigated.

Keywords: betaine aldehyde dehydrogenase 2, fragrant rice, homology modeling, molecular dynamics simulations

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Authors: Abdelbary Prince, Said Moussa, Hyungkyu Kim, Eman Gouda, Jin Han

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We compared the dynamic alterations of mitochondrial proteome of control, ischemia-reperfusion (IR) and ischemic preconditioned (IPC) rabbit hearts. Using 2-DE, we identified 29 mitochondrial proteins that were differentially expressed in the IR heart compared with the control and IPC hearts. For two of the spots, the expression patterns were confirmed by Western blotting analysis. These proteins included succinate dehydrogenase complex, Acyl-CoA dehydrogenase, carnitine acetyltransferase, dihydrolipoamide dehydrogenase, Atpase, ATP synthase, dihydrolipoamide succinyltransferase, ubiquinol-cytochrome c reductase, translation elongation factor, acyl-CoA dehydrogenase, actin alpha, succinyl-CoA Ligase, dihydrolipoamide S-succinyltransferase, citrate synthase, acetyl-Coenzyme A dehydrogenase, creatine kinase, isocitrate dehydrogenase, pyruvate dehydrogenase, prohibitin, NADH dehydrogenase (ubiquinone) Fe-S protein, enoyl Coenzyme A hydratase, superoxide dismutase [Mn], and 24-kDa subunit of complex I. Interestingly, most of these proteins are associated with the mitochondrial respiratory chain, antioxidant enzyme system, and energy metabolism. The results provide clues as to the cardioprotective mechanism of ischemic preconditioning at the protein level and may serve as potential biomarkers for detection of ischemia-induced cardiac injury.

Keywords: ischemic preconditioning, mitochondria, proteome, cardioprotection

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Authors: Sergio Alejandro Cuevas, Catherine Etchebest, Fernando Luis Barroso Da Silva

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The flavivirus genus has several organisms responsible for generating various diseases in humans. Special in Brazil, Zika (ZIKV), Dengue (DENV) and Yellow Fever (YFV) viruses have raised great health concerns due to the high number of cases affecting the area during the last years. Diagnostic is still a difficult issue since the clinical symptoms are highly similar. The understanding of their common structural/dynamical and biomolecular interactions features and differences might suggest alternative strategies towards differential serological diagnostics and therapeutic intervention. Due to their immunogenicity, the primary focus of this study was on the ZIKV, DENV and YFV non-structural proteins 1 (NS1) protein. By means of computational studies, we calculated the main physical chemical properties of this protein from different strains that are directly responsible for the biomolecular interactions and, therefore, can be related to the differential infectivity of the strains. We also mapped the electrostatic differences at both the sequence and structural levels for the strains from Uganda to Brazil that could suggest possible molecular mechanisms for the increase of the virulence of ZIKV. It is interesting to note that despite the small changes in the protein sequence due to the high sequence identity among the studied strains, the electrostatic properties are strongly impacted by the pH which also impact on their biomolecular interactions with partners and, consequently, the molecular viral biology. African and Asian strains are distinguishable. Exploring the interfaces used by NS1 to self-associate in different oligomeric states, and to interact with membranes and the antibody, we could map the strategy used by the ZIKV during its evolutionary process. This indicates possible molecular mechanisms that can explain the different immunological response. By the comparison with the known antibody structure available for the West Nile virus, we demonstrated that the antibody would have difficulties to neutralize the NS1 from the Brazilian strain. The present study also opens up perspectives to computationally design high specificity antibodies.

Keywords: zika, biomolecular interactions, electrostatic interactions, molecular mechanisms

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Authors: Habib Onsori, Somayeh Akrami, Mohammad Rahmati

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Deafness is the most common sensory disorder with the frequency of 1/1000 in many populations. Mutations in the GJB2 (CX26) gene at the DFNB1 locus on chromosome 13q12 are associated with congenital hearing loss. Approximately 80% of congenital hearing loss cases are recessively inherited and 15% dominantly inherited. Mutations of the GJB2 gene, encoding gap junction protein Connexin 26 (Cx26), are the most common cause of hereditary congenital hearing loss in many countries. This report presents two cases of different mutations from Iranian patients with bilateral hearing loss. DNA studies were performed for the GJB2 gene by PCR and sequencing methods. In one of them, direct sequencing of the gene showed a heterozygous T→C transition at nucleotide 604 resulting in a cysteine to arginine amino acid substitution at codon 202 (C202R) in the fourth extracellular domain (TM4) of the protein. The analyses indicate that the C202R mutation appeared de novo in the proband with a possible dominant effect (GenBank: KF 638275). In the other one, DNA sequencing revealed a compound heterozygous mutation (35delG, 363delC) in the Cx26 gene that is strongly associated with congenital non-syndromic hearing loss (NSHL). So screening the mutations for hearing loss individuals referring to genetics counseling centers before marriage and or pregnancy is recommended.

Keywords: CX26, deafness, GJB2, mutation

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Authors: Enhui Wang, Qingzhu Ou, Yan Tang, Xiaodong Guo

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As one of most popular polyanionic compounds in lithium-ion cathode materials, Li3V2(PO4)3 has always suffered from the low rate capability especially during 3~4.8V, which is considered to be related with the ion diffusion resistance and structural transformation during the Li+ de/intercalation. Here, as the change of cut-off voltages, cycling numbers and current densities, the process of SEI interfacial film’s formation-growing- destruction-repair on the surface of the cathode, the structural transformation during the charge and discharge, the de/intercalation kinetics reflected by the electrochemical impedance and the diffusion coefficient, have been investigated in detail. Current density, cycle numbers and cut-off voltage impacting on interfacial film and structure was studied specifically. Firstly, the matching between electrolyte and material was investigated, it turned out that the batteries with high voltage electrolyte showed the best electrochemical performance and high voltage electrolyte would be the best electrolyte. Secondly, AC impedance technology was used to study the changes of interface impedance and lithium ion diffusion coefficient, the results showed that current density, cycle numbers and cut-off voltage influenced the interfacial film together and the one who changed the interfacial properties most was the key factor. Scanning electron microscopy (SEM) analysis confirmed that the attenuation of discharge specific capacity was associated with the destruction and repair process of the SEI film. Thirdly, the X-ray diffraction was used to study the changes of structure, which was also impacted by current density, cycle numbers and cut-off voltage. The results indicated that the cell volume of Li3V2 (PO4 )3 increased as the current density increased; cycle numbers merely influenced the structure of material; the cell volume decreased first and moved back gradually after two Li-ion had been deintercalated as the charging cut-off voltage increased, and increased as the intercalation number of Li-ion increased during the discharging process. Then, the results which studied the changes of interface impedance and lithium ion diffusion coefficient turned out that the interface impedance and lithium ion diffusion coefficient increased when the cut-off voltage passed the voltage platforms and decreased when the cut-off voltage was between voltage platforms. Finally, three-electrode system was first adopted to test the activation energy of the system, the results indicated that the activation energy of the three-electrode system (22.385 KJ /mol) was much smaller than that of two-electrode system (40.064 KJ /mol).

Keywords: cut-off voltage, de/intercalation kinetics, solid electrolyte interphase film, structural transformation

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Authors: Arifur Rahman, Ardeshir Amirkhani, Honghua Hu, Mark Molloy, Karen Vickery

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Introduction and Objectives: Staphylococcus aureus and coagulase-negative staphylococci comprises approximately 65% of infections associated with medical devices and are well known for their biofilm formatting ability. Biofilm-related infections are extremely difficult to eradicate owing to their high tolerance to antibiotics and host immune defences. Currently, there is no efficient method for early biofilm detection. A better understanding to enable detection of biofilm specific proteins in vitro and in vivo can be achieved by studying planktonic and different growth phases of biofilms using a proteome analysis approach. Our goal was to construct a reference map of planktonic and biofilm associated proteins of S. aureus. Methods: S. aureus reference strain (ATCC 25923) was used to grow 24 hours planktonic, 3-day wet biofilm (3DWB), and 12-day wet biofilm (12DWB). Bacteria were grown in tryptic soy broth (TSB) liquid medium. Planktonic growth was used late logarithmic bacteria, and the Centres for Disease Control (CDC) biofilm reactor was used to grow 3 days, and 12-day hydrated biofilms, respectively. Samples were subjected to reduction, alkylation and digestion steps prior to Multiplex labelling using Tandem Mass Tag (TMT) 10-plex reagent (Thermo Fisher Scientific). The labelled samples were pooled and fractionated by high pH RP-HPLC which followed by loading of the fractions on a nanoflow UPLC system (Eksigent UPLC system, AB SCIEX). Mass spectrometry (MS) data were collected on an Orbitrap Elite (Thermo Fisher Scientific) Mass Spectrometer. Protein identification and relative quantitation of protein levels were performed using Proteome Discoverer (version 1.3, Thermo Fisher Scientific). After the extraction of protein ratios with Proteome Discoverer, additional processing, and statistical analysis was done using the TMTPrePro R package. Results and Discussion: The present study showed that a considerable proteomic difference exists among planktonic and biofilms from S. aureus. We identified 1636 total extracellular secreted proteins, of which 350 and 137 proteins of 3DWB and 12DWB showed significant abundance variation from planktonic preparation, respectively. Of these, simultaneous up-regulation in between 3DWB and 12DWB proteins such as extracellular matrix-binding protein ebh, enolase, transketolase, triosephosphate isomerase, chaperonin, peptidase, pyruvate kinase, hydrolase, aminotransferase, ribosomal protein, acetyl-CoA acetyltransferase, DNA gyrase subunit A, glycine glycyltransferase and others we found in this biofilm producer. On the contrary, simultaneous down-regulation in between 3DWB and 12DWB proteins such as alpha and delta-hemolysin, lipoteichoic acid synthase, enterotoxin I, serine protease, lipase, clumping factor B, regulatory protein Spx, phosphoglucomutase, and others also we found in this biofilm producer. In addition, we also identified a big percentage of hypothetical proteins including unique proteins. Therefore, a comprehensive knowledge of planktonic and biofilm associated proteins identified by S. aureus will provide a basis for future studies on the development of vaccines and diagnostic biomarkers. Conclusions: In this study, we constructed an initial reference map of planktonic and various growth phase of biofilm associated proteins which might be helpful to diagnose biofilm associated infections.

Keywords: bacterial biofilms, CDC bioreactor, S. aureus, mass spectrometry, TMT

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Authors: Nebiyou Masebo

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The severities of land degradation in southern Ethiopia has been increasing due to high population density, replacement of an age-old agroforestry (AF) based agricultural system with monocropping. The consequences of these activities combined with climate change have been impaired soil biota, soil organic carbon (SOC), soil glomalin, soil aggregation and aggregate stability. The AF systems could curb these problems due it is an ecologically and economically sustainable. This study was aimed to determine the effect of agroforestry practices (AFPs) on soil glomalin, soil aggregate stability (SAS), and aggregate association with SOC. Soil samples (from two depth level: 0-30 & 30-60 cm) and woody species were collected from homegarden based agroforestry practice (HAFP), cropland based agroforestry practice (ClAFP), woodlot based agroforestry practice (WlAFP) and trees on soil and water conservation based agroforestry practice (TSWAFP) using systematic sampling. In this study, both easily extractable glomalin related soil protein (EEGRSP) and total glomalin related soil protein (TGRSP) were significantly (p<0.05) higher in HAFP compared to others, with decreasing order HAFP>WlAFP>TSWAFP>ClAFP at upper surface but in subsurface in decreasing order: WlAFP>HAFP>TSWAFP>ClAFP. On the other hand, the macroaggregate fraction of AFPs ranged from 22.64-36.51% where the lowest was in ClAFP, while the highest was in HAFP, moreover, the order for subsurface was also the same but SAS decreased with the increasing of soil depths. The micro-aggregate fraction ranged from 15.9–24.56%, where the lowest was in HAFP, but the highest was in ClAFP. Besides, the association of OC with both macro-and micro-aggregates was greatest in HAFP and followed by WlAFP. The findings also showed that both glomalin and SAS were significantly high with woody species diversity and richness. Thus, AFP with good management practice can play role on maintenance of biodiversity, glomalin content and other soil quality parameters with future implications for a stable ecosystem.

Keywords: agroforestry, soil aggregate stability, glomalin, aggregate-associated carbon, HAFP, ClAFP, WlAFP, TSWAFP.

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Authors: Adedokun Mathew Adewale, Ayandiran Samuel Kola, Adekunle Ibironke

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Pigeon pea (Cajanus cajan) seeds contain about 20–22 percent protein and appreciable amounts of essential amino acids and minerals. Hence, this study evaluated the blood profile of weaner rabbits fed Cajanus cajan meal (CCM) as a replacement for groundnut cake. Forty weaner rabbits of mixed breed aged 5 - 6 weeks were used for the study, which lasted for 8 weeks. The rabbits were randomly allocated to four treatments (10 rabbits per treatment) in a completely randomized design. Four concentrate diets were compounded by direct replacement of groundnut cake with Cajanus cajan meal (CCM) at 0, 50, 75, and 100%, respectively. There were no significant differences (p>0.05) among the mean counts of packed cell volume, red blood cell, haemoglobin, and monocyte. The 75% CCM diet had significantly the highest (p<0.05). However, rabbits fed diets containing CCM had significantly higher (p<0.05) eosinophil than 0%CCM. Rabbits fed diets containing 100%CCM had significantly highest (p<0.05) total protein followed by 0%CCM, 75%CCM, and least 50%CCM, while 0%CCM and 75%CCM diets were significantly higher (p<0.05) in albumin. However, animals fed diets containing CCM had significantly lower (p<0.05) cholesterol content than 0%CCM diet. It could be concluded that Cajanus cajan meal could replace groundnut cake up to 100% in the diets of rabbits without any deleterious effect on the blood profile of the animals.

Keywords: blood profile, groundnut cake, pigeon pea, weaner rabbits

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Authors: Stephen Adeniyi Adefegha, Vitor Mostardeiro, Vera Maria Morsch, Ademir F. Morel, Ivana Beatrice Manica Da Cruz, Sabrina Somacal Maria Rosa Chitolina Schetinger

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Moringa stenopetala is often consumed as food and used in folkloric medicine for the management of several diseases. Purpose: This study was set up in order to assess the effect of aqueous extract of Moringa stenopetala on cell viability and oxidative stress biomarkers in BV-2 microglial cells. Aqueous extracts of M. stenopetala were prepared, lyophilized and reconstituted in 0.5% dimethylsulphoxide (DMSO). Cells were treated with M. stenopetala extracts (0.1 - 100 µg/ml) for cell viability and nitric oxide (NO) production tests. However, M. stenopetala extract (50 µg/ml) was used in the treatment of cells for the determination of protein carbonyl content and reactive oxygen species (ROS) level. Incubation of BV-2 microglia cell with M. stenopetala extract maintained cell viability, diminished NO and ROS levels, and reduced protein carbonyl contents Chlorogenic acid, rutin, kaempferol and quercetin derivatives were the main phenolic compounds identified in M. stenopetala leaf extract. These phenolic compounds present in M. stenopetala may be responsible for the mitigation of oxidative stress in BV-2 microglial cells.

Keywords: oxidative stress, BV-2 microglial cell, Moringa stenopetala, cell viability, antioxidant

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Authors: Tajudin Ahmed

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Malnutrition is a significant problem in developing countries, particularly among children, due to inadequate diets, lack of proper care, and unequal distribution of food within households. High rates of malnutrition have been shown in Ethiopia, including stunting, underweight, and wasting. This study aims to assess the prevalence and associated factors of Protein-Energy Malnutrition (PEM) among children aged 6-59 months in Babile Town. The study utilized a community-based cross-sectional design conducted in Babile Town, Eastern Ethiopia. Two kebeles were randomly selected, and a census was conducted to identify eligible households. A total of 391 households with children aged 6-59 months were included in the study. Data was collected using structured questionnaires, and anthropometric measurements were taken to assess the weight and height of the children. The study found that a majority of the mothers (72.34%) and fathers (43%) had no formal education. Among the mothers who could read and write, a small percentage had completed primary (14%) or secondary (14%) education, and even fewer had higher education (2.7%). Similarly, among the fathers who could read and write, a majority had completed primary (46.15%) or secondary (27.22%) education, with smaller percentages completing preparatory (8.4%) or higher education (6.29%). The prevalence of malnutrition in the study area was high, with 38.85% of children experiencing stunting (8.2% severely stunted), 50.13% wasting (9% severely wasted), and 41.43% underweight (6.65% severely underweight). These findings indicate a significant burden of malnutrition in Babile Town, likely exacerbated by the high prevalence of infectious diseases such as diarrhea. The study concludes that the prevalence of malnutrition, particularly stunting, wasting, and underweight, is high in Babile Town. The findings indicate the urgent need for interventions to address malnutrition and improve nutrition and healthcare practices in the study area. These results can serve as a baseline for future studies and inform policymakers and healthcare providers in their efforts to combat childhood malnutrition.

Keywords: protein-energy malnutrition, children 6-59 month age babble town, Marasmus

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Authors: Yugander Arra, Florence Auguy, Melissa Stiebner, Sophie Chéron, Michael M. Wudick, Van Schepler-Luu, Sébastien Cunnac, Wolf B. Frommer, Laurence Albar

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Rice yellow mottle virus (RYMV) is one of the most important diseases affecting rice in Africa. The most promising strategy to reduce yield losses is the use of highly resistant varieties. The resistance gene RYMV2 is homolog of the Arabidopsis constitutive expression of pathogenesis related protein-5 (AtCPR5) nucleoporin gene. Resistance alleles are originating from African cultivated rice Oryza glaberrima, rarely cultivated, and are characterized by frameshifts or early stop codons, leading to a non-functional or truncated protein. Rice possesses two paralogs of CPR5 and function of these genes are unclear. Here, we evaluated the role of the two rice candidate nucleoporin paralogs OsCPR5.1 (pathogenesis-related gene 5; RYMV2) and OsCPR5.2 by CRISPR/Cas9 genome editing. Despite striking sequence and structural similarity, only loss-of-function of OsCPR5.1 led to full resistance, while loss-of-function oscpr5.2 mutants remained susceptible. Short N-terminal deletions in OsCPR5.1 also did not lead to resistance. In contrast to Atcpr5 mutants, neither OsCPR5.1 nor OsCPR5.2 knock out mutants showed substantial growth defects. Taken together, the candidate nucleoporin OsCPR5.1, but not its close homolog OsCPR5.2, plays a specific role for the susceptibility to RYMV, possibly by impairing the import of viral RNA or protein into the nucleus. Whereas gene introgression from O. glaberrima to high yielding O. sativa varieties is impaired by strong sterility barriers and the negative impact of linkage drag, genome editing of OsCPR5.1, while maintaining OsCPR5.2 activity, thus provides a promising strategy to generate O. sativa elite lines that are resistant to RYMV.

Keywords: CRISPR Cas9, genome editing, knock out mutant, recessive resistance, rice yellow mottle virus

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Authors: Sunidhi Syal, Rasangi Tennakoon, Patrick O'Donoghue

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Polyglutamine (polyQ) diseases are a group of diseases related to neurodegeneration caused by repeats of the amino acid glutamine (Q) in the DNA, which translates into an elongated polyQ tract in the protein. The pathological explanation is that the polyQ tract forms cytotoxic aggregates in the neurons, leading to their degeneration. There are no cures or preventative efforts established for these diseases as of today, although the symptoms of these diseases can be relieved. This study specifically focuses on Huntington's disease, which is a type of polyQ disease in which aggregation is caused by the extended cytosine, adenine, guanine (CUG) codon repeats in the huntingtin (HTT) gene, which encodes for the huntingtin protein. Using this principle, we attempted to create six models, which included mutating wildtype tRNA alanine variant tRNA-AGC-8-1 to have glutamine anticodons CUG and UUG so serine is incorporated at glutamine sites in poly Q tracts. In the process, we were successful in obtaining tAla-8-1 CUG mutant clones in the HTTexon1 plasmids with a polyQ tract of 23Q (non-pathogenic model) and 74Q (disease model). These plasmids were transfected into mouse neuroblastoma cells to characterize protein synthesis and aggregation in normal and mistranslating cells and to investigate the effects of glutamines replaced with alanines on the disease phenotype. Notably, we observed no noteworthy differences in mean fluorescence between the CUG mutants for 23Q or 74Q; however, the Triton X-100 assay revealed a significant reduction in insoluble 74Q aggregates. We were unable to create a tAla-8-1 UUG mutant clone, and determining the difference in the effects of the two glutamine anticodons may enrich our understanding of the disease phenotype. In conclusion, by generating structural disruption with the amino acid alanine, it may be possible to find ways to minimize the toxicity of Huntington's disease caused by these polyQ aggregates. Further research is needed to advance knowledge in this field by identifying the cellular and biochemical impact of specific tRNA variants found naturally in human genomes.

Keywords: Huntington's disease, polyQ, tRNA, anticodon, clone, overlap PCR

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Authors: A. M. S. S. Amarasiri, A. P. Attanayake, K. A. P. W. Jayatilaka, L. K. B. Mudduwa

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Adriamycin (ADR) is an effective anthracyclin antitumor drug, but its clinical use is limited due to renal toxicity. The leaves of Asparagus falcatus (Family: Liliaceae) have been used in the management of renal diseases since antiquity. In the present investigation, the aqueous leaf extract of A. falcatus was evaluated for acute nephroprotective activity in ADR induced nephrotoxic rats. Nephrotoxicity was induced in healthy male Wistar rats by intraperitoneal administration of ADR 20 mg/kg. The lyophilized powder of the aqueous refluxed (4h) leaf extract of A. falcatus was administered orally at three selected doses; 200, 400 and 600 mg/kg for three consecutive days. Fosinopril sodium (0.09 mg/kg) was used as the standard drug. Administration of the plant extract and the standard drug was commenced 24 hours after the induction of nephrotoxicity to rats. The nephroprotective effect was determined by selected biochemical parameters and by the assessment of histopathology on H and E stained kidney sections. The results were compared to a group of control rats with ADR induced nephrotoxicity. A group of rats administered with the equivalent volume of normal saline served as the healthy control. Administration of ADR 20 mg/kg produced a significant increase in the concentrations of serum creatinine (61%) and urine protein (73%) followed by a significant decrease in serum total protein (21%) and albumin (44%) of the plant extract treated animals compared to the healthy control group (p < 0.05). The aqueous extract of Asparagus falcatus at the three doses; 200, 400 and 600 mg/kg and the standard drug were found to decrease the elevation of concentrations of serum creatinine (33%, 51%, 54% and 42%) and urine protein (8%, 63%, 80% and 86%) respectively. The serum concentrations of total protein (12%, 17%, 29% and 12%) and albumin (3%, 17%, 17% and 16%) were significantly increased compared to the nephrotoxic control group respectively. Assessment of histopathology on H and E stained kidney sections demonstrated that ADR induced renal injury, as evidenced by loss of brush border, cytoplasmic vacuolization, pyknosis in renal tubular epithelial cells, haemorrhages, glomerular congestion and presence of hyaline casts. Treatment with the plant extract and the standard drug resulted in attenuation of the morphological destruction in rats. The results of the present study revealed that the aqueous leaf extract of A. falcatus possesses significant nephroprotective activity against adriamycin induced acute nephrotoxicity. The improved kidney functions were supported with the results of selected biochemical parameters and histological changes observed on H and E stained sections of the kidney tissues in Wistar rats.

Keywords: adriamycin induced nephrotoxicity, asparagus falcatus, biochemical assessment, histopathological assessment, nephroprotective activity

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Authors: Agnieszka Sobczak-Kupiec, Dagmara Malina, Klaudia Pluta, Wioletta Florkiewicz, Bozena Tyliszczak

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Introduction: Request for compound of phosphorus grows continuously, thus, it is searched for alternative sources of this element. One of these sources could be by-products from meat industry which contain prominent quantity of phosphorus compounds. Hydroxyapatite, which is natural component of animal and human bones, is leading material applied in bone surgery and also in stomatology. This is material, which is biocompatible, bioactive and osteoinductive. Methodology: Hydroxyapatite preparation: As a raw material was applied deproteinized and defatted bone pulp called bone sludge, which was formed as waste in deproteinization process of bones, in which a protein hydrolysate was the main product. Hydroxyapatite was received in calcining process in chamber kiln with electric heating in air atmosphere in two stages. In the first stage, material was calcining in temperature 600°C within 3 hours. In the next stage unified material was calcining in three different temperatures (750°C, 850°C and 950°C) keeping material in maximum temperature within 3.0 hours. Bone sludge: Bone sludge was formed as waste in deproteinization process of bones, in which a protein hydrolysate was the main product. Pork bones coming from the partition of meat were used as a raw material for the production of the protein hydrolysate. After disintegration, a mixture of bone pulp and water with a small amount of lactic acid was boiled at temperature 130-135°C and under pressure4 bar. After 3-3.5 hours boiled-out bones were separated on a sieve, and the solution of protein-fat hydrolysate got into a decanter, where bone sludge was separated from it. Results of the study: The phase composition was analyzed by roentgenographic method. Hydroxyapatite was the only crystalline phase observed in all the calcining products. XRD investigation was shown that crystallization degree of hydroxyapatite was increased with calcining temperature. Conclusion: The researches were shown that phosphorus content is around 12%, whereas, calcium content amounts to 28% on average. The conducted researches on bone-waste calcining at the temperatures of 750-950°C confirmed that thermal utilization of deproteinized bone-waste was possible. X-ray investigations were confirmed that hydroxyapatite is the main component of calcining products, and also XRD investigation was shown that crystallization degree of hydroxyapatite was increased with calcining temperature. Contents of calcium and phosphorus were distinctly increased with calcining temperature, whereas contents of phosphorus soluble in acids were decreased. It could be connected with higher crystallization degree of material received in higher temperatures and its stable structure. Acknowledgements: “The authors would like to thank the The National Centre for Research and Development (Grant no: LIDER//037/481/L-5/13/NCBR/2014) for providing financial support to this project”.

Keywords: bone by-products, bone sludge, calcination, hydroxyapatite

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Authors: Ahmed Kamil Hasan Al-Ali, Bouchra Senadji, Ganesh Naik

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We propose a system to real environmental noise and channel mismatch for forensic speaker verification systems. This method is based on suppressing various types of real environmental noise by using independent component analysis (ICA) algorithm. The enhanced speech signal is applied to mel frequency cepstral coefficients (MFCC) or MFCC feature warping to extract the essential characteristics of the speech signal. Channel effects are reduced using an intermediate vector (i-vector) and probabilistic linear discriminant analysis (PLDA) approach for classification. The proposed algorithm is evaluated by using an Australian forensic voice comparison database, combined with car, street and home noises from QUT-NOISE at a signal to noise ratio (SNR) ranging from -10 dB to 10 dB. Experimental results indicate that the MFCC feature warping-ICA achieves a reduction in equal error rate about (48.22%, 44.66%, and 50.07%) over using MFCC feature warping when the test speech signals are corrupted with random sessions of street, car, and home noises at -10 dB SNR.

Keywords: noisy forensic speaker verification, ICA algorithm, MFCC, MFCC feature warping

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Authors: Cheng-Cao Sun, Shu-Jun Li, Cuili Yang, Yongyong Xi, Liang Wang, Feng Zhang, De-Jia Li

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Hsa-miRNA-326 (miR-326) has recently been discovered having anticancer efficacy in different organs. However, the role of miR-326 on non-small cell lung cancer (NSCLC) is still ambiguous. In this study, we investigated the role of miR-326 on the development of NSCLC. The results indicated that miR-326 was significantly down-regulated in primary tumor tissues and very low levels were found in NSCLC cell lines. Ectopic expression of miR-326 in NSCLC cell lines significantly suppressed cell growth as evidenced by cell viability assay, colony formation assay and BrdU staining, through inhibition of cyclin D1, cyclin D2, CDK4, and up-regulation of p57(Kip2) and p21(Waf1/Cip1). In addition, miR-326 induced apoptosis, as indicated by concomitantly with up-regulation of key apoptosis protein cleaved caspase-3, and down-regulation of anti-apoptosis protein Bcl2. Moreover, miR-326 inhibited cellular migration and invasiveness through inhibition of matrix metalloproteinases (MMP)-7 and MMP-9. Further, oncogene CCND1 was revealed to be a putative target of miR-326, which was inversely correlated with miR-326 expression in NSCLC. Taken together, our results demonstrated that miR-326 played a pivotal role on NSCLC through inhibiting cell proliferation, migration, invasion, and promoting apoptosis by targeting oncogenic CCND1.

Keywords: hsa-miRNA-326 (miR-326), cyclin D1, non-small cell lung cancer (NSCLC), proliferation, apoptosis

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Authors: Faizul Azly Abd Dzubir, Muhammad F. Othman

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Conventional structural health monitoring approach for mechanical equipment uses inspection data from Non-Destructive Testing (NDT) during plant shut down window and fitness for service evaluation to estimate the integrity of the equipment that is prone to crack damage. Yet, this forecast is fraught with uncertainty because it is often based on assumptions of future operational parameters, and the prediction is not continuous or online. Advanced Diagnostic and Prognostic Technology (ADaPT) uses Acoustic Emission (AE) technology and a stochastic prognostic model to provide real-time monitoring and prediction of mechanical defects or cracks. The forecast can help the plant authority handle their cracked equipment before it ruptures, causing an unscheduled shutdown of the facility. The ADaPT employs process historical data trending, finite element analysis, fitness for service, and probabilistic statistical analysis to develop a prediction model for crack initiation and growth due to mechanical damage. The prediction model is combined with live equipment operating data for real-time prediction of the remaining life span owing to fracture. ADaPT was devised at a hot combined feed exchanger (HCFE) that had suffered creep crack damage. The ADaPT tool predicts the initiation of a crack at the top weldment area by April 2019. During the shutdown window in April 2019, a crack was discovered and repaired. Furthermore, ADaPT successfully advised the plant owner to run at full capacity and improve output by up to 7% by April 2019. ADaPT was also used on a coke drum that had extensive fatigue cracking. The initial cracks are declared safe with ADaPT, with remaining crack lifetimes extended another five (5) months, just in time for another planned facility downtime to execute repair. The prediction model, when combined with plant information data, allows plant operators to continuously monitor crack propagation caused by mechanical damage for improved maintenance planning and to avoid costly shutdowns to repair immediately.

Keywords: mechanical damage, cracks, continuous monitoring tool, remaining life, acoustic emission, prognostic model

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Authors: M. Benhaliliba, A. Ben Ahmed, C.E. Benouis, A.Ayeshamariam

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The properties of nonlinear optical NLOs are examined, and the results confirm the 2.19 eV HOMO-LUMO mismatch. In the Al-Pc cluster, certain functional bond lengths and bond angles have been observed. The Quantum chemical method (DFT and TD-DFT) and Vibrational spectra properties of AlPc are studied. X-ray pattern reveals the crystalline structure along with the (242) orientation of the AlPc organic thin layer. UV-Vis shows the frequency selective behavior of the device. The absorbance of such layer exhibits a high value within the UV range and two consecutive peaks within visible range. Spin coating is used to make an organic diode based on the Aluminium-phthalocynanine (AlPc-Cl) molecule. Under dark and light conditions, electrical characterization of Ag/AlPc/Si/Au is obtained. The diode's high rectifying capability (about 1x104) is subsequently discovered. While the height barrier is constant and saturation current is greatly reliant on light, the ideality factor of such a diode increases to 6.9 which confirms the non-ideality of such a device. The Cheung-Cheung technique is employed to further the investigation and gain additional data such as series resistance and barrier height.

Keywords: AlPc-Cl organic material, nonlinear optic, optical filter, diode

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Authors: Mardiana Ahamad Zabidi, Akmalluddin Md. Yunus

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Soursop (Anona muricata) is one of the underutilized tropical fruits containing nutrients, particularly dietary fibre and antioxidant properties that are beneficial to human health. This objective of this study is to investigate the feasibility of matured soursop pulp flour (SPF) to be substituted with high-protein wheat flour in bread. Bread formulation was substituted with different levels of SPF (0%, 5%, 10% and 15%). The effect on physicochemical properties and sensory attributes were evaluated. Higher substitution level of SPF resulted in significantly higher (p<0.05) fibre, protein and ash content, while fat and carbohydrate content reduced significantly (p<0.05). FESEM showed that the bread crumb surface of control and 5% SPF appeared to distribute evenly and coalesced by thin gluten film. However, higher SPF substitution level in bread formulation exhibited a deleterious effect by formation of discontinuous gluten network. For texture profile analysis, 5% SPF bread resulted in the lowest value of hardness. The score of sensory evaluation showed that 5% SPF bread received good acceptability and is comparable with control bread.

Keywords: soursop pulp flour, bread, physicochemical properties, sensory attributes, scanning electron microscopy (SEM)

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Authors: Chin-Yuan Hsu, Yu-Lung Chuang

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The expression, concentration, and activity of mitochondrial energy-utilized molecules and cellular energy-regulated molecules decreased with age in the trophocytes and oenocytes of honeybees (Apis mellifera), but those of cellular energy-metabolized molecules is unknown. In this study, the expression, concentration, and activity of cellular energy-metabolized molecules were assayed in the trophocytes and fat cells of young and old worker bees by using the techniques of cell and biochemistry. The results showed that (i) the •-hydroxylacyl-coenzyme A dehydrogenase (HOAD) activity/citrate synthase (CS) activity ratio, non-esterified fatty acids concentrations, the expression of eukaryotic initiation factor 4E, and the expression of phosphorylated eIF4E binding protein 1 decreased with age; (ii) fat and glycogen accumulation increased with age; and (iii) the pyruvate dehydrogenase (PDH) activity/citrate synthase (CS) activity ratio was not correlated with age. These finding indicated that •-oxidation (HOAD/CS) and protein synthsis decreased with age. Glycolysis (PDH/CS) was unchanged with age. The most likely reason is that sugars are the vital food of worker bees. Taken together these data reveal that young workers have higher cellular energy metabolism than old workers and that aging results in a decline in the cellular energy metabolism in worker honeybees.

Keywords: aging, energy, honeybee, metabolism

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Authors: Sarah A. Goodchild, Rachel Gao, Philip N. Bartlett

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A novel detection approach using immobilized DNA probes labeled with Anthraquinone (AQ) as an electrochemically active reporter moiety has been successfully developed as a new, simple, reliable method for the detection of DNA. This method represents a step forward in DNA detection as it can discriminate between multiple nucleotide polymorphisms within target DNA strands without the need for any additional reagents, reporters or processes such as melting of DNA strands. The detection approach utilizes single-stranded DNA probes immobilized on gold surfaces labeled at the distal terminus with AQ. The effective immobilization has been monitored using techniques such as AC impedance and Raman spectroscopy. Simple voltammetry techniques (Differential Pulse Voltammetry, Cyclic Voltammetry) are then used to monitor the reduction potential of the AQ before and after the addition of complementary strand of target DNA. A reliable relationship between the shift in reduction potential and the number of base pair mismatch has been established and can be used to discriminate between DNA from highly related pathogenic organisms of clinical importance. This indicates that this approach may have great potential to be exploited within biosensor kits for detection and diagnosis of pathogenic organisms in Point of Care devices.

Keywords: Anthraquinone, discrimination, DNA detection, electrochemical biosensor

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Authors: L. K. Davis

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The effects of the evolution force are observable in nature at all structural levels ranging from small molecular systems to conversely enormous biospheric systems. However, the evolution force and work associated with formation of biological structures has yet to be described mathematically or theoretically. In addressing the conundrum, we consider evolution from a unique perspective and in doing so we introduce the “Fundamental Theory of the Evolution Force: FTEF”. We utilized synthetic evolution artificial intelligence (SYN-AI) to identify genomic building blocks and to engineer 14-3-3 ζ docking proteins by transforming gene sequences into time-based DNA codes derived from protein hierarchical structural levels. The aforementioned served as templates for random DNA hybridizations and genetic assembly. The application of hierarchical DNA codes allowed us to fast forward evolution, while dampening the effect of point mutations. Natural selection was performed at each hierarchical structural level and mutations screened using Blosum 80 mutation frequency-based algorithms. Notably, SYN-AI engineered a set of three architecturally conserved docking proteins that retained motion and vibrational dynamics of native Bos taurus 14-3-3 ζ.

Keywords: 14-3-3 docking genes, synthetic protein design, time-based DNA codes, writing DNA code from scratch

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Authors: Mihai Palade, Gina Cecilia Pistol, Mariana Stancu, Veronica Chedea, Ionelia Taranu

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Nutrition is an important determinant of general health status, with especially focus on prevention and/or attenuation of the inflammatory-associated pathologies. People with chronic kidney disease can experience chronic inflammation that can lead to cardiovascular disease and even an increased rate of death. There are important links between chronic kidney diseases, inflammation and nutritional strategies that may prevent or protect against undesirable inflammation and oxidative stress. The grape by-products either seeds or pomace are rich in polyphenols which may be beneficial in prevention of inflammatory, antioxidant and antimicrobial processes. As a model for studying the impact of grape seeds on renal inflammation and oxidative stress, we used in this study weaned piglets. After a feeding trial of 30 days with a control diet and an experimental diet containing 5% grape seed (GS), kidney samples were collected. In renal tissues were determined the expression and activity of important markers of immune respose and oxidative stress: pro-inflammatory cytokines (TNF-alpha, IL-1 beta, IL-6, IL-8, IFN-gamma), anti-inflammatory cytokines (IL-4, IL-10), anti-oxidant enzymes (catalase CAT, superoxide dismutase SOD, glutathione peroxidise GPx) and important mediators belonging to nuclear receptors (NF-kB1, Nrf-2 and PPAR-gamma). Gene expression was evaluated by qPCR, whereas protein concentration was determined using proteomic techniques (ELISA). The activity of anti-oxidant enzymes was determined using specific kits. Our results showed that GS enriched in polyphenols does not have effect on TNF-alpha, IL-6 and IL-1 beta gene expression and protein concentration in kidney. By contrast, the gene expression and protein level of IL-8 and IFN-gamma were decreased in GS kidney. Anti-inflammatory cytokines IL-4 and IL-10 gene levels were increased in kidneys collected from GS piglets in comparison with controls, with no modification of protein levels between the two groups. The activities of anti-oxidant enzymes CAT and GPx were increased in kidney by GS, whereas SOD activity was unmodified in comparison with control samples. Also, the GS diet was associated with no modulation of mRNAs for nuclear receptors NF-kB1, Nrf-2 and PPAR-gamma gene expressions in kidneys. In conclusion, our results demonstrated that GS enriched in bioactive compounds such polyphenols could modulate inflammation and oxidative stress markers in kidney tissues. Further studies are necessary to elucidate the mechanism of action of GS compounds in case kidney inflammation associated with oxidative stress, and signalling molecules involved in these mechanisms.

Keywords: animal model, kidney inflammation, oxidative stress, grape seed

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Authors: Adrian Sebastian, Chris Gillespie

Abstract:

We describe a case of diffuse rectal involvement with cap polyposis, manifesting with a protein-losing colopathy and occurring in the setting of advanced mechanical pelvic floor dysfunction. A 59-year-old male with a 5-year history of persistent excessive flatulence, defecatory difficulties, and diarrhea. He had extensive cap polyposis of the entire rectum endoscopically. His symptoms progressed to severe fecal incontinence with mucus leakage, pelvic pain, weight loss, and hypoalbuminemia. Clinical examination exhibited severe perineal descent, a large rectocele, poor anal squeeze, and a poor defecatory technique. After a trial of nonoperative therapies addressing his defecatory dysfunction, and Helicobacter pylori eradication, surgical resection was offered due to severe symptoms with ongoing incontinence and protein loss with no other reasonable options. A robotic abdominoperineal resection with a permanent colostomy was performed, followed by an uncomplicated recovery. Our observation of coexisting mechanical pelvic floor changes in this patient lends weight to the concept of a prolapse-related phenomenon in the pathophysiology of this rare condition.

Keywords: cap polyposis, pelvic dysfunction, fecal incontinence, case report

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Authors: Ramalingam Boobalan, Chinpiao Chen, Jui-I. Chiao

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A series of erlotinib analogues that have structural modification at 6,7-alkoxyl positions is efficiently synthesized. The key reactions that involved in synthesis are one-pot oxime formation-dehydration for the formation of nitrile, quinazoline ring formation reaction between aniline and o-cyanoaniline via formamidine intermediate, Fe/NH4Cl catalyzed reduction-hetereocyclization-reductive ring opening reaction for the formation of o-aminobenzamide, high yielding seal tube reactions for O-demethylation, sodium iodide substitution, ammonia substitution. The in vitro anti-tumor activity of synthesized compounds is studied in two non-small cell lung cancer (NSCLC) cell lines (A549 and H1975). Among the synthesized compounds, the iodo compound 6 (ETN-6) exhibits higher anti-cancer activity compared to erlotinib. An efficient method is developed for the conjugation of erlotinib analogue-4, alcohol compound, with protein, bovine serum albumin (BSA), via succinic acid linker. The in vitro anti-tumor activity of the protein attached erlotinib analogue, 8 (ETN-4-Suc-BSA), showed stronger inhibitory activity in both A549 and H1975 NSCLC cell lines.

Keywords: anti-cancer, BSA, EGFR, Erlotinib

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Authors: Reem Al-Shidhani, Isabelle S. R. Storer, Michael J. Bromley, Lydia Tabernero

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Invasive fungal diseases are an increasing global health concern that contributes to the high mortality rates in immunocompromised patients. The rising of antifungal resistance severely lowers the efficacy of the limited antifungal agents available. New antifungal drugs that target new mechanisms are necessary to tackle the current shortfalls. Amongst post- modifications, phosphorylation is a predominant and an outstanding protein alteration in all eukaryotes. In fungi, protein phosphorylation plays a vital role in many signal transduction pathways, including cell cycle, cell growth, metabolism, transcription, differentiation, proliferation, and virulence. The investigation of Aspergillus fumigatus phosphatases revealed seven genes essential for viability. Inhibiting one of these phosphatases is a new interesting route to develop novel antifungal drugs. In this study, we carried out an early drug discovery process targeting oneessential phosphatase, GlcA. Here, we report the identification of new GlcA inhibitors that show antifungal activity. These important finding open a new avenue to the development of novel antifungals to expand the current narrow arsenal of clinical candidates.

Keywords: invasive fungal diseases, phosphatases, GlcA, competitive inhibitors

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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

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