Search results for: fusion proteins

Authors: Farideh Halakou, Emel Sen, Attila Gursoy, Ozlem Keskin

Abstract:

Protein–Protein Interactions (PPIs) mediate major biological processes in living cells. The study of PPIs as networks and analyze the network properties contribute to the identification of genes and proteins associated with diseases. In this study, we have created the sub-networks of brain and lung metastasis from primary tumor in breast cancer. To do so, we used seed genes known to cause metastasis, and produced their interactions through a network-topology based prioritization method named GUILDify. In order to have the experimental support for the sub-networks, we further curated them using STRING database. We proceeded by modeling structures for the interactions lacking complex forms in Protein Data Bank (PDB). The functional enrichment analysis shows that KEGG pathways associated with the immune system and infectious diseases, particularly the chemokine signaling pathway, are important for lung metastasis. On the other hand, pathways related to genetic information processing are more involved in brain metastasis. The structural analyses of the sub-networks vividly demonstrated their difference in terms of using specific interfaces in lung and brain metastasis. Furthermore, the topological analysis identified genes such as RPL5, MMP2, CCR5 and DPP4, which are already known to be associated with lung or brain metastasis. Additionally, we found 6 and 9 putative genes that are specific for lung and brain metastasis, respectively. Our analysis suggests that variations in genes and pathways contributing to these different breast metastasis types may arise due to change in tissue microenvironment. To show the benefits of using structural PPI networks instead of traditional node and edge presentation, we inspect two case studies showing the mutual exclusiveness of interactions and effects of mutations on protein conformation which lead to different signaling.

Keywords: breast cancer, metastasis, PPI networks, protein conformational changes

Procedia PDF Downloads 233

Authors: Donald Zullick, Cihan Varol

Abstract:

This research paper begins with a look at current work in business continuity as it relates to the cloud and small to medium business (SMB). While cloud services are an emerging paradigm that is quickly making an impact on business, there has been no substantive research applied to SMB. Seeing this lapse, we have taken a fusion of continuity and cloud research with application to the SMB market. It is an initial reflection with base framework guidelines as a starting point for implementation. In this approach, our research ties together existing work and fill the gap with an SMB outlook.

Keywords: business continuity, cloud services, medium size business, risk assessment, small business

Procedia PDF Downloads 387

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

Abstract:

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

Procedia PDF Downloads 48

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

Abstract:

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 33

Authors: David De Smet, Myriam Vanneste

Abstract:

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 697

Authors: Svitlana Antonenko, Gennady Telegeev

Abstract:

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

Procedia PDF Downloads 59

Authors: Muñiz-Lino Marcos A.

Abstract:

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. A 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 that has not erupted and contains liquid. The treatment of odontogenic tumors and cysts usually involves a partial or total removal of the jaw, causing important secondary co-morbidities. However, molecules implicated in DC pathogenesis, as well as in its development into odontogenic tumors, remain 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 follicles, indicating that DeCy-1 cells are 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 for 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, ameloblastoma, MMP-2, odontogenic tumors

Procedia PDF Downloads 13

Authors: Seynabou Toure, Oumar Diop, Kidiyo Kpalma, Amadou S. Maiga

Abstract:

Color and texture are the two most determinant elements for perception and recognition of the objects in an image. For this reason, color and texture analysis find a large field of application, for example in image classification and segmentation. But, the pioneering work in texture analysis was conducted on grayscale images, thus discarding color information. Many grey-level texture descriptors have been proposed and successfully used in numerous domains for image classification: face recognition, industrial inspections, food science medical imaging among others. Taking into account color in the definition of these descriptors makes it possible to better characterize images. Color texture is thus the subject of recent work, and the analysis of color texture images is increasingly attracting interest in the scientific community. In optical remote sensing systems, sensors measure separately different parts of the electromagnetic spectrum; the visible ones and even those that are invisible to the human eye. The amounts of light reflected by the earth in spectral bands are then transformed into grayscale images. The primary natural colors Red (R) Green (G) and Blue (B) are then used in mixtures of different spectral bands in order to produce RGB images. Thus, good color texture discrimination can be achieved using RGB under controlled illumination conditions. Some previous works investigate the effect of using different color space for color texture classification. However, the selection of the best performing color space in land-sea segmentation is an open question. Its resolution may bring considerable improvements in certain applications like coastline detection, where the detection result is strongly dependent on the performance of the land-sea segmentation. The aim of this paper is to present the results of a study conducted on different color spaces in order to show the best-performing color space for land-sea segmentation. In this sense, an experimental analysis is carried out using five different color spaces (RGB, XYZ, Lab, HSV, YCbCr). For each color space, the Haar wavelet decomposition is used to extract different color texture features. These color texture features are then used for Fusion of Over Segmentation (FOOS) based classification; this allows segmentation of the land part from the sea one. By analyzing the different results of this study, the HSV color space is found as the best classification performance while using color and texture features; which is perfectly coherent with the results presented in the literature.

Keywords: classification, coastline, color, sea-land segmentation

Procedia PDF Downloads 232

Authors: Shamim Mushtaq, Moazzam Shahid

Abstract:

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 32

Authors: Joshua Buli, David Pietrowski, Samuel Britton

Abstract:

Processing SAR data usually requires constraints in extent in the Fourier domain as well as approximations and interpolations onto a planar surface to form an exploitable image. This results in a potential loss of data requires several interpolative techniques, and restricts visualization to two-dimensional plane imagery. The data can be interpolated into a ground plane projection, with or without terrain as a component, all to better view SAR data in an image domain comparable to what a human would view, to ease interpretation. An alternate but computationally heavy method to make use of more of the data is the basis of this research. Pre-processing of the SAR data is completed first (matched-filtering, motion compensation, etc.), the data is then range compressed, and lastly, the contribution from each pulse is determined for each specific point in space by searching the time history data for the reflectivity values for each pulse summed over the entire collection. This results in a per-3D-point reflectivity using the entire collection domain. New advances in GPU processing have finally allowed this rapid projection of acquired SAR data onto any desired reference surface (called backprojection). Mathematically, the computations are fast and easy to implement, despite limitations in SAR phase history data size and 3D-point cloud size. Backprojection processing algorithms are embarrassingly parallel since each 3D point in the scene has the same reflectivity calculation applied for all pulses, independent of all other 3D points and pulse data under consideration. Therefore, given the simplicity of the single backprojection calculation, the work can be spread across thousands of GPU threads allowing for accurate reflectivity representation of a scene. Furthermore, because reflectivity values are associated with individual three-dimensional points, a plane is no longer the sole permissible mapping base; a digital elevation model or even a cloud of points (collected from any sensor capable of measuring ground topography) can be used as a basis for the backprojection technique. This technique minimizes any interpolations and modifications of the raw data, maintaining maximum data integrity. This innovative processing will allow for SAR data to be rapidly brought into a common reference frame for immediate exploitation and data fusion with other three-dimensional data and representations.

Keywords: backprojection, data fusion, exploitation, three-dimensional, visualization

Procedia PDF Downloads 63

Authors: Dalia G. Aseel

Abstract:

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 127

Authors: Shivam Yadav, Neelam Atri

Abstract:

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 122

Authors: F. Tang, S. Boskovich, A. Raheja, Z. Aliyazicioglu, S. Bhandari, N. Tsuchiya

Abstract:

Calpoly Autonomous Transportation Experience (CATE) is a driverless vehicle that we are developing to provide safe, accessible, and efficient transportation of passengers throughout the Cal Poly Pomona campus for events such as orientation tours. Unlike the other self-driving vehicles that are usually developed to operate with other vehicles and reside only on the road networks, CATE will operate exclusively on walk-paths of the campus (potentially narrow passages) with pedestrians traveling from multiple locations. Safety becomes paramount as CATE operates within the same environment as pedestrians. As driverless vehicles assume greater roles in today’s transportation, this project will contribute to autonomous driving with pedestrian traffic in a highly dynamic environment. The CATE project requires significant interdisciplinary work. Researchers from mechanical engineering, electrical engineering and computer science are working together to attack the problem from different perspectives (hardware, software and system). In this abstract, we describe the software aspects of the project, with a focus on the requirements and the major components. CATE shall provide a GUI interface for the average user to interact with the car and access its available functionalities, such as selecting a destination from any origin on campus. We have developed an interface that provides an aerial view of the campus map, the current car location, routes, and the goal location. Users can interact with CATE through audio or manual inputs. CATE shall plan routes from the origin to the selected destination for the vehicle to travel. We will use an existing aerial map for the campus and convert it to a spatial graph configuration where the vertices represent the landmarks and edges represent paths that the car should follow with some designated behaviors (such as stay on the right side of the lane or follow an edge). Graph search algorithms such as A* will be implemented as the default path planning algorithm. D* Lite will be explored to efficiently recompute the path when there are any changes to the map. CATE shall avoid any static obstacles and walking pedestrians within some safe distance. Unlike traveling along traditional roadways, CATE’s route directly coexists with pedestrians. To ensure the safety of the pedestrians, we will use sensor fusion techniques that combine data from both lidar and stereo vision for obstacle avoidance while also allowing CATE to operate along its intended route. We will also build prediction models for pedestrian traffic patterns. CATE shall improve its location and work under a GPS-denied situation. CATE relies on its GPS to give its current location, which has a precision of a few meters. We have implemented an Unscented Kalman Filter (UKF) that allows the fusion of data from multiple sensors (such as GPS, IMU, odometry) in order to increase the confidence of localization. We also noticed that GPS signals can easily get degraded or blocked on campus due to high-rise buildings or trees. UKF can also help here to generate a better state estimate. In summary, CATE will provide on-campus transportation experience that coexists with dynamic pedestrian traffic. In future work, we will extend it to multi-vehicle scenarios.

Keywords: driverless vehicle, path planning, sensor fusion, state estimate

Procedia PDF Downloads 131

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

Abstract:

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 298

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

Abstract:

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

Procedia PDF Downloads 372

Authors: M. I. Tyletc, O. I. Podgornya, T. G. Shaposhnikova, S. V. Shabelnikov, A. G. Mittenberg, M. A. Daugavet

Abstract:

Ascidiacea is the most numerous class of the Tunicata subtype. These chordates' distinctive feature of the anatomical structure is a tunic consisting of cellulose fibrils, protein molecules, and single cells. The mechanisms of the tunic formation are not known in detail; tunic formation could be used as the model system for studying the interaction of cells with the extracellular matrix. Our model species is the ascidian Styela rustica, which is prevalent in benthic communities of the White Sea. As previously shown, the tunic formation involves morula blood cells, which contain the major 48 kDa protein p48. P48 participation in the tunic formation was proved using antibodies against the protein. The nature of the protein and its function remains unknown. The current research aims to determine the amino acid sequence of p48, as well as to clarify its role in the tunic formation. The peptides that make up the p48 amino acid sequence were determined by mass spectrometry. A search for peptides in protein sequence databases identified sequences homologous to p48 in Styela clava, Styela plicata, and Styela canopus. Based on sequence alignment, their level of similarity was determined as 81-87%. The correspondent sequence of ascidian Styela canopus was used for further analysis. The Styela rustica p48 sequence begins with a signal peptide, which could indicate that the protein is secretory. This is consistent with experimentally obtained data: the contents of morula cells secreted in the tunic matrix. The isoelectric point of p48 is 9.77, which is consistent with the experimental results of acid electrophoresis of morula cell proteins. However, the molecular weight of the amino acid sequence of ascidian Styela canopus is 103 kDa, so p48 of Styela rustica is a shorter homolog. The search for conservative functional domains revealed the presence of two Ca-binding EGF-like domains, thrombospondin (TSP1) and tyrosinase domains. The p48 peptides determined by mass spectrometry fall into the region of the sequence corresponding to the last two domains and have amino acid substitutions as compared to Styela canopus homolog. The tyrosinase domain (pfam00264) is known to be part of the phenoloxidase enzyme, which participates in melanization processes and the immune response. The thrombospondin domain (smart00209) interacts with a wide range of proteins, and is involved in several biological processes, including coagulation, cell adhesion, modulation of intercellular and cell-matrix interactions, angiogenesis, wound healing and tissue remodeling. It can be assumed that the tyrosinase domain in p48 plays the role of the phenoloxidase enzyme, and TSP1 provides a link between the extracellular matrix and cell surface receptors, and may also be responsible for the repair of the tunic. The results obtained are consistent with experimental data on p48. The domain organization of protein suggests that p48 is an enzyme involved in the tunic tunning and is an important regulator of the organization of the extracellular matrix.

Keywords: ascidian, p48, thrombospondin, tyrosinase, tunic, tunning

Procedia PDF Downloads 100

Authors: Ahmed F. Elaksher, Islam Omar

Abstract:

In this project, we used MOLA-based DEMs to orthorectify HiRISE optical images. The MOLA data was interpolated using the kriging interpolation technique. Corresponding tie points were then digitized from both datasets. These points were employed in co-registering both datasets using GIS analysis tools. Different transformation models, including the affine and projective transformation models, were used with different sets and distributions of tie points. Additionally, we evaluated the use of the MOLA elevations in co-registering the MOLA and HiRISE datasets. The planimetric RMSEs achieved for each model are reported. Results suggested the use of 3D-2D transformation models.

Keywords: photogrammetry, Mars, MOLA, HiRISE

Procedia PDF Downloads 63

Authors: Norica Godja, Andreas Schindel, Luka Payrits, Zsolt Pasztor, Bálint Hegedüs, Petr Homola, Jan Horňas, Jiří Běhal, Roman Ruzek, Martin Holzleitner, Sascha Senck

Abstract:

In order to reduce fuel consumption and CO₂ emissions in the aviation sector, innovative solutions are being sought to reduce the weight of aircraft, including additive manufacturing (AM). Of particular importance are the excellent mechanical properties that are required for aircraft structures. Ti6Al4V alloys, with their high mechanical properties in relation to weight, can reduce the weight of aircraft structures compared to structures made of steel and aluminium. Currently, conventional processes such as casting and CNC machining are used to obtain the desired structures, resulting in high raw material removal, which in turn leads to higher costs and impacts the environment. Additive manufacturing (AM) offers advantages in terms of weight, lead time, design, and functionality and enables the realisation of alternative geometric shapes with high mechanical properties. However, there are currently technological shortcomings that have led to AM not being approved for structural components with high safety requirements. An assessment of damage tolerance for AM parts is required, and quality control needs to be improved. Pores and other defects cannot be completely avoided at present, but they should be kept to a minimum during manufacture. The mechanical properties of the manufactured parts can be further improved by various treatments. The influence of different treatment methods (heat treatment, CNC milling, electropolishing, chemical polishing) and operating parameters were investigated by scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM/EDX), X-ray diffraction (XRD), electron backscatter diffraction (EBSD) and measurements with a focused ion beam (FIB), taking into account surface roughness, possible anomalies in the chemical composition of the surface and possible cracks. The results of the characterisation of the constructed and treated samples are discussed and presented in this paper. These results were generated within the framework of the 3TANIUM project, which is financed by EU with the contract number 101007830.

Keywords: Ti6Al4V alloys, laser powder bed fusion, damage tolerance, heat treatment, electropolishing, potential cracking

Procedia PDF Downloads 69

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

Abstract:

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

Procedia PDF Downloads 130

Authors: Atena Sadat Hosseini, Mohammadhossein Habibi

Abstract:

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

Procedia PDF Downloads 85

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

Abstract:

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

Procedia PDF Downloads 232

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

Abstract:

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)

Procedia PDF Downloads 265

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

Abstract:

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

Procedia PDF Downloads 141

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

Abstract:

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

Procedia PDF Downloads 126

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

Abstract:

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

Procedia PDF Downloads 128

Authors: Barun K. De

Abstract:

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

Procedia PDF Downloads 394

Authors: Kuladip Jana

Abstract:

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

Procedia PDF Downloads 247

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

Abstract:

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

Procedia PDF Downloads 178

Authors: Zaida Perez-Bassart, Berta Polanco-Estibalez, Maria Jose Fabra, Amparo Lopez-Rubio, Antonio Martinez-Abad

Abstract:

Mushroom production has enormously increased in recent years, not only as food products but also for applications in pharmaceuticals, nutraceuticals, and cosmetics. Consequently, interest in its chemical composition, nutritional value, and therapeutic properties has also increased. Fungi are rich in bioactive compounds such as polysaccharides, polyphenols, glycopeptides, and ergosterol, of great medicinal value, but within polysaccharides, β-glucans are the most prominent molecules. They are formed by D-glucose monomers, linked by β-glucosidic bonds β-(1,3) with side chains linked by β-(1,6) bonds. The number and position of the β-(1,6) branches strongly influence the arrangement of the tertiary structure, which, together with the molecular weight, determine the different attributed bioactivities (immunostimulating, anticancer, antimicrobial, prebiotic, etc.) and physico-chemical properties (solubility, bioaccessibility, viscosity or emulsifying). On the other hand, there is a growing interest in the study of fungi as an alternative source of chitin obtained from the by-products of the fungal industry. In this work, a cascade extraction process using aqueous neutral and alkaline treatments was carried out for Grifola frondosa and Lentinula edodes, and the compositional analysis and functional properties of each fraction were characterized. Interestingly, the first fraction obtained by using aqueous treatment at room temperature was the richest in polysaccharides, proteins, and polyphenols, thus obtaining a greater antioxidant capacity than in the other fractions. In contrast, the fractions obtained by alkaline treatments showed a higher degree of β-glucans purification compared to aqueous extractions but a lower extraction yield. Results revealed the different structural recalcitrance of β-glucans, preferentially linked to proteins or chitin depending on the fungus type, which had a direct impact on the functionalities and bioactivities of each fraction.

Keywords: fungi, mushroom, β-glucans, chitin

Procedia PDF Downloads 126

Authors: Narges Shayesteh Moghaddam

Abstract:

Tungsten (W), a refractory metal known for its remarkably high melting temperature, offers tremendous potential for use in challenging environments prevalent in sectors such as space exploration, defense, and nuclear industries. Additive manufacturing, especially the Laser Powder-Bed Fusion (LPBF) technique, emerges as a beneficial method for fabricating tungsten parts. This technique enables the production of intricate components while simultaneously reducing production lead times and associated costs. However, the inherent brittleness of tungsten and its tendency to crack under high-temperature conditions pose significant challenges to the manufacturing process. Our research primarily focuses on the process of rolling tungsten parts in a layer-by-layer manner in LPBF and the subsequent changes in microstructure. Our objective is not only to identify the alterations in the microstructure but also to assess their implications on the physical properties and performance of the fabricated tungsten parts. To examine these aspects, we conducted an extensive series of experiments that included the fabrication of tungsten samples through LPBF and subsequent characterization using advanced materials analysis techniques. These investigations allowed us to scrutinize shifts in various microstructural features, including, but not limited to, grain size and grain boundaries occurring during the rolling process. The results of our study provide crucial insights into how specific factors, such as plastic deformation occurring during the rolling process, influence the microstructural characteristics of the fabricated parts. This information is vital as it provides a foundation for understanding how the parameters of the layer-by-layer rolling process affect the final tungsten parts. Our research significantly broadens the current understanding of microstructural evolution in tungsten parts produced via the layer-by-layer rolling process in LPBF. The insights obtained will play a pivotal role in refining and optimizing manufacturing parameters, thus improving the mechanical properties of tungsten parts and, therefore, enhancing their performance. Furthermore, these findings will contribute to the advancement of manufacturing techniques, facilitating the wider application of tungsten parts in various high-demand sectors. Through these advancements, this research represents a significant step towards harnessing the full potential of tungsten in high-temperature and high-stress applications.

Keywords: additive manufacturing, rolling, tungsten, refractory materials

Procedia PDF Downloads 79