Search results for: hyaluronan synthase 3

Authors: Alvin Chun Man Kwok, Wai Sun Chan, Wei Yuan, Joseph Tin Yum Wong

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Cellulose, the most abundant biopolymer, is the major constituent of plant and dinoflagellate cell walls. Thecate dinoflagellates, in particular, are renowned for their remarkable capacity to synthesize intricate cellulosic thecal plates (CTPs). Unlike the extracellular two-dimensional structure of plant cell walls, these CTPs are three-dimensional and reside within the cellular structure itself. The deposition of CTPs occurs with remarkable precision, and their arrangement serves as crucial taxonomic markers. It is noteworthy that these plates possess the hardness of wood, despite the absence of lignin. Partial and prolonged hydrolysis of CTPs results in the formation of uniform long bundles and lowdimensional, modular crystalline whiskers. This observation aligns with the consistent nanomechanical properties, suggesting a CTPboard structure. The unique composition and structural characteristics of CTPs distinguish them from other cellulose-based materials in the natural world. Spectroscopic studies using Raman and FTIR methods indicate a clear low crystallinity index, with the OH shift becoming more distinct following SDS treatment. Birefringence imaging confirms the highly organized structure of CTPs, demonstrating varying degrees of anisotropy in different regions, including both seaward and cytosolic passages. The knockdown of a cellulose synthase enzyme in dinoflagellates resulted in severe malformation of CTPs and hindered the life-cycle transition. Unlike certain other microalgal groups, these unique circum-spherical depositions of CTPs were not pre-fabricated and transported "to site," but synthesized within alveolar sacs at the specific site. Our research is particularly focused on unraveling the mechanisms underlying the biodeposition of CTPs and exploring their potential biotechnological applications. Understanding the processes involved in CTP formation can pave the way for harnessing their unique properties for various practical applications. Dinoflagellates play a crucial role as major agents of algal blooms and are also known for producing anti-greenhouse sulfur compounds such as DMS/DMSP, highlighting the significance of CTPs as a carbon-neutral source of cellulose. Grant acknowledgement: Research in the laboratory are supported by GRF16104523 from Research Grant Council to JTYW.

Keywords: cellulosic thecal plates, dinoflagellates, cellulose, cell wall

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Authors: Lalita Subedi, Jae Kyoung Chae, Yong Un Park, Cho Kyo Hee, Lee Jae Hyuk, Kang Min Cheol, Sun Yeou Kim

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Neuroinflammation may mediate the relationship between low levels of estrogens and neurodegenerative disease. Estrogens are neuroprotective and anti-inflammatory in neurodegenerative disease models. Due to the long term side effects of estrogens, researches have been focused on finding an effective phytoestrogens for biological activities. Daidzein present in soybeans and its active metabolite equol (7-hydroxy-3-(4'-hydroxyphenyl)-chroman) bears strong antioxidant and anticancer showed more potent anti-inflammatory and neuroprotective role in neuroinflammatory model confirmed its in vitro activity with molecular mechanism through NF-κB pathway. Three major CNS cells Microglia (BV-2), Astrocyte (C6), Neuron (N2a) were used to find the effect of equol in inducible nitric oxide synthase (iNOS), cyclooxygenase (COX-2), MAPKs signaling proteins, apoptosis related proteins by western blot analysis. Nitric oxide (NO) and prostaglandin E2 (PGE2) was measured by the Gries method and ELISA, respectively. Cytokines like tumor necrosis factor-α (TNF-α) and IL-6 were also measured in the conditioned medium of LPS activated cells with or without equol. Equol inhibited the NO production, PGE-2 production and expression of COX-2 and iNOS in LPS-stimulated microglial cells at a dose dependent without any cellular toxicity. At the same time Equol also showed promising effect in modulation of MAPK’s and nuclear factor kappa B (NF-κB) expression with significant inhibition of the production of proinflammatory cytokine like interleukin -6 (IL-6), and tumor necrosis factor -α (TNF-α). Additionally, it inhibited the LPS activated microglia-induced neuronal cell death by downregulating the apoptotic phenomenon in neuronal cells. Furthermore, equol increases the production of neurotrophins like NGF and increase the neurite outgrowth as well. In conclusion the natural daidzein metabolite equol are more active than daidzein, which showed a promising effectiveness as an anti-neuroinflammatory and neuroprotective agent via downregulating the LPS stimulated microglial activation and neuronal apoptosis. This work was supported by Brain Korea 21 Plus project and High Value-added Food Technology Development Program 114006-4, Ministry of Agriculture, Food and Rural Affairs.

Keywords: apoptosis, equol, neuroinflammation, phytoestrogen

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Authors: Eun-Young Kwon, Myung-Sook Choi, Su-Jung Cho, Ji-Young Choi, So Young Kim, Youngji Han

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Overweight and obesity have been linked to a low-grade chronic inflammatory response and an increased risk of developing metabolic syndrome including insulin resistance, type 2 diabetes mellitus and certain types of cancers. Luteolin is a dietary flavonoid with anti-inflammatory, anti-oxidant, anti-cancer and anti-diabetic properties. However, little is known about the detailed mechanism associated with the effect of luteolin on inflammation-related obesity and its complications. The aim of the present study was to reveal the anti-diabetic effect of luteolin in diet-induced obesity mice using “transcriptomics” tool. Thirty-nine male C57BL/6J mice (4-week-old) were randomly divided into 3 groups and were fed normal diet, high-fat diet (HFD, 20% fat) and HFD+0.005% (w/w) luteolin for 16 weeks. Luteolin improved insulin resistance, as measured by HOMA-IR and glucose tolerance, along with preservation action of pancreatic β-cells, compared to the HFD group. Luteoiln was significantly decreased the levels of leptin and ghrelin that play a pivotal role in energy balance, and the macrophage low-grade inflammation marker sCD163 (soluble Cd antigen 163) in plasma. Activities of hepatic anti-oxidant enzymes (catalase and glutathione peroxidase) were increased, while the levels of plasma transaminase (GOT and GPT) and oxidative damage markers (hepatic mitochondria H2O2 and TBARS) were markedly decreased by luteolin supplementation. In addition, luteolin increased oxidative capacity and fatty acid utilization by presenting decrease in enzyme activities of citrate synthase, cytochrome C oxidase and β-hydroxyacyl CoA dehydrogenase and UCP3 gene expression compared to high-fat diet. Moreover, our microarray results of muscle also revealed down-regulated gene expressions associated with TCA cycle by HFD were reversed to normal level by luteolin treatment. Taken together, our results indicate that luteolin is one of bioactive components for improving insulin resistance by increasing oxidative capacity, modulating anti-oxidant metabolism and suppressing inflammatory signaling cascades in diet-induced obese mice. These results provide possible therapeutic targets for prevention and treatment of diet-induced obesity and its complications.

Keywords: anti-oxidant metabolism, diabetes, luteolin, oxidative capacity

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Authors: Franco Van De Velde, Debora Esposito, Maria E. Pirovani, Mary A. Lila

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The physiology of various inflammatory diseases is a complex process mediated by inflammatory and immune cells such as macrophages and monocytes. Chronic inflammation, as observed in many cardiovascular and autoimmune disorders, occurs when the low-grade inflammatory response fails to resolve with time. Because of the complexity of the chronic inflammatory disease, major efforts have focused on identifying novel anti-inflammatory agents and dietary regimes that prevent the pro-inflammatory process at the early stage of gene expression of key pro-inflammatory mediators and cytokines. The ability of the extracts of three blackberry cultivars (‘Jumbo’, ‘Black Satin’ and ‘Dirksen’), and one strawberry cultivar (‘Camarosa’) to inhibit four well-known genetic biomarkers of inflammation: inducible nitric oxide synthase (iNOS), cyclooxynase-2 (Cox-2), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in an in vitro lipopolysaccharide-stimulated murine RAW 264.7 macrophage model were investigated. Moreover, the effect of latter extracts on the intracellular reactive oxygen species (ROS) and nitric oxide (NO) production was assessed. Assay was conducted with 50 µg/mL crude extract concentration, an amount that is easily achievable in the gastrointestinal tract after berries consumption. The mRNA expression levels of Cox-2 and IL-6 were reduced consistently (more than 30%) by extracts of ‘Jumbo’ and ‘Black Satin’ blackberries. Strawberry extracts showed high reduction in mRNA expression levels of IL-6 (more than 65%) and exhibited moderate reduction in mRNA expression of Cox-2 (more than 35%). The latter behavior mirrors the intracellular ROS production of the LPS stimulated RAW 264.7 macrophages after the treatment with blackberry ‘Black Satin’ and ‘Jumbo’, and strawberry ‘Camarosa’ extracts, suggesting that phytochemicals from these fruits may play a role in the health maintenance by reducing oxidative stress. On the other hand, effective inhibition in the gene expression of IL-1β and iNOS was not observed by any of blackberry and strawberry extracts. However, suppression in the NO production in the activated macrophages among 5–25% was observed by ‘Jumbo’ and ‘Black Satin’ blackberry extracts and ‘Camarosa’ strawberry extracts, suggesting a higher NO suppression property by phytochemicals of these fruits. All these results suggest the potential beneficial effects of studied berries as functional foods with antioxidant and anti-inflammatory roles. Moreover, the underlying role of phytochemicals from these fruits in the protection of inflammatory process will deserve to be further explored.

Keywords: cyclooxygenase-2, functional foods, interleukin-6, reactive oxygen species

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Authors: Ruwani N. Nugara, Masashi Inafuku, Kensaku Takara, Hironori Iwasaki, Hirosuke Oku

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Pteryxin from the partially purified hexane phase (HP) of Peucedanum japonicum Thunb (PJT) was identified as the active compound related to anti-obesity. Thus, in this study we investigated the mechanisms related to anti-obesity activity in-vitro. The HP was fractionated, and effect on the triglyceride (TG) content was evaluated in 3T3-L1 and HepG2 cells. Comprehensive spectroscopic analyses were used to identify the structure of the active compound. The dose dependent effect of active constituent on the TG content, and the gene expressions related to adipogenesis, fatty acid catabolism, energy expenditure, lipolysis and lipogenesis (20 μg/mL) were examined in-vitro. Furthermore, higher dosage of pteryxin (50μg/mL) was tested against 20μg/mL in 3T3-L1 adipocytes. The mRNA were subjected to SOLiD next generation sequencer and the obtained data were analyzed by Ingenuity Pathway Analysis (IPA). The active constituent was identified as pteryxin, a known compound in PJT. However, its biological activities against obesity have not been reported previously. Pteryxin dose dependently suppressed TG content in both 3T3-L1 adipocytes and HepG2 hepatocytes (P < 0.05). Sterol regulatory element-binding protein-1 (SREBP1 c), Fatty acid synthase (FASN), and acetyl-CoA carboxylase-1 (ACC1) were downregulated in pteryxin-treated adipocytes (by 18.0, 36.1 and 38.2%; P < 0.05, respectively) and hepatocytes (by 72.3, 62.9 and 38.8%, respectively; P < 0.05) indicating its suppressive effects on fatty acid synthesis. The hormone-sensitive lipase (HSL), a lipid catabolising gene was upregulated (by 15.1%; P < 0.05) in pteryxin-treated adipocytes suggesting improved lipolysis. Concordantly, the adipocyte size marker gene, paternally expressed gene1/mesoderm specific transcript (MEST) was downregulated (by 42.8%; P < 0.05), further accelerating the lipolytic activity. The upregulated trend of uncoupling protein 2 (UCP2; by 77.5%; P < 0.05) reflected the improved energy expenditure due to pteryxin. The 50μg/mL dosage of pteryxin completely suppressed PPARγ, MEST, SREBP 1C, HSL, Adiponectin, Fatty Acid Binding Protein (FABP) 4, and UCP’s in 3T3-L1 adipocytes. The IPA suggested that pteryxin at 20μg/mL and 50μg/mL suppress obesity in two different pathways, whereas the WNT signaling pathway play a key role in the higher dose of pteryxin in preadipocyte stage. Pteryxin in PJT play the key role in regulating lipid metabolism related gene network and improving energy production in vitro. Thus, the results suggests pteryxin as a new natural compound to be used as an anti-obesity drug in pharmaceutical industry.

Keywords: obesity, peucedanum japonicum thunb, pteryxin, food science

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Authors: Ammar Boudaka, Maryam Al-Suleimani, Hajar BaOmar, Intisar Al-Lawati, Fahad Zadjali

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Background: Hypertension is increasingly becoming a matter of medical and public health importance. The maintenance of normal blood pressure requires a balance between cardiac output and total peripheral resistance. The endothelium, through the release of vasodilating factors, plays an important role in the control of total peripheral resistance and hence blood pressure homeostasis. Transient Receptor Potential Vanilloid type 4 (TRPV4) is a mechanosensitive non-selective cation channel that is expressed on the endothelium and contributes to endothelium-mediated vasodilation. So far, no data are available about the morphological and functional status of this channel in hypertensive cases. Objectives: This study aimed to investigate whether there is any difference in the morphological and functional features of TRPV4 in the mesenteric artery of normotensive and hypertensive rats. Methods: Functional feature of TRPV4 in four experimental animal groups: young and adult Wistar-Kyoto rats (WKY-Y and WKY-A), young and adult spontaneously hypertensive rats (SHR-Y and SHR-A), was studied by adding 5 µM 4αPDD (TRPV4 agonist) to mesenteric arteries mounted in a four-chamber wire myograph and pre-contracted with 4 µM phenylephrine. The 4αPDD-induced response was investigated in the presence and absence of 1 µM HC067047 (TRPV4 antagonist), 100 µM L-NAME (nitric oxide synthase inhibitor), and endothelium. The morphological distribution of TRPV4 in the wall of rat mesenteric arteries was investigated by immunostaining. Real-time PCR was used in order to investigate mRNA expression level of TRPV4 in the mesenteric arteries of the four groups. The collected data were expressed as mean ± S.E.M. with n equal to the number of animals used (one vessel was taken from each rat). To determine the level of significance, statistical comparisons were performed using the student’s t-test and considered to be significantly different at p<0.05. Results: 4αPDD induced a relaxation response in the mesenteric arterial preparations (WKY-Y: 85.98% ± 4.18; n = 5) that was markedly inhibited by HC067047 (18.30% ± 2.86; n= 5; p<0.05), endothelium removal (19.93% ± 1.50; n = 5; p<0.05) and L-NAME (28.18% ± 3.09; n = 5; p<0.05). The 4αPDD-induced relaxation was significantly lower in SHR-Y compared to WKY-Y (SHR-Y: 70.96% ± 3.65; n = 6, WKY-Y: 85.98% ± 4.18; n = 5-6, p<0.05. Moreover, the 4αPDD-induced response was significantly lower in WKY-A than WKY-Y (WKY-A: 75.58 ± 1.30; n = 5, WKY-Y: 85.98% ± 4.18; n = 5, p<0.05). Immunostaining study showed immunofluorescent signal confined to the endothelial layer of the mesenteric arteries. The expression of TRPV4 mRNA in SHR-Y was significantly lower than in WKY-Y (SHR-Y; 0.67RU ± 0.34; n = 4, WKY-Y: 2.34RU ± 0.15; n = 4, p<0.05). Furthermore, TRPV4 mRNA expression in WKY-A was lower than its expression in WKY-Y (WKY-A: 0.62RU ± 0.37; n = 4, WKY-Y: 2.34RU ± 0.15; n = 4, p<0.05). Conclusion: Stimulation of TRPV4, which is expressed on the endothelium of rat mesenteric artery, triggers an endothelium-mediated relaxation response that markedly decreases with hypertension and growing up changes due to downregulation of TRPV4 expression.

Keywords: hypertension, endothelium, mesenteric artery, TRPV4

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Authors: Laura Helena Caicedo Lopez, Luis Miguel Contreras Medina, Ramon Gerardo Guevara Gonzales, Juan E. Andrade

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In this study, we evaluated the effect of three different hydric stress conditions: Low (LHS), medium (MHS), and high (HHS) on capsaicinoid content and enzyme regulation of C. annuum plants. Five main peaks were detected using a 2 Hz resolution vibrometer laser (Polytec-B&K). These peaks or “characteristic frequencies” were used as acoustic emissions (AEs) treatment, transforming these signals into audible sound with the frequency (Hz) content of each hydric stress. Capsaicinoids (CAPs) are the main, secondary metabolites of chili pepper plants and are known to increase during hydric stress conditions or short drought-periods. The AEs treatments were applied in two plant stages: the first one was in the pre-anthesis stage to evaluate the genes that encode the transcription of enzymes responsible for diverse metabolic activities of C. annuum plants. For example, the antioxidant responses such as peroxidase (POD), superoxide dismutase (Mn-SOD). Also, phenyl-alanine ammonia-lyase (PAL) involved in the biosynthesis of the phenylpropanoid compounds. The chalcone synthase (CHS) related to the natural defense mechanisms and species-specific aquaporin (CAPIP-1) that regulate the flow of water into and out of cells. The second stage was at 40 days after flowering (DAF) to evaluate the biochemical effect of AEs related to hydric stress on capsaicinoids production. These two experiments were conducted to identify the molecular responses of C. annuum plants to AE. Moreover, to define AEs could elicit any increase in the capsaicinoids content after a one-week exposition to AEs treatments. The results show that all AEs treatment signals (LHS, MHS, and HHS) were significantly different compared to the non-acoustic emission control (NAE). Also, the AEs induced the up-regulation of POD (~2.8, 2.9, and 3.6, respectively). The gene expression of another antioxidant response was particularly treatment-dependent. The HHS induced and overexpression of Mn-SOD (~0.23) and PAL (~0.33). As well, the MHS only induced an up-regulation of the CHs gene (~0.63). On the other hand, CAPIP-1 gene gas down-regulated by all AEs treatments LHS, MHS, and HHS ~ (-2.4, -0.43 and -6.4, respectively). Likewise, the down-regulation showed particularities depending on the treatment. LHS and MHS induced downregulation of the SOD gene ~ (-1.26 and -1.20 respectively) and PAL (-4.36 and 2.05, respectively). Correspondingly, the LHS and HHS showed the same tendency in the CHs gene, respectively ~ (-1.12 and -1.02, respectively). Regarding the elicitation effect of AE on the capsaicinoids content, additional treatment controls were included. A white noise treatment (WN) to prove the frequency-selectiveness of signals and a hydric stressed group (HS) to compare the CAPs content. Our findings suggest that WN and NAE did not present differences statically. Conversely, HS and all AEs treatments induced a significant increase of capsaicin (Cap) and dihydrocapsaicin (Dcap) after one-week of a treatment. Specifically, the HS plants showed an increase of 8.33 times compared to the NAE and WN treatments and 1.4 times higher than the MHS, which was the AEs treatment with a larger induction of Capsaicinoids among treatments (5.88) and compared to the controls.

Keywords: acoustic emission, capsaicinoids, elicitors, hydric stress, plant signaling

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

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

Keywords: lead, quercetin, neuroinflammation, memory

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Authors: Yuriko Takayama, Norihiro Kato

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Quorum sensing (QS) is a cell-to-cell communication system in bacteria to regulate expression of target genes. In gram-negative bacteria, activation on QS is controlled by a concentration increase of N-acylhomoserine lactone (AHL), which can diffuse in and out of the cell. Effective control of QS is expected to avoid virulence factor production in infectious pathogens, biofilm formation, and antibiotic production because various cell functions in gram-negative bacteria are controlled by AHL-mediated QS. In this research, we applied cyclodextrins (CDs) as artificial hosts for the AHL signal to reduce the AHL concentration in the culture broth below its threshold for QS activation. The AHL-receptor complex induced under the high AHL concentration activates transcription of the QS-target gene. Accordingly, artificial reduction of the AHL concentration is one of the effective strategies to inhibit the QS. A hydrophobic cavity of the CD can interact with the acyl-chain of the AHL due to hydrophobic interaction in aqueous media. We studied N-hexanoylhomoserine lactone (C6HSL)-mediated QS in Serratia marcescens; accumulation of C6HSL is responsible for regulation of the expression of pig cluster. Inhibitory effects of added CDs on QS were demonstrated by determination of prodigiosin amount inside cells after reaching stationary phase, because production of prodigiosin depends on the C6HSL-mediated QS. By adding approximately 6 wt% hydroxypropyl-β-CD (HP-β-CD) in Luria-Bertani (LB) medium prior to inoculation of S. maecescens AS-1, the intracellularly accumulated prodigiosin was drastically reduced to 7-10%, which was determined after the extraction of prodigiosin in acidified ethanol. The AHL retention ability of HP-β-CD was also demonstrated by Chromobacterium violacuem CV026 bioassay. The CV026 strain is an AHL-synthase defective mutant that activates QS solely by adding AHLs from outside of cells. A purple pigment violacein is induced by activation of the AHL-mediated QS. We demonstrated that the violacein production was effectively suppressed when the C6HSL standard solution was spotted on a LB agar plate dispersing CV026 cells and HP-β-CD. Physico-chemical analysis was performed to study the affinity between the immobilized CD and added C6HSL using a quartz crystal microbalance (QCM) sensor. The COOH-terminated self-assembled monolayer was prepared on a gold electrode of 27-MHz AT-cut quartz crystal. Mono(6-deoxy-6-N, N-diethylamino)-β-CD was immobilized on the electrode using water-soluble carbodiimide. The C6HSL interaction with the β-CD cavity was studied by injecting the C6HSL solution to a cup-type sensor cell filled with buffer solution. A decrement of resonant frequency (ΔFs) clearly showed the effective C6HSL complexation with immobilized β-CD and its stability constant for MBP-SpnR-C6HSL complex was on the order of 102 M-1. The CD has high potential for engineered control of QS because it is safe for human use.

Keywords: acylhomoserine lactone, cyclodextrin, intracellular signaling, quorum sensing

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Authors: Iñaki Milton-Laskibar, Leixuri Aguirre, Maria P. Portillo

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Introduction: Energy restriction is an effective approach in preventing liver steatosis. However, due to social and economic reasons among others, compliance with this treatment protocol is often very poor, especially in the long term. Resveratrol, a natural polyphenolic compound that belongs to stilbene group, has been widely reported to imitate the effects of energy restriction. Objective: To analyze the effects of resveratrol under normoenergetic feeding conditions and under a mild energy restriction on liver fat accumulation and hepatic fatty acid oxidation. Methods: 36 male six-week-old rats were fed a high-fat high-sucrose diet for 6 weeks in order to induce steatosis. Then, rats were divided into four groups and fed a standard diet for 6 additional weeks: control group (C), resveratrol group (RSV, resveratrol 30 mg/kg/d), restricted group (R, 15 % energy restriction) and combined group (RR, 15 % energy restriction and resveratrol 30 mg/kg/d). Liver triacylglycerols (TG) and total cholesterol contents were measured by using commercial kits. Carnitine palmitoyl transferase 1a (CPT 1a) and citrate synthase (CS) activities were measured spectrophotometrically. TFAM (mitochondrial transcription factor A) and peroxisome proliferator-activator receptor alpha (PPARα) protein contents, as well as the ratio acetylated peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α)/Total PGC1α were analyzed by Western blot. Statistical analysis was performed by using one way ANOVA and Newman-Keuls as post-hoc test. Results: No differences were observed among the four groups regarding liver weight and cholesterol content, but the three treated groups showed reduced TG when compared to the control group, being the restricted groups the ones showing the lowest values (with no differences between them). Higher CPT 1a and CS activities were observed in the groups supplemented with resveratrol (RSV and RR), with no difference between them. The acetylated PGC1α /total PGC1α ratio was lower in the treated groups (RSV, R and RR) than in the control group, with no differences among them. As far as TFAM protein expression is concerned, only the RR group reached a higher value. Finally, no changes were observed in PPARα protein expression. Conclusions: Resveratrol administration is an effective intervention for liver triacylglycerol content reduction, but a mild energy restriction is even more effective. The mechanisms of action of these two strategies are different. Thus resveratrol, but not energy restriction, seems to act by increasing fatty acid oxidation, although mitochondriogenesis seems not to be induced. When both treatments (resveratrol administration and a mild energy restriction) were combined, no additive or synergic effects were appreciated. Acknowledgements: MINECO-FEDER (AGL2015-65719-R), Basque Government (IT-572-13), University of the Basque Country (ELDUNANOTEK UFI11/32), Institut of Health Carlos III (CIBERobn). Iñaki Milton is a fellowship from the Basque Government.

Keywords: energy restriction, fat, liver, oxidation, resveratrol

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Authors: Reem Alajmi, Hind Al Harbi, Tahany Ayaad, Zainab Al Musawi

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Hard ticks Hyalomma spp. (family: Ixodidae) are obligate ectoparasite in their all life stages on some domestic animals mainly camels and cattle. Ticks may lead to many economic and public health problems because of their blood feeding behavior. Also, they act as vectors for many bacterial, viral and protozoan agents which may cause serious diseases such as tick-born encephalitis, Rocky-mountain spotted fever, Q-fever and Lyme disease which can affect human and/or animals. In the present study, molecular identification of ticks that attack camels in Riyadh region, Saudi Arabia based on the partial sequence of mitochondrial 16s rRNA gene was applied. Also, the present study aims to detect natural infections of collected camel ticks with Rickessia spp. and Borelia spp. using PCR/hybridization of Citrate synthase encoding gene present in bacterial cells. Hard ticks infesting camels were collected from different camels located in a farm in Riyadh region, Saudi Arabia. Results of the present study showed that the collected specimens belong to two species: Hyalomma dromedari represent 99% of the identified specimens and Hyalomma marginatum which account for 1 % of identified ticks. The molecular identification was made through blasting the obtained sequence of this study with sequences already present and identified in GeneBank. All obtained sequences of H. dromedarii specimens showed 97-100% identity with the same gene sequence of the same species (Accession # L34306.1) which was used as a reference. Meanwhile, no intraspecific variations of H. marginatum mesured because only one specimen was collected. Results also had shown that the intraspecific variability between individuals of H. dromedarii obtained in 92 % of samples ranging from 0.2- 6.6%, while the remaining 7 % of the total samples of H. dromedarii showed about 10.3 % individual differences. However, the interspecific variability between H. dromedarii and H. marginatum was approximately 18.3 %. On the other hand, by using the technique of PCR/hybridization, we could detect natural infection of camel ticks with Rickettsia spp. and Borrelia spp. Results revealed the natural presence of both bacteria in collected ticks. Rickettsial spp. infection present in 29% of collected ticks, while 35% of collected specimen were infected with Borrelia spp. The valuable results obtained from the present study are a new record for the molecular identification of camel ticks in Riyadh, Saudi Arabia and their natural infection with both Rickettsia spp. and Borrelia spp. These results may help scientists to provide a good and direct control strategy of ticks in order to protect one of the most important economic animals which are camels. Also results of this project spotlight on the disease that might be transmitted by ticks to put out a direct protective plan to prevent spreading of these dangerous agents. Further molecular studies are needed to confirm the results of the present study by using other mitochondrial and nuclear genes for tick identification.

Keywords: Camel ticks, Rickessia spp. , Borelia spp. , mitochondrial 16s rRNA gene

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Authors: Qi Liu, Jiqin Yao, Xiaohu Zhou, Bo Zheng

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The first artificial cell was produced by Thomas Chang in the 1950s when he was trying to make a mimic of red blood cells. Since then, many different types of artificial cells have been constructed from one of the two approaches: a so-called bottom-up approach, which aims to create a cell from scratch, and a top-down approach, in which genes are sequentially knocked out from organisms until only the minimal genome required for sustaining life remains. In this project, bottom-up approach was used to build a new cell-free expression system which mimics artificial cell that capable of protein expression and communicate with each other. The artificial cells constructed from the bottom-up approach are usually lipid vesicles, polymersomes, hydrogels or aqueous droplets containing the nucleic acids and transcription-translation machinery. However, lipid vesicles based artificial cells capable of communication present several issues in the cell communication research: (1) The lipid vesicles normally lose the important functions such as protein expression within a few hours. (2) The lipid membrane allows the permeation of only small molecules and limits the types of molecules that can be sensed and released to the surrounding environment for chemical communication; (3) The lipid vesicles are prone to rupture due to the imbalance of the osmotic pressure. To address these issues, the hydrogel-based artificial cells were constructed in this work. To construct the artificial cell, polyacrylamide hydrogel was functionalized with Acrylate PEG Succinimidyl Carboxymethyl Ester (ACLT-PEG2000-SCM) moiety on the polymer backbone. The proteinaceous factors can then be immobilized on the polymer backbone by the reaction between primary amines of proteins and N-hydroxysuccinimide esters (NHS esters) of ACLT-PEG2000-SCM, the plasmid template and ribosome were encapsulated inside the hydrogel particles. Because the artificial cell could continuously express protein with the supply of nutrients and energy, the artificial cell-artificial cell communication and artificial cell-natural cell communication could be achieved by combining the artificial cell vector with designed plasmids. The plasmids were designed referring to the quorum sensing (QS) system of bacteria, which largely relied on cognate acyl-homoserine lactone (AHL) / transcription pairs. In one communication pair, “sender” is the artificial cell or natural cell that can produce AHL signal molecule by synthesizing the corresponding signal synthase that catalyzed the conversion of S-adenosyl-L-methionine (SAM) into AHL, while the “receiver” is the artificial cell or natural cell that can sense the quorum sensing signaling molecule form “sender” and in turn express the gene of interest. In the experiment, GFP was first immobilized inside the hydrogel particle to prove that the functionalized hydrogel particles could be used for protein binding. After that, the successful communication between artificial cell-artificial cell and artificial cell-natural cell was demonstrated, the successful signal between artificial cell-artificial cell or artificial cell-natural cell could be observed by recording the fluorescence signal increase. The hydrogel-based artificial cell designed in this work can help to study the complex communication system in bacteria, it can also be further developed for therapeutic applications.

Keywords: artificial cell, cell-free system, gene circuit, synthetic biology

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Authors: Emma Castiglioni, Nigel Scrutton, Derren Heyes, Alistair Fielding

Abstract:

By using light as trigger, it is possible to study many biological processes, such as the activity of genes, proteins, and other molecules, with precise spatiotemporal control. Caged compounds, where biologically active molecules are generated from an inert precursor upon laser photolysis, offer the potential to initiate such biological reactions with high temporal resolution. As light acts as the trigger for cleaving the protecting group, the ‘caging’ technique provides a number of advantages as it can be intracellular, rapid and controlled in a quantitative manner. We are developing caging strategies to study the catalytic cycle of a number of enzyme systems, such as nitric oxide synthase and ethanolamine ammonia lyase. These include the use of caged substrates, caged electrons and the possibility of caging the enzyme itself. In addition, we are developing a novel freeze-quench instrument to study these reactions, which combines rapid mixing and flashing capabilities. Reaction intermediates will be trapped at low temperatures and will be analysed by using electron paramagnetic resonance (EPR) spectroscopy to identify the involvement of any radical species during catalysis. EPR techniques typically require relatively long measurement times and very often, low temperatures to fully characterise these short-lived species. Therefore, common rapid mixing techniques, such as stopped-flow or quench-flow are not directly suitable. However, the combination of rapid freeze-quench (RFQ) followed by EPR analysis provides the ideal approach to kinetically trap and spectroscopically characterise these transient radical species. In a typical RFQ experiment, two reagent solutions are delivered to the mixer via two syringes driven by a pneumatic actuator or stepper motor. The new mixed solution is then sprayed into a cryogenic liquid or surface, and the frozen sample is then collected and packed into an EPR tube for analysis. The earliest RFQ instrument consisted of a hydraulic ram unit as a drive unit with direct spraying of the sample into a cryogenic liquid (nitrogen, isopentane or petroleum). Improvements to the RFQ technique have arisen from the design of new mixers in order to reduce both the volume and the mixing time. In addition, the cryogenic isopentane bath has been coupled to a filtering system or replaced by spraying the solution onto a surface that is frozen via thermal conductivity with a cryogenic liquid. In our work, we are developing a novel RFQ instrument which combines the freeze-quench technology with flashing capabilities to enable the studies of both thermally-activated and light-activated biological reactions. This instrument also uses a new rotating plate design based on magnetic couplings and removes the need for mechanical motorised rotation, which can otherwise be problematic at cryogenic temperatures.

Keywords: caged compounds, freeze-quench apparatus, photolysis, radicals

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Authors: Natalia V. Naryzhnaya, Alexandr V. Mukhomedzyanov, Ivan A. Derkachev, Boris K. Kurbatov, Leonid N. Maslov

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Background: Techniques of adaptation to hypoxia and remote postconditioning (RPost) have great prospects for use in the clinic. However, recent studies have shown low efficacy of remote postconditioning in patients with AMI. We hypothesize that the reasons for this inefficiency may be metabolic disorders, which are very common, especially in patients with cardiovascular disease, and age of patients. The purpose of the study was to reveal the effectiveness of adaptation to chronic hypoxia and RPost. To determine the possible relationship between the decrease in the effectiveness of projective impacts and disorders of carbohydrate and lipid metabolism. Design: The study was carried out on Wistar rats 60 day old. MetS was induced by high-carbohydrate, high-fat diet (HСHFD). Modeling MS led to the formation of obesity, hypertension, impaired lipid and carbohydrate metabolism, hyperleptinemia, and moderate stress. Groups with adaptation to chronic hypoxia were subjected to hypoxia for 21 days at 12% O2 and 0.3% CO2 after complete of HСHFD. All animals were subjected to 45 min coronary occlusion and 120 min reperfusion. Groups with RPost, immediately after the end of ischemia, tourniquets were applied to the hind limbs in the area of the hip joint (3 times in the mode of 5 min ischemia, 5 min reperfusion). Results: RPost led to a twofold reduction of infarct size in rats with intact metabolism (р < 0.0001), while in rats with MetS, a decrease in infarct size during RPost was 25 % (p = 0.00003). A direct correlation was found between of infarct size during RPost and the serum leptin level of rats with MetC (r = 0.85). The presented data suggested that a decrease in the efficiency of remote postconditioning in rats with diet-induced metabolic syndrome depends on serum leptin. Chronic hypoxia resulted in a 38% reduced in infarct size in metabolically intact rats. The decrease of cardioprotection was observed in rats with chronic hypoxia and MetS. Infarct size showed a direct correlation with impaired glucose tolerance (AUC, glucose tolerance test, r = 0.034) and serum triglyceride levels (r = 0.39). Our study showed the dependence of cardioprotection in rats with metabolic syndrome during chronic hypoxia and DPost on opioids in the blood serum and myocardium, protein kinase C and NO synthase activity. Conclusion: The results obtained showed that the infarct-limiting efficiency of adaptation to hypoxia and remote postconditioning is reduced or completely absent in animals with metabolic syndrome. The increase in the infarction, in this case, directly depends on the disturbances in carbohydrate. lipid metabolism and opioids signaling. Funding: Investigation of effectiveness of chronic hypoxia at the metabolic syndrome was carried out within the support of Russian Science Foundation Grant 22-15-00048. Studies of the mechanisms of arterial hypertension in induced metabolic syndrome were carried out within the framework of the state assignment (122020300042-4). The work was performed using the Center for Collective Use "Medical Genomics".

Keywords: chronic hypoxia, opioids, remote postconditioning, metabolic syndrome

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Authors: Heba M. Saad Eldien, Ola Abdel-Tawab Hussein, Ahmed Yassein Nassar

Abstract:

Background: Indomethacin is a non-steroidal anti inflammatory drug. Indomethacin induces an injury to gastrointestinal mucosa in experimental animals and humans and their use is associated with a significant risk of hemorrhage, erosions and perforation of both gastric and intestinal ulcers. The anti-inflammatory action of copper complexes is an important activity of their anti-ulcer effect achieved by their intermediary role as a transport form of copper that allow activation of the several copper-dependent enzymes. Therefore, several copper complexes were synthesized and investigated as promising alternative anti-ulcer therapy. Aim of the work: The purpose of this study was to evaluate a copper chelating complex consisting of egg albumin and copper as one of the copper peptides that can be used as anti-inflammatory agent and effective in ameliorates the hazards of the indomethacin on the histological structure of the fundus of the stomach that could be added to raise the efficacy of the currently used simple and cheap gastric anti-inflammatory drug mucogel. Material &methods: This study was carried out on 40 adult male albino rats,divided equally into 4 groups;Group I(control group) received distilled water,Group II(indomethacin treated group) received (25 mg/kg body weight, oral intubation) once, Group III (mucogel treated group)2 mL/rat once daily, oral incubation, Group IV(copper complex group) 1 mL /rat of 30 gm of copper albumin complex was mixed uniformly with mucogel to 100 mL. Treatment has been started six hour after Induction of Ulcers and continued till the 3rd day. The animals sacrificed and was processed for light, transmission electron microscopy(TEM) and immunostaining for inducible nitric oxide synthase(iNOS). Results: Fundic mucosa of group II, showed exfoliation of epithelial cells lining the gland, discontinuity of surface epithelial cells (ulcer formation), vacuolation and detachment of cells, eosinophilic infiltration and congestion of blood vessels in the lamina propria and submucosa. There was thickening and disarrangement of mucosa, weak positive reaction for PAS and marked increase in the collagen fibers lamina propria and the submucosa of the fundus. TEM revealed degeneration of cheif and parietal cells.Marked increase positive reactive of iNOS in all cells of the fundic gland. Group III showed reconstruction of gastric gland with cystic dilatation and vacuolation, moderate decrease of collagen fibers, reduced the intensity of iNOS while in Group IV healthy mucosa with normal surface lining epithelium and fundic glands, strong positive reaction for PAS, marked decrease of collagen fibers and positive reaction for iNOS. TEM revealed regeneration of cheif and parietal cells. Conclusion: Co treatment of copper-albumin complex seems to be useful for gastric ulcer treatment and ameliorates most of hazards of indomethacin.

Keywords: copper complex, gastric ulcer, indomethacin, rat

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Authors: Sandy Elsayed, Aya Mohamed, Noha Nassar

Abstract:

Introduction: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by social deficits and repetitive behavior. Multiple studies suggest that oxidative stress and neuroinflammation are key factors in the etiology of ASD and often associated with worsening of ASD-related behaviors. Nuclear factor erythroid 2-related factor 2 (NRF-2) is a transcription factor that promotes expression of antioxidant response element genes in oxidative stress. In ASD subjects, decreased expression of NRF-2 in frontal cortex shifted the redox homeostasis towards oxidative stress, and resulted in inflammation evidenced by elevation of nuclear factor kappa B (NF-κB) transcriptional activity. Dimethyl fumarate (DMF) is a NRF-2 activator that is used in the treatment of psoriasis and multiple sclerosis. It participates in the transcriptional control of inflammatory factors via inhibition of NF-κB and its downstream targets. This study aimed to investigate the role of DMF in alleviating the cognitive impairments and behavior deficits associated with ASD through mitigation of oxidative stress and inflammation in prenatal valproic acid (VPA) rat model of autism. Methods: Pregnant female Wistar rats received a single intraperitoneal injection of VPA (600 mg/kg) to induce autistic-like-behavioral and neurobiological alterations in their offspring. Chronic oral gavage of DMF (150mg/kg/day) started from postnatal day (PND) 24 till PND62 (39 days). Prenatal VPA exposure elicited autistic behaviors including decreased social interaction and stereotyped behavior. Social interaction was evaluated using three-chamber sociability test and calculation of sociability index (SI), while stereotyped repetitive behavior and anxiety associated with ASD were assessed using marble burying test (MBT). Biochemical analyses were done on prefrontal cortex homogenates including NRF-2, and NF-κB expression. Moreover, inducible nitric oxide synthase (iNOS) gene expression and tumor necrosis factor (TNF-) protein expression were evaluated as markers of inflammation. Results: Prenatal VPA elicited decreased social interaction shown by decreased SI compared to control group (p < 0.001) and DMF enhanced SI (p < 0.05). In MBT, prenatal injection of VPA manifested stereotyped behavior and enhanced number of buried marbles compared to control (p < 0.05) and DMF reduced the anxiety-related behavior in rats exhibiting ASD-like behaviors (p < 0.05). In prefrontal cortex, NRF-2 expression was downregulated in prenatal VPA model (p < 0.0001) and DMF reversed this effect (p < 0.0001). The inflammatory transcription factor NF-κB was elevated in prenatal VPA model (p < 0.0001) and reduced (p < 0.0001) upon NRF-2 activation by DMF. Prenatal VPA expressed higher levels of proinflammatory cytokine TNF- compared to control group (p < 0.0001) and DMF reduced it (p < 0.0001). Finally, the gene expression of iNOS was downregulated upon NRF-2 activation by DMF (p < 0.01). Conclusion: This study proposes that DMF is a potential agent that can be used to ameliorate autistic-like-changes through NRF-2 activation along with NF-κB downregulation and therefore, it is a promising novel therapy for ASD.

Keywords: autism spectrum disorders, dimethyl fumarate, neuroinflammation, NRF-2

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

Abstract:

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

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

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Authors: Batul Kagalwala

Abstract:

The nervous system is made up of complex delicate structures such as the spinal cord, peripheral nerves and the brain. These are prone to various types of injury ranging from neurodegenerative diseases to trauma leading to diseases like Parkinson's, Alzheimer's, multiple sclerosis, amyotrophic lateral sclerosis (ALS), multiple system atrophy etc. Unfortunately, because of the complicated structure of nervous system, spontaneous regeneration, repair and healing is seldom seen due to which brain damage, peripheral nerve damage and paralysis from spinal cord injury are often permanent and incapacitating. Hence, innovative and standardized approach is required for advance treatment of neurological injury. Nigella sativa (N. sativa), an annual flowering plant native to regions of southern Europe and Asia; has been suggested to have neuroprotective and anti-seizures properties. Neuroregeneration is found to occur in damaged cells when treated using extract of N. sativa. Due to its proven health benefits, lots of experiments are being conducted to extract all the benefits from the plant. The flowers are delicate and are usually pale blue and white in color with small black seeds. These seeds are the source of active components such as 30–40% fixed oils, 0.5–1.5% essential oils, pharmacologically active components containing thymoquinone (TQ), ditimoquinone (DTQ) and nigellin. In traditional medicine, this herb was identified to have healing properties and was extensively used Middle East and Far East for treating diseases such as head ache, back pain, asthma, infections, dysentery, hypertension, obesity and gastrointestinal problems. Literature studies have confirmed the extract of N. sativa seeds and TQ have inhibitory effects on inducible nitric oxide synthase and production of nitric oxide as well as anti-inflammatory and anticancer activities. Experimental investigation will be conducted to understand which ingredient of N. sativa causes neuroregeneration and roots to its healing property. An aqueous/ alcoholic extract of N. sativa will be made. Seed oil is also found to have used by researchers to prepare such extracts. For the alcoholic extracts, the seeds need to be powdered and soaked in alcohol for a period of time and the alcohol must be evaporated using rotary evaporator. For aqueous extracts, the powder must be dissolved in distilled water to obtain a pure extract. The mobile phase will be the extract while the suitable stationary phase (substance that is a good adsorbent e.g. silica gels, alumina, cellulose etc.) will be selected. Different ingredients of N. sativa will be separated using High Performance Liquid Chromatography (HPLC) for treating damaged cells. Damaged brain cells will be treated individually and in different combinations of 2 or 3 compounds for different intervals of time. The most suitable compound or a combination of compounds for the regeneration of cells will be determined using DOE methodology. Later the gene will also be determined and using Polymerase Chain Reaction (PCR) it will be replicated in a plasmid vector. This plasmid vector shall be inserted in the brain of the organism used and replicated within. The gene insertion can also be done by the gene gun method. The gene in question can be coated on a micro bullet of tungsten and bombarded in the area of interest and gene replication and coding shall be studied. Investigation on whether the gene replicates in the organism or not will be examined.

Keywords: black cumin, brain cells, damage, extract, neuroregeneration, PCR, plasmids, vectors

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Authors: Jayanwita Sarkar, Usha Chakraborty, Bishwanath Chakraborty

Abstract:

Plants are constantly interacting with various abiotic and biotic stresses. In changing climate scenario plants are continuously modifying physiological processes to adapt to changing environmental conditions which profoundly affect plant-pathogen interactions. Spot blotch in wheat is a fast-rising disease in the warmer plains of South Asia where the rise in minimum average temperature over most of the year already affecting wheat production. Hence, the study was undertaken to explore the role of elevated temperature in spot blotch disease development and modulation of antioxidative responses by plant growth promoting rhizobacteria (PGPR) for biocontrol of spot blotch at high temperature. Elevated temperature significantly increases the susceptibility of wheat plants to spot blotch causing pathogen Bipolaris sorokiniana. Two PGPR Bacillus safensis (W10) and Ochrobactrum pseudogrignonense (IP8) isolated from wheat (Triticum aestivum L.) and blady grass (Imperata cylindrical L.) rhizophere respectively, showing in vitro antagonistic activity against Bipolaris sorokiniana were tested for growth promotion and induction of resistance against spot blotch in wheat. GC-MS analysis showed that Bacillus safensis (W10) and Ochrobactrum pseudogrignonense (IP8) produced antifungal and antimicrobial compounds in culture. Seed priming with these two bacteria significantly increase growth, modulate antioxidative signaling and induce resistance and eventually reduce disease incidence in wheat plants at optimum as well as elevated temperature which was further confirmed by indirect immunofluorescence assay using polyclonal antibody raised against Bipolaris sorokiniana. Application of the PGPR led to enhancement in activities of plant defense enzymes- phenylalanine ammonia lyase, peroxidase, chitinase and β-1,3 glucanase in infected leaves. Immunolocalization of chitinase and β-1,3 glucanase in PGPR primed and pathogen inoculated leaf tissue was further confirmed by transmission electron microscopy using PAb of chitinase, β-1,3 glucanase and gold labelled conjugates. Activity of ascorbate-glutathione redox cycle related enzymes such as ascorbate peroxidase, superoxide dismutase and glutathione reductase along with antioxidants such as carotenoids, glutathione and ascorbate and osmolytes like proline and glycine betain accumulation were also increased during disease development in PGPR primed plant in comparison to unprimed plants at high temperature. Real-time PCR analysis revealed enhanced expression of defense genes- chalcone synthase and phenyl alanineammonia lyase. Over expression of heat shock proteins like HSP 70, small HSP 26.3 and heat shock factor HsfA3 in PGPR primed plants effectively protect plants against spot blotch infection at elevated temperature as compared with control plants. Our results revealed dynamic biochemical cross talk between elevated temperature and spot blotch disease development and furthermore highlight PGPR mediated array of antioxidative and molecular alterations responsible for induction of resistance against spot blotch disease at elevated temperature which seems to be associated with up-regulation of defense genes, heat shock proteins and heat shock factors, less ROS production, membrane damage, increased expression of redox enzymes and accumulation of osmolytes and antioxidants.

Keywords: antioxidative enzymes, defense enzymes, elevated temperature, heat shock proteins, PGPR, Real-Time PCR, spot blotch, wheat

Procedia PDF Downloads 171

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

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Authors: Sajeli A. Begum, Ameer Basha, Kirti Hira, Rukaiyya Khan

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Cyclic dipeptides are simple diketopiperazine derivatives being investigated by several scientists for their biological effects which include anticancer, antimicrobial, haematological, anticonvulsant, immunomodulatory effect, etc. They are potentially active microbial metabolites having been synthesized too, for developing into drug candidates. Cultures of Pseudomonas species have earlier been reported to produce cyclic dipeptides, helping in quorum sensing signals and bacterial–host colonization phenomena during infections, causing cell anti-proliferation and immunosuppression. Fluorescing Pseudomonas species have been identified to secrete lipid derivatives, peptides, pyrroles, phenazines, indoles, aminoacids, pterines, pseudomonic acids and some antibiotics. In the present work, results of investigation on the cyclic dipeptide metabolites secreted by the culture broth of Pseudomonas species as potent pro-inflammatory cytokine inhibitors are discussed. The bacterial strain was isolated from the rhizospheric soil of groundnut crop and identified as Pseudomonas aeruginosa by 16S rDNA sequence (GenBank Accession No. KT625586). Culture broth of this strain was prepared by inoculating into King’s B broth and incubating at 30 ºC for 7 days. The ethyl acetate extract of culture broth was prepared and lyophilized to get a dry residue (EEPA). Lipopolysaccharide (LPS)-induced ELISA assay proved the inhibition of tumor necrosis factor-alpha (TNF-α) secretion in culture supernatant of RAW 264.7 cells by EEPA (IC50 38.8 μg/mL). The effect of oral administration of EEPA on plasma TNF-α level in rats was tested by ELISA kit. The LPS mediated plasma TNF-α level was reduced to 45% with 125 mg/kg dose of EEPA. Isolation of the chemical constituents of EEPA through column chromatography yielded ten cyclic dipeptides, which were characterized using nuclear magnetic resonance and mass spectroscopic techniques. These cyclic dipeptides are biosynthesized in microorganisms by multifunctional assembly of non-ribosomal peptide synthases and cyclic dipeptide synthase. Cyclo (Gly-L-Pro) was found to be more potentially (IC50 value 4.5 μg/mL) inhibiting TNF-α production followed by cyclo (trans-4-hydroxy-L-Pro-L-Phe) (IC50 value 14.2 μg/mL) and the effect was equal to that of standard immunosuppressant drug, prednisolone. Further, the effect was analyzed by determining mRNA expression of TNF-α in LPS-stimulated RAW 264.7 macrophages using quantitative real-time reverse transcription polymerase chain reaction. EEPA and isolated cyclic dipeptides demonstrated diminution of TNF-α mRNA expression levels in a dose-dependent manner under the tested conditions. Also, they were found to control the expression of other pro-inflammatory cytokines like IL-1β and IL-6, when tested through their mRNA expression levels in LPS-stimulated RAW 264.7 macrophages under LPS-stimulated conditions. In addition, significant inhibition effect was found on Nitric oxide production. Further all the compounds exhibited weak toxicity to LPS-induced RAW 264.7 cells. Thus the outcome of the study disclosed the effectiveness of EEPA and the isolated cyclic dipeptides in down-regulating key cytokines involved in pathophysiology of autoimmune diseases.In another study led by the investigators, microbial cyclic dipeptides were found to exhibit excellent antimicrobial effect against Fusarium moniliforme which is an important causative agent of Sorghum grain mold disease. Thus, cyclic dipeptides are emerging small molecular drug candidates for various autoimmune diseases.

Keywords: cyclic dipeptides, cytokines, Fusarium moniliforme, Pseudomonas, TNF-alpha

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Authors: Kuladip Jana, Bhaswati Banerjee, Parimal C. Sen

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 along with carcinogenesis in skin, prostate, ovary, lung and mammary glands. Our study was focused on elucidating the molecular mechanism of B(a)P induced male reproductive toxicity and its prevention with phytochemical like resveratrol. 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 Wistar 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 Reactive Oxygen Species (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 p38 mitogen activated protein kinase (p38MAPK), cytosolic translocation of cytochrome-c, upregulation of Bax and inducible nitric oxide synthase (iNOS) with cleavage of poly ADP ribose polymerase (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 steroidogenic acute regulatory protein (StAR), cytochrome P450 IIA1 (CYPIIA1), 3β hydroxy steroid dehydrogenase (3β HSD), 17β hydroxy steroid dehydrogenase (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. Next we investigated the role of resveratrol on B(a)P induced male reproductive toxicity. Our study highlighted that resveratrol co-treatment with B(a)P maintained testicular redox potential, increased serum testosterone level and prevented steroidogenic dysfunction with enhanced expression of major testicular steroidogenic proteins (CYPIIA1, StAR, 3β HSD,17β HSD) relative to treatment with B(a)P only. Resveratrol suppressed B(a)P-induced testicular activation of p38 MAPK, ATF2, iNOS and ROS production; cytosolic translocation of Cytochome c and Caspase 3 activation thereby prevented oxidative stress of testis and inhibited apoptosis. Resveratrol co-treatment also decreased B(a)P-induced AhR protein level, its nuclear translocation and subsequent CYP1A1 promoter activation, thereby decreased protein and mRNA levels of testicular cytochrome P4501A1 (CYP1A1) and prevented BPDE-DNA adduct formation. Our findings cumulatively suggest that resveratrol prevents activation of B(a)P by modulating the transcriptional regulation of CYP1A1 and acting as an antioxidant thus prevents B(a)P-induced oxidative stress and testicular apoptosis.

Keywords: benzo(a)pyrene, resveratrol, testis, apoptosis, cytochrome P450 1A1 (CYP1A1), aryl hydrocarbon receptor (AhR), p38 MAPK/ATF2/iNOS

Procedia PDF Downloads 232