Search results for: cage cultured

Authors: Karem H. Alzoubi, Ahmad S. Alkofahi, Omar F. Khabour, Nizar M. Mhaidat

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Herbal medicinal products represent a major focus for drug development and industry and it holds a significant share in drug-market all over the globe. In here, selected medicinal plant extracts from Jordan with high antioxidative capacity were tested for their protective effect against oxidative DNA damage using in vitro 8-hydroxydeoxyguanisine and sister chromatid exchanges (SCEs) assays in cultured human lymphocytes. The following plant extracts were tested Cupressus sempervirens L., Psidium guajava (L.) Gaerth., Silybum marianum L., Malva sylvestris L., Varthemia iphionoides Boiss., Eminium spiculatum L. Blume, Pistachia palaestina Boiss., Artemisia herba-alba Asso, Ficus carica L., Morus alba Linn , Cucumis sativus L., Eucalyptus camaldulensis Dehnh., Salvia triloba L., Zizyphus spina-christi L. Desf., and Laurus nobilis L. A fractionation scheme for the active plant extracts of the above was followed. Plants extract fractions with best protective properties against DNA damage included hexane fraction of S. marianum L. (aerial parts), chloroform fractions of P. palaestina Boiss. (Fruits), ethanolic fractions of E. camaldulensis Dehnh (leaves), S. triloba L. (leaves), and ethanolic fractions of Z. spina-christi L. Desf. (Fruits/leaves). On the other hand, the ethanolic extracts of V. iphionoides Boiss was found to increase oxidative DNA damage. Results of the SCEs are undergoing. In conclusion, plant extracts with antioxidative DNA damage properties might have clinical applications in cancer prevention.

Keywords: medicinal plants extract, fractionation, oxidative DNA damage, 8-hydroxydeoxyguanisine, SCEs, Jordan

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Authors: Tadesse Eguale, Ephrem Engdawork, Wondwossen Gebreyes, Dainel Asrat, Hile Alemayehu, John Gunn

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Salmonella is one of the major zoonotic pathogens affecting wide range of vertebrates and humans worldwide. Consumption of contaminated dairy products and contact with dairy cattle represent the common sources of non-typhoidal Salmonella infection in humans. Fecal samples were collected from 132 dairy herds in central Ethiopia and cultured for Salmonella to determine the prevalence, serotype distribution and antimicrobial susceptibility. Salmonella was recovered from the feces of at least one cattle in 10(7.6%) of the dairy farms. Out of 1193 fecal samples 30(2.5%) were positive for Salmonella. Large farm size, detection of diarrhea in one or more animals during sampling and keeping animals completely indoor compared to occasional grazing outside were associated with Salmonella positivity of the farms. Farm level prevalence of Salmonella was significantly higher in young animals below 6 months of age compared to other age groups(X2=10.24; p=0.04). Nine different serotypes were isolated. The four most frequently recovered serotypes were S. Typhimurium (23.3%),S. Saintpaul (20%) and S. Kentucky and S. Virchow (16.7%) each. All isolates were resistant or intermediately resistant to at least one of the 18 drugs tested. Twenty-six (86.7%), 20(66.7%), 18(60%), 16(53.3%) of the isolates were resistant to streptomycin, nitrofurantoin, sulfisoxazole and tetracycline respectively. Resistance to 2 drugs was detected in 93.3% of the isolates. Resistance to 3 or more drugs were detected in 21(70%) of the total isolates while multi-drug resistance (MDR) to 7 or more drugs were detected in 12 (40%) of the isolates. The rate of occurrence of MDR in Salmonella strains isolated from dairy farms in Addis Ababa was significantly higher than those isolated from farms outside of Addis Ababa((p= 0.009). The detection of high MDR in Salmonella isolates originating from dairy farms warrants the need for strict pathogen reduction strategy in dairy cattle and spread of these MDR strains to human population.

Keywords: salmonella, antimicrobial resistance, fecal prevalence

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Authors: Yazeed Al-Sheikh

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Pregnancy represents a risk factor in the occurrence of vulvovaginal candidiasis. To investigate the prevalence rate of vaginal carriage of Candida species in Saudi pregnant and non-pregnant women, high vaginal swab (HVS) specimens (707) were examined by direct microscopy (10% KOH and Giemsa staining) and parallel cultured on Sabouraud Dextrose Agar (SDA) as well as on “CHROM agar Candida” medium. As expected, Candida-positive cultures were frequently observed in pregnant-test group (24%) than in non-pregnant group (17%). The frequency of culture positive was correlated to pregnancy (P=0.047), parity (P=0.001), use of contraceptive (P=0.146), or antibiotics (P=0.128), and diabetic-patients (P < 0.0001). Out of 707 HVS examined specimens, 157 specimens were yeast-positive culture (22%) on Sabouraud Dextrose Agar or “CHROM agar Candida”. In comparison, the sensitivities of the direct 10% KOH and the Giemsa stain microscopic examination methods were 84% (132/157) and 95% (149/157) respectively but both with 100% specificity. As for the identity of recovered 157 yeast isolates, based on API 20C biotype carbohydrate assimilation, germ tube and chlamydospore formation, C. albicansand C. glabrata constitute 80.3 and 12.7% respectively. Rates of C. tropicalis, C. kefyr, C. famata or C. utilis were 2.6, 1.3, and 0.6% respectively. Sachromyces cerevisiae and Rhodotorula mucilaginosa yeasts were also encountered at a frequency of 1.3 and 0.6% respectively. Finally, among all recovered 157 yeast-isolates, strains resistant to ketoconazole were not detected, whereas 5% of the C. albicans and as high as 55% of the non-albicans yeast isolates (majority C. glabrata) showed resistance to fluconazole. Our findings may prove helpful for continuous determination of the existing vaginal candidiasis causative species during pregnancy, its lab-diagnosis and/or control and possible measures to minimize the incidence of the disease-associated pre-term delivery.

Keywords: vaginal candidiasis, Candida spp., pregnancy, risk factors, API 20C-yeast biotypes, giemsa stain, antifungal agents

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Authors: Nwafia Ifeyinwa Nkeiruka, Nwafia Walter Chukwuma

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Since the early 1980’s fungi have emerged as major causes of human diseases, especially among immunocompromised. The most commonly isolated yeast is Candida albicans and constitutes the 4th most common nosocomial BSI in humans. It is progressive and cumulative and become more complex over time.It can even lead to leaky gut syndrome that causes food and environmental allergies. It is worthy of note that all the available data on oral Candida risk factors in humans were documented essentially using data from studies conducted in other areas, hence there is need for comparative and complementary information from the South eastern part of Nigeria. Method: 200 subjects of all age groups of both sexes were randomly examined,by swabbing their palatine mucosa and dorsal tongue with sterile cotton wool,then cultured into Sabouraud dextrose agar plates supplemented with antibiotics and incubated aerobically at 37 degree for 48 hrs. Identification of Candida albicans was done by germ tubes tests, chlamydospores production on cornmeal agar supplemented with 1% Tween 80.Sugar and nitrogen assimilation test using API 20C Auxanogram and potassium nitrate agar. Results: Out of 30 samples that were positive for candida, 15 (50%) were candida albicans. Using the anova test (P < 0.05) this variation is significant (P = 0016). followed by C. dublinensis 3 (13%), C. tropicalis 3 (10%), C. pseudotropicalis 3 (10%), C, glabrata 2 (7%), C. parapsilosis 2 (7%) and lastly C. krusei 1 (3%).However, 53% of the patients were female while 47% were male. Among the HIV positive isolates.67% were HIV isolates not on drugs while 33% positives isolates were on drugs and the percentages of candida species in these patients were as follows C. albicans were 45% followed by C. glabrata and C.tropicalis which were 17% each, C.parapsilosis, C.dubliensis and C.pseudotropicalis were all 8% each. Conclusion: Oral Candidiasis is a marker of systemic diseases and in some cases, it may be the first clinical presentation. There is need for more intensive clinical and laboratory monitoring and possible early intervention to prevent the reoccurrence and resistance to treatment.

Keywords: oral cavity, Candida species, oral Candidiasis, risk factors

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Authors: Nurul Hamirah Kamsani, Mohd Hamim Rajikin, Nor Ashikin Mohamed Noor Khan, Sharaniza Abdul Rahim

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Cytoskeletal structures, in particular actin and tubulin, provide a fundamental framework in all cells, including embryos. Under influence of nicotine, the cytoskeletal organization may be subjected to oxidative stress (OS) insult and cause alteration. Tocotrienol-rich fraction (TRF) is proven to enhance fertility better than the other sub-group of Vitamin E, tocopherols (TCPs). The objective of this study was to evaluate the effects of TRF on 1) actin and tubulin of 2- and 8-cell murine embryos and 2) the regulation of reactive oxygen species (ROS)-scavenging enzymes; induced by nicotine. Twenty four female Balb/C were subjected to either subcutaneous (sc) injection of 0.9% NaCl; sc injection of 3.0 mg/kg bw/day nicotine; sc injection of 3.0 mg/kg bw/day nicotine + oral gavage (OG) of 60 mg/kg bw/day TRF; or OG of 60 mg/kg bw/day TRF for 7 consecutive days. After superovulation and mating, animals were euthanized. 2-cell developing embryos were retrieved. 50% of the retrieved embryos were visualized under confocal laser staining microscopy (CLSM) for alterations of actin and tubulin. The remaining amount of embryos was cultured in vitro until 8-cell stage followed by CLSM visualization. Blood plasma was subjected to OS assays. Plasma malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were determined and analysed accordingly. At both 2- and 8-cell developing stages, actin intensities were significantly reduced in the nicotine group (p<0.001). After the intervention, actin intensity was significantly increased compared to that of the nicotine group (p<0.001). The same trend was seen in tubulin at both cell stages. TRF has minimized the deleterious effects of nicotine in actin and tubulin of both 2- and 8-cell developmental stages during pre-implantation embryonic development in mice in vitro. Levels of endogenous anti-oxidative enzymes were sustained close to control accompanied by decreased levels of OS biomarker.

Keywords: actin, nicotine, pre-implantation embryos, tocotrienol rich fraction, tubulin

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Authors: Chukwuma S. Ezeonu, Ikechukwu N. E. Onwurah, Uchechukwu U. Nwodo, Chibuike S. Ubani, Chigozie M. Ejikeme

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Trichophyton soudanense and Trichophyton mentagrophyte were isolated from the rice mill environment, cultured and used singly and as di-culture in the treatment of measure quantities of preheated rice husk. Optimized conditions studied showed that carboxymethylcellulase (CMCellulase) activity of 57.61 µg/ml/min was optimum for Trichophyton mentagrophyte heat pretreated rice husk crude enzymes at 50oC and 80oC respectively. Duration of 120 hours (5 days) gave the highest CMcellulase activity of 75.84 µg/ml/min for crude enzyme of Trichophyton mentagrophyte heat pretreated rice husk. However, 96 hours (4 days) duration gave maximum activity of 58.21 µg/ml/min for crude enzyme of Trichophyton soudanense heat pretreated rice husk. Highest CMCellulase activities of 67.02 µg/ml/min and 69.02 µg/ml/min at pH of 5 were recorded for crude enzymes of monocultures of Trichophyton soudanense (TS) and Trichophyton mentagrophyte (TM) heat pretreated rice husk respectively. Biomass components showed that rice husk cooled after heating followed by treatment with Trichophyton mentagrophyte gave 44.50 ± 10.90 (% ± Standard Error of Mean) cellulose as the highest yield. Maximum total lignin value of 28.90 ± 1.80 (% ± SEM) was obtained from pre-heated rice husk treated with di-culture of Trichophyton soudanense and Trichophyton mentagrophyte (TS+TM). The hemicellulose content of 30.50 ± 2.12 (% ± SEM) from pre-heated rice husk treated with Trichophyton soudanense (TS); lignin value of 28.90 ± 1.80 from pre-heated rice husk treated with di-culture of Trichophyton soudanense and Trichophyton mentagrophyte (TS+TM); also carbohydrate content of 16.79 ± 9.14 (% ± SEM) , reducing and non-reducing sugar values of 2.66 ± 0.45 and 14.13 ± 8.69 (% ± SEM) were all obtained from for pre- heated rice husk treated with Trichophyton mentagrophyte (TM). All the values listed above were the highest values obtained from each rice husk treatment. The pre-heated rice husk treated with Trichophyton mentagrophyte (TM) fermented with palmwine yeast gave bio-ethanol value of 11.11 ± 0.21 (% ± Standard Deviation) as the highest yield.

Keywords: Trichophyton soudanense, Trichophyton mentagrophyte, biomass, bioethanol, rice husk

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Authors: Benjamin Bonsu Bruce, Marian Dorcas Quain David Appiah-Kubi, Gertrude Osei-Diko, Harrison Kwame Dapaah

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Taro [Colocasia esculenta (L.) Schott] is a major staple food and remains a significant crop to many cultural and agricultural customs worldwide. In Ghana, taro is mostly propagated using vegetative material, which is conserved in field collection and recycled from their farms to establish new fields. However, this practice promotes the accumulation of systemic pathogens. Prior exposure to pests and subsequent expression of disease symptoms can also be a huge constraint to sustainable conservation and utilization of taro genetic resources. In vitro, slow growth is one of the most promising techniques to be utilized for conservation. The objective of this study was to find a medium-term in vitro conservation protocol for local and exotic taro genotypes. The medium-term conservation study was conducted using actively growing shoots obtained from in vitro cultures. Explants were cultured to full strength in complete Murashige and Skoog medium supplemented with Mannitol at different concentrations (0g/l, 20g/l, 25g/l, and 30g/l). Another medium that was tested as an additional treatment is the White’s medium. The highest number of shoots (6.33) and leaves (22.67) occurred on medium containing 20 and 25g/l mannitol in genotype SAO 006 as compared to other genotypes, whereas 30g/l mannitol was the best to restrict growth for the entire 6 months period in terms of shoot height (22.50cm). The study reveals that mannitol supplemented culture media could reduce the growth of Colocasia plantlets, especially in stem height. Culture growth following 6 months of conservation, showed that healthy shoot cultures of Taro were obtained after 6 months of storage in a medium containing 20gl⁻¹ and 25gl⁻¹ mannitol.

Keywords: complete murashige, skoog medium, culture conditions, mannitol, slow growth conservation

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Authors: Seyede Sanaz Seyedebrahimi, Shima Rahimi, Shohreh Fakhari, Ali Jalili

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Background: CXCR4, as a chemokine receptor, plays well-known roles in various types of cancers. Several studies have been conducted to overcome CXCR4 axis acts in multiple myeloma (MM) pathogenesis and progression. Dexamethasone, a standard treatment for multiple myeloma, has been shown to increase CXCR4 levels in multiple myeloma cell lines. Herein, we focused on the effects of metformin and dexamethasone on CXCR4 at the cellular level and the migration rate of cell lines after exposure to a combination compared to single-agent models. Materials and Method: Multiple myeloma cell lines (U266 and RPMI8226) were cultured with different metformin and dexamethasone concentrations in single-agent and combination models. The simultaneous combination doses were calculated by CompuSyn software. Cell surface and mRNA expression of CXCR4 were determined using flow cytometry and the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay, respectively. The Transwell cell migration assay evaluated the migration ability. Results: In concurred with previous studies, our results showed a dexamethasone up-regulation effect on CXCR4 in a dose-dependent manner. Although, the metformin single-agent model could reduce CXCR4 expression of U266 and RPMI8226 in cell surface and mRNA expression level. Moreover, the administration of metformin and dexamethasone simultaneously exerted a higher suppression effect on CXCR4 expression than the metformin single-agent model. The migration rate through the combination model's matrigel membrane was remarkably lower than the metformin and dexamethasone single-agent model. Discussion: According to our findings, the combination of metformin and dexamethasone effectively inhibited dexamethasone-induced CXCR4 expression in multiple myeloma cell lines. As a result, metformin may be counted as an alternative medicine combined with other chemotherapies to combat multiple myeloma. However, more research is required.

Keywords: CXCR4, dexamethasone, metformin, migration, multiple myeloma

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Authors: Mudassar Mohiuddin, Zahid Iqbal

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Introduction: Clostridium perfringens is an important pathogen responsible for causing enteric diseases in both human and animals. The bacteria produce several toxins. These toxins play vital role in the pathogenesis of various fatal enteric diseases and are classified into five types, on the basis of the differential production of Alpha, Beta, Epsilon and Iota toxins. In addition to the so-called major toxins, there are other toxins like beta2 toxin, produced by some strains of C. perfringens which may play a role in the pathogenesis of disease. Aim of the study: In this study a multiplex PCR assay was developed and used for detection of cpb2 gene to identify the Beta2 harboring isolates among different types of C. perfringens. Objectives: The primary objective of this study was to identify the prevalence of β2-toxin gene in local isolates of Clostridium perfringens. Methodology: This was an experimental study. Random sampling technique was used. A total of 97 sheep and goats were included in this study. All were Pakistani local breeds. The samples were collected during the period from Sep, 2014 to Mar, 2015 from selected districts of Punjab province (Pakistan). Faecal samples were cultured in cooked meat media. The identification of Clostridium perfringens was made on the basis of biochemical tests. Multiplex PCR was performed to identify the toxin genes. Results: A total of 43 C. perfringens isolates were genotyped using multiplex PCR assay. The gene encoding C. perfringens β2-toxin (cpb2) was present in more than 50% of the isolates genotyped. However, the prevalence of this gene varied between sheep and goat isolates. Conclusion: The present study suggests the high occurrence of C. perfringens b2-toxin (cpb2) in the local isolates of Pakistan. As β2-toxin is present in both healthy and diseased animals, so further studies are suggested to establish the role of β2-toxin in pathogenesis of the clostridial enteric diseases.

Keywords: beta 2 toxin gene, clostridium perfringens, enteric diseases, goats, multiplex PCR, sheep

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Authors: Sineenat Kempubpha, Phornpa-Ngan Muadmuang, Putthamas Phetmuangprab, Surin Promphet, Sopita Bandit

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Introduction: Discarded pericarp of mangosteen (Garcinia mangostana) is a benefit to be developed as veterinary phytopharmacal products since it made up of abundance pharmacological active compounds. The active compounds of mangosteen pericarp not only act as an antihistamine, an anti-inflammatory, heart disease and HIV therapeutic substances but also act as antibacterial and antifungal agents. Aim: This study was an in vitro procedural attempt to determine the antibacterial effects of mangosteen pericarp 95% ethanol extract on the main causative pathogen of canine superficial pyoderma, Staphylococcus pseudintermedius. Methods: S. pseudintermedius were collected from various sites of the skin of canine superficial pyoderma dogs and were revived and lawn cultured. The S. pseudintermedius growth inhibition study was determined by disc diffusion technique, the mangosteen pericarp crude extracted was dissolved in 3 types of solvents (95% ethanol, 2% DMSO and distilled water, respectively). The micro broth dilution technique was used for determining both minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values. Statistical analysis was done by calculating the mean of the zones of inhibition of tested microorganisms. Results: S. pseudintermedius growth inhibition study showed that the inhibition efficacy of 95% ethanol was greater than the inhibition efficacy of 2% DMSO and distilled water (9.10±0.18 mm, 6.95±0.60 mm and 6.80±0.18 mm, respectively). The MIC value was 125 µg/ml and the MBC value was 1 mg/ml. Conclusion: Mangosteen pericarp extract dissolved with 95% ethanol showed the highest zone of inhibition against the tested microorganisms. The MIC value was 125 µg/ml and the MBC value was 1 mg/ml which suggests its potent antibacterial action against S. pseudintermedius. However, further analytical studies are needed to isolate the key molecules of mangosteen pericarp for higher effect on canine superficial pyoderma microorganism therapeutic products.

Keywords: mangosteen, Garcinia mangostana, Staphylococcus pseudintermedius, canine superficial pyoderma, in vitro study

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Authors: Gülcan Taşkıran, Ayla Bayık Temel

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Background: Florence Nightingale, the modern founder of the nursing, is most famous for her role as a nurse. But not so much known about her contributions as a mathematician and statistician. Aim: In this conceptual article it is aimed to examine Florence Nightingale's statistics education, how she used her passion for statistics and applied statistical data in nursing care and her scientific contributions to statistical science. Design: Literature review method was used in the study. The databases of Istanbul University Library Search Engine, Turkish Medical Directory, Thesis Scanning Center of Higher Education Council, PubMed, Google Scholar, EBSCO Host, Web of Science were scanned to reach the studies. The keywords 'statistics' and 'Florence Nightingale' have been used in Turkish and English while being screened. As a result of the screening, totally 41 studies were examined from the national and international literature. Results: Florence Nightingale has interested in mathematics and statistics at her early ages and has received various training in these subjects. Lessons learned by Nightingale in a cultured family environment, her talent in mathematics and numbers, and her religious beliefs played a crucial role in the direction of the statistics. She was influenced by Quetelet's ideas in the formation of the statistical philosophy and received support from William Farr in her statistical studies. During the Crimean War, she applied statistical knowledge to nursing care, developed many statistical methods and graphics, so that she made revolutionary reforms in the health field. Conclusions: Nightingale's interest in statistics, her broad vision, the statistical ideas fused with religious beliefs, the innovative graphics she has developed and the extraordinary statistical projects that she carried out has been influential on the basis of her professional achievements. Florence Nightingale has also become a model for women in statistics. Today, using and teaching of statistics and research in nursing care practices and education programs continues with the light she gave.

Keywords: Crimean war, Florence Nightingale, nursing, statistics

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Authors: Maryam Beiranvand, Sajad Yaghoubi

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Background: Some microbes can colonize plants’ inner tissues without causing obvious damage and can even produce useful bioactive substances. In the present study, the diversity of the endophytic bacteria associated with medicinal plants from Iran was investigated by culturing techniques, molecular gene identification, as well as measuring them for antibacterial activity. Results: In the spring season from 2013 to 2014, 35 herb pharmacology samples were collected, sterilized, meshed, and then cultured on selective media culture. A total of 199 endophytic bacteria were successfully isolated from 35 tissue cultures of medical plants, and sixty-seven out of 199 bacterial isolates were subjected to identification by the 16S rRNA gene sequence analysis method. Based on the sequence similarity gene and phylogenetic analyses, these isolates were grouped into five classes, fourteen orders, seventeen families, twenty-one genera, and forty strains. The most abundant group of endophytic bacteria was actinobacterial, consisting of thirty-two (47%) out of 67 bacterial isolates. Ten (22.3%) out of 67 bacterial isolates remained unidentified and classified at the genus level. The signature of the 16S rRNA gene formed a distinct line in a phylogenetic tree showing that they might be new species of bacteria. One (5.2%) out of 67 bacterial isolates was still not well categorized. Forty-two out of 67 strains were candidates for antimicrobial activity tests. Nineteen (45%) out of 42 strains showed antimicrobial activity multidrug resistance (MDR); thirteen (68%) out of 19 strains were allocated to classes actinobacteria. Four (21%) out of 19 strains belonged to the Bacillaceae family, one (5.2%) out of 19 strains was the Paenibacillaceae family, and one (5.2%) out of 19 strains belonged to the Pseudomonadaceae family. The other twenty-three strains did not show inhibitory activities. Conclusions: Our research showed a high-level phylogenetic diversity and the intoxicating antibiotic activity of endophytic bacteria in the herb pharmacology of Iran.

Keywords: Antibacterial activity, endophytic bacteria, multidrug-resistant bacteria, whole genom sequencing

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Authors: Muhammad Aasim, Mehmet Karataş, Fatih Erci, Şeyma Bakırcı, Ecenur Korkmaz, Burak Kahveci

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Water hyssop (Bacopa monnieri L. Wettst.) is an important medicinal aquatic/semi aquatic plant native to India where it is used in traditional medicinal system. The plant contains bioactive compounds mainly Bacosides which are the main ingridient of commercial drug available as memory enhancer tonic. The local name of water hyssop is Brahmi and brahmi based drugs are available against for curing chronic diseases and disorders Alzheimer’s disease, anxiety, asthma, cancer, mental illness, respiratory ailments, and stomach ulcers. The plant is not a cultivated plant and collection of plant from nature make palnt threatened to endangered. On the other hand, low seed viability and availability make it difficult to propagate plant through traditional techniques. In recent years, plant tissue culture techniques have been employed to propagate plant for its conservation and production for continuous availability of secondary metabolites. On the other hand, application of nanoparticles has been reported for increasing biomass, in vitro regeneration and secondary metabolites production. In this study, silver nanoparticles (AgNPs) were applied at the rate of 2, 4, 6, 8 and 10 ppm to Murashihe and Skoog (MS) medium supplemented with 1.0 mg/l Benzylaminopurine (BAP), 3.0% sucrose and 0.7% agar. Leaf explants of water hyssop were cultured on AgNPs containing medium. Shoot induction from leaf explants were relatively slow compared to medium without AgNPs. Multiple shoot induction was recorded after 3-4 weeks of culture comapred to control that occured within 10 days. Regenerated shoots were rooted successfully on MS medium supplemented with 1.0 mg/l IBA and acclimatized in the aquariums for further studies.

Keywords: Water hyssop, Silver nanoparticles, In vitro, Regeneration, Secondary metabolites

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Authors: Dwi Arisandi Wijaya, Ernawati Arifin Giri-Rachman, Neni Nurainy

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Typhoid fever disease can be prevented by using a polysaccharide-based vaccine Vi which is a virulence factor of S.typhi. To produce high yield Vi polysaccharide from bacteria, it is important to know the biosynthesis of Vi polysaccharide and the regulators involved. In the In vivo condition, S. typhi faces different osmolarity, and the bacterial two-component system OmpR-EnvZ, regulate by up and down Capsular Vi polysaccharide biosynthesis. A high yielded Vi Polysaccharide strain, S. typhi strain C6524 used to study the effect of media osmolarity on Vi polysaccharide biosynthesis and the osmoregulation pattern of S. typhi strain C6524. The methods were performed by grown S. typhi strain C6524 grown on medium with 50 mM, 100 mM, and 150 mM osmolarity with the batch system. Vi polysaccharide concentration was measured by ELISA method. For further investigation of the osmoregulation pattern of strain C6524, the osmoregulator gene, OmpR, has been isolated and sequenced using the specific primer of the OmpR gene. Nucleotide sequence analysis is done with BLAST and Lallign. Amino Acid sequence analysis is done with Prosite and Multiple Sequence Alignment. The results of cultivation showed the average content of polysaccharide Vi for 50 mM, 100 mM, and 150 mM osmolarities 11.49 μg/mL, 12.06 μg/mL, and 14.53 μg/mL respectively. Analysis using Anova stated that the osmolarity treatment of 150 mM significantly affects Vi content. Analysis of nucleotide sequences shows 100% identity between S. typhi strain C6524 and Ty2. Analysis of amino acid sequences shows that the OmpR response regulator protein of the C6524 strain also has a α4-β5-α5 motif which is important for the regulatory activation system when phosphorylation occurs by domain kinase. This indicates that the regulator osmolarity response of S. typhi strain C6524 has no difference with the response regulator owned by S. typhi strain Ty2. A high Vi response rate in the 150 mM osmolarity treatment requires further research for RcsB-RcsC, another two-component system involved in Vi Biosynthesis.

Keywords: osmoregulator, OmpR, Salmonella, Vi polysaccharide

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Authors: Kedar Hardikar, Joe Varghese

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Conductive adhesives have found wide-ranging applications in electronics industry ranging from fixing a defective conductor on printed circuit board (PCB) attaching an electronic component in an assembly to protecting electronics components by the formation of “Faraday Cage.” The reliability requirements for the conductive adhesive vary widely depending on the application and expected product lifetime. While the conductive adhesive is required to maintain the structural integrity, the electrical performance of the associated sub-assembly can be affected by the degradation of conductive adhesive. The degradation of the adhesive is dependent upon the highly varied use case. The conventional approach to assess the reliability of the sub-assembly involves subjecting it to the standard environmental test conditions such as high-temperature high humidity, thermal cycling, high-temperature exposure to name a few. In order to enable projection of test data and observed failures to predict field performance, systematic development of an acceleration factor between the test conditions and field conditions is crucial. Common acceleration factor models such as Arrhenius model are based on rate kinetics and typically rely on an assumption of linear degradation in time for a given condition and test duration. The application of interest in this work involves conductive adhesive used in an electronic circuit of a capacitive sensor. The degradation of conductive adhesive in high temperature and humidity environment is quantified by the capacitance values. Under such conditions, the use of established models such as Hallberg-Peck model or Eyring Model to predict time to failure in the field typically relies on linear degradation rate. In this particular case, it is seen that the degradation is nonlinear in time and exhibits a square root t dependence. It is also shown that for the mechanism of interest, the presence of moisture is essential, and the dominant mechanism driving the degradation is the diffusion of moisture. In this work, a framework is developed to incorporate nonlinear degradation of the conductive adhesive for the development of an acceleration factor. This method can be extended to applications where nonlinearity in degradation rate can be adequately characterized in tests. It is shown that depending on the expected product lifetime, the use of conventional linear degradation approach can overestimate or underestimate the field performance. This work provides guidelines for suitability of linear degradation approximation for such varied applications

Keywords: conductive adhesives, nonlinear degradation, physics of failure, acceleration factor model.

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Authors: Himanshu Lal, Bipasha Ghosh, Arun Srivastava

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A pilot study of indoor air quality in terms of bioaerosol (fungus and bacteria) and few selective microbial volatile organic compounds (MVOCs) was carried out in different indoor sections of a library for two seasons, namely monsoon and post monsoon. Bioaerosol sampling was carried out using Anderson six stage viable sampler at a flow rate of 28.3 L/min while MVOCs were collected on activated charcoal tubes ORBOTM 90 Carboxen 564.Collected MVOCs were desorbed using carbon disulphide (CS2) and analysed by GC-FID. Microscopic identification for fungus was only carried out. Surface dust was collected by sterilised buds and cultured to identify fungal contaminants. Unlike bacterial size distribution, fungal bioaerosol concentration was found to be highest in the fourth stage in different sections of the library. In post monsoon season both fungal bioaerosol (710 to 3292cfu/m3) and bacterial bioaerosol (298 to 1475cfu/m3) were fund at much greater concentration than in monsoon. In monsoon season unlike post monsoon, I/O ratio for both the bioaerosol fractions was more than one. Rain washout could be the reason of lower outdoor concentration in monsoon season. On the contrary most of the MVOCs namely 1-hexene, 1-pentanol and 1-octen-3-ol were found in the monsoon season instead of post monsoon season with the highest being 1-hexene with 7.09µg/m3 concentration. Among the six identified fungal bioaerosol Aspergillus, Cladosporium and Penicillium were found in maximum concentration while Aspergillus niger, Curvuleria lunata, Cladosporium cladosporioides and Penicillium sp., was indentified in surface dust samples. According to regression analysis apart from environmental factors other factors also played an important role. Thus apart from outdoor infiltration and human sources, accumulated surface dust mostly on organic materials like books, wooden furniture and racks can be attributed to being one of the major sources of both fungal bioaerosols as well as MVOCs found in the library.

Keywords: bacteria, Fungi, indoor air, MVOCs

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Authors: Maryam Beiranvand, Sajad Yaghoubi

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Background: Some microbes can colonize plants’ inner tissues without causing obvious damage and can even produce useful bioactive substances. In the present study, the diversity of the endophytic bacteria associated with medicinal plants from Iran was investigated by culturing techniques, molecular gene identification, as well as measuring them for antibacterial activity. Results: In the spring season from 2013 to 2014, 35 herb pharmacology samples were collected, sterilized, meshed, and then cultured on selective media culture. A total of 199 endophytic bacteria were successfully isolated from 35 tissue cultures of medical plants, and sixty-seven out of 199 bacterial isolates were subjected to identification by the 16S rRNA gene sequence analysis method. Based on the sequence similarity gene and phylogenetic analyses, these isolates were grouped into five classes, fourteen orders, seventeen families, twenty-one genera, and forty strains. The most abundant group of endophytic bacteria was actinobacterial, consisting of thirty-two (47%) out of 67 bacterial isolates. Ten (22.3%) out of 67 bacterial isolates remained unidentified and classified at the genus level. The signature of the 16S rRNA gene formed a distinct line in a phylogenetic tree showing that they might be new species of bacteria. One (5.2%) out of 67 bacterial isolates was still not well categorized. Forty-two out of 67 strains were candidates for antimicrobial activity tests. Nineteen (45%) out of 42 strains showed antimicrobial activity multidrug-resistance (MDR); thirteen (68%) out of 19 strains were allocated to classes actinobacteria. Four (21%) out of 19 strains belonged to the Bacillaceae family, one (5.2%) out of 19 strains was the Paenibacillaceae family, and one (5.2%) out of 19 strains belonged to the Pseudomonadaceae family. The other twenty-three strains did not show inhibitory activities. Conclusions: Our research showed a high-level phylogenetic diversity and the intoxicating antibiotic activity of endophytic bacteria in the herb pharmacology of Iran.

Keywords: medical plant, endophytic bacteria, antimicrobial activity, whole genome sequencing analysis

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Authors: Kairat Uteulin, Azhar Iskakova, Serik Mukhambetzhanov, Bayan Yesbolayeva, Gabit Bari, Aslan Zheksenbai, Kabyl Zhambakin, Chingis Dzhabykbayev, Vladimir Piven, Izbasar Rakhimbaiev

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To explore the potential for in vitro rapid regeneration of Russian dandelion (Taraxacum kok-saghyz Rodin), different concentrations of 6-Benzylaminopurine (BAP), 2,4-Dichlorophenoxyacetic acid (2.4-D) and BAP combined with Indole-3-acetic acid (IAA) were evaluated for their effects on the induction of somatic embryos from leaf, seed stem and root explants. Different explants were cultured on MS medium supplemented with various concentrations (0, 0.5, 1, 1.5, 2, 2.5 and 3 mg/l) of each kind of hormone. Callus induction percentage, fresh weight, color and texture of the callus were assessed after 14 and 28 days of culture. The optimum medium for the proliferation of embryogenic calli from leaf and root explants was MS supplemented with 2.5 mg/L BAP and 0.5 mg/L 2.4-D. Concentrations of 2.5 mg/L BAP and 1.5 mg/L IAA also had a remarkable effect on root and stem explants. The best concentration to produce callus from stem explants was 0.5 mg/L BAP and 1 mg/L IAA. Results of mean comparison showed that BAP and 2.4-D were more effective on different explants than BAP and IAA. Results of the double staining method proved that somatic embryogenesis occurred in the most concentrations of BAP and 2.4-D. Under microscopic observations, the different developmental stages of the embryos (globular, heart, torpedo and cotyledonary) were revealed together in callus cells, indicating that the most tested hormone combinations were effective for somatic embryogenesis formation in this species. Seed explants formed torpedo and cotyledonary stages faster than leaf and root explants in the most combinations. Most calli from seed explants were cream colored and friable, while calli were compact and light green from leaf and root explants. Some combinations gave direct regeneration and (3 mg/L BAP and 2 mg/L IAA) in seed explants and (0.5 mg/L BAP and 2.5 mg/L IAA) in leaf explants had the highest number of shoots with average of 21 and 27 shoots per callus. The developed protocol established the production of different callus types from seed, leaf, and root explants and plant regeneration through somatic embryogenesis.

Keywords: taraxacum kok-saghyz Rodin, callus, somatic embryogenesis

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Authors: Z. Nourmohammadi, F. Farahani, M. Shaker

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Aloe is an important commercial crop available in a wide range of species and varieties in international markets. The applications of this plant have been recorded in the ancient cultures of India, Egypt, Greece, Rome and China. Aloe has been used for centuries and is currently being actively studied for medicinal purposes. Aloe is propagated through lateral buds, which is slow, very expensive and low income practice. Nowadays, it has been cultured by in vitro propagation for rapid multiplication of plants, genetic improvement of crops, obtaining disease-free clones and for progressive valuable germplasm. The present study focused on the influence of different phytohormones on rapid in vitro propagation of Aloe plants. We also investigated the effect of gamma radiation on biochemical characters as well as genetic changes. Shoot tip of 2-3 cm were collected from offshoot of Aloe barbadensis and Aloe littoralis, and were inoculated with MS medium containing various concentrations of BA (0.5, 1, 2 mg/l), IAA (0.5, 1 mg/l). The best treatment for a highest shoot number and bud proliferation was MS medium containing 2 mg/l BAP and 0.5 mg/l IAA in A. barbadensis and A. littoralis. Maximum percentage of proliferated shoot buds (90% and 95%) from a single explant were obtained in MS medium after 4-5 weeks of the second and the first subcultures, respectively. Different genome sizes were also indicated among treatments and subcultures. The mixoploids identified in flow cytometery histograms in different treatments. The effect of gamma radiation on A. littoralis showed that by increasing the dose of gamma radiation, amounts of chlorophyll A, B, carotenoids, total protein content and superoxide dismutase were significantly increased compared to control plants. Genetic variation analysis also revealed significant genetic differences between control and gamma radiation treated regenerated plants by AMOVA test. Higher genetic heterozygocity was observed in radiation treated plants. Our findings may provide useful method for improving of Aloe plant proliferation with increasing of useful material such as antioxidant enzymes.

Keywords: aloe, antioxidant enzyme, micropropagation, gamma radiation, genetic variation

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Authors: Afiq Mohd Fahmi, Tony Gutierrez, Sebastian Hennige

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Increasing amounts of CO₂ entering the marine environment, also known as ocean acidification, is documented as having harmful impacts on a variety of marine organisms. When considering the future risk of hydrocarbon pollution, which is generally detrimental to marine life as well, this needs to consider how OA-induced changes to microbial communities will compound this since hydrocarbon degradation is influenced by the community-level microbial response. This study aims to evaluate the effects of increased atmospheric CO₂ conditions and oil enrichment on the phytoplankton-associated bacterial communities. Faroe Shetland Channel (FSC) is a subarctic region in the northeast Atlantic where crude oil extraction has recently been expanded. In the event of a major oil spill in this region, it is vital that we understand the response of the bacterial community and its consequence on primary production within this region—some phytoplankton communities found in the ocean harbor hydrocarbon-degrading bacteria that are associated with its psychosphere. Surface water containing phytoplankton and bacteria from FSC were cultured in ambient and elevated atmospheric CO₂ conditions for 4 days of acclimation in microcosms before introducing 1% (v/v) of crude oil into the microcosms to simulate oil spill conditions at sea. It was found that elevated CO₂ conditions do not significantly affect the chl a concentration, and exposure to crude oil detrimentally affected chl a concentration up to 10 days after exposure to crude oil. The diversity and richness of the bacterial community were not significantly affected by both CO₂ treatment and oil enrichment. The increase in the relative abundance of known hydrocarbon degraders such as Oleispira, Marinobacter and Halomonas indicates potential for biodegradation of crude oil, while the resilience of dominant taxa Colwellia, unclassified Gammaproteobacteria, unclassified Rnodobacteria and unclassified Halomonadaceae could be associated with the recovery of microalgal community 13 days after oil exposure. Therefore, the microbial community from the subsurface of FSC has the potential to recover from crude oil pollution even under elevated CO₂ (750 ppm) conditions.

Keywords: phytoplankton, bacteria, crude oil, ocean acidification

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Authors: Dona Pamoda W. Jayatunga, Veer Bala Gupta, Eugene Hone, Ralph N. Martins

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Being a neurodegenerative disorder, Alzheimer’s disease (AD) affects a majority of the elderly demented worldwide. The key risk factors for AD are age, metabolic syndrome, allele status of APOE gene, head injuries and lifestyle. The progressive nature of AD is characterized by symptoms of multiple cognitive deficits exacerbated over time, leading to death within a decade from clinical diagnosis. However, it is revealed that AD originates via a prodromal phase that spans from one to few decades before symptoms first manifest. The key pathological hallmarks of AD brains are deposition of amyloid beta (Aβ) plaques and neurofibrillary tangles (NFT). However, the yet unknown etiology of the disease fails to distinguish mitochondrial dysfunction between a cause or an outcome. The absence of early diagnosis tools and definite therapies for AD have permitted recruits of nutraceutical-based approaches aimed at reducing the risk of AD by modulating lifestyle or be used as preventive tools during AD prodromal state before widespread neurodegeneration begins. The objective of the present study was to investigate beneficial effects of luteolin, a plant-based flavone compound, against AD. The neuroprotective effects of luteolin on amyloid beta (Aβ) (1-42)-induced neurotoxicity was measured using cultured human neuroblastoma BE(2)-M17 cells. After exposure to 20μM Aβ (1-42) for 48 h, the neuroblastoma cells exhibited marked apoptotic death. Co-treatment of 20μM Aβ (1-42) with luteolin (0.5-5μM) significantly protected the cells against Aβ (1-42)-induced toxicity, as assessed by the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4sulfophenyl)-2H-tetrazolium, inner salt; MTS] reduction assay and the lactate dehydrogenase (LDH) cell death assay. The results suggest that luteolin prevents Aβ (1-42)-induced apoptotic neuronal death. However, further studies are underway to determine its protective mechanisms in AD including the activity against tau hyperphosphorylation and mitochondrial dysfunction.

Keywords: Aβ (1-42)-induced toxicity, Alzheimer’s disease, luteolin, neuroblastoma cells

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Authors: M. H. Rajikin, S. M. M. Syairah, A. R. Sharaniza

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Effects of nicotine on pre-partum body weight and preimplantation embryonic development has been reported previously. Present study was conducted to determine the effects of annatto (Bixa orellana)-derived delta-tocotrienol (TCT) (with presence of 10% gamma-TCT isomer) on the nicotine-induced reduction in body weight and 8-cell embryonic growth in mice. Twenty four 6-8 weeks old (23-25g) female balb/c mice were randomly divided into four groups (G1-G4; n=6). Those groups were subjected to the following treatments for 7 consecutive days: G1 (control) were gavaged with 0.1 ml tocopherol stripped corn oil, G2 was subcutaneously (s.c.) injected with 3 mg/kg/day of nicotine, G3 received concurrent treatment of nicotine (3 mg/kg/day) and 60 mg/kg/day of δ-TCT mixture (contains 90% delta & 10% gamma isomers) and G4 was given 60 mg/kg/day of δ-TCT mixture alone. Body weights were recorded daily during the treatment. On Day 8, females were superovulated with 5 IU Pregnant Mare’s Serum Gonadotropin (PMSG) for 48 hours followed with 5 IU human Chorionic Gonadotropin (hCG) before mated with males at the ratio of 1:1. Females were sacrificed by cervical dislocation for embryo collection 48 hours post-coitum. Collected embryos were cultured in vitro. Results showed that throughout Day 1 to Day 7, the body weight of nicotine treated group (G2) was significantly lower (p<0.05) than that of G1, G3 and G4. Intervention with δ-TCT mixture (G3) managed to increase the body weight close to the control group. This is also observed in the group treated with δ-TCT mixture alone (G4). The development of 8-cell embryos following in vitro culture (IVC) was totally inhibited in G2. Intervention with δ-TCT mixture (G3) resulted in the production of 8-cell embryos, although it was not up to that of the control group. Treatment with δ-TCT mixture alone (G4) caused significant increase in the average number of produced 8-cell embryo compared to G1. Present data indicated that δ-TCT mixture was able to reverse the body weight loss in nicotine treated mice and the development of 8-cell embryos was also improved.

Keywords: δ-tocotrienol, body weight, nicotine, preimplantation embryonic development

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Authors: Maryam Parsian, Pelin Mutlu, Ufuk Gunduz

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Two-dimensional cell culture affords simplicity and low cost, but it has serious limitations; lacking cell-cell and cell-matrix interactions that are present in tissues. Cancer cells grown in 3D culture systems have distinct phenotypes of adhesion, growth, migration, invasion as well as profiles of gene and protein expression. These interactions cause the 3D-cultured cells to acquire morphological and cellular characteristics relevant to in vivo tumors. Palbociclib is a chemotherapeutic agent for the treatment of ER-positive and HER-negative metastatic breast cancer. Poly-amidoamine (PAMAM) dendrimer is a well-defined, special three-dimensional structure and has a multivalent surface and internal cavities that can play an essential role in drug delivery systems. In this study, palbociclib is loaded onto the magnetic PAMAM dendrimer. Hanging droplet method was used in order to form 3D spheroids. The possible toxic effects of both free drug and drug loaded nanoparticles were evaluated in 2D and 3D MCF-7, MD-MB-231 and SKBR-3 breast cancer cell culture models by performing MTT cell viability and Alamar Blue assays. MTT analysis was performed with six different doses from 1000 µg/ml to 25 µg/ml. Drug unloaded PAMAM dendrimer did not demonstrate significant toxicity on all breast cancer cell lines. The results showed that 3D spheroids are clearly less sensitive than 2D cell cultures to free palbociclib. Also, palbociclib loaded PAMAM dendrimers showed more toxic effect than free palbociclib in all cell lines at 2D and 3D cultures. The results suggest that the traditional cell culture method (2D) is insufficient for mimicking the actual tumor tissue. The response of the cancer cells to anticancer drugs is different in the 2D and 3D culture conditions. This study showed that breast cancer cells are more resistant to free palbociclib in 3D cultures than in 2D cultures. However, nanoparticle loaded drugs can be more cytotoxic when compared to free drug.

Keywords: 2D and 3D cell culture, breast cancer, palbociclibe, PAMAM magnetic nanoparticles

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Authors: Scott Nielsen, Luca Longanesi, Chris Chuck

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Palm oil is obtained from the flesh and kernel of the fruit of oil palms and is the most productive and inexpensive oil crop. The global demand for palm oil is approximately 75 million metric tonnes, a 29% increase in global production of palm oil since 2016. This expansion of oil palm cultivation has resulted in mass deforestation, vast biodiversity destruction and increasing net greenhouse gas emissions. One possible alternative is to produce a saturated oil, similar to palm, from microbes such as oleaginous yeast. The yeasts can be cultured on sugars derived from second-generation sources and do not compete with tropical forests for land. One highly promising oleaginous yeast for this application is Metschnikowia pulcherrima. However, recent techno-economic modeling has shown that cell lysis and standard lipid extraction are major contributors to the cost of the oil. Typical cell disruption techniques to extract either single cell oils or proteins have been based around bead-beating, homogenization and acid lysis. However, these can have a detrimental effect on lipid quality and are energy-intensive. In this study, a vortex separator, which produces high sheer with minimal energy input, was investigated as a potential low energy method of lysing cells. This was compared to four more traditional methods (thermal lysis, acid lysis, alkaline lysis, and osmotic lysis). For each method, the yeast loading was also examined at 1 g/L, 10 g/L and 100 g/L. The quality of the cell disruption was measured by optical cell density, cell counting and the particle size distribution profile comparison over a 2-hour period. This study demonstrates that the vortex separator is highly effective at lysing the cells and could potentially be used as a simple apparatus for lipid recovery in an oleaginous yeast process. The further development of this technology could potentially reduce the overall cost of microbial lipids in the future.

Keywords: palm oil substitute, metschnikowia pulcherrima, cell disruption, cell lysis

Procedia PDF Downloads 172

Authors: Abdul Matin, Muazzama Akhtar, Shahid Nisar, Saddaf Mazzar, Umer Rashid

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Antibiotic resistance is an increasing concern worldwide now a day. Neisseria gonorrhea and Staphylococcus aureus are known to cause major human sexually transmitted and respiratory diseases respectively. Nanotechnology is an emerging discipline and its application in various fields especially in medical sciences is gigantic. In the present study, we synthesized multi-walled carbon nanotubes (MWNTs) using acid oxidation method and solubilized MWNTs were with length predominantly >500 nm and diameters ranging from 40 to 50 nm. The locally synthesized MWNTs were used against gram positive and negative bacteria to determine their impact on bacterial growth. Clinical isolates of Neisseria gonorrhea (isolate: 4C-11) and Staphylococcus aureus (isolate: 38541) were obtained from local hospital and normally cultured in LB broth at 37°C. Both clinical strains can be obtained on request from University of Gujarat. Spectophometric assay was performed to determine the impact of MWNTs on bacterial growth in vitro. To determine the effect of MWTNs on test organisms, various concentration of MWNTs were used and recorded observation on various time intervals to understand the growth inhibition pattern. Our results demonstrated that MWNTs exhibited toxic effects to Staphylococcus aureus while showed very limited growth inhibition to Neisseria gonorrhea, which suggests the resistant potential of Neisseria against nanoparticles. Our results clearly demonstrate the gradual decrease in bacterial numbers with passage of time when compared with control. Maximum bacterial inhibition was observed at maximum concentration (50 µg/ml). Our future work will include further characterization and mode of action of our locally synthesized MWNTs. In conclusion, we investigated and reported for the first time the inhibitory potential of locally synthesized MWNTs on local clinical isolates of Staphylococcus aureus and Neisseria gonorrhea.

Keywords: antibacterial activity, multi walled carbon nanotubes, Neisseria gonorrhea, spectrophotometer assay, Staphylococcus aureus

Procedia PDF Downloads 293

Authors: Binh T. T. Nguyen, Zhenyu Li, Eric Yap, Yi Zhang, Ai-Qun Liu

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Silicon-photonic-sensor system is an emerging class of analytical technologies that use evanescent field wave to sensitively measure the slight difference in the surrounding environment. The wavelength shift induced by local refractive index change is used as an indicator in the system. These devices can be served as sensors for a wide variety of chemical or biomolecular detection in clinical and environmental fields. In our study, a system including a silicon-based micro-ring resonator, microfluidic channel, and optical processing is designed, fabricated for biomolecule detection. The system is demonstrated to detect Clostridium botulinum type A neurotoxin (BoNT) in different water sources. BoNT is one of the most toxic substances known and relatively easily obtained from a cultured bacteria source. The toxin is extremely lethal with LD50 of about 0.1µg/70kg intravenously, 1µg/ 70 kg by inhalation, and 70µg/kg orally. These factors make botulinum neurotoxins primary candidates as bioterrorism or biothreat agents. It is required to have a sensing system which can detect BoNT in a short time, high sensitive and automatic. For BoNT detection, silicon-based micro-ring resonator is modified with a linker for the immobilization of the anti-botulinum capture antibody. The enzymatic reaction is employed to increase the signal hence gains sensitivity. As a result, a detection limit to 30 pg/mL is achieved by our silicon-photonic sensor within a short period of 80 min. The sensor also shows high specificity versus the other type of botulinum. In the future, by designing the multifunctional waveguide array with fully automatic control system, it is simple to simultaneously detect multi-biomaterials at a low concentration within a short period. The system has a great potential to apply for online, real-time and high sensitivity for the label-free bimolecular rapid detection.

Keywords: biotoxin, photonic, ring resonator, sensor

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Authors: Namratha Pai, K. Vasantharaj, K. Haribabu

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Our Team is developing an innovative cost effective biological technique to desulfurize crude oil. ’Sulphur’ is found to be present in crude oil samples from .05% - 13.95% and its elimination by industrial methods is expensive currently. Materials required :- Alicyclobacillus acidoterrestrius, potato dextrose agar, oxygen, Pyragallol and inert gas(nitrogen). Method adapted and proposed:- 1) Growth of bacteria studied, energy needs. 2) Compatibility with crude-oil. 3) Reaction rate of bacteria studied and optimized. 4) Reaction development by computer simulation. 5) Simulated work tested by building the reactor. The method being developed requires the use of bacteria Alicyclobacillus acidoterrestrius - an acidothermophilic heterotrophic, soil dwelling aerobic, Sulfur bacteria. The bacteria are fed to crude oil in a unique manner. Its coated onto potato dextrose agar beads, cultured for 24 hours (growth time coincides with time when it begins reacting) and fed into the reactor. The beads are to be replenished with O2 by passing them through a jacket around the reactor which has O2 supply. The O2 can’t be supplied directly as crude oil is inflammable, hence the process. Beads are made to move around based on the concept of fluidized bed reactor. By controlling the velocity of inert gas pumped , the beads are made to settle down when exhausted of O2. It is recycled through the jacket where O2 is re-fed and beads which were inside the ring substitute the exhausted ones. Crude-oil is maintained between 1 atm-270 M Pa pressure and 45°C treated with tartaric acid (Ph reason for bacteria growth) for optimum output. Bacteria being of oxidising type react with Sulphur in crude-oil and liberate out SO4^2- and no gas. SO4^2- is absorbed into H2O. NaOH is fed once reaction is complete and beads separated. Crude-oil is thus separated of SO4^2-, thereby Sulphur, tartaric acid and other acids which are separated out. Bio-corrosion is taken care of by internal wall painting (phenolepoxy paints). Earlier methods used included use of Pseudomonas and Rhodococcus species. They were found to be inefficient, time and energy consuming and reduce the fuel value as they fed on skeleton.

Keywords: alicyclobacillus acidoterrestrius, potato dextrose agar, fluidized bed reactor principle, reaction time for bacteria, compatibility with crude oil

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Authors: Akram Abi, Clarissa Müller, Hans-Joachim Jördening

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Laminaribiose is a beta-1,3-glycoside which is used in the medical field for the treatment of dermatitis and also can be used as a building block for new pharmaceutics. The conventional process of laminaribiose production is the uneconomical process of hydrolysis of laminarin extracted from natural polysaccharides of plant origin. A more economical approach however is attainable by enzymatically synthesis of laminaribiose via a reverse phosphorylase reaction catalyzed by laminaribiose phosphorylase (LP) from Euglena gracilis. Different cultivation methods of Euglena gracilis and the effect on LP production have been investigated. Buffered/unbuffered heterotrophic and mixotrophic cultivations of Euglena gracilis has been carried out. Changes of biomass and LP production, glucose level and pH, cell count and shape has been monitored in the course of time. The results obtained from experiments each in three repetitions, show that in the heterotrophic cultivation of Euglena gracilis not only more biomass is produced compared to mixotrophic cultivation, but also higher specific protein concentration is achieved. Furthermore, the LP activity test showed that the protein extracted from heterotrophically cultured cells has a higher LP activity. It was also observed that the cells develop in a distinctive different shape between these two cultures and have different length to width ratios. Taking the heterotrophic culture as the more efficient cultivation method in LP production, another comparative experiment between buffered and unbuffered heterothrophic culture was carried out that showed the unbuffered culture has advantages over the other one in respect of both LP production and resulting activity. A hetrotrophic cultivation of Euglena gracilis in a 5L bioreactor with controlled operating conditions showed a distinctive improvement of all the aspects of culture compared to the shaking flask cultivations. Biomass production was improved from 5 to more than 8 g/l (dry weight) which resulted in a specific protein concentration of 45 g/l in the heterotrophic cultivation in the bioreactor. In further attempts to improve LP production, different purification methods were tested and each method was checks through an activity assay. A laminaribiose yield of 35% was achieved which was by far the highest amount amongst different methods tested.

Keywords: euglena gracilis, heterotrophic culture, laminaribiose production, mixotrophic culture

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Authors: Yemima Dani Riani, Anggraini Barlian

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Green turtle (Chelonia mydas) is one of well known long-lived turtle. Its age can reach 100 years old. Senescence in green turtle is an interesting process to study because until now no clear explanation has been established about senescence at cellular or molecular level in this species. Since 1999, green turtle announced as an endangered species. Hence, establishment of fibroblast skin cell culture of green turtle may be material for future study of senescence. One common marker used for detecting senescence is telomere shortening. Reduced telomerase activity, the reverse transcriptase enzyme which adds TTAGGG DNA sequence to telomere end, may also cause senescence. The purpose of this research are establish and identify green turtle fibroblast skin cell culture and also compare telomere length and telomerase activity from passage 5 and 14. Primary cell culture made with primary explant method then cultured in Leibovitz-15 (Sigma) supplemented by 10% Fetal Bovine Serum (Sigma) and 100 U/mL Penicillin/Streptomycin (Sigma) at 30 ± 1oC. Cells identified with Rabbit Anti-Vimentin Polyclonal Antibody (Abcam) and Goat Polyclonal Antibody (Abcam) using confocal microscope (Zeiss LSM 170). Telomere length obtained using TeloTAGGG Telomere Length Assay (Roche) while telomerase activity obtained using TeloTAGGG Telomerase PCR ElisaPlus (Roche). Primary cell culture from green turtle skin had fibroblastic morphology and immunocytochemistry test with vimentin antibody proved the culture was fibroblast cell. Measurement of telomere length and telomerase activity showed that telomere length and telomerase activity of passage 14 was greater than passage 5. However, based on morphology, green turtle fibroblast skin cell culture showed senescent morphology. Based on the analysis of telomere length and telomerase activity, suspected fibroblast skin cell culture of green turtles is not undergo aging through telomere shortening.

Keywords: cell culture, chelonia mydas, telomerase, telomere, senescence

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Authors: Aliya Sekenova, Vyacheslav Ogay

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The derivation of human induced pluripotent stem cells (iPSCs) from somatic cells by direct reprogramming opens wide perspectives in the regenerative medicine. It means the possibility to develop the personal and, consequently, any immunologically compatible cells for applications in cell-based therapy. The purpose of our study was to develop the technology for the production of NK cells from T cell-derived induced pluripotent stem cells (TiPSCs) for subsequent application in adoptive cancer immunotherapy. Methods: In this study iPSCs were derived from peripheral blood T cells using Sendai virus vectors expressing Oct4, Sox2, Klf4 and c-Myc. Pluripotent characteristics of TiPSCs were examined and confirmed with alkaline phosphatase staining, immunocytochemistry and RT-PCR analysis. For NK cell differentiation, embryoid bodies (EB) formed from (TiPSCs) were cultured in xenogeneic serum-free medium containing human serum, IL-3, IL-7, IL-15, SCF, FLT3L without using M210-B4 and AFT-024 stromal feeder cells. After differentiation, NK cells were characterized with immunofluorescence analysis, flow cytometry and cytotoxicity assay. Results: Here, we for the first time demonstrate that TiPSCs can effectively differentiate into functionally active NK cells without M210-B4 and AFT-024 xenogeneic stroma cells. Immunofluorescence and flow cytometry analysis showed that EB-derived cells can differentiate into a homogeneous population of NK cell expressing high levels of CD56, CD45 and CD16 specific markers. Moreover, these cells significantly express killing activation receptors such as NKp44 and NKp46. In the comparative analysis, we observed that NK cells derived using feeder-free culture system have more high killing activity against K-562 tumor cells, than NK cells derived by feeder-dependent method. Thus, we think that our obtained data will be useful for the development of large-scale production of NK cells for translation into cancer immunotherapy.

Keywords: induced pluripotent stem cells, NK cells, T cells, cell diffentiation, feeder cell-free culture system

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