Search results for: GFP-LC3 dot formation assay

Authors: Gulcan Sahal, Isil Seyis Bilkay

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Candida species which often exist as commensal microorganisms in healthy individuals are major causes of important infections, especially in AIDS and immunocompromised patients, by means of their biofilm formation abilities. Therefore, in this study, determination of biofilm formation in different clinical strains of Candida species, investigation of strong biofilm forming Candida strains, examination of clinical information of each strong and weak biofilm forming Candida strains and investigation of some plant substances’ effects on biofilm formation of strong biofilm forming strains were aimed. In this respect, biofilm formation of Candida strains was analyzed via crystal violet binding assay. According to our results, biofilm levels of strains belong to different Candida species were different from each other. Additionally, it is also found that some plant substances effect biofilm formation. All these results indicate that, as well as C. albicans strains, other non-albicans Candida species also emerge as causative agents of infections and have biofilm formation abilities. In addition, usage of some plant substances in different concentrations may provide a new treatment against biofilm related Candida infections.

Keywords: anti-biofilm, biofilm formation, Candida species, biosystems engineering

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Authors: Gulcan Sahal, Nermin Hande Avcioglu, Isil Seyis Bilkay

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Klebsiella species which are natural colonizers of human upper respiratory and human gastrointestinal tracts are also responsible for every reoccurring nosocomial infections by means of having ability to form slimy layers known as biofilm on many surfaces. Therefore, in this study, investigation of biofilm formation in K. pneumoniae and K. rhinoscleromatis and examination of each Klebsiella strains’ clinical information in the light of their biofilm formation results were aimed. In this respect, biofilm formation of Klebsiella strains was analyzed via crystal violet binding assay. According to our results, biofilm formation levels of K. pneumoniae and K. rhinoscleromatis strains were different from each other. Additionally, in comparison to K. rhinoscleromatis strains, K. pneumoniae was observed to include higher amounts of strong biofilm forming strains. Besides, it was also seen that clinical information of patients from which strong biofilm forming Klebsiella strains were isolated were similar to each other. Our results indicate that there should be more precautions against K. pneumoniae which includes higher amount of strong biofilm forming strains.

Keywords: biofilm formation, Klebsiella pneumoniae, Klebsiella rhinoscleromatis, biosystems engineering

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Authors: Gulcan Sahal, Behzad Nasseri, Ali Akbar Ebrahimi, Isil Seyis Bilkay

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Proteus mirabilis strains which are natural colonizers of healthy individuals’ gastrointestinal tract are also known as common causes of catheter-associated urinary tract infections. Nowadays, as a result of an increased resistance to various antimicrobial drugs, there has been a growing interest in natural products. Therefore, the aim of this study is to investigate biofilm formation of P. mirabilis strains on lemongrass oil containing polycaprolactone nanofibers. Polycaprolactone nanofibers with different lemongrass oil concentrations were successfully prepared by electrospinning and biofilm formation of P. mirabilis on these nanofibers were determined by ‘Crystal Violet Staining Assay’. According to our results, polycaprolactone nanofibers with some lemongrass oil concentrations, decreased biofilm formation of P. mirabilis and this effect increased in parallel with the increase in lemongrass oil concentration. Our results indicate that, polycaprolactone nanofibers with some concentrations of lemongrass oil may provide a treatment against catheter-associated urinary tract infections by means of causing an inhibition on biofilm formation of P. mirabilis.

Keywords: anti-biofilm, biofilm formation, essential oils, nanofibers, proteus mirabilis

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Authors: Azna Zuberi

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Biofilm is a sessile bacterial accretion in which bacteria adapts different physiological and morphological behavior from planktonic form. It is the root cause of about 80% microbial infections in human. Among them, E. coli biofilms are most prevalent in medical devices associated nosocomial infections. The objective of this study was to inhibit biofilm formation by targeting LuxS gene, involved in quorum sensing using CRISPRi. luxS is a synthase, involved in the synthesis of Autoinducer-2(AI-2), which in turn guides the initial stage of biofilm formation. To implement CRISPRi system, we have synthesized complementary sgRNA to target gene sequence and co-expressed with dCas9. Suppression of luxS was confirmed through qRT-PCR. The effect of luxS gene on biofilm inhibition was studied through crystal violet assay, XTT reduction assay and scanning electron microscopy. We conclude that CRISPRi system could be a potential strategy to inhibit bacterial biofilm through mechanism base approach.

Keywords: biofilm, CRISPRi, luxS, microbial

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Authors: Benjaporn Buranrat, Nootchanat Mairuae

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Cratoxylum formosum (Jack) Dyer (CF) has been used for the traditional medicines in South East Asian and Thailand. Normally, northeast Thai vegetables have proven cytotoxic to many cancer cells. Therefore, the present study aims to explore the molecular mechanisms underlying CF-induced cancer cell death and apoptosis on breast and liver cancer cells. The cytotoxicity and antiproliferative effects of CF on the human breast MCF-7 and liver HepG2 cancer cell lines were evaluated using sulforhodamine B assay and colony formation assay. Cell migration assay was measured using wound healing assay. The apoptosis induction mechanisms were investigated through reactive oxygen species formation, caspase 3 activity, and JC-1 activity. Gene expression by real-time PCR and apoptosis related protein levels by Western blot analysis. CF induced MCF-7 and HepG2 cell death by time- and dose-dependent manner. Furthermore, CF had the greater cytotoxic potency on MCF-7 more than HepG2 cells with IC50 values of 85.70+4.52 μM and 219.03±9.96 μM respectively, at 24 h. Treatment with CF also caused a dose-dependent decrease in colony forming ability and cell migration, especially on MCF-7 cells. CF induced ROS formation, increased caspase 3 activities, and decreased the mitochondrial membrane potential, and causing apoptotic body production and DNA fragmentation. CF significantly decreased expression of the cell cycle regulatory protein RAC1 and downstream proteins, cdk6. Additionally, CF enhanced p21 and reduced cyclin D1 protein levels. CF leaf extract induced cell death, apoptosis, antimigration in both of MCF-7 and HepG2 cells. CF could be useful for developing to anticancer drug candidate for breast and liver cancer therapy.

Keywords: cratoxylum formosum (jack) dyer, breast cancer, liver cancer, cell death

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Authors: Dalia Jomehpour, Sara Sheikhlary, Esmaeil Heydari, Mohammad Hossien Majles Ara

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In this study, we report fulvic acid-coated iron oxide nanoparticles of 10.7 ± 2.7 nm size, which serve to inhibit amyloid fibrillation formation. Although the effect of fulvic acid on tau fibrils was investigated, to our best knowledge, its inhibitory impacts on amyloid aggregation formation have been assessed neither in-vitro nor in-vivo. On the other hand, iron oxide nanoparticles exhibit anti-amyloid activity on their own. This study investigates the inhibitory effect of fulvic acid coated iron oxide nanoparticles on amyloid aggregations formed from the commonly used in-vitro model, lysozyme from chicken egg white. FESEM, XRD, and FTIR characterization confirmed that fulvic acid was coated onto the surface of the nanoparticles. The inhibitory effects of the fulvic acid coated iron oxide nanoparticles were verified by Thioflavin T assay, circular dichroism (CD), and FESEM analysis. Furthermore, the toxicity of the nanoparticles on the neuroblastoma SH-SY5Y human cell line was assessed through an MTT assay. Our results indicate that fulvic acid coated iron oxide nanoparticles can efficiently inhibit the formation of amyloid aggregations while exhibiting negligible in-vitro toxicity; thus, they can be used as anti-amyloid agents in the development of the potential drug for neurodegenerative diseases.

Keywords: Alzheimer’s disease, fulvic acid coated iron oxide nanoparticles, fulvic acid, amyloid inhibitor, polyphenols

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Authors: Siobhan O’Shea, Sangeetha Vijaysri Nair, Hee Cheol Kim, Charles Thomas Nugent, Cheuk Yan William Tong, Sam Douthwaite, Andrew Worlock

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The Aptima® HIV-1 Quant Dx Assay is a fully automated assay on the Panther system. It is based on Transcription-Mediated Amplification and real time detection technologies. This assay is intended for monitoring HIV-1 viral load in plasma specimens and for the detection of HIV-1 in plasma and serum specimens. Nine-hundred and seventy nine specimens selected at random from routine testing at St Thomas’ Hospital, London were anonymised and used to compare the performance of the Aptima HIV-1 Quant Dx assay and Roche COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Test, v2.0. Two-hundred and thirty four specimens gave quantitative HIV-1 viral load results in both assays. The quantitative results reported by the Aptima Assay were comparable those reported by the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, v2.0 with a linear regression slope of 1.04 and an intercept on -0.097. The Aptima assay detected HIV-1 in more samples than the Roche assay. This was not due to lack of specificity of the Aptima assay because this assay gave 99.83% specificity on testing plasma specimens from 600 HIV-1 negative individuals. To understand the reason for this higher detection rate a side-by-side comparison of low level panels made from the HIV-1 3rd international standard (NIBSC10/152) and clinical samples of various subtypes were tested in both assays. The Aptima assay was more sensitive than the Roche assay. The good sensitivity, specificity and agreement with other commercial assays make the HIV-1 Quant Dx Assay appropriate for both viral load monitoring and detection of HIV-1 infections.

Keywords: HIV viral load, Aptima, Roche, Panther system

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Authors: Fadime Eroglu

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Acanthamoeba genus belongs to kingdom protozoa, and it is known as free-living amoebae. Acanthamoeba genus has been isolated from human bodies, swimming pools, bottled mineral water, contact lens solutions, dust, and soil. The members of the genus Acanthamoeba causes Acanthamoeba Keratitis which is a painful sight-threatening disease of the eyes. In recent years, the prevalence of Acanthamoeba keratitis has been high rate reported. The eight different Acanthamoeba species are known to be effective in Acanthamoeba keratitis. These species are Acanthamoeba castellanii, Acanthamoeba polyphaga, Acanthamoeba griffini, Acanthamoeba hatchetti, Acanthamoeba culbertsoni and Acanhtamoeba rhysodes. The conventional diagnosis of Acanthamoeba Keratitis has relied on cytological preparations and growth of Acanthamoeba in culture. However molecular methods such as real-time PCR has been found to be more sensitive. The real-time PCR has now emerged as an effective method for more rapid testing for the diagnosis of infectious disease in decade. Therefore, a real-time PCR assay for the detection of Acanthamoeba keratitis and Acanthamoeba species have been developed in this study. The 18S rRNA sequences from Acanthamoeba species were obtained from National Center for Biotechnology Information and sequences were aligned with MEGA 6 programme. Primers and probe were designed using Custom Primers-OligoPerfectTMDesigner (ThermoFisherScientific, Waltham, MA, USA). They were also assayed for hairpin formation and degree of primer-dimer formation with Multiple Primer Analyzer ( ThermoFisherScientific, Watham, MA, USA). The eight different ATCC Acanthamoeba species were obtained, and DNA was extracted using the Qiagen Mini DNA extraction kit (Qiagen, Hilden, Germany). The DNA of Acanthamoeba species were analyzed using newly designed primer and probe set in real-time PCR assay. The early definitive laboratory diagnosis of Acanthamoeba Keratitis and the rapid initiation of suitable therapy is necessary for clinical prognosis. The results of the study have been showed that new primer and probes could be used for detection and distinguish for Acanthamoeba species. These new developing methods are helpful for diagnosis of Acanthamoeba Keratitis.

Keywords: Acathamoeba Keratitis, Acanthamoeba species, fast diagnosis, Real-Time PCR

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Authors: P. Singh, R. M. Banik

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Artificial neural network (ANN) was employed to optimize assay parameters viz., time, temperature, pH of reaction mixture, enzyme volume and substrate concentration of L-glutaminase from Bacillus cereus MTCC 1305. ANN model showed high value of coefficient of determination (0.9999), low value of root mean square error (0.6697) and low value of absolute average deviation. A multilayer perceptron neural network trained with an error back-propagation algorithm was incorporated for developing a predictive model and its topology was obtained as 5-3-1 after applying Levenberg Marquardt (LM) training algorithm. The predicted activity of L-glutaminase was obtained as 633.7349 U/l by considering optimum assay parameters, viz., pH of reaction mixture (7.5), reaction time (20 minutes), incubation temperature (35˚C), substrate concentration (40mM), and enzyme volume (0.5ml). The predicted data was verified by running experiment at simulated optimum assay condition and activity was obtained as 634.00 U/l. The application of ANN model for optimization of assay conditions improved the activity of L-glutaminase by 1.499 fold.

Keywords: Bacillus cereus, L-glutaminase, assay parameters, artificial neural network

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Authors: Z. Kadivar, M. Vahidinia, A. Mousavinia

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Kopet-Dagh Mountain Range is located in the north and northeast of Iran. Mozduran Formation in the east of Kopet-Dagh is mainly composed of limestone, dolomite, with shale and sandstone interbedded. Mozduran Formation is reservoir rock of the Khangiran gas field. The location of the study was east Kopet-Dagh basin (Northeast Iran) where the deliberate thickness of formation is 418 meters. In the present study, a total of 57 samples were gathered. Moreover, 100 thin sections were made out of 52 samples. According to the findings of the thin section study, 18 genera and nine species of foraminifera and algae were identified. Based on the index fossils, the age of the Mozduran Formation was identified as Upper Jurassic (Kimmerdgian-Tithonian) in the east of Kopet-Dagh basin. According to the magnetite data (total intensity and RTP map), there is a disconformity (low intensity) between the Kashaf-Rood Formation and Mozduran Formation. At the top, where among Mozduran Formation and Shurijeh Formation, is high intensity and a widespread disconformity (high intensity).

Keywords: upper jurassic, magnetometre, mozduran formation, stratigraphy

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Authors: Shabnam Abdi, Behzad Toosi

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Background: Melanoma is the most lethal type of malignant skin cancer in humans and dogs since it spreads rapidly throughout the body. Despite significant advances in treatment, cancer at an advanced stage has a poor prognosis. Hence, more effective treatments are needed to enhance outcomes with fewer side effects. Erythropoietin-producing hepatocellular receptors are the largest family of receptor tyrosine kinases and are divided into two subfamilies, EphA and EphB, both of which play a significant role in disease, especially cancer. Due to their association with proliferation and invasion in many aggressive types of cancer, Eph receptor tyrosine kinases (Eph RTKs) are promising cancer therapy molecules. Because these receptors have not been studied in canine melanoma, we investigated how EphA2 influences survival and tumorigenicity of melanoma cells. Methods: Expression of EphA2 protein in canine melanoma cell lines and human melanoma cell line was evaluated by Western blot. Melanoma cells were transduced with lentiviral particles encoding Eph-targeting shRNAs or non-silencing shRNAs (control) for silencing the expression of EphA2 receptor, and silencing was confirmed by Western blotting and immunofluorescence. The effect of siRNA treatment on cellular proliferation, colony formation, tumorsphere assay, invasion was analyzed by Resazurin assay Matrigel invasion assay, respectively. Results: Expression of EphA2 was detected in canine and human melanoma cell lines. Moreover, stably silencing EphA2 by specific shRNAs significantly and consistently decreased the expression of EphA2 protein in both human and canine melanoma cells. Proliferation, colony formation, tumorsphere and invasion of melanoma cells were significantly decreased in EphA2 siRNA-treated cells compared to control. Conclusion: Our data provide the first functional evidence that the EphA2 receptor plays a critical role in the malignant cellular behavior of melanoma in both human and dogs.

Keywords: ephA2, targeting, melanoma, human, canine

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Authors: Cheng-Cao Sun, Shu-Jun Li, De-Jia Li

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Through analysis of a published micro-array-based high-throughput assessment, we discovered that miR-1275 was markedly down-regulated in nasopharyngeal carcinoma (NPC) tissues. However, little is known about its effect and mechanism involved in NPC development and progression. In this study, we investigated the role of miR-1275 on the development of NPC. The results indicated that miR-1275 was significantly down-regulated in primary NPC tissues, and very low levels were found in NPC cell lines. Ectopic expression of miR-1275 in NPC cell lines significantly suppressed cell growth as evidenced by cell viability assay and colony formation assay, through inhibition of HOXB5. In addition, miR-1275 suppresses G1/S transition through inhibition of HOXB5. Further, oncogene HOXB5 was revealed to be a putative target of miR-1275, which was inversely correlated with miR-1275 expression in NPC. Collectively, our study demonstrates that as a tumor suppressor, miR-1275 played a pivotal role on NPC through inhibiting cell proliferation, and suppressing G1/S transition by targeting oncogenic HOXB5.

Keywords: microRNA-1275 (miR-1275), HOXB5, nasopharyngeal carcinoma, proliferation

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Authors: Changhan Ouyang, Zhonglin Xie

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In response to proatherosclerotic factors such as oxidized lipids, or to therapeutic interventions such as angioplasty, stents, or bypass surgery, vascular smooth muscle cells (VSMCs) migrate from the media to the intima, resulting in intimal hyperplasia, restenosis, graft failure, or atherosclerosis. These proatherosclerotic factors also activate autophagy in VSMCs. However, the functional role of autophagy in vascular health and disease remains poorly understood. In the present study, we determined the role of autophagy in the regulation of VSMC migration. Autophagy activity in cultured human aortic smooth muscle cells (HASMCs) and mouse carotid arteries was measured by Western blot analysis of microtubule-associated protein 1 light chain 3 B (LC3B) and P62. The VSMC migration was determined by scratch wound assay and transwell migration assay. Ex vivo smooth muscle cell migration was determined using aortic ring assay. The in vivo SMC migration was examined by staining the carotid artery sections with smooth muscle alpha actin (alpha SMA) after carotid artery ligation. To examine the relationship between autophagy and neointimal hyperplasia, C57BL/6J mice were subjected to carotid artery ligation. Seven days after injury, protein levels of Atg5, Atg7, Beclin1, and LC3B drastically increased and remained higher in the injured arteries three weeks after the injury. In parallel with the activation of autophagy, vascular injury-induced neointimal hyperplasia as estimated by increased intima/media ratio. The en face staining of carotid artery showed that vascular injury enhanced alpha SMA staining in the intimal cells as compared with the sham operation. Treatment of HASMCs with platelet-derived growth factor (PDGF), one of the major factors for vascular remodeling in response to vascular injury, increased Atg7 and LC3 II protein levels and enhanced autophagosome formation. In addition, aortic ring assay demonstrated that PDGF treated aortic rings displayed an increase in neovessel formation compared with control rings. Whole mount staining for CD31 and alpha SMA in PDGF treated neovessels revealed that the neovessel structures were stained by alpha SMA but not CD31. In contrast, pharmacological and genetic suppression of autophagy inhibits VSMC migration. Especially, gene silencing of Atg7 inhibited VSMC migration induced by PDGF. Furthermore, three weeks after ligation, markedly decreased neointimal formation was found in mice treated with chloroquine, an inhibitor of autophagy. Quantitative morphometric analysis of the injured vessels revealed a marked reduction in the intima/media ratio in the mice treated with chloroquine. Conclusion: Autophagy activation increases VSMC migration while autophagy suppression inhibits VSMC migration. These findings suggest that autophagy suppression may be an important therapeutic strategy for atherosclerosis and intimal hyperplasia.

Keywords: autophagy, vascular smooth muscle cell, migration, neointimal formation

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Authors: Hadda Krarcha, S. Messaasdi

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In order to investigate Hafnium transition metal alloys HfM (M= Co, Ir, Os,Pt, Rh, Ru) phase diagrams in the region of 50/50% atomic ratio, we performed ab initio Full-Potential Linearized Augmented Plane Waves calculations of the enthalpies of formation of HfM compounds at B2 (CsCl) structure type. The obtained enthalpies of formation are discussed and compared to some of the existing models and available experimental data.

Keywords: enthalpy of formation, transition metal, binarry compunds, hafnium

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Authors: Puchita Chokcharoenying

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Polyphenols are the most abundant antioxidants found in plants, and they are highly effective at scavenging oxidative free radicals. Antioxidants are substances found in medicinal plants to help prevent heart disease, stroke, and some cancers. This study focused on evaluating the flavonoids content of Chrysanthemum Indicum and determine their antioxidant capacity by using DPPH and ABTS radical scavenging capacity assay. The total flavonoid content of C. indicumextract was determined and expressed as quercetin equivalents (QE)/g measured by an aluminiumchloride colorimetric method. The results showed that the IC50 of C. indicum extract were 83.57μg/mL ± 0.875 and52.57μg/mL ± 0.632for DPPH and ABTS, respectively. C. indicumextract exhibited antioxidant activities as a concentration dependent manner. In the DPPH assay, vitamin C was used as a positive control, whereas Trolox was used as a positive control in the ABTS assay. In summary, C. indicum extract is rich in flavonoids, which have potent antioxidant properties. Thus, C. indicum extract is a good source of antioxidants and can be developed for medicinal purposes. Nevertheless, more research on the antioxidant activity of C. indicum extract and in vivo antioxidant studies are still needed.

Keywords: ABTS assay, antioxidant, chrysanthemum indicum, DPPH assay, total flavonoid content

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Authors: Ajay Pratap Singh Lodhi, Deepak Kumar

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Vegetable oil-based environmentally friendly metalworking fluids (MWFs) are formulated. The tribological performance, cytotoxicity, and corrosion resistance of the formulated fluids (FFs) are evaluated and benchmarked with commercial mineral oil-based MWFs (CF). Results show that FFs exhibited better machining characteristics (roughness, cutting forces, and surface morphology) during machining than CF. MTT assay and Live dead cell assay confirm the cytocompatibility nature of the FFs relative to the toxic CF. Electrochemical analysis shows that FFs and CF exhibited comparable corrosion current density.

Keywords: corrosion inhibitors, cytotoxicity, machining, MTT assay, Taguchi method, vegetable oil

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Authors: Dawit Gebreegzabher Hagos, Yazezew Kebede Kiro, Mahmud Abdulkader, Henk H. D. F. Schallig, Dawit Wolday

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Rapid and accurate visceral leishmaniasis (VL) diagnosis is needed to initiate prompt treatment to reduce morbidity and mortality. Here, we evaluated the performance of loop-mediated isothermal amplification (LAMP) assay for the diagnosis of VL from blood in an endemic area in Ethiopia. LAMP was positive in 117/122 confirmed VL cases and negative in 149/152 controls, resulting in a sensitivity of 95.9% (95% CI: 90.69–98.66) and a specificity of 98.0% (95% CI: 94.34–99.59), respectively. The sensitivity of the LAMP assay was 95.0% (95% CI: 88.61–98.34) in HIV-negatives and 100% (95% CI: 85.18–100.0) in HIV-positives. Compared with microscopy, LAMP detected 82/87 (94.3%, 95% CI: 87.10–98.11) of the microscopy1 cases and was negative in 11/27 (40.7%, 95% CI: 22.39–61.20) of the microscopy2 cases. Compared with the rK39 serology, LAMP detected 113/120 (94.2%, 95% CI: 88.35–97.62) of the rK391 cases and was negative in 149/154 (96.8%, 95% CI: 92.59–98.94) of the rK392 cases. However, when compared with microscopy only, rK39 detected 83/87 (95.4%, 95% CI: 88.64–98.73) of the microscopy1 cases and negative in only 12/27 (44.4%, 95% CI: 25.48–64.67) of the microscopy– cases. There was an excellent agreement between rK39 and LAMP (Kappa 5 0.91, 95% CI: 0.86–0.96). Furthermore, an algorithm using rK39 followed by LAMP would yield a sensitivity of 99.2% (95%CI: 95.52–99.89) and a specificity of 98.0% (95% CI: 94.34–99.59). The findings demonstrate that the LAMP assay is an accurate and rapid molecular assay for VL diagnosis, including in HIV-1 co-infected patients, in an endemic setting.

Keywords: visceral leishmaniasis, HIV, diagnosis, LAMP, Ethiopia

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Authors: Jirathan Pongchababnapa

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Polyphenols are the most common antioxidant found in plants and are efficient in capturing oxidative free radicals. Antioxidants are substances found in medicinal plants which may have a protective role to play in certain conditions such as heart disease, stroke and some cancers. By relying on these benefits, we have traced out the presence of antioxidant in Camellia sinensis leaves extract. This study aims to evaluate flavonoids content in C. sinensisextract and investigate antioxidant activities by using DPPH and ABTS radical scavenging capacity assay. The total flavonoid content of C. Sinensis extract was determined and expressed as quercetin equivalents (QE)/g measured by the aluminum chloride colorimetric method. The results showed that the IC₅₀ of C. Sinensis leaves extract were 40.90 μg/mL ± 0.755 and32.96 μg/mL ± 0.679 for DPPH and ABTS, respectively. C. Sinensis extract at increasing concentration showed antioxidant activities as a concentration dependent manner. In the DPPH assay, vitamin C was used as a positive control, whereas Trolox was used as a positive control in the ABTS assay. In conclusion, C. Sinensis extract consisted of a high amount of flavonoids content which possesses potent antioxidant activity. However, further investigation on the identification of pure compound of this plant and molecular antioxidant assays are still required.

Keywords: ABTS assay, antioxidant, camellia sinensis, DPPH assay, total flavonoid content

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Authors: Tawqeer Ali Syed, Prakash Chandra

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This study aims to evaluate the antileishmanial activity of Nigella sativa black seed extract. This well-known plant extract was taken from the botanical garden of Kashmir. Materials and Methods: The methanolic extracts of these plants were screened for their antileishmanial activity against Leishmania major using 3‑(4.5‑dimethylthiazol‑2yl)‑2.5‑diphenyltetrazolium bromide assay or MTT assay. Results: The methanolic extract of Nigella sativa showed potential antileishmanial activity at an inhibition% value of 80.29% ± 0.65%. IC 50 was calculated after 48 hours to be 964.3 µg/ml. Conclusion: Considering these results, these medicinal plants from Kashmir could serve as potential drug sources for antileishmanial compounds.

Keywords: MTT assay, antileishmanial, cell viability, Nigella sativa

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Authors: Stanley Dula, Oluwatosini A. Ijabadeniyi

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Biofilm formation is a major source of materials and foodstuffs contamination, contributing to occurrence of pathogenic and spoilage microbes in food processing resulting in food spoilage, transmission of diseases and significant food hygiene and safety issues. This study elucidates biofilm formation of E. coli O157:H7 and L. monocytogenes ATCC 7644 grown under food related environmental stress conditions of varying pH (5.0;7.0; and 8.5) and temperature (15, 25 and 37 ℃). Both strains showed confluent biofilm formation at 25 ℃ and 37 ℃, at pH 8.5 after 5 days. E. coli showed curli fimbriae production at various temperatures, while L. monocytogenes did not show pronounced expression. Swarm, swimming and twitching plate assays were used to determine strain motilities. Characterization of cell hydrophobicity was done using the microbial adhesion to hydrocarbons (MATH) assay using n-hexadecane. Both strains showed hydrophilic characteristics as they fell within a < 20 % interval. FT-IR revealed COOH at 1622 cm-1, and a strong absorption band at 3650 cm-1 – 3200 cm-1 indicating the presence of both -OH and -NH groups. Both strains were hydrophilic and could form biofilm at different combinations of temperature and pH. EPS produced in both species proved to be an acidic hetero-polysaccharide.

Keywords: biofilm, pathogens, hydrophobicity, motility

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Authors: Hoo Cheol Lee, Dae Seung Kim, Sang Myung Jung, Gwang Heum Yoon, Hwa Sung Shin

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Loss of bone tissue can occur due to a bone tissue disease and aging or fracture. Renewable formation of bone is mainly made by its differentiation and metabolism. For this reason, osteoblasts have been studied for regeneration of bone tissue. So, tissue engineering has attracted attention as a recovery means. In tissue engineering, a particularly important factor is a scaffold that supports cell growth. For osteoblast scaffold, we used the cellulose nanowhisker (CNW) extracted from marine organism. CNW is one of an abundant material obtained from a number of plants and animals. CNW is polymer consisting of monomer cellulose and this composition offers biodegradability and biocompatibility to CNW. Mechanical strength of CNW is superior to the existing natural polymers. In addition, substances of marine origin have a low risk of secondary infection by bacteria and pathogen in contrast with those of land-derived. For evaluating its suitability as an osteoblast scaffold, we fabricate CNW film for osteoblast culture and performed the MTT assay and ALP assay to confirm its cytotoxicity and effect on differentiation. Taking together these results, we assessed CNW is a potential candidate of a material for bone tissue regeneration.

Keywords: bone regeneration, cellulose nanowhisker, marine derived material, osteoblast

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Authors: Hadiatullah, T. S. D. Desak Ketut, A. A. Ayu, A. N. Isna, D. P. Ririn

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Spirogyra sp. is genus of microalgae which has a high carbohydrate content that used as a best medium for bacterial fermentation to produce cellulose. This study objective to determine the effect of pulp banana in the fermented paper production process using Spirogyra sp. and characterizing of the paper product. The method includes the production of bacterial cellulose, assay of the effect fermented paper interpolating with banana pulp using Spirogyra sp., and the assay of paper characteristics include gram-mage paper, water assay absorption, thickness, power assay of tensile resistance, assay of tear resistance, density, and organoleptic assay. Experiments were carried out with completely randomized design with a variation of the concentration of sewage treatment in the fermented paper production interpolating banana pulp using Spirogyra sp. Each parameter data to be analyzed by Anova variance that continued by real difference test with an error rate of 5% using the SPSS. Nata production results indicate that different carbon sources (glucose and sugar) did not show any significant differences from cellulose parameters assay. Significantly different results only indicated for the control treatment. Although not significantly different from the addition of a carbon source, sugar showed higher potency to produce high cellulose. Based on characteristic assay of the fermented paper showed that the paper gram-mage indicated that the control treatment without interpolation of a carbon source and a banana pulp have better result than banana pulp interpolation. Results of control gram-mage is 260 gsm that show optimized by cardboard. While on paper gram-mage produced with the banana pulp interpolation is about 120-200 gsm that show optimized by magazine paper and art paper. Based on the density, weight, water absorption assays, and organoleptic assay of paper showing the highest results in the treatment of pulp banana interpolation with sugar source as carbon is 14.28 g/m2, 0.02 g and 0.041 g/cm2.minutes. The conclusion found that paper with nata material interpolating with sugar and banana pulp has the potential formulation to produce super-quality paper.

Keywords: cellulose, fermentation, grammage, paper, Spirogyra sp.

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Authors: Monthon Tangjitmungman

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Numerous diseases have been linked to oxidative stress, in which a disproportion of free radicals in the body leads to tissue or cell damage. Polyphenols are the most abundant antioxidants found in plants, and they are highly effective at scavenging oxidative free radicals. Due to the presence of phenolic compounds in Caesalpinia sappan has been discovered to have antioxidant activity. It has several health benefits, the most important of which is preventing cardiovascular and cancer diseases. This study aimed to determine the phenolic content and antioxidant activity of C. sappan extract using a variety of antioxidant assays. The extract of C. sappan was made using a mixture of solvents (ethyl alcohol: water in ratio 8:2). The total phenolic content of C. sappan extract was determined and expressed as gallic acid equivalents using the Folin-Cioucalteu method (GAE). The antioxidant activity of C. sappan extract was assessed using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay and the ABTS radical scavenging capacity assay. An association was found between antioxidant activity and total phenol content. The antioxidant activity of C. sappan extract was also determined by DPPH and ABTS assays. The IC50 values for C. sappan extract from DPPH and ABTS assays were 54.48 μg/mL ± 0.545 and 25.46 μg/mL ± 0.790, respectively, in the DPPH assay. In the DPPH assay, vitamin C was used as a positive control, whereas Trolox was used as a positive control in the ABTS assay. In conclusion, C. sappan extract contains a high level of total phenolics and exhibits significant antioxidant activity. Nevertheless, more research should be done on the antioxidant activity, such as SOD and ROS scavenging assays and in vivo experiments, to determine whether the compound has antioxidant activity.

Keywords: ABTS assay, antioxidant activity, Caesalpinia sappan, DPPH assays, total phenolic content

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Authors: Ibrahim Aly, Rabab Zalat, Bahaa EL Deen W. El Aswad, Ismail M. Moharm, Basam M. Masoud, Tarek Diab

Abstract:

The objective of the present study was to evaluate the novel nanomagnetic beads based–latex agglutination assay (NMB-LAT) as a simple test for diagnosis of S. haematobium as well as standardize the novel nanomagnetic beads based –ELISA (NMB-ELISA). According to urine examination this study included 85 S. haematobium infected patients, 30 other parasites infected patients and 25 negative control samples. The sensitivity of novel NMB-LAT was 82.4% versus 96.5% and 88.2% for NMB-ELISA and currently used sandwich ELISA respectively. The specificity of NMB-LAT was 83.6% versus 96.3% and 87.3% for NMB-ELISA and currently used sandwich ELISA respectively. In conclusion, the novel NMB-ELISA is a valuable applicable diagnostic technique for diagnosis of human schistosomiasis haematobium. The novel NMB-ELISA assay is a suitable applicable diagnostic method in field survey especially when followed by ELISA as a confirmatory test in query false negative results. Trials are required to increase the sensitivity and specificity of NMB-ELISA assay.

Keywords: diagnosis, iatex agglutination, nanomagnetic beads, sandwich ELISA

Procedia PDF Downloads 342

Authors: Shayma Munqith Al-Baker, Fadhl Ahmed Saeed Al-Gasha’a, Samira Hamid Hanash, Ahmed Ali Al-Hazmi

Abstract:

A total of 200 samples (180 fecal materials and 20 organ samples) were collected from (5 different poultry farms, 10 local poultry shops, 5 houses poultry, 5 Eggs stores shops and 5 hand slaughters centers) in Ibb city, Yemen, 2014. According to morphological, cultural, as well as biochemical characterization and serological tests, 59 29.5% isolates were identified as Salmonella spp. and all Salmonella isolates were categorized by serotype, which comprised of, 37 62.71% Salmonella Typhimurium serovar, 21 35.59%. Salmonella Enteritidis serovar and 11.69% Salmonella Heidelberg serovar. Antibiotic sensitivity test was done for bacterial isolates and the results showed there were clear differences in antibiotic resistant. Antimicrobial susceptibility of the isolates varies as follows: Ofloxacin 79.66%, Ciprofloxacin 67.80%, Colistin 59.32% and Gentamycin 52.54%. All of isolates were resistant to Erythromycin, Penicillin and Lincomycin. Antibacterial activity was done for both aqueous and ethanol extracts of Dodonaea viscosa plant by using well and disc diffusion assay. The results indicated that well diffusion assay had best results than disc diffusion assay, the highest inhibition zone was 22 mm for well diffusion and 15 mm for disc diffusion assay, the results observed that ethanol extract had best antibacterial effect than aqueous extract which the percentage of bacterial isolates affected with ethanol extract was 71.19% comparing with aqueous extract 28.81% by using disc diffusion assay, while the percentage of bacterial isolates affected with ethanol extract was 88.13% comparing with aqueous extract 52.54% by using will diffusion assay.

Keywords: Salmonella spp, Dodonaea viscosa, antimicrobial and salmonellosis

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Authors: Ayman K. El Essawy, Amal M. Hosny, Hala M. Abu Shady

Abstract:

The rapid detection of TB and drug resistance, both optimizes treatment and improves outcomes. In the current study, respiratory specimens were collected from 155 patients. Conventional susceptibility testing and MIC determination were performed for rifampicin (RIF) and isoniazid (INH). Genotype MTBDRplus assay, which is a molecular genetic assay based on the DNA-STRIP technology and specific gene sequencing with primers for rpoB, KatG, and mab-inhA genes were used to detect mutations associated with resistance to rifampicin and isoniazid. In comparison to other categories, most of rifampicin resistant (61.5%) and isoniazid resistant isolates (47.1%) were from patients relapsed in treatment. The genotypic profile (using Genotype MTBDRplus assay) of multi-drug resistant (MDR) isolates showed missing of katG wild type 1 (WT1) band and appearance of mutation band katG MUT2. For isoniazid mono-resistant isolates, 80% showed katG MUT1, 20% showed katG MUT1, and inhA MUT1, 20% showed only inhA MUT1. Accordingly, 100% of isoniazid resistant strains were detected by this assay. Out of 17 resistant strains, 16 had mutation bands for katG distinguished high resistance to isoniazid. The assay could clearly detect rifampicin resistance among 66.7% of MDR isolates that showed mutation band rpoB MUT3 while 33.3% of them were considered as unknown. One mono-resistant rifampicin isolate did not show rifampicin mutation bands by Genotype MTBDRplus assay, but it showed an unexpected mutation in Codon 531 of rpoB by DNA sequence analysis. Rifampicin resistance in this strain could be associated with a mutation in codon 531 of rpoB (based on molecular sequencing), and Genotype MTBDRplus assay could not detect the associated mutation. If the results of Genotype MTBDRplus assay and sequencing were combined, this strain shows hetero-resistance pattern. Gene sequencing of eight selected isolates, previously tested by Genotype MTBDRplus assay, could detect resistance mutations mainly in codon 315 (katG gene), position -15 in inhA promotes gene for isoniazid resistance and codon 531 (rpoB gene) for rifampicin resistance. Genotyping techniques allow distinguishing between recurrent cases of reinfection or reactivation and supports epidemiological studies.

Keywords: M. tuberculosis, rpoB, KatG, inhA, genotype MTBDRplus

Procedia PDF Downloads 115

Authors: Khaled Ramadan Elbeydi

Abstract:

This study is carried out to provide an insight into the analysis of the impact of selected macro-economic variables on gross fixed capital formation in Libya using annual data over the period (1970-2010). The importance of this study comes from the ability to show the relative important factors that impact the Libyan gross fixed capital formation. This understanding would give indications to decision makers on which policy they must focus to stimulate the economy. An Auto-Regressive Distributed Lag (ARDL) modeling process is employed to investigate the impact of the gross domestic product, monetary base, and trade openness on gross fixed capital formation in Libya. The results of this study reveal that there is an equilibrium relationship between capital formation and its determinants. The results also indicate that GDP and trade openness largely explain the pattern of capital formation in Libya. The findings and recommendations provide vital information relevant for policy formulation and implementation aimed to improve capital formation in Libya.

Keywords: ARDL, bounds test, capital formation, co-integration, Libya

Procedia PDF Downloads 327

Authors: Aqeela Ashraf, Muhammad Imran, Tahir Yaqub, Muhammad Tayyab, Yung Fu Chang

Abstract:

For the identification of bovine mastitic pathogen, an economical, rapid and sensitive molecular diagnostic assay is developed by PCR multiplexing of gene and pathogenic species specific DNA sequences. The multiplex PCR assay is developed for detecting nine important bacterial pathogens causing mastitis Worldwide. The bacterial species selected for this study are Streptococcus agalactiae, Streptococcus dysagalactiae, Streptococcus uberis, Staphylococcus aureus, Escherichia coli, Staphylococcus haemolyticus, Staphylococcus chromogenes Mycoplasma bovis and Staphylococcus epidermidis. A single reaction assay was developed and validated by 27 reference strains and further tested on 276 bacterial strains obtained from culturing mastitic milk. The multiplex PCR assay developed here is further evaluated by applying directly on genomic DNA isolated from 200 mastitic milk samples. It is compared with bacterial culturing method and proved to be more sensitive, rapid, economical and can specifically identify 9 bacterial pathogens in a single reaction. It has detected the pathogens in few culture negative mastitic samples. Recognition of disease is the foundation of disease control and prevention. This assay can be very helpful for maintaining the udder health and milk monitoring.

Keywords: multiplex PCR, bacteria, mastitis, milk

Procedia PDF Downloads 293

Authors: Faraj M. Elkhatri, Hana Ellafi

Abstract:

In recent years, there has been a growing recognition of the potential of marine-based functional foods and combination therapies in promoting a healthy lifestyle and exploring their effectiveness in preventing or treating diseases. The combination of marine bioactive compounds or extracts offers synergistic or enhancement effects through various mechanisms, including multi-target actions, improved bioavailability, enhanced bioactivity, and mitigation of potential adverse effects. Both the green-lipped mussel (GLM) and fucoidan derived from brown seaweed are rich in bioactivities. These two, mussel and fucoidan, have not been previously formulated together. This study aims to combine GLM oil from Perna canaliculus with low molecular weight fucoidan (LMWF) extracted from Undaria pinnatifida to investigate the unique mixture’s anti-inflammatory and antioxidant properties. The cytotoxicity of individual compounds and combinations was assessed using the MTT assay in (THP-1 and RAW264.7) cell lines. The anti-inflammatory activity of mussel-fucoidan was evaluated by treating LPS-stimulated human monocyte and macrophage (THP1-1) cells. Subsequently, the inflammatory cytokines released into the supernatant of these cell lines were quantified via ELISA. Antioxidant activity was determined by using the free radical scavenging assay (DPPH). DPPH assay demonstrated that the radical scavenging activity of the combinations, particularly at concentrations exceeding 1 mg/ml, showed a significantly higher percentage of inhibition when compared to the individual component. This suggests an enhancement effect when the two compounds are combined, leading to increased antioxidant activity. In terms of immunomodulatory activity, the individual compounds exhibited distinct behaviors. GLM oil displayed a higher ability to suppress the cytokine TNF- compared to LMWF. Interestingly, the LMWF fraction, when used individually, did not demonstrate TNF- suppression. However, when combined with GLM, the TNF- suppression (anti-inflammatory) activity of the combination was better than GLM or LWMF alone. This observation underscores the potential for enhancement interactions between the two components in terms of anti-inflammatory properties. This study revealed that each individual compound, LMWF, and GLM, possesses unique and notable bioactivity. The combination of these two individual compounds results in an enhancement effect, where the bioactivity of each is enhanced, creating a superior combination. This suggests that the combination of LMWF and GLM has the potential to offer a more potent and multifaceted therapeutic effect, particularly in the context of antioxidant and anti-inflammatory activities. These findings hold promise for the development of novel therapeutic interventions or supplements that harness the enhancement effects.

Keywords: formation damage, porosity loses, pore throat, quartz cement

Procedia PDF Downloads 34

Authors: J. Hidalgo de Quintana, I. Stoner, M. Tackett, G. Doran, C. Rafferty, A. Windemuth, J. Tytell, D. Pregibon

Abstract:

We have developed the Multiplexed Circulating microRNA assay that allows the detection of up to 68 microRNA targets per sample. The assay combines particle­based multiplexing, using patented Firefly hydrogel particles, with single­ step RT-PCR signal. Thus, the Circulating microRNA assay leverages PCR sensitivity while eliminating the need for separate reverse transcription reactions and mitigating amplification biases introduced by target­-specific qPCR. Furthermore, the ability to multiplex targets in each well eliminates the need to split valuable samples into multiple reactions. Results from the Circulating microRNA assay are interpreted using Firefly Analysis Workbench, which allows visualization, normalization, and export of experimental data. To aid discovery and validation of biomarkers, we have generated fixed panels for Oncology, Cardiology, Neurology, Immunology, and Liver Toxicology. Here we present the data from several studies investigating circulating and tumor microRNA, showcasing the ability of the technology to sensitively and specifically detect microRNA biomarker signatures from fluid specimens.

Keywords: biomarkers, biofluids, miRNA, photolithography, flowcytometry

Procedia PDF Downloads 321