Search results for: rice bran proteins and peptides

Authors: Qing Zhang, Janak L. Pathak, Macro N. Helder, Richard T. Jaspers, Yin Xiao

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Introduction: Nanomaterial-based bone regeneration is greatly influenced by the immune microenvironment. Tissue-engineered nanomaterials mediate the inflammatory response of macrophages to regulate bone regeneration. Silica nanoparticles have been widely used in tissue engineering-related preclinical studies. However, the effect of topological features on the surface of silica nanoparticles on the immune response of macrophages remains unknown. Purposes: The aims of this research are to compare the influences of normal and pollen-like silica nano-surface topography on macrophage immune responses and to obtain insight into their potential regulatory mechanisms. Method: Macrophages (RAW 264.7 cells) were exposed to mesoporous silica nanoparticles with normal morphology (MSNs) and pollen-like morphology (PMSNs). RNA-seq, RT-qPCR, and LSCM were used to assess the changes in expression levels of immune response-related genes and proteins. SEM and TEM were executed to evaluate the contact and adherence of silica nanoparticles by macrophages. For the assessment of the immunomodulation-mediated osteogenic potential, BMSCs were cultured with conditioned medium (CM) from LPS pre-stimulated macrophage cultures treated with MSNs or PMSNs. Osteoimmunomodulatory potential of MSNs and PMSNs in vivo was tested in a mouse cranial bone osteolysis model. Results: The results of the RNA-seq, RT-qPCR, and LSCM assays showed that PMSNs inhibited the expression of pro-inflammatory genes and proteins in macrophages. SEM images showed distinct macrophage membrane surface binding patterns of MSNs and PMSNs. MSNs were more evenly dispersed across the macrophage cell membrane, while PMSNs were aggregated. PMSNs-induced macrophage anti-inflammatory response was associated with upregulation of the cell surface receptor CD28 and inhibition of ERK phosphorylation. TEM images showed that both MSNs and PMSNs could be phagocytosed by macrophages, and inhibiting nanoparticle phagocytosis did not affect the expression of anti-inflammatory genes and proteins. Moreover, PMSNs-induced conditioned medium from macrophages enhanced BMP-2 expression and osteogenic differentiation mBMSCs. Similarly, PMSNs prevented LPS-induced bone resorption via downregulation of inflammatory reaction. Conclusions: PMSNs can promote bone regeneration by modulating osteoimmunological processes through surface topography. The study offers insights into how surface physical contact cues can modulate the regulation of osteoimmunology and provides a basis for the application of nanoparticles with pollen-like morphology to affect immunomodulation in bone tissue engineering and regeneration.

Keywords: physical contact, osteoimmunology, macrophages, silica nanoparticles, surface morphology, membrane receptor, osteogenesis, inflammation

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Authors: Dibakar Chandra Deka, Anamika Kalita Deka

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Fermented alcoholic beverage production has its own stand among the tribal communities of North-East India. This biological oxidation method is followed by Ahom, Dimasa, Nishi, Miri, Bodo, Rabha tribes of this region. Bodo tribes among them not only prepare fermented alcoholic beverage but also store it for various time periods like 3 months, 6 months, 9 months, 12 months and 15 months etc. They prepare alcoholic beverage Jou (rice beer) following the fermentation of Oryza sativa with traditional yeast culture Amao. Saccharomyces cerevisiae is the main domain strain present in Amao. Dongphangrakep (Scoparia dulcis), Mwkhna (Clerodendrum viscosum), Thalir (Musa balbisina) and Khantal Bilai (Ananas cosmos) are the main plants used for Amao preparation. The stored Jou is known as Joufinai. They store the fermented mixture (rice and Amao) in anaerobic conditions for the preparation of Joufinai. We observed a successive increase in alcohol content from 3 months of storage period with 11.79 ± 0.010 (%, v/v) to 15.48 ± 0.070 (%, v/v) at 15 months of storage by a simple, reproducible and solution based colorimetric method. A positive linear correlation was also observed between pH and ethanol content with storage having correlation coefficient 0.981. Here, we optimised the detection of change in constituents of Joufinai during storage using reverse phase HPLC method. We found acetone, ethanol, acetic acid, glycerol as main constituents present in Joufinai. A very good correlation was observed from 3 months to 15 months of storage periods with its constituents. Increase in glycerol content was also detected with storage periods and hence Joufinai can be use as a precursor of above stated compounds. We also observed antioxidant activities increase from 0.056 ±2.80 mg/mL for 3 months old to 0.078± 5.33 mg/mL (in ascorbic acid equivalents) for 15 month old beverage by DPPH radical scavenging method. Therefore, we aimed for scientific validation of storage procedure used by Bodos in Joufinai production and to convert the Bodos’ traditional alcoholic beverage to a commercial commodity through our study.

Keywords: Amao, correlation, beverage, joufinai

Procedia PDF Downloads 298

Authors: Iliyasu Musa Omoyine, Musa Sunday Abraham, Oladele Sunday Blessing, Iliya Ibrahim Abdullahi, Ibegbu Augustine Oseloka, Nuhu Nana-Hawau, Animoku Abdulrazaq Amoto, Yusuf Abdullateef Onoruoiza, Sambo Sohnap James, Akpulu Steven Peter, Ajayi Abayomi

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The neuroprotective role of inflammation from detrimental intrinsic and extrinsic factors has been reported. However, the overactivation of astrocytes and microglia due to lead toxicity produce excessive pro-inflammatory cytokines, mediating neurodegenerative diseases. The present study investigated the mitigatory effects of quercetin on neuroinflammation, correlating with memory function in lead-exposed rats. In this study, Wistar rats were administered orally with Quercetin (Q: 60 mg/kg) and Succimer as a standard drug (S: 10 mg/kg) for 21 days after lead exposure (Pb: 125 mg/kg) of 21 days or in combination with Pb, once daily for 42 days. Working and reference memory was assessed using an Eight-arm radial water maze (8-ARWM). The changes in brain lead level, the neuronal nitric oxide synthase (nNOS) activity, and the level of neuroinflammatory markers such as tumour necrosis factor-alpha (TNF-α) and Interleukin 1 Beta (IL-1β) were determined. Immunohistochemically, astrocyte expression was evaluated. The results showed that the brain level of lead was increased significantly in lead-exposed rats. The expression of astrocytes increased in the CA3 and CA1 regions of the hippocampus, and the levels of brain TNF-α and IL-1β increased in lead-exposed rats. Lead impaired reference and working memory by increasing reference memory errors and working memory incorrect errors in lead-exposed rats. However, quercetin treatment effectively improved memory and inhibited neuroinflammation by reducing astrocytes’ expression and the levels of TNF-α and IL-1β. The expression of astrocytes and the levels of TNF-α and IL-1β correlated with memory function. The possible explanation for quercetin’s anti-neuroinflammatory effect is that it modulates the activity of cellular proteins involved in the inflammatory response; inhibits the transcription factor of nuclear factor-kappa B (NF-κB), which regulates the expression of proinflammatory molecules; inhibits kinases required for the synthesis of Glial fibrillary acidic protein (GFAP) and modifies the phosphorylation of some proteins, which affect the structure and function of intermediate filament proteins; and, lastly, induces Cyclic-AMP Response Element Binding (CREB) activation and neurogenesis as a compensatory mechanism for memory deficits and neuronal cell death. In conclusion, the levels of neuroinflammatory markers negatively correlated with memory function. Thus, quercetin may be a promising therapy in neuroinflammation and memory dysfunction in populations prone to lead exposure.

Keywords: lead, quercetin, neuroinflammation, memory

Procedia PDF Downloads 28

Authors: David De Smet, Myriam Vanneste

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There is a constant need for the improvement of materials applied in textile industries. Nowadays there is a tendency for “bio, eco, natural and environmental friendly” consciousness of the consumer resulting in various textile labels. Materials, totally based on CO2-neutral renewable resources (biopolymers), respond very well to this tendency. Proteins and PLA were evaluated as binders for textile coatings. Much attention is paid to the functionalization of textiles, therefore bio-additves are examined to introduce abrasion resistance, antimicrobial and flame retardant properties.

Keywords: biomaterial, textile, coating, finishing

Procedia PDF Downloads 691

Authors: D. Jablonskytė-Raščė, A. Mankevičienė, S. Supronienė, I. Kerienė, S. Maikštėnienė, S. Bliznikas, R. Česnulevičienė

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Organic farming does not allow the use of conventional mineral fertilizers and crop protection products. As a result, in our experiments we chose to grow different species of cereals and to see how cereal species affects mycotoxin accumulation. From the phytopathological and entomological viewpoint, the glumes of spelt grain perform a positive role since they protect grain from the infection of pathogenic microorganisms. On the background of the above-mentioned infection, there were more Fusarium–affected grains of spelt than of common wheat. It can be assumed that spelt is more susceptible to the Fusarium fungi infection than common wheat. This study describes the occurrence of DON, ZEA and T2/HT2 toxin in a survey of spelt and common wheat and their bran as well as flour. The analysis was conducted using the enzyme-linked immunosorbent assay (ELISA) method. The concentrations of DON, ZEA, and T2/HT2 in Triticum spelta and Triticum aestivum are influenced by species, cereal type and year interaction. The highest concentration of mycotoxin was found in spelt grain with glumes. The obtained results indicate the significantly higher concentrations of Fusarium toxins in glumes than in dehulled grain which implicate the possible protective effect of spelt wheat glumes. The lowest DON, ZEA, and T2/HT2 concentration was determined in spelt grain without glumes.

Keywords: Fusarium mycotoxins, organic farming, spelt

Procedia PDF Downloads 302

Authors: Svitlana Antonenko, Gennady Telegeev

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Introductive statement: The development of chronic myeloid leukemia (CML) is caused by Bcr-Abl oncoprotein. Modern treatments with tyrosine kinase inhibitors are greatly complicated by the mutational variability of the Bcr-Abl oncoprotein, which causes drug resistance. Therefore, there is an urgent need to develop new approaches to the treatment of the disease, which will allow modeling the level of Bcr-Abl oncoprotein in the cell. Promising in this direction is the identification of proteases that can selectively promote cellular proteolysis of oncoproteins. The aim of the study was to study the effect of the interaction of Bcr-Abl with deubiquitinase USP1 on the level of oncoprotein in CML cells. Methodology: K562 cells were selected for the experiment. Сells were incubated with ML323 inhibitor for 24 hours. Precipitation of endogenous proteins from K562 cell lysate was performed using anti-Bcr-Abl antibodies. Cell lysates and precipitation results were studied by Western blot. Subcellular localization of proteins was studied by immunofluorescence analysis followed by confocal microscopy. The results were analyzed quantitatively and statistically. Major findings: The Bcr-Abl/USP1 protein complex was detected in CML cells, and it was found that inhibition of USP1 deubiquitinating activity by the compound ML323 leads to disruption of this protein complex and a decrease in the level of Bcr-Abl oncoprotein in cells. The interaction of Bcr-Abl with USP1 may result in deubiquitination of the oncoprotein, which disrupts its proteasomal degradation and leads to the accumulation of CML in cells. Conclusion: We believe that the interaction of oncoprotein with USP1 may be one of the prerequisites that contribute to malignant cell transformation due to the deubiquitination of oncoprotein, which leads to its accumulation and disease progression. A correlation was found between the deubiquitinating activity of USP1 and the level of oncoprotein in CML cells. Thus, we identify deubiquitinase USP1 as a promising therapeutic target for the development of a new strategy for the treatment of CML by modulating the level of Bcr-Abl in the cell.

Keywords: chronic myeloid leukemia, Bcr-Abl, USP1, deubiquitination Bcr-Abl, K562 cell

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Authors: Liaqat Ali, Furqan Ahmad

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The heat losses should be controlled during the high temperature processes from energy conservation point of view. For this purpose, refractories with low thermal conductivity, high porosity and good mechanical strength along with low price are desirable. In this work, various combinations of naturally occurring, locally available, cheap raw materials, namely, clay, rice husk and saw dust were used. Locally produced insulating firebricks (IFBs) cannot be used at higher than a few hundred °C and possess low strength as well. Various process parameters were studied and the refractories with desirable properties were produced, which can be used up to 1200 °C.

Keywords: firebricks, mechanical strength, thermal conductivity, refractory bricks

Procedia PDF Downloads 312

Authors: Eman H. El-Gamal, Maher E. Saleh, Mohamed Rashad, Ibrahim Elsokkary, Mona M. Abd El-Latif

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Biochar application to calcareous soils could potentially influence the nitrogen dynamics that affect the bioavailability of plants. This study was carried out to investigate the effect of incubation periods on the changes of nitrogen levels (total nitrogen TN and exchangeable ammonium NH₄⁺ and nitrate NO₃⁻) in biochar-treated calcareous soil. The incubation course was extended to 144 days at 30 ± 3 ℃ and at 50% of soil water holding capacity (WHC). Two types of biochars were obtained by pyrolysis at 500 ℃ from rice husk (RHB) and sugarcane bagasse (SCBB). The experiment was planned in a factorial experimental design with three factors (6 periods '24 days for each period' × 3 biochar types 'un-amended, RHB and SCBB' × 3 nitrogen fertilizers 'control, ammonium nitrate; AN and animal manure; AM') in a completely randomized design. The results obtained showed that the highest level of TN was found in the first 24 days of the incubation period in all treatments. However, the amount of TN was decreased with proceeding incubation period up to 144 days and reached to the lowest level at the end of incubation with values of change rate was 17.5, 16.6, and 14.6 g kg⁻¹ day⁻¹ for the un-amended, RHB and SCBB treated soil, respectively. The values of change rate in biochar-soils treated with nitrogen fertilizers were decreased gradually through the whole incubation time from 127.22 to 12.45 g kg⁻¹ day⁻¹ and from 65.00 to 13.43 g kg⁻¹ day⁻¹ for AN and AM respectively, in the case of RHB-soil. While in SCBB-soil, these values were decreased from 70.83 to 12.13 g kg⁻¹ day⁻¹ and from 59.17 to 11.48 g kg⁻¹ day⁻¹ for AN and AM treatments, respectively. The lowest concentration of exchangeable NH₄⁺ was generally found through the period from 24-48 days of incubation. However, the addition of nitrogen fertilizers, enhanced NH₄⁺ production through incubation periods. In the case of RHB-soil, the value of change rate in NH₄⁺ level in the first 24 days of incubation was 0.43 mg kg⁻¹ day⁻¹ and with the addition of AN and AM this value increased to 1.54 and 4.38 mg kg⁻¹ day⁻¹, respectively. In the case of SCBB-soil, the value of change rate in NH₄⁺ level was 0.29 mg kg⁻¹ day⁻¹ which increased to 1.04 mg kg⁻¹ day⁻¹ at the end of incubation, and due to the addition of AN and AM this value increased to 2.78 and 1.90 mg kg⁻¹ day⁻¹ in the first 24 days of incubation period, respectively. However, as compared to the control treatment, the lowest rate of change in NH₄⁺ level was found at the end of incubation. On the other hand, incubation of all biochars-amended soil and treated with AN and AM decreased the concentration levels of NO₃⁻, especially through the first 24-72 days of incubation period. As a result, the values of change rate in NO₃⁻ concentrations in all treatments were almost negative.

Keywords: ammonium nitrate, animal manure, biochar, rice husk, sugarcane bagasse

Procedia PDF Downloads 111

Authors: Shamim Mushtaq, Moazzam Shahid

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Breast cancer (BC) claims the lives of half a million women every year and is the most common cause of death in the developing world. In 2019, it was estimated that BC alone accounts for 15% of all cancer deaths in younger women (aged < 45 years old) with advanced-stage lung metastasis. According to the World Health Organization & International Union against Cancer, in Asia, a high number of cancer-related deaths will be observed in 2020, whereas the burden will be reduced in Western countries due to awareness about the disease, better health facilities and advanced treatments. In the last 15 years, it has been reported that the incidence of BC has increased by 1.1% among Asian compared to the US population from 2003 to 2012. To date, several BC biological subtypes have been reported so far, which are associated with different treatment responses. The heterogeneity and diversity of BC reflected these different subtypes, including Luminal A (23.7% prevalence) and B (38.8% prevalence) that have pathological estrogen receptor (ER+)-positive tumors, the human epidermal growth factor receptor 2 (HER2) (11.2% prevalence) and triple-negative breast cancer (TNBC) (25% prevalence). According to Shaukat Khanum Memorial Cancer Hospital and Research Centre – Pakistan, ten years of data showed that among 636 BC patients, 30.5% had TNBC who were <40 years of age, which is an extremely alarming situation. Therefore, there is a dire need to explore and develop therapeutic targets for the treatment of early TNBC. Since the last decade, unfortunately, there has been little success in understanding the complexity of TNBC and in discovering new biological therapeutic targets. However, conventional chemotherapy is the only choice of treatment for TNBC patients. Many investigators revealed advances in multi-omics (multiple "omes", e.g., genome, proteome, transcriptome, epigenome, and microbiome) which were later identified as actionable targets and increased prevalence in TNBC patients. However, various drugs have been identified so far which are related to a particular diagnostic and prognostic biomarker. For example, Epidermal growth factor receptor ( EGFR or ErbB-1), HER-2/neu (ErbB-2), HER-3 (ErbB-3), and HER-4 (ErbB-4). Protein Transglin-2 (TAGLN 2 ) and Profilins-1 (Pfn-1 ) are the ubiquitously expressed large family of proteins present in all eukaryotes, enabling actin cytoskeletal reorganization. It is known that the oncogenic transformation of cells is accompanied by alteration in the actin cytoskeleton. There are causal connections between altered expression of actin cytoskeletal regulators and cancer progression. Our case-control study identified TAGLN-2 and Pfn-1 proteins in TNBC blood by mass spectrometry. Both TAGLN-2 and Pfn-1 proteins are differentially expressed in early and advanced stages of TNBS patients, which could be potential predictors or therapeutic targets for TNBC.

Keywords: TNBC, blood biomarkers, mass spectrometry, qPCR, ELISA

Procedia PDF Downloads 27

Authors: Md. Shoffikul Islam, Hongqing Hu

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Reducing trace elements' mobility and bioavailability through amendment addition, especially biochar (BC), is a cost-effective and efficient method to address their toxicity in the soil environment. However, the low molecular weight organic acids (LMWOAs) in the rhizosphere could affect BC's efficiency to stabilize trace metals as the LMWOAs could either mobilize or fix metals in the soils. Therefore, understanding the BC's and LMWOAs' interaction mechanisms on metals stabilization in the rhizosphere is crucial. The present study explored the impact of BC derived from rice husk and citric acid (CA) and the combination of BC and CA on the redistribution of cadmium (Cd), lead (Pb), and zinc (Zn) among their geochemical forms through incubation experiment. The changes of zeta potential and X-ray diffraction (XRD) pattern of BC and BC-amended soils to investigate the probable mechanisms of trace elements' immobilization by BC under the CA attack were also examined. The rice husk BC at 5% (w/w) was mixed with the air-dry soil (an Anthrosols) contaminated with Cd, Pb, and Zn in the plastic pot. The 2, 5, 10, and 20 mM kg-1 (w/v) of CA were added separately into the pot. All the ingredients were mixed thoroughly with the soil. A control (CK) treatment was also prepared without BC and CA addition. After 7, 15, and 60 days of incubation with 60% (w/v) moisture level at 25 °C, the incubated soils were determined for pH and EC and were sequentially extracted to assess the metals' transformation in soil. The electronegative charges and XRD peaks of BC and BC-amended soils were also measured. Compared to CK, the application of BC, low level of CA (2 mM kg-1 soil) (CA2), and BC plus the low concentration of CA (BC-CA2) considerably declined the acid-soluble Cd, Pb, and Zn in which BC-CA2 was found to be the most effective treatment. The reversed trends were observed concerning the high levels of CA (>5-20 mM kg-1 soil) and the BC plus high concentrations of CA treatments. BC-CA2 changed the highest amounts of acid-soluble and reducible metals to the oxidizable and residual forms with time. The most increased electronegative charges of BC-CA2 indicate its (BC-CA2) highest Cd, Pb, and Zn immobilizing efficiency, probably through metals adsorption and fixation with the negative charge sites. The XRD study revealed the presence of P, O, CO32-, and Cl1- in BC, which might be responsible for the precipitation of CdCO3, pyromorphite, and hopeite in the case of Cd, Pb, and Zn immobilization, respectively. The findings depicted that the low concentration of CA increased metals' stabilization, whereas the high levels of CA enhanced their mobilization. The BC-CA2 emerged as the best amendment among treatments for metals stabilization in contaminated soils.

Keywords: Biochar, citric acid, immobilization, trace elements contaminated soil

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Authors: Rutuja Upadhyay, Anurag Mehra

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Solid food foams/cellular foods are colloidal systems which impart structure, texture and mouthfeel to many food products such as bread, cakes, ice-cream, meringues, etc. Their heterogeneous morphology makes the quantification of structure/mechanical relationships complex. The porous structure of solid food foams is highly influenced by the processing conditions, ingredient composition, and their interactions. Sensory perceptions of food foams are dependent on bubble size, shape, orientation, quantity and distribution and determines the texture of foamed foods. The state and structure of the solid matrix control the deformation behavior of the food, such as elasticity/plasticity or fracture, which in turn has an effect on the force-deformation curves. The obvious step in obtaining the relationship between the mechanical properties and the porous structure is to quantify them simultaneously. Here, we attempt to research food foams such as bread dough, baked bread and steamed rice cakes to determine the link between ingredients and the corresponding effect of each of them on the rheology, microstructure, bubble size and texture of the final product. Dynamic rheometry (SAOS), confocal laser scanning microscopy, flatbed scanning, image analysis and texture profile analysis (TPA) has been used to characterize the foods studied. In all the above systems, there was a common observation that when the mean bubble diameter is smaller, the product becomes harder as evidenced by the increase in storage and loss modulus (G′, G″), whereas when the mean bubble diameter is large the product is softer with decrease in moduli values (G′, G″). Also, the bubble size distribution affects texture of foods. It was found that bread doughs with hydrocolloids (xanthan gum, alginate) aid a more uniform bubble size distribution. Bread baking experiments were done to study the rheological changes and mechanisms involved in the structural transition of dough to crumb. Steamed rice cakes with xanthan gum (XG) addition at 0.1% concentration resulted in lower hardness with a narrower pore size distribution and larger mean pore diameter. Thus, control of bubble size could be an important parameter defining final food texture.

Keywords: food foams, rheology, microstructure, texture

Procedia PDF Downloads 318

Authors: B. D. Nkosi, T. F. Mutavhatsindi, J. J. Baloyi, R. Meeske, T. M. Langa, I. M. M. Malebana, M. D. Motiang

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Potato hash (PH), a by-product from food production industry, contains 188.4 g dry matter (DM)/kg and 3.4 g water soluble carbohydrate (WSC)/kg DM, and was mixed with wheat bran (70:30 as is basis) to provide 352 g DM/kg and 315 g WSC/kg DM. The materials were ensiled with or without silage additives in 1.5L anaerobic jars (3 jars/treatment) that were kept at 25-280 C for 3 months. Four types of silages were produced which were: control (no additive, denoted as T1), celluclast enzyme (denoted as T2), emsilage bacterial inoculant (denoted as T3) and silosolve bacterial inoculant (denoted as T4). Three jars per treatment were opened after 3 months of ensiling for the determination of nutritive values, fermentation characteristics and aerobic stability. Aerobic stability was done by exposing silage samples to air for 5 days. The addition of enzyme (T2) was reduced (P<0.05) silage pH, fiber fractions (NDF and ADF) while increasing (P < 0.05) residual WSC and lactic acid (LA) production, compared to other treatments. Silage produced had pH of < 4.0, indications of well-preserved silage. Bacterial inoculation (T3 and T4) improved (P < 0.05) aerobic stability of the silage, as indicated by increased number of hours and lower CO2 production, compared to other treatments. However, the aerobic stability of silage was worsen (P < 0.05) with the addition of an enzyme (T2). Further work to elucidate these effects on nutrient digestion and growth performance on ruminants fed the silage is needed.

Keywords: by-products, digestibility, feeds, inoculation, ruminants, silage

Procedia PDF Downloads 423

Authors: Dalia G. Aseel

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The potato (Solanum tubersum, L.) has become one of the major vegetable crops in Egypt and all over the world. Potato leafroll virus(PLRV) was observed on potato plants collected from different governorates in Egypt. Three cultivars, Spunta, Diamont, and Cara, infected with PLRV were collected; RNA was extracted and subjected to Real-Time PCR using the coat protein gene primers. The results showed that the expression of the coat protein was 39.6-fold, 12.45-fold, and 47.43-fold, respectively, for Spunta, Diamont, and Cara cultivars. Differential Display Polymerase Chain Reaction (DD-PCR) using pathogenesis-related protein 1 (PR-1), β-1,3-glucanases (PR-2), chitinase (PR-3), peroxidase (POD), and polyphenol oxidase (PPO) forward primers for pathogenesis-related proteins (PR). The obtained data revealed different banding patterns depending on the viral type and the region of infection. Regarding PLRV, 58 up-regulated and 19 down-regulated genes were detected. Sequence analysis of the up-and down-regulated genes revealed that infected plants were observed in comparison with the healthy control. Sequence analysis of the up-regulated gene was performed, and the encoding sequence analysis showed that the obtained genes include: induced stolen tip protein. On the other hand, two down-regulated genes were identified: disease resistance RPP-like protein and non-specific lipid-transfer protein. In this study, the expressions of PR-1, PR-2, PR-3, POD, and PPO genes in the infected leaves of three potato cultivars were estimated by quantitative real-time PCR. We can conclude that the PLRV-infection of potato plants inhibited the expression of the five PR genes. On the contrary, infected leaves by PLRV elevated the expression of some defense genes. This interaction may also induce and/or suppress the expression of some genes responsible for the plant's defense mechanisms.

Keywords: PLRV, pathogenesis-related proteins (PRs), DD-PCR, sequence, real-time PCR

Procedia PDF Downloads 121

Authors: Shivam Yadav, Neelam Atri

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Cyanobacteria are photoautotrophic prokaryotes able to grow in diverse ecological habitats, originated 2.5 - 3.5 billion years ago and brought oxygenic photosynthesis. Since then superoxide dismutases (SODs) acquired great significance due to their ability to catalyze detoxification of byproducts of oxygenic photosynthesis, i.e. superoxide radicals. Sequence information from several cyanobacterial genomes offers a unique opportunity to conduct a comprehensive comparative analysis of the superoxide dismutases family. In the present study, we extracted information regarding SODs from species of sequenced cyanobacteria and investigated their diversity, conservation, domain structure, and evolution. 144 putative SOD homologues were identified. SODs are present in all cyanobacterial species reflecting their significant role in survival. However, their distribution varies, fewer in unicellular marine strains whereas abundant in filamentous nitrogen-fixing cyanobacteria. Motifs and invariant amino acids typical in eukaryotic SODs were conserved well in these proteins. These SODs were classified into three major families according to their domain structures. Interestingly, they lack additional domains as found in proteins of other family. Phylogenetic relationships correspond well with phylogenies based on 16S rRNA and clustering occurs on the basis of structural characteristics such as domain organization. Similar conserved motifs and amino acids indicate that cyanobacterial SODs make use of a similar catalytic mechanism as eukaryotic SODs. Gene gain-and-loss is insignificant during SOD evolution as evidenced by absence of additional domain. This study has not only examined an overall background of sequence-structure-function interactions for the SOD gene family but also revealed variation among SOD distribution based on ecophysiological and morphological characters.

Keywords: comparative genomics, cyanobacteria, phylogeny, superoxide dismutases

Procedia PDF Downloads 118

Authors: Md. Shoffikul Islam, Hongqing Hu

Abstract:

Immobilizing trace elements by reducing their mobility and bioavailability through amendment application, especially biochar (BC), is a cost-effective and efficient method to address their toxicity in the soil environment. However, the low molecular weight organic acids (LMWOAs) in the rhizosphere could affect BC's efficiency to immobilize trace metals as the LMWOAs could either mobilize or fix metals in the soils. Therefore, understanding the BC's and LMWOAs' interaction mechanisms on metals stabilization in the rhizosphere is crucial. The present study examined the impact of BC derived from rice husk, tartaric acid (TA), and oxalic acid (OA), and the combination of BC and TA/OA on the changes of cadmium (Cd), lead (Pb), and zinc (Zn) among their geochemical forms through incubation experiment. The changes of zeta potential and X-ray diffraction (XRD) pattern of BC and BC-amended soils to investigate the probable mechanisms of trace elements' immobilization by BC under the attacks of TA and OA were also examined. The rice husk BC at 5% (w/w) was mixed with the air-dry soil (an Anthrosols) contaminated with Cd, Pb, and Zn in the plastic pot. The TA and OA each at 2, 5, 10, and 20 mM kg-1 (w/v) were added separately into the pot. All the ingredients were mixed thoroughly with the soil. A control (CK) treatment was also prepared without BC, TA, and OA addition. After 7, 15, and 60 days of incubation with 60% (w/v) moisture level at 25 °C, the incubated soils were determined for pH and EC and were sequentially extracted to assess the metals' transformation in soil. The electronegative charges and XRD peaks of BC and BC-amended soils were also measured. The BC, low level of TA (2 mM kg-1 soil), and BC plus the low concentration of TA (BC-TA2) addition considerably declined the acid-soluble Cd, Pb, and Zn in which BC-TA2 was found to be the most effective treatment. The trends were reversed concerning the high levels of TA (>5-20 mM kg-1 soil), all levels of OA (2-20 mM kg-1 soil), and the BC plus high levels of TA/OA treatments. BC-TA2 changed the highest amounts of acid-soluble and reducible metals to the oxidizable and residual fractions with time. The most increased electronegative charges of BC-TA2 indicate its (BC-TA2) highest metals' immobilizing efficiency, probably through metals adsorption and fixation with the negative charge sites. The XRD study revealed the presence of P, O, CO32-, and Cl1- in BC, which might be responsible for the precipitation of CdCO3, pyromorphite, and hopeite concerning Cd, Pb, and Zn immobilization, respectively. The findings demonstrated that the low level of TA increased metals immobilization, while the high levels of TA and all levels of OA enhanced their mobilization. The BC-TA2 was the best treatment in stabilizing metals in soil.

Keywords: biochar, immobilization, low molecular weight organic acids, trace elements contaminated soil

Procedia PDF Downloads 61

Authors: Sarmiza Stanca, Wolfgang Fritzsche, Christoph Krafft, Jürgen Popp

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Independent of the cell type a thin layer of a few nanometers thickness surrounds the cell interior as the cellular membrane. The transport of ions and molecules through the membrane is achieved in a very precise way by pores. Understanding the process of opening and closing the pores due to an electrochemical gradient across the membrane requires knowledge of the pore constitutive proteins. Recent reports prove the access to the molecular level of the cellular membrane by atomic force microscopy (AFM). This technique also permits an electrochemical study in the immediate vicinity of the tip. Specific molecules can be electrochemically localized in the natural cellular membrane. Our work aims to recognize the protein domains of the pores using an AFM probe as a miniaturized amperometric sensor, and to follow the protein behavior while changing the applied potential. The intensity of the current produced between the surface and the AFM probe is amplified and detected simultaneously with the surface imaging. The AFM probe plays the role of the working electrode and the substrate, a conductive glass on which the cells are grown, represent the counter electrode. For a better control of the electric potential on the probe, a third electrode Ag/AgCl wire is mounted in the circuit as a reference electrode. The working potential is applied between the electrodes with a programmable source and the current intensity in the circuit is recorded with a multimeter. The applied potential considers the overpotential at the electrode surface and the potential drop due to the current flow through the system. The reported method permits a high resolved electrochemical study of the protein domains on the living cell membrane. The amperometric map identifies areas of different current intensities on the pore depending on the applied potential. The reproducibility of this method is limited by the tip shape, the uncontrollable capacitance, which occurs at the apex and a potential local charge separation.

Keywords: AFM, sensor, membrane, pores, proteins

Procedia PDF Downloads 296

Authors: Clotilde Guyon, Nada Jmari, Yen-Chin Li, Jean Denoyel, Noriyuki Fujikado, Christophe Blanchet, David Root, Matthieu Giraud

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Aire induces ectopic expression of a large repertoire of tissue-specific antigen (TSA) genes in thymic medullary epithelial cells (MECs), driving immunological self-tolerance in maturing T cells. Although important mechanisms of Aire-induced transcription have recently been disclosed through the identification and the study of Aire’s partners, the fine transcriptional functions underlied by a number of them and conferred to Aire are still unknown. Alternative cleavage and polyadenylation (APA) is an essential mRNA processing step regulated by the termination complex consisting of 85 proteins, 10 of them have been related to Aire. We evaluated APA in MECs in vivo by microarray analysis with mRNA-spanning probes and RNA deep sequencing. We uncovered the preference of Aire-dependent transcripts for short-3’UTR isoforms and for proximal poly(A) site selection marked by the increased binding of the cleavage factor Cstf-64. RNA interference of the 10 Aire-related proteins revealed that Clp1, a member of the core termination complex, exerts a profound effect on short 3’UTR isoform preference. Clp1 is also significantly upregulated in the MECs compared to 25 mouse tissues in which we found that TSA expression is associated with longer 3’UTR isoforms. Aire-dependent transcripts escape a global 3’UTR lengthening associated with MEC differentiation, thereby potentiating the repressive effect of microRNAs that are globally upregulated in mature MECs. Consistent with these findings, RNA deep sequencing of actinomycinD-treated MECs revealed the increased stability of short 3’UTR Aire-induced transcripts, resulting in TSA transcripts accumulation and contributing for their enrichment in the MECs.

Keywords: Aire, central tolerance, miRNAs, transcription termination

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Authors: Caroline Kim

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The designation autism spectrum disorder (ASD) groups complex disorders in the development of the brain. Autism is defined essentially as a condition in which an individual lacks a theory of mind. The theory of mind, in this sense, explains the ability of an individual to attribute feelings, emotions, or thoughts to another person. An autistic patient is characteristically unable to determine what an interlocutor is feeling, or to understand the beliefs of others. However, it is possible that autism cannot plausibly characterized as the lack of theory of mind in an individual. Genes, the bran, and its interplay with environmental factors may also cause autism. A mutation in a gene may be hereditary, or instigated by diseases such as mumps. Though an autistic patient may experience abnormalities in the cerebellum and the cortical regions, these are in fact only possible theories as to a biochemical explanation behind the disability. The prevailing theory identifying autism with lacking the theory of mind is supported by behavioral observation, but this form of observation is itself determined by socially constructed standards, limiting the possibility for empirical verification. The theory of mind infers that the beliefs and emotions of people are causally based on their behavior. This paper demonstrates the fallacy of this inference, critiquing its basis in socially constructed values, and arguing instead for a biochemical approach free from the conceptual apparatus of language and social expectation.

Keywords: autism spectrum disorder, sociology of psychology, social construction, the theory of mind

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Authors: Sri E. Wati, D. Roswidyatmoko, N. Maslahatun, Gunawan, Andhika B. Taji

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Natural resource is the main sources to fulfill human needs. Its utilization must consider not only human prosperity but also sustainability. Balance of natural resources is a tool to manage natural wealth and to control land use change. This tool is needed to organize land use planning as stated on spatial plan in a certain region. Balance of natural resources can be calculated by comparing two-series of natural resource data obtained at different year. In this case, four years data period of land and forest were used (2010 and 2014). Land use data were acquired through satellite image interpretation and field checking. By means of GIS analysis, its result was then assessed with land use plan. It is intended to evaluate whether existing land use is suitable with land use plan. If it is improper, what kind of efforts and policies must be done to overcome the situation. Subosukawonosraten is rapid developed areas in Central Java Province. This region consists of seven regencies/cities which are Sukoharjo Regency, Boyolali Regency, Surakarta City, Karanganyar Regency, Wonogiri Regency, Sragen Regency, and Klaten Regency. This region is regarding to several former areas under Karasidenan Surakarta and their location is adjacent to Surakarta. Balance of forest resources show that width of forest area is not significantly changed. Some land uses within the area are slightly changed. Some rice field areas are converted into settlement (0.03%) whereas water bodies become vacant areas (0.09%). On the other hand, balance of land resources state that there are many land use changes in this region. Width area of rice field decreases 428 hectares and more than 50% of them have been transformed into settlement area and 11.21% is converted into buildings such as factories, hotels, and other infrastructures. It occurs mostly in Sragen, Sukoharjo, and Karanganyar Regency. The results illustrate that land use change in this region is mostly influenced by increasing of population number. Some agricultural lands have been converted into built-up area since demand of settlement, industrial area, and other infrastructures also increases. Unfortunately, recent utilization of more than a half of total area is not appropriate with land use plan declared in spatial planning document. It means, local government shall develop a strict regulation and law enforcement related to any violation in land use management.

Keywords: balance, forest, land, spatial plan

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Authors: K. Cartelier, D. Aime, V. Vernoud, J. Buitink, J. M. Prosperi, K. Gallardo, C. Le Signor

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Legumes can produce protein-rich seeds without nitrogen fertilizer through root symbiosis with nitrogen-fixing rhizobia. Rich in lysine, these proteins are used for human nutrition and animal feed. However, the instability of seed protein yield and quality due to environmental fluctuations limits the wider use of legumes such as pea. Breeding efforts are needed to optimize and stabilize seed nutritional value, which requires to identify the genetic determinism of seed protein plasticity in response to the environment. Towards this goal, we have studied the plasticity of protein content and composition of seeds from a collection of 200 Medicago truncatula ecotypes grown under four controlled conditions (optimal, drought, and winter/spring sowing). A quantitative analysis of one-dimensional protein profiles of these mature seeds was performed and plasticity indices were calculated from each abundant protein band. Genome-Wide Association Studies (GWAS) from these data identified major GWAS hotspots, from which a list of candidate genes was obtained. A Gene Ontology Enrichment Analysis revealed an over-representation of genes involved in several amino acid metabolic pathways. This led us to propose that environmental variations are likely to modulate amino acid balance, thus impacting seed protein composition. The selection of candidate genes for controlling the plasticity of seed protein composition was refined using transcriptomics data from developing Medicago truncatula seeds. The pea orthologs of key genes were identified for functional studies by mean of TILLING (Targeting Induced Local Lesions in Genomes) lines in this crop. We will present how this study highlighted mechanisms that could govern seed protein plasticity, providing new cues towards the stabilization of legume seed quality.

Keywords: GWAS, Medicago truncatula, plasticity, seed, storage proteins

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Authors: Atena Sadat Hosseini, Mohammadhossein Habibi

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Acute myeloid leukemia (AML) is the most typically diagnosed leukemia. In older adults, AML imposes a dismal outcome. AML originates with a dominant mutation, then adds collaborative, transformative mutations leading to myeloid transformation and clinical/biological heterogeneity. Several chemotherapeutic drugs are used for this cancer. These drugs are naturally associated with several side effects, and finding a more accurate molecular mechanism of these drugs can have a significant impact on the selection and better candidate of drugs for treatment. In this study, we evaluated bortezomibin myeloma cells using bioinformatics analysis and evaluation of RNA-Seq data. Then investigated the molecular pathways proteins- proteins interactions associated with this chemotherapy drug. A total of 658upregulated genes and 548 downregulated genes were sorted.AUF1 (hnRNP D0) binds and destabilizes mRNA, degradation of GLI2 by the proteasome, the role of GTSE1 in G2/M progression after G2 checkpoint, TCF dependent signaling in response to WNT demonstrated in upregulated genes. Besides insulin resistance, AKT phosphorylates targets in the nucleus, cytosine methylation, Longevity regulating pathway, and Signal Transduction of S1P Receptor were related to low expression genes. With respect to this results, HIST2H2AA3, RP11-96O20.4, ZED9, PRDX1, and DOK2, according to node degrees and betweenness elements candidates from upregulated genes. in the opposite side, PYURF, NRSN1, FGF23, UPK3BL, and STAG3 were a prominent role in downregulated genes. Sum up, Using in silico analysis in the present study, we conducted a precise study ofbortezomib molecular mechanisms in myeloma cells. so that we could take further evaluation to discovermolecular cancer therapy. Naturally, more additional experimental and clinical procedures are needed in this survey.

Keywords: myeloma cells, acute myeloid leukemia, bioinformatics analysis, bortezomib

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Authors: V. Konjik, J. Schwartz, R. Sandhoff, M. Mack

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The rising number of multi-resistant pathogens demands the development of new antibiotics in order to reduce the lethal risk of infections. Here, we investigate roseoflavin, a vitamin B2 analogue which is produced by Streptomyces davawensis and Streptomyces cinnabarinus. We consider roseoflavin to be a 'Trojan horse' compound. Its chemical structure is very similar to riboflavin but in fact it is a toxin. Furthermore, it is a clever strategy with regard to the delivery of an antibiotic to its site of action but also with regard to the production of this chemical: The producer cell has only to convert a vitamin (which is already present in the cytoplasm) into a vitamin analog. Roseoflavin inhibits the activity of Flavin depending proteins, which makes up to 3.5 % of predicted proteins in organisms sequenced so far. We sequentially knocked out gene clusters and later on single genes in order to find the ones which are involved in the roseoflavin biosynthesis. Consequently, we identified the gene rosB, coding for the protein carrying out the first step of roseoflavin biosynthesis, starting form Flavin mononucleotide. Here we show, that the protein RosB has so far unknown features. It is per se an oxidoreductase, a decarboxylase and an aminotransferase, all rolled into one enzyme. A screen of cofactors revealed needs of oxygen, NAD+, thiamine and glutamic acid to carry out its function. Surprisingly, thiamine is not only needed for the decaboxylation step, but also for the oxidation of 8-demethyl-8-formyl Flavin mononucleotide. We had managed to isolate three different Flavin intermediates with different oxidation states, which gave us a mechanistic insight of RosB functionality. Our work points to a so far new function of thiamine in Streptomyces davawensis. Additionally, RosB could be extremely useful for chemical synthesis. Careful engineering of RosB may allow the site-specific replacement of methyl groups by amino groups in polyaromatic compounds of commercial interest. Finally, the complete clarification of the roseoflavin biosynthesis opens the possibility of engineering cost-effective roseoflavin producing strains.

Keywords: antibiotic, flavin analogue, roseoflavin biosynthesis, vitamin B2

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Authors: Matej Buzgo, Erico Himawan, Ksenija JašIna, Aiva Simaite

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Electrospinning is a versatile and efficient technology for producing nanofibers for biomedical applications. One of the most common polymers used for the preparation of nanofibers for regenerative medicine and drug delivery applications is polycaprolactone (PCL). PCL is a biocompatible and bioabsorbable material that can be used to stimulate the regeneration of various tissues. It is also a common material used for the development of drug delivery systems by blending the polymer with small active molecules. However, for many drug delivery applications, e.g. cancer immunotherapy, PCL biodegradation rate that may exceed 9 months is too long, and faster nanofiber dissolution is needed. In this paper, we investigate the dissolution and small molecule release rates of PCL blends with two hydrophilic polymers: polyethylene oxide (PEO) or polyvinylpyrrolidone (PVP). We show that adding hydrophilic polymer to the PCL reduces the water contact angle, increases the dissolution rate, and strengthens the interactions between the hydrophilic drug and polymer matrix that further sustain its release. Finally using this method, we were also able to increase the nanofiber degradation rate when PCL-PEO and PCL-PVP were used as a shell in the electrospun core-shell nanofibers and spread up the release of active proteins from their core. Electrospinning can be used for the preparation of the core-shell nanofibers, where active ingredients are encapsulated in the core and their release rate is regulated by the shell. However, such fibers are usually prepared by coaxial electrospinning that is an extremely low-throughput technique. An alternative is emulsion electrospinning that could be upscaled using needleless blades. In this work, we investigate the possibility of using emulsion electrospinning for encapsulation and sustained release of the growth factors for the development of the organotypic skin models. The core-shell nanofibers were prepared using the optimized formulation and the release rate of proteins from the fibers was investigated for 2 weeks – typical cell culture conditions.

Keywords: electrospinning, polycaprolactone (PCL), polyethylene oxide (PEO), polyvinylpyrrolidone (PVP)

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Authors: Lorena Coelho, David Ramada, Catarina Nobre, Joaquim Gaião, Juliana Duarte

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The development of processes that allows the valorization of waste and by-products generated by industries is crucial to promote symbiotic relationships between different sectors and is mandatory to “close the loop” in the circular economy paradigm. In recent years, by-products and waste from agro-food and forestry sector have attracted attention due to their potential application and technical characteristics. The extraction of bio-based active compounds to be reused is in line with the circular bioeconomy concept trends, combining the use of renewable resources with the process’s circularity, aiming the waste reduction and encouraging reuse and recycling. Among different types of bio-based materials, which are being explored and can be extracted, proteins fractions are becoming an attractive new raw material. Within this context, BioTrace4Leather project, a collaboration between two Technological Centres – CeNTI and CTIC, and a company of Tanning and Finishing of Leather – Curtumes Aveneda, aims to develop innovative and biologically sustainable solutions for leather industry and accomplish the market circularity trends. Specifically, it aims to the valorisation of waste and by-products from the tannery industry through proteins extraction and the development of an innovative and biologically sustainable materials. The achieved results show that keratin, gelatine, and collagen fractions can be successfully extracted from hair and leather bovine waste. These products could be reintegrated into the industrial manufacturing process to attain innovative and functional textile and leather substrates. ACKNOWLEDGEMENT This work has been developed under BioTrace4Leather scope, a project co-funded by Operational Program for Competitiveness and Internationalization (COMPETE) of PORTUGAL2020, through the European Regional Development Fund (ERDF), under grant agreement Nº POCI-01-0247-FEDER-039867.

Keywords: leather by-products, circular economy, sustainability, protein fractions

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Authors: Malgorzata Krysiak, Anna Wegrzyn, Maciej Garstka, Radoslaw Mazur

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Under constantly fluctuating environmental conditions, the thylakoid membrane protein network evolved the ability to dynamically respond to changing biotic and abiotic factors. One of the most important protective mechanism is rearrangement of the chlorophyll-protein (CP) complexes, induced by protein phosphorylation. In a temperate climate, low temperature is one of the abiotic stresses that heavily affect plant growth and productivity. The aim of this study was to determine the role of LHCII antenna complex phosphorylation in the dark-chilling response. The study included an experimental model based on dark-chilling at 4 °C of detached chilling sensitive (CS) runner bean (Phaseolus coccineus L.) and chilling tolerant (CT) garden pea (Pisum sativum L.) leaves. This model is well described in the literature as used for the analysis of chilling impact without any additional effects caused by light. We examined changes in thylakoid membrane protein phosphorylation, interactions between phosphorylated LHCII (P-LHCII) and CP complexes, and their impact on the dynamics of photosystem II (PSII) under dark-chilling conditions. Our results showed that the dark-chilling treatment of CS bean leaves induced a substantial increase of phosphorylation of LHCII proteins, as well as changes in CP complexes composition and their interaction with P-LHCII. The PSII photochemical efficiency measurements showed that in bean, PSII is overloaded with light energy, which is not compensated by CP complexes rearrangements. On the contrary, no significant changes in PSII photochemical efficiency, phosphorylation pattern and CP complexes interactions were observed in CT pea. In conclusion, our results indicate that different responses of the LHCII phosphorylation to chilling stress take place in CT and CS plants, and that kinetics of LHCII phosphorylation and interactions of P-LHCII with photosynthetic complexes may be crucial to chilling stress response. Acknowledgments: presented work was financed by the National Science Centre, Poland grant No.: 2016/23/D/NZ3/01276

Keywords: LHCII, phosphorylation, chilling stress, pea, runner bean

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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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Authors: Muñiz-Lino Marcos Agustín, Vazquez Borbolla Jessica, Licéaga-Escalera Carlos

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The ectomesenchymal tissues involved in tooth development and their remnants are the origin of different odontogenic lesions, including tumors and cysts of the jaws, with a wide range of clinical behaviors. Dentigerous cyst (DC) represents approximately 20% of all cases of odontogenic cysts, and it has been demonstrated that it can develop benign and malignant odontogenic tumors. DC is characterized by bone destruction of the area surrounding the crown of a tooth which has not erupted and it contain is liquid. The treatment of odontogenic tumors and cysts usually are partial or total removal of the jaw, causing important secondary co-morbidities. However, molecules implicated in DC pathogenesis as well in its development to odontogenic tumors remains unknown. A cellular model may be useful to study these molecules, but that model has not been established yet. Here, we reported the establishment of a cell culture derived from a dentigerous cyst. This cell line was named DeCy-1. In spite of its ectomesenchymal morphology, DeCy-1 cells express epithelial markers such as cytokeratins 5, 6, and 8. Furthermore, these cells express the ODAM protein, which is present in odontogenesis and in dental follicle, indicating that DeCy-1 cells derived from odontogenic epithelium. Analysis by electron microscopy of this cell line showed that it has a high vesicular activity, suggesting that DeCy-1 could secrete molecules that may be involved in DC pathogenesis. Thus, secreted proteins were analyzed by PAGE-SDS, where we observed approximately 11 bands. In addition, the capacity of these secretions to degrade proteins was analyzed by gelatin substrate zymography. A degradation band of about 62 kDa was found in these assays. Western blot assays suggested that the matrix metalloproteinase 2 (MMP-2) is responsible of this protease activity. Thus, our results indicate that the establishment of a cell line derived from DC is a useful in vitro model to study the biology of this odontogenic lesion and its participation in the development of odontogenic tumors.

Keywords: dentigerous cyst, MMP20, cancer, cell culture

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Authors: Barun K. De

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Acquired immunodeficiency syndrome (AIDS) is caused by two types of human immunodeficiency viruses, collectively designated HIV. HIV infection is spreading globally particularly in developing countries. Before an individual is diagnosed with HIV, the disease goes through different phases. First there is an acute early phase that is followed by an established or chronic phase. Subsequently, there is a latency period after which the individual becomes immunodeficient. It is in the acute phase that an individual is highly infectious due to a high viral load. Presently, HIV diagnosis involves use of tests that do not detect the acute phase infection during which both the viral RNA and p24 antigen are expressed. Instead, these less sensitive tests detect antibodies to viral antigens which are typically sero-converted later in the disease process following acute infection. These antibodies are detected in both asymptomatic HIV-infected individuals as well as AIDS patients. Studies indicate that early diagnosis and treatment of HIV infection can reduce medical costs, improve survival, and reduce spreading of infection to new uninfected partners. Newer 4th generation combination antigen/antibody tests are highly sensitive and specific for detection of acute and established HIV infection (HIV1 and HIV2) enabling immediate linkage to care. The CDC (Center of Disease Control, USA) recently recommended an algorithm involving three different tests to screen and diagnose acute and established infections of HIV-1 and HIV-2 in a general population. Initially a 4th generation combo test detects a viral antigen p24 and specific antibodies against HIV -1 and HIV-2 envelope proteins. If the test is positive it is followed by a second test known as a differentiation assay which detects antibodies against specific HIV-1 and HIV-2 envelope proteins confirming established infection of HIV-1 or HIV-2. However if it is negative then another test is performed that measures viral load confirming an acute HIV-1 infection. Screening results of a Phoenix area population detected 0.3% new HIV infections among which 32.4% were acute cases. Studies in the U.S. indicate that this algorithm effectively reduces HIV infection through immediate treatment and education following diagnosis.

Keywords: new algorithm, HIV, diagnosis, infection

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Authors: Kuladip Jana

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Benzo(a)pyrene [B(a)P] is an environmental toxicant present mostly in cigarette smoke and car exhaust, is an aryl hydrocarbon receptor (AhR) ligand that exerts its toxic effects on both male and female reproductive systems. In this study, the effect of B(a)P at different doses (0.1, 0.25, 0.5, 1 and 5 mg /kg body weight) was studied on male reproductive system of rat. A significant decrease in cauda epididymal sperm count and motility along with the presence of sperm head abnormalities and altered epididymal and testicular histology were documented following B(a)P treatment. B(a)P treatment resulted apoptotic sperm cells as observed by TUNEL and Annexin V-PI assay with increased ROS, altered sperm mitochondrial membrane potential (ΔΨm) with a simultaneous decrease in the activity of antioxidant enzymes and GSH status. TUNEL positive apoptotic cells also observed in testis as well as isolated germ and Leydig cells following B(a)P exposure. Western Blot analysis revealed the activation of p38MAPK, cytosolic translocation of cytochrome-c, up-regulation of Bax and inducible nitric oxide synthase (iNOS) with cleavage of PARP and down-regulation of BCl2 in testis upon B(a)P treatment. The protein and mRNA levels of testicular key steroidogenesis regulatory proteins like StAR, cytochrome P450 IIA1 (CYPIIA1), 3β HSD, 17β HSD showed a significant decrease in a dose dependent manner while an increase in the expression of cytochrome P450 1A1 (CYP1A1), Aryl hydrocarbon Receptor (AhR), active caspase- 9 and caspase- 3 following B(a)P exposure. We conclude that exposure of benzo(a)pyrene caused testicular gamatogenic and steroidogenic disorders by induction of oxidative stress, inhibition of StAR and other steroidogenic enzymes along with activation of p38MAPK and initiated caspase-3 mediated germ and Leydig cell apoptosis.The possible protective role of naturally occurring phytochemicals against B(a)P induced testicular toxicity needs immediate consideration. Curcumin and resveratrol separately were found to protect against B(a)P induced germ cell apoptosis, and their combinatorial effect was more significant. Our present study in isolated testicular germ cell population from adult male Wistar rats, highlighted their synergistic protective effect against B(a)P induced germ cell apoptosis. Curcumin-resveratrol co-treatment decreased the expression of pro-apoptotic proteins like cleaved caspase 3,8,9, cleaved PARP, Apaf1, FasL, tBid. Curcumin-resveratrol co-treatment decreased Bax/Bcl2 ratio, mitochondria to cytosolic translocation of cytochrome c and activated the survival protein Akt. Curcumin-resveratrol decreased the expression of p53 dependent apoptotic genes like Fas, FasL, Bax, Bcl2, Apaf1.Curcumin-resveratrol co-treatment thus prevented B(a)P induced germ cell apoptosis. B(a)P induced testicular ROS generation and oxidative stress were significantly ameliorated with curcumin and resveratrol. Curcumin-resveratrol co-treatment prevented B(a)P induced nuclear translocation of AhR and CYP1A1 production. The combinatorial treatment significantly inhibited B(a)P induced ERK 1/2, p38 MAPK and JNK 1/2 activation. B(a)P treatment increased the expression of p53 and its phosphorylation (p53 ser 15). Curcumin-resveratrol co-treatment significantly decreased p53 level and its phosphorylation (p53 ser 15). The study concludes that curcumin-resveratrol synergistically modulated MAPKs and p53, prevented oxidative stress, regulated the expression of pro and anti-apoptotic proteins as well as the proteins involved in B(a)P metabolism thus protected germ cells from B(a)P induced apoptosis.

Keywords: benzo(a)pyrene, germ cell, apoptosis, oxidative stress, resveratrol, curcumin

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Authors: Maysam Mard-Soltani, Mohamad Javad Rasaee, Saeed Khalili, Abdol Karim Sheikhi, Mehdi Hedayati

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Production of specific antibody responses against hTSH is a cumbersome process due to the high identity between the hTSH and the other members of the glycoprotein hormone family (FSH, LH and HCG) and the high identity between the human hTSH and host animals for antibody production. Therefore, two polyclonal antibodies were purified against two recombinant proteins. Four possible ELISA tests were designed based on these antibodies. These ELISA tests were checked against hTSH and other glycoprotein hormones, and their sensitivity and specificity were assessed. Bioinformatics tools were used to analyze the immunological properties. After the immunogen region selection from hTSH protein, c terminal of B hTSH was selected and applied. Two recombinant genes, with these cut pieces (first: two repeats of C terminal of B hTSH, second: tetanous toxin+B hTSH C terminal), were designed and sub-cloned into the pET32a expression vector. Standard methods were used for protein expression, purification, and verification. Thereafter, immunizations of the white New Zealand rabbits were performed and the serums of them were used for antibody titration, purification and characterization. Then, four ELISA tests based on two antibodies were employed to assess the hTSH and other glycoprotein hormones. The results of these assessments were compared with standard amounts. The obtained results indicated that the desired antigens were successfully designed, sub-cloned, expressed, confirmed and used for in vivo immunization. The raised antibodies were capable of specific and sensitive hTSH detection, while the cross reactivity with the other members of the glycoprotein hormone family was minimum. Among the four designed tests, the test in which the antibody against first protein was used as capture antibody, and the antibody against second protein was used as detector antibody did not show any hook effect up to 50 miu/l. Both proteins have the ability to induce highly sensitive and specific antibody responses against the hTSH. One of the antibody combinations of these antibodies has the highest sensitivity and specificity in hTSH detection.

Keywords: hTSH, bioinformatics, protein expression, cross reactivity

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