Search results for: serum proteins

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 142

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 133

Authors: Wei Qu, Cassandra Agius, Nikki Varvazovsky, Angela Meade

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The goals of the case study are to address the importance of early diagnosis of osmotic demyelination syndrome (ODS) and to analyse the types, duration, and intensities of the rehabilitation program to promote neurological and functional recovery. It can be associated with biphasic course of disease and severe neurological and neuropsychiatric symptoms. Although a few treatment modalities, such as plasmapheresis, immunoglobulin therapy, steroid, and thyrotrophin-releasing hormone, have been suggested, there is no effective treatment for ODS. The overall prognosis of established ODS is generally poor. A high proportion of patients have a severe permanent disability, which has led to social, economic, and emotional burdens to carers and societies. In this case, a 69-year-old retired pensioner with chronic alcoholism was admitted to the hospital with a reduced level of consciousness and tonic-clonic seizure. He had severe hyponatraemia (serum sodium 118 mmol/L) and hypokalemia (serum potassium 2.8 mmol/L). He was treated with anticonvulsants, 150ml 3% hypertonic saline over one hour, and 40 mmol potassium chloride over one hour, and his sodium was increased by 11 mmol/L in the first 24 hours. However, he had worsened neurological symptoms with quadriplegia, dysphagia, anarthria, and confusion, and the radiological features suggested the diagnosis of ODS. He had minimal neurological recovery during the first four weeks of hospital admission. He was treated with seven weeks of a multi-disciplinary intensive rehabilitation program. On discharge, he had made a significant cognitive and functional recovery and could mobilize independently without a walking aid. In conclusion, ODS can still occur despite correcting sodium following the current clinical guidelines. Patients with severe neurological deficits in the context of osmotic demyelination syndrome would benefit from intensive rehabilitation to facilitate their functional improvement and to promote their quality of life.

Keywords: osmotic demyelination syndrome, hyponatremia, central pontine and extrapontine myelinolysis, rehabilitation

Procedia PDF Downloads 108

Authors: Ampe Mohottige Sachinthi Sandaruwani Amarasiri, Anoja Priyadarshani Attanayake, Kamani Ayoma Perera Wijewardana Jayatilaka, Lakmini Kumari Boralugoda Mudduwa

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The search for alternative pharmacological therapies based on natural extracts for renal failure has become an urgent need, due to paucity of effective pharmacotherapy. The current study was undertaken to evaluate the acute nephroprotective effect of aqueous leaf extract of Plectranthus amboinicus (Roxb.) (Family: Lamiaceae), a medicinal plant used in traditional Ayurvedic medicine for the management of renal diseases in Sri Lanka. The study was performed in adriamycin (ADR) induced nephrotoxic in Wistar rats. Wistar rats were randomly divided into four groups each with six rats. A single dose of ADR (20 mg/kg body wt., ip) was used for the induction of nephrotoxicity in all groups of rats except group one. The treatments were started 24 hours after induction of nephrotoxicity and continued for three days. Group one and two served as healthy and nephrotoxic control rats and were administered equivalent volumes of normal saline (0.9% NaCl) orally. Group three and four nephrotoxic rats were administered the lyophilized powder of the aqueous extract of P. amboinicus (400 mg/ kg body wt.; equivalent human therapeutic dose) and the standard drug, fosinopril sodium (0.09 mg/ kg body wt.) respectively. Urine and blood samples were collected from rats in each group at the end of the period of intervention for the estimation of selected renal parameters. H and E stained sections of the kidney tissues were examined for histopathological changes. Rats treated with the plant extract showed significant improvement in biochemical parameters and histopathological changes compared to ADR induced nephrotoxic group. The elevation of serum concentrations of creatinine and β2-microglobulin were decreased by 38%, and 66% in plant extract treated nephrotoxic rats respectively (p < 0.05). In addition, serum concentrations of total protein and albumin were significantly increased by 25% and 14% in rats treated with P. amboinicus respectively (p < 0.05). The results of β2 –microglobulin and serum total protein demonstrated a significant reduction in the elevated values in rats administered with the plant extract (400 mg/kg) compared to that of fosinopril (0.09 mg/kg). Urinary protein loss in 24hr urine samples was significantly decreased in rats treated with both fosinopril (86%) and P. ambonicus (56%) at the end of the intervention (p < 0.01). Accordingly, an attenuation of morphological destruction was observed in the H and E stained sections of the kidney with the treatments of plant extract and fosinopril. The results of the present study revealed that the aqueous leaf extract of P. amboinicus possesses significant nephroprotective activity at the equivalent therapeutic dose of 400 mg/ kg against adriamycin induced acute nephrotoxicity.

Keywords: biochemical assessment, histopathological assessment, nephroprotective activity, Plectranthus amboinicus

Procedia PDF Downloads 146

Authors: T. Vijay Bhasker, N. K. S Gowda, P. Krishnamoorthy, D. T. Pal, A. K. Pattanaik, A. K. Verma

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Boron (B) is a newer trace element and its biological importance and dietary essentiality is unclear in animals. The available literature suggests its putative role in bone mineralization, antioxidant status and steroid hormone synthesis. A feeding trial was conducted in Wister strain (Rattus norvegicus) albino rats for duration of 90 days. A total of 84 healthy weaned (3-4 weeks) experimental rats were randomly divided into 7 dietary groups (4 replicates of three each) viz., A (Basal diet/ Control), B (Basal diet + 5 ppm B), C (Basal diet + 10 ppm B), D (Basal diet + 20 ppm B), E (Basal diet + 40 ppm B), F (Basal diet-Ca 50%), G (Basal diet-Ca 50% + 40 ppm B). Dietary level of calcium (Ca) was maintained at two levels, 100% and 50% of requirement. Sodium borate was used as source of boron along with other ingredients of basal diet while preparing the pelletized diets. All the rats were kept in proper ventilated laboratory animal house maintained at temperature (23±2º C) and humidity (50 to 70%). At the end of experiment digestibility trial was conducted for 5 days to estimate nutrient digestibility and gut absorption of minerals. Eight rats from each group were sacrificed to collect the vital organs (liver, kidney and spleen) to study histopathology. Blood sample was drawn by heart puncture to determine biochemical profile. The average daily feed intake (g/rat/day), water intake (ml/rat/day) and body weight gain (g/rat/day) were similar among the dietary groups. The digestibility (%) of organic matter and crude fat were significantly improved (P < 0.05) was by B supplementation. The gut absorption (%) Ca was significantly increased (P < 0.01) in B supplemented groups compared to control. However, digestibility of dry matter and crude protein, gut absorption of magnesium and phosphorus showed a non-significant increasing trend with B supplementation. The gut absorption (%) of B (P < 0.01) was significantly lowered (P<0.05) in supplemented groups compared to un-supplemented ones. The serum level of triglycerides (mg/dL), HDL-cholesterol (mg/dL) and alanine transaminase (IU/L) were significantly lowered (P < 0.05) in B supplemented groups. While serum level of glucose (mg/dL) and alkaline phosphatase (KA units) showed a non-significant decreasing trend with B supplementation. However the serum levels of total cholesterol (mg/dL) and aspartate transaminase (IU/L) were similar among dietary groups. The histology sections of kidney and spleen revealed no significant changes among the dietary groups and were observed to be normal in anatomical architecture. However, the liver histology revealed cell degenerative changes with vacuolar degeneration and nuclear condensation in Ca deficient groups. But the comparative degenerative changes were mild in 40 ppm B supplemented Ca deficient group. In conclusion, dietary supplementation of graded levels of boron in rats had a positive effect on metabolism and health by improving nutrient digestibility and gut absorption of Ca. This indicates the beneficial role of dietary boron supplementation.

Keywords: boron, calcium, nutrient utilization, histopathology

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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

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Authors: Saad Al-Shibli, Nasser Amjad, Muna Al Kubaisi, Norra Harun, Shaikh Mizan

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Leptin is a multifunctional hormone produced mainly by adipocyte. Leptin and its receptor have long been found associated with breast cancer. The main aim of this study is to investigate the correlation between Leptin/Leptin receptor and the clinicopathological features of breast cancer. Blood samples for ELISA, tissue samples from tumors and adjacent breast tissue were taken from 51 women with breast cancer with a control group of 40 women with a negative mammogram. Leptin and Leptin receptor in the tissues were estimated by immunohistochemistry (IHC). They were localized at the subcellular level by immunocytochemistry using transmission electron microscopy (TEM). Our results showed significant difference in serum leptin level between control and the patient group, but no difference between pre and post-operative serum leptin levels in the patient group. By IHC, we found that the majority of the breast cancer cells studied, stained positively for leptin and leptin receptors with co-expression of leptin and its receptors. No significant correlation was found between leptin/leptin receptors expression with the race, menopausal status, lymph node metastasis, estrogen receptor expression, progesterone receptor expression, HER2 expression and tumor size. Majority of the patients with distant metastasis were associated with high leptin and leptin receptor expression. TEM views both Leptin and Leptin receptor were found highly concentrated within and around the nucleus of the cancer breast cells, indicating nucleus is their principal seat of actions while the adjacent breast epithelial cells showed that leptin gold particles are scattered all over the cell with much less than that of the cancerous cells. However, presence of high concentration of leptin does not necessarily prove its over-expression, because it could be internalized from outside by leptin receptor in the cells. In contrast, leptin receptor is definitely over-expressed in the ductal breast cancer cells. We conclude that reducing leptin levels, blocking its downstream tissue specific signal transduction, and/or blocking the upstream leptin receptor pathway might help in prevention and therapy of breast cancer.

Keywords: breast cancer, expression, leptin, leptin receptors

Procedia PDF Downloads 139

Authors: Zahra Bahadoran, Parvin Mirmiran, Hanieh-Sadat Ejtahed, Maryam Tohidi, Fereidoun Azizi

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Background and Aim: Broccoli sprouts, rich source of bioactive compounds specially sulforaphane (SFN), have unique functional properties. This study was conducted to investigate the possible treatment effects of high-SFN broccoli sprouts powder on metabolic and liver disorders in rats fed with high-fructose corn syrup. Methods: Thirty-two male wistar rats, pretreated with an eight-week high-fructose diet (water containing 30% fructose), were randomly allocated into three groups: Baseline control (BC), control (C) (normal diet), and BSP-diet (normal diet+5% BSP). The duration of the study was 6 weeks. Biochemical measurements, liver weight and triglyceride content were evaluated and histopathological examination of liver was performed. Results: After 6-weeks, the liver weight was significantly lower in BSP group compared to controls (13.4 g vs. 11.4 g, P<0.05). After 6 weeks, a significant decrease was observed in fasting serum glucose, total cholesterol and triglyceride levels in both experimental groups (P<0.05). Compared to controls, serum levels of HDL-C were significantly higher in BSP group. The liver TG content in BSP compared to control group was lower (14.6 vs. 16.4 mg/mg tissue). The hepatic levels of alanine aminotransferase, aspartate aminotransferase and γ-glutamyl transferase had not considerable changes in the groups after the intervention period but the level of alkaline phosphatase significantly decreased in BSP group (P<0.05). The histopathological examination of liver confirmed a decrease lobular and portal inflammation and ballooning in BSP group compared to control. Conclusion: High-SFN broccoli sprouts powder has beneficials effect on metabolic and liver changes-induced by high fructose corn syrup.

Keywords: broccoli sprouts, metabolic disorders, fatty liver, food science

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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

Procedia PDF Downloads 383

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

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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 115

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: Bahar Tunctan, Sefika Pinar Kucukkavruk, Meryem Temiz-Resitoglu, Demet Sinem Guden, Ayse Nihal Sari, Seyhan Sahan-Firat

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We hypothesized that rexinoids such as bexarotene, a selective retinoid X receptor α (RXRα) agonist, may be beneficial for preventing mortality due to inflammation associated with increased expression/activity of inducible nitric oxide synthase (iNOS) induced by lipopolysaccharide (LPS). Therefore, we investigated effects of bexarotene on the changes in circulating protein levels of iNOS (an index for systemic iNOS expression), myeloperoxidase (MPO) (an index for systemic inflammation), and lactate dehydrogenase (LDH) (an index for systemic tissue injury) in LPS-induced systemic inflammation model resulting in septic shock in rats. Rats were injected with saline (4 ml/kg; i.p.), LPS (10 mg/kg; i.p.), dimethylsulphoxide (4 ml/kg, 0.1%; s.c.) at time 0. Mean arterial blood pressure and heart rate were measured using a tail-cuff device. Bexarotene (0.03, 0.1, 0.3, and 1 mg/kg; s.c.) was administered to separate groups of rats 1 h after injection of saline or LPS. The rats were sacrificed 4 h after saline or LPS injection and blood was collected for measurement of serum iNOS, MPO, and LDH protein levels. Blood pressure decreased by 31 mmHg and heart rate increased by 63 bpm in the LPS-treated rats. Bexarotene at 0.3 and 1 mg/kg doses caused 20% mortality 4 h after LPS injection. In the LPS-treated rats, serum iNOS, MPO, and LDH protein levels were increased. Bexarotene only at 0.1 mg/kg dose prevented the LPS-induced hypotension and increased in iNOS, MPO, and LDH protein levels. These data are consistent with the view that a decrease in systemic iNOS levels contributes to the beneficial effect of bexarotene to prevent the hypotension associated with inflammation and tissue injury during rat endotoxemia. [This work was financially supported by The Scientific and Technological Research Council of Turkey (SBAG-109S121)].

Keywords: bexarotene, inflammation, iNOS, lipopolisaccharide, RXRa

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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: Suneeta Gireesh Panicker

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Aim: Aminopeptidase P (APP) is an enzyme that has specificity for proline, can specifically cleave Xaa-Proline peptides and is a metallo-aminopeptidase. The bonds nearby to the imino acid proline are tough to cleave by many peptidases, but APP can specifically break peptide bonds engaged with proline. Membrane-bound form and a cytosolic form are the two forms in which this enzyme exists. The exact physiological function of APP remains unclear and hence the present work attempts to determine it. Methods: In the present study, the expression pattern of cytosolic Aminopeptidase P (DAP) was determined in all the embryonic stages and larval stages of wild-type Drosophila by using polyclonal monospecific antibodies. To show the presence of DAP RNA in embryonic and larval stages, RNA in situ hybridization was performed. DAP promoter-LacZ fusion reporter gene vector was used to construct transgenic embryos to study the regulation pattern of DAP. To study the DAP expression profile, a transgenic fly consisting of a DAP promoter with β-gal and GFP reporter genes in front of it was constructed. Results: DAP protein expression was observed in neuroectodermal cells, posterior midgut primordium, proctodeum, ventral neuroblast and primordial stomatogastric nervous system. It was observed in the ventral cord and midgut in stage 12. The completely developed embryos showed the intense occurrence of it in the ventral cord and gut region. The eye-antennal disc, wing disc and leg disc also showed the presence of DAP protein. LacZ expression in transgenic embryos also showed the same pattern. Conclusion: Similar to various known multiple-functional proteins, DAP could be one with different functions at different stages and in different cells. Data presented here designates DAP functions in the early embryonic and imaginal dics differentiation and development, suggesting that it may be required for the metabolism of proteins like neuropeptides and tachykinins.

Keywords: aminopeptidase P, in situ hybridization, transgenic fly, embryonic stages

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Authors: Madelyn N. Moawad, Hermine R. Z. Tadros, Mary G. Ghobrial, Ahmad R. Bassiouny, Kamal M. Kandeel, Athar Ata

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Seaweeds are potential renewable resources in marine environment. They provide an excellent source of bioactive substances such as dietary fibers and various functional polysaccharides that could potentially be used as ingredients for both human and animal health applications. The observations suggested that these bioactive compounds have strong antioxidant properties, which have beneficial effects on human health. The present research aimed at finding new chemical products from local marine macroalgae for natural medicinal uses and consumption for their nutritional values. Macroalgae samples were collected manually mainly from the Mediterranean Sea at shallow subtidal zone of Abu Qir Bay, Alexandria, Egypt. The chemical compositions of lyophilized materials of six selected macroalgal species; Colpomenia sinuosa, Sargassum linifolium, Padina pavonia, Pterocladiella capillacea, Laurencia pinnatifidia, and Caulerpa racemosa, were investigated for proteins using bovine serum albumin, and carbohydrates were assayed by phenol-sulfuric acid reaction. The macroalgae lipid was extracted with chloroform, methanol and phosphate buffer. Vitamins were extracted using trichloroacetic acid. Chlorophylls and total carotenoids were determined spectrophotometrically and total phenols were extracted with methanol. In addition, lipid-soluble, and water-soluble antioxidant, and anti α-glucosidase activities were measured spectrophotometrically. The antioxidant activity of hexane extracts was investigated using phosphomolybdenum reagent. The anti-α-glucosidase effect measurement was initiated by mixing α-glucosidase solution with p-nitrophenyl α-D-glucopyranoside. The results showed that the ash contents varied from 11.2 to 35.4 % on dry weight basis for P. capillacea and Laurencia pinnatifidia, respectively. The protein contents ranged from 5.63 % in brown macroalgae C. sinuosa to 8.73 % in P. pavonia. A relative wide range in carbohydrate contents was observed (20.06–46.75 %) for the test algal species. The highest lipid percentage was found in green alga C. racemosa (5.91%) followed by brown algae P. pavonia (3.57%) and C. sinuosa (2.64%). The phenolic contents varied from 1.32 mg GAE/g for C. sinuosa to 4.00 mg GAE/g in P. pavonia. The lipid-soluble compounds exhibited higher antioxidant capacity (73.18-145.95 µM/g) than that of the water-soluble ones ranging from 24.83 µM/g in C. racemosa to 74.07 µM/g in S. linifolium. The most potent anti-α-glucosidase activity was observed for P. pavonia with IC50 of 17.12 μg/ml followed by S. linifolium (IC50 = 71.75 μg/ml), C. racemosa (IC50 = 84.73 μg/ml), P. capillacea (IC50 = 92.16 μg/ml), C. sinuosa (IC50 = 112.44 μg/ml), and L. pinnatifida (IC50 = 115.11 μg/ml).

Keywords: α-glucosidase, lyophilized, macroalgae, spectrophotometrically

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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

Procedia PDF Downloads 243

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)

Procedia PDF Downloads 273

Authors: Angela O. Ugwu, Sunday Ocheni

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Background: Cancer-associated thrombosis (CAT) is a cause of morbidity and mortality among cancer patients. Several risk factors for developing venous thromboembolism (VTE) also coexist with cancer patients, such as chemotherapy and immobilization, thus contributing to the higher risk of VTE in cancer patients when compared to non-cancer patients. This study aimed to determine if there is any correlation between levels of some inflammatory cytokines/haematological parameters and Khorana scores of newly diagnosed chemotherapy naïve ambulatory cancer patients (CNACP). Methods: This was a cross-sectional analytical study carried out from June 2021 to May 2022. Eligible newly diagnosed cancer patients 18 years and above (case group) were enrolled consecutively from the adult Oncology Clinics of the University of Nigeria Teaching Hospital, Ituku/Ozalla (UNTH). The control group was blood donors at UNTH Ituku/Ozalla, Enugu blood bank, and healthy members of the Medical and Dental Consultants Association of Nigeria (MDCAN), UNTH Chapter. Blood samples collected from the participants were assayed for IL-6, TNF-Alpha, and haematological parameters such as haemoglobin, white blood cell count (WBC), and platelet count. Data were entered into an Excel worksheet and were then analyzed using Statistical Package for Social Sciences (SPSS) computer software version 21.0 for windows. A P value of < 0.05 was considered statistically significant. Results: A total of 200 participants (100 cases and 100 controls) were included in the study. The overall mean age of the participants was 47.42 ±15.1 (range 20-76). The sociodemographic characteristics of the two groups, including age, sex, educational level, body mass index (BMI), and occupation, were similar (P > 0.05). Following One Way ANOVA, there were significant differences between the mean levels of interleukin-6 (IL-6) (p = 0.036) and tumor necrotic factor-α (TNF-α) (p = 0.001) in the three Khorana score groups of the case group. Pearson’s correlation analysis showed a significant positive correlation between the Khorana scores and IL-6 (r=0.28, p = 0.031), TNF-α (r= 0.254, p= 0.011), and PLR (r= 0.240, p=0.016). The mean serum levels of IL-6 were significantly higher in CNACP than in the healthy controls [8.98 (8-12) pg/ml vs. 8.43 (2-10) pg/ml, P=0.0005]. There were also significant differences in the mean levels of the haemoglobin (Hb) level (P < 0.001)); white blood cell (WBC) count ((P < 0.001), and platelet (PL) count (P = 0.005) between the two groups of participants. Conclusion: There is a significant positive correlation between the serum levels of IL-6, TNF-α, and PLR and the Khorana scores of CNACP. The mean serum levels of IL-6, TNF-α, PLR, WBC, and PL count were significantly higher in CNACP than in the healthy controls. Ambulatory cancer patients with high-risk Khorana scores may benefit from anti-inflammatory drugs because of the positive correlation with inflammatory cytokines. Recommendations: Ambulatory cancer patients with 2 Khorana scores may benefit from thromboprophylaxis since they have higher Khorana scores. A multicenter study with a heterogeneous population and larger sample size is recommended in the future to further elucidate the relationship between IL-6, TNF-α, PLR, and the Khorana scores among cancer patients in the Nigerian population.

Keywords: thromboprophylaxis, cancer, Khorana scores, inflammatory cytokines, haematological parameters

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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

Procedia PDF Downloads 158

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: Ammara Khan

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Sepsis is characterized by an overwhelming surge of cytokines and oxidative stress to one of many factors, gram-negative bacteria commonly implicated. Despite major expansion and elaboration of sepsis pathophysiology and therapeutic approach; death rate remains very high in septic patients due to multiple organ damages including hepatotoxicity.The present study was aimed to ascertain the adequacy of three different drugs delivered separately and collectively- low dose steroid-dexamethasone (3mg/kg i.p) ,antioxidant-melatonin(10 mg/kg i.p) ,and phosphodiesterases inhibitor - pentoxifylline (75 mg/kg i.p)in endotoxin-induced hepatotoxicity in mice. Endotoxin/lipopolysaccharides induced hepatotoxicity was reproduced in mice by giving lipopolysaccharide of serotype E.Coli intraperitoneally. The preventive role was questioned by giving the experimental agent half an hour prior to LPS injection whereas the therapeutic potential of the experimental agent was searched out via post-LPS delivering. The extent of liver damage was adjudged via serum alanine aminotransferases (ALT) and aspartate aminotransferase (AST) estimation along with a histopathological examination of liver tissue. Dexamethasone is given before (Group 3) and after LPS (group 4) significantly attenuated LPS generated liver injury.Pentoxifylline generated similar results and serum ALT; AST histological alteration abated considerably (p≤ 0.05) both in animals subjected to pentoxifylline pre (Group 5) and post-treatment(Group 6). Melatonin was also prosperous in aversion (Group 7) and curation (Group 8) of LPS invoked hepatotoxicity as evident by lessening of augmented ALT (≤0.01) and AST (≤0.01) along with restoration of pathological changes in liver sections (p≤0.05). Combination therapies with dexamethasone in conjunction with melatonin (Group 9), dexamethasone together with pentoxifylline (Group 10), and pentoxifylline along with melatonin (Group 11) after LPS administration tapered LPS evoked hepatic dysfunction statistically considerably. In conclusion, both melatonin and pentoxifylline set up promising results in endotoxin-induced hepatotoxicity and can be used therapeutic adjuncts to conventional treatment strategies in sepsis-induced liver failure.

Keywords: endotoxin/lipopolysacchride, dexamethasone, hepatotoxicity, melatonin, pentoxifylline

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Authors: Farhan Ahmad Atif, Abd Ur Rehman, Muhammad Babir

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Dietary cation anion difference (DCAD) is an important aspect of dairy nutrition, especially in the transition period. Sahiwal cattle is the highest milk producing breed among Zebu cattle. We planned first study on transition Sahiwal cattle to determine the effects of feeding varying levels of negative DCAD. For this purpose, twenty pregnant cows (at the 250th day of gestation) were selected and randomly divided into 5 groups comprising four animals each. Five iso-caloric (2100 Kcal) and iso-nitrogenous (12%) diets were formulated and each diet was allotted to each group. The animals received positive DCAD diet served as control. Diets were supplemented with NutriCAB® to attain 0, -15, -30 and -45 DCAD levels. Experimental diets were fed at ad-libitum upto parturition and data regarding feed intake were recorded on daily. Post-partum incidence of milk fever, dystocia, retention of placenta (RP), mastitis as well as milk production, milk fat percentage and serum Ca levels were recorded. Urine and blood pH were determined weekly during the last month of pregnancy. Results showed that prepartum feed intake and blood pH were not affected (P > 0.05), while urine pH was significantly reduced (P < 0.05) by lowering DCAD levels. Post parturient blood calcium level linearly increased (P < 0.05) with decreasing DCAD. Pre-partum negative DCAD feeding had no effect (P > 0.05) on post-parturient milk production and fat percentage. However, parturient related problems decreased with decreasing DCAD feeding. It was concluded that negative DCAD feeding raised serum calcium level and reduced the incidence of post-parturient problems in Sahiwal cattle.

Keywords: dairy cattle, transition, metabolic diseases, reproductive disorders, incidence

Procedia PDF Downloads 186

Authors: Vadanasundari Vedarethinam, Kun Qian

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The metabolic analysis is more distal over proteomics and genomics engaging in clinics and needs rationally distinct techniques, designed materials, and device for clinical diagnosis. Conventional techniques such as spectroscopic techniques, biochemical analyzers, and electrochemical have been used for metabolic diagnosis. Currently, there are four major challenges including (I) long-term process in sample pretreatment; (II) difficulties in direct metabolic analysis of biosamples due to complexity (III) low molecular weight metabolite detection with accuracy and (IV) construction of diagnostic tools by materials and device-based platforms for real case application in biomedical applications. Development of chips with nanomaterial is promising to address these critical issues. Mass spectroscopy (MS) has displayed high sensitivity and accuracy, throughput, reproducibility, and resolution for molecular analysis. Particularly laser desorption/ ionization mass spectrometry (LDI MS) combined with devices affords desirable speed for mass measurement in seconds and high sensitivity with low cost towards large scale uses. We developed a plasmonic chip for clinical metabolic fingerprinting as a hot carrier in LDI MS by series of chips with gold nanoshells on the surface through controlled particle synthesis, dip-coating, and gold sputtering for mass production. We integrated the optimized chip with microarrays for laboratory automation and nanoscaled experiments, which afforded direct high-performance metabolic fingerprinting by LDI MS using 500 nL of serum, urine, cerebrospinal fluids (CSF) and exosomes. Further, we demonstrated on-chip direct in-vitro metabolic diagnosis of early-stage lung cancer patients using serum and exosomes without any pretreatment or purifications. To our best knowledge, this work initiates a bionanotechnology based platform for advanced metabolic analysis toward large-scale diagnostic use.

Keywords: plasmonic chip, metabolic fingerprinting, LDI MS, in-vitro diagnostics

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Authors: Amar Ranjan, Julieana Durai, Pranay Tanwar

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Background &Introduction: Ovarian cancer is one of the most common malignant tumor in the female. 70% of the cases of ovarian cancer are diagnosed at an advanced stage. The five-year survival rate associated with ovarian cancer is less than 30%. The early diagnosis of ovarian cancer becomes a key factor in improving the survival rate of patients. Presently, CAl25 (carbohydrate antigen125) is used for the diagnosis and therapeutic monitoring of ovarian cancer, but its sensitivity and specificity is not ideal. The introduction of HE4, human epididymis protein 4 has attracted much attention. HE4 has a sensitivity and specificity of 72.9% and 95% for differentiating between benign and malignant adnexal masses, which is better than CA125 detection.  Methods: Serum HE4 and CA -125 were estimated using the chemiluminescence method. Our cases were 40 epithelial ovarian cancer, 9 benign ovarian tumor, 29 benign gynaecological diseases and 13 healthy individuals. This group include healthy woman those who have undergoing family planning and menopause-related medical consultations and they are negative for ovarian mass. Optimal cut off values for HE4 and CA125 were 55.89pmol/L and 40.25U/L respectively (determined by statistical analysis). Results: The level of HE4 was raised in all ovarian cancer patients (n=40) whereas CA125 levels were normal in 6/40 ovarian cancer patients, which were the cases of OC confirmed by histopathology. There is a significant decrease in the level of HE4 with comparison to CA125 in benign ovarian tumor cases. Both the levels of HE4 and CA125 were raised in the nonovarian cancer group, which includes cancer of endometrium and cervix. In the healthy group, HE4 was normal in all patients except in one case of the rudimentary horn, and the reason for this raised HE4 level is due to the incomplete development of uterus whereas CA125 was raised in 3 cases. Conclusions: Findings showed that the serum level of HE4 is an important indicator in the diagnosis of ovarian cancer, and it also distinguishes between benign and malignant pelvic masses. However, a combination of HE4 and CA125 panel will be extremely valuable in improving the diagnostic efficiency of ovarian cancer. These findings of our study need to be validated in the larger cohort of patients.

Keywords: human epididymis protein 4, ovarian cancer, diagnosis, benign lesions

Procedia PDF Downloads 131

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

Procedia PDF Downloads 135

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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Authors: Wen-Ting Wang, Wei-An Tsai, Rong-Hong Hsieh

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Long term taking high fat diet can lead to over production of energy, result in accumulation of body fat, dyslipidemia and increased lipid metabolism in the body. Over metabolism of lipid results in excessive reactive oxygen species and oxidative stress, may also cause mitochondrial dysfunction and cell death. Krill oil, fish oil and linseed oil are good sources of n-3 polyunsaturated fatty acids (PUFA). The present study investigated the effect of high fat diet and various oil rich of n-3 fatty acids on mitochondrial function and cell membrane composition. Six-weeks old male Spraque-Dawley rats were randomly divided into 8 groups including: control group, high fat diet group, low dosage and high dosage krill oil group, low dosage and high dosage fish oil group, and low dosage and high dosage linseed oil group. After 12 weeks of experimental period, the low dosage krill oil, fish oil group and linseed oil group with different dosage prevented mitochondrial dysfunction caused by high fat diet. The supplementation of different oils increased plasma, erythrocyte and mitochondrial n-3/n-6 ratio and further increased the proportion of PUFA in erythrocyte and mitochondrial membrane. It also decreased serum triglyceride (TG) and low density lipoprotein cholesterol (LDL-C) concentration. However, there was no significant change in serum total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), biomarker of liver function, glucose, insulin, homeostasis model assessment-insulin resistance (HOMA-IR) and plasma malonadialdehyde (MDA) concentration when compared with high fat diet group. The supplementation of different sources of n-3 PUFA can maintain mitochondrial function and modulate cell membrane fatty acid composition in high fat diet conditions, and there is a positive relationship between mitochondrial function and mitochondrial membrane composition.

Keywords: fish oil, linseed oil, mitochondria, n-3 PUFA

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Authors: Sahar M. El-Gowilly, Mai M. Helmy, Hanan M. El-Gowelli

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Methotrexate (MTX) is widely used in treatment of cancers and autoimmune diseases. However, nephrotoxicity is one of the most important side effects of MTX. The peroxisome proliferator-activated receptor gamma agonist, pioglitazone (PIO), is known to exert anti-inflammatory and reno-protective effects in various kidney injuries. The purpose of this study was to investigate the potential involvement of endothelial damage in MTX-induced renal injury and to elaborate the possible protective effect of PIO against MTX-induced nephropathy. Compared with saline-treated rats, treatment with MTX (7 mg/kg for 3 day) caused significant elevations in serum levels of urea and creatinine, increased renal nitrate/nitrite level and impaired renovascular responsiveness of isolated perfused kidney to endothelium-dependent vasodilations induced by acetylcholine (0.01-2.43 nmol) and isoprenaline (1µmol). These effects were abolished by concurrent treatment with PIO (2.5 mg/kg, for 5 days starting two days before MTX). Alternatively, MTX treatment did not affect endothelium-independent renovascular relaxation induced by sodium nitroprusside (1-30 μmole). The possibility that alterations in renal antioxidants, circulating cytokine and apoptotic factor (Fas) levels contributed to MTX-PIO interaction was assessed. PIO treatment abrogated renal oxidative stress (decreased reduced glutathione and catalase activity and increased malondialdehyde), elevated serum cytokine (interleukin-6, interleukin-10, tumor necrosis factor-alpha and transforming growth factor-beta1) and Fas induced by MTX. Histologically, MTX caused defused tubular cells swelling and vacuolization associated with endothelial damage in renal arterioles. These effects disappeared upon co-treated with PIO. Collectively, PIO abolished MTX-induced endothelium dysfunction and nephrotoxicity via ameliorating oxidative stress and rectifying cytokines and Fas abnormalities caused by MTX.

Keywords: methotrexate, pioglitazone, endothelium, kidney

Procedia PDF Downloads 312