Search results for: anticancer vaccine
Commenced in January 2007
Frequency: Monthly
Edition: International
Paper Count: 518

Search results for: anticancer vaccine

98 Effector and Memory Immune Responses Induced by Total Extracts of Naegleria fowleri Co-Administered with Cholera Toxin

Authors: Q. B. Maria de la Luz Ortega Juárez, Saúl Rojas Hernández, Itzel Berenice Rodríguez Mera, María Maricela Carrasco Yépez, Mara Gutierrez Sánchez

Abstract:

Naegleria fowleri is a free-living amoeba found mainly in temperate freshwater and is the etiologic agent of primary amebic meningoencephalitis (PAM), a fatal acute disease with a mortality rate greater than 95%. At present, there are no treatments available for MAP, and the development of effective vaccines that generate long-term immunological memory allowing protection against MAP would be of great importance. The objective of this work was to analyze the effector and memory immune response in BALB/c mice immunized with total extract of N. fowleri co-administered with cholera toxin. In this study, BALB/c mice were immunized four times intranasally with ET of N. fowleri adjuvanted with CT with or without booster at three months and were challenged or not with the lethal dose of N. fowleri, determining survival, the humoral, effector and memory response, by ELISA and flow cytometry techniques. The results obtained showed that the survival of mice immunized with booster had 60% protection compared to the group without booster, which obtained 20% protection. Evaluating the humoral response, it was found that both IgG and IgA levels were higher in sera than in nasal washes in both treatments. In the cellular response, the increase in the percentage of positive cells was found for effector T and B lymphocytes in the nasal passages (NP) in the group with boost and nasopharynx-associated lymphoid tissue (NALT) in the group without boost and lymphocytes only. B in both treatments, as well as in memory cells treatment with boost T lymphocytes in PN and NALT and without boost in cervical lymph nodes (CG) with respect to B lymphocytes, in PN, GC and NALT in treatment with boost and NALT in treatment without booster. Therefore, the involvement of the effector immune response and memory play a fundamental role for protection against N. fowleri and for the development of vaccine candidates.

Keywords: immune response, immunological memory, naegleria fowleri, primary amebic meningoencephalitis

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97 Comparison of Susceptibility to Measles in Preterm Infants versus Term Infants

Authors: Joseph L. Mathew, Shourjendra N. Banerjee, R. K. Ratho, Sourabh Dutta, Vanita Suri

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Background: In India and many other developing countries, a single dose of measles vaccine is administered to infants at 9 months of age. This is based on the assumption that maternal transplacentally transferred antibodies will protect infants until that age. However, our previous data showed that most infants lose maternal anti-measles antibodies before 6 months of age, making them susceptible to measles before vaccination at 9 months. Objective: This prospective study was designed to compare susceptibility in pre-term vs term infants, at different time points. Material and Methods: Following Institutional Ethics Committee approval and a formal informed consent process, venous blood was drawn from a cohort of 45 consecutive term infants and 45 consecutive pre-term infants (both groups delivered by the vaginal route); at birth, 3 months, 6 months and 9 months (prior to measles vaccination). Serum was separated and anti-measles IgG antibody levels were measured by quantitative ELISA kits (with sensitivity and specificity > 95%). Susceptibility to measles was defined as antibody titre < 200mIU/ml. The mean antibody levels were compared between the two groups at the four time points. Results: The mean gestation of term babies was 38.5±1.2 weeks; and pre-term babies 34.7±2.8 weeks. The respective mean birth weights were 2655±215g and 1985±175g. Reliable maternal vaccination record was available in only 7 of the 90 mothers. Mean anti-measles IgG antibody (±SD) in terms babies was 3165±533 IU/ml at birth, 1074±272 IU/ml at 3 months, 314±153 IU/ml at 6 months, and 68±21 IU/ml at 9 months. The corresponding levels in pre-term babies were 2875±612 IU/ml, 948±377 IU/ml, 265±98 IU/ml, and 72±33 IU/ml at 9 months (p > 0.05 for all inter-group comparisons). The proportion of susceptible term infants at birth, 3months, 6months and 9months was 0%, 16%, 67% and 96%. The corresponding proportions in the pre-term infants were 0%, 29%, 82%, and 100% (p > 0.05 for all inter-group comparisons). Conclusion: Majority of infants are susceptible to measles before 9 months of age suggesting the need to anticipate measles vaccination, but there was no statistically significant difference between the proportion of susceptible term and pre-term infants, at any of the four-time points. A larger study is required to confirm these findings and compare sero-protection if vaccination is anticipated to be administered between 6 and 9 months.

Keywords: measles, preterm, susceptibility, term infant

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96 Solid Lipid Nanoparticles of Levamisole Hydrochloride

Authors: Surendra Agrawal, Pravina Gurjar, Supriya Bhide, Ram Gaud

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Levamisole hydrochloride is a prominent anticancer drug in the treatment of colon cancer but resulted in toxic effects due poor bioavailability and poor cellular uptake by tumor cells. Levamisole is an unstable drug. Incorporation of this molecule in solid lipids may minimize their exposure to the aqueous environment and partly immobilize the drug molecules within the lipid matrix-both of which may protect the encapsulated drugs against degradation. The objectives of the study were to enhance bioavailability by sustaining drug release and to reduce the toxicities associated with the therapy. Solubility of the drug was determined in different lipids to select the components of Solid Lipid Nanoparticles (SLN). Pseudoternary phase diagrams were created using aqueous titration method. Formulations were subjected to particle size and stability evaluation to select the final test formulations which were characterized for average particle size, zeta potential, and in-vitro drug release and percentage transmittance to optimize the final formulation. SLN of Levamisole hydrochloride was prepared by Nanoprecipitation method. Glyceryl behenate (Compritol 888 ATO) was used as core comprising of Tween 80 as surfactant and Lecithin as co-surfactant in (1:1) ratio. Entrapment efficiency (EE) was found to be 45.89%. Particle size was found in the range of 100-600 nm. Zeta potential of the formulation was -17.0 mV revealing the stability of the product. In-vitro release study showed that 66 % drug released in 24 hours in pH 7.2 which represent that formulation can give controlled action at the intestinal environment. In pH 5.0 it showed 64% release indicating that it can even release drug in acidic environment of tumor cells. In conclusion, results revealed SLN to be a promising approach to sustain the drug release so as to increase bioavailability and cellular uptake of the drug with reduction in toxic effects as dose has been reduced with controlled delivery.

Keywords: SLN, nanoparticulate delivery of levamisole, pharmacy, pharmaceutical sciences

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95 Molecular Detection of Acute Virus Infection in Children Hospitalized with Diarrhea in North India during 2014-2016

Authors: Ali Ilter Akdag, Pratima Ray

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Background:This acute gastroenteritis viruses such as rotavirus, astrovirus, and adenovirus are mainly responsible for diarrhea in children below < 5 years old. Molecular detection of these viruses is crucially important to the understand development of the effective cure. This study aimed to determine the prevalence of common these viruses in children < 5 years old presented with diarrhea from Lala Lajpat Rai Memorial Medical College (LLRM) centre (Meerut) North India, India Methods: Total 312 fecal samples were collected from diarrheal children duration 3 years: in year 2014 (n = 118), 2015 (n = 128) and 2016 (n = 66) ,< 5 years of age who presented with acute diarrhea at the Lala Lajpat Rai Memorial Medical College (LLRM) centre(Meerut) North India, India. All samples were the first detection by EIA/RT-PCR for rotaviruses, adenovirus and astrovirus. Results: In 312 samples from children with acute diarrhea in sample viral agent was found, rotavirus A was the most frequent virus identified (57 cases; 18.2%), followed by Astrovirus in 28 cases (8.9%), adenovirus in 21 cases (6.7%). Mixed infections were found in 14 cases, all of which presented with acute diarrhea (14/312; 4.48%). Conclusions: These viruses are a major cause of diarrhea in children <5 years old in North India. Rotavirus A is the most common etiological agent, follow by astrovirus. This surveillance is important to vaccine development of the entire population. There is variation detection of virus year wise due to differences in the season of sampling, method of sampling, hygiene condition, socioeconomic level of the entire people, enrolment criteria, and virus detection methods. It was found Astrovirus higher then Rotavirus in 2015, but overall three years study Rotavirus A is mainly responsible for causing severe diarrhea in children <5 years old in North India. It emphasizes the required for cost-effective diagnostic assays for Rotaviruses which would help to determine the disease burden.

Keywords: adenovirus, Astrovirus, hospitalized children, Rotavirus

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94 Preparation Nanocapsules of Chitosan Modified With Selenium Extracted From the Lactobacillus Acidophilus and Their Anticancer Properties

Authors: Akbar Esmaeili, Mahnoosh Aliahmadi

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This study synthesized a modified imaging of gallium@deferoxamine/folic acid/chitosan/polyaniline/polyvinyl alcohol (Ga@DFA/FA/CS/PANI/PVA). It contains Morus nigra extract by selenium nanoparticles prepared from Lactobacillus acidophilus. Using the impregnation method, Se nanoparticles were then deposited on (Ga@DFA/FA/ CS/PANI/PVA). The modified contrast agents were mixed with M. nigra extract, and investigated their antibacterial activities by applying to L929 cell lines. The influence of variable factors, including 1. surfactant, 2. solvent, 3. aqueous phase, 4. pH, 5. buffer, 6. minimum Inhibitory concentration (MIC), 7. minimum bactericidal concentration (MBC), 8. cytotoxicity on cancer cells., 9. antibiotic, 10. antibiogram, 11. release and loading, 12. the emotional effect, 13. the concentration of nanoparticles, 14. olive oil, and 15. they have investigated thermotical methods. The structure and morphology of the synthesized contrast agents were characterized by zeta potential sizer analysis (ZPS), X-Ray diffraction (XRD), Fourier-transform infrared (FT-IR), energy dispersive X-ray (EDX), ultraviolet–visible (UV–Vis) spectra, and scanning electron microscope (SEM). The experimental section was conducted and monitored by response surface methods (RSM), MTT, MIC, MBC, and cancer cytotoxic conversion assay. Antibiogram testing of NCs on Pseudomonas aeruginosa bacteria was successful and obtained MIC = 2 factors with less harmful effect. All experimental sections confirmed that our synthesized particles have potent antioxidant properties. Antibiogram testing revealed that NPS could kill P. aeruginosa and P. aeruginosa. A variety of synthetic conditions were done by diffusion emulsion method by varying parameters, the optimum state of DFA release Ga@DFA/FA/CS/PANI/PVA NPs (6 ml) with pH = 5.5, time = 3 h, NCs and DFA (3 mg), and achieved buffer (20 ml). DFA in Ga@DFA/FA/ CS/PANI/PVA was released and showed an absorption peak at 378 nm by applying a 300-rpm magnetic rate. In this report, Ga decreased the harmful effect on the human body.

Keywords: nanocapsules, technolgy, biology, nano

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93 Evaluation of Genetic Fidelity and Phytochemical Profiling of Micropropagated Plants of Cephalantheropsis obcordata: An Endangered Medicinal Orchid

Authors: Gargi Prasad, Ashiho A. Mao, Deepu Vijayan, S. Mandal

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The main objective of the present study was to optimize and develop an efficient protocol for in vitro propagation of a medicinally important orchid Cephalantheropsis obcordata (Lindl.) Ormerod along with genetic stability analysis of regenerated plants. This plant has been traditionally used in Chinese folk medicine and the decoction of whole plant is known to possess anticancer activity. Nodal segments used as explants were inoculated on Murashige and Skoog (MS) medium supplemented with various concentrations of isopentenyl adenine (2iP). The rooted plants were successfully acclimatized in the greenhouse with 100% survival rate. Inter-simple sequence repeats (ISSR) markers were used to assess the genetic fidelity of in vitro raised plants and the mother plant. It was revealed that monomorphic bands showing the absence of polymorphism in all in vitro raised plantlets analyzed, confirming the genetic uniformity among the regenerants. Phytochemical analysis was done to compare the antioxidant activities and HPLC fingerprinting assay of 80% aqueous ethanol extract of the leaves and stem of in vitro and in vivo grown C. obcordata. The extracts of the plants were examined for their antioxidant activities by using free radical 1, 1-diphenyl-2-picryl hydrazyl (DPPH) scavenging method, 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging ability, reducing power capacity, estimation of total phenolic content, flavonoid content and flavonol content. A simplified method for the detection of ascorbic acid, phenolic acids and flavonoids content was also developed by using reversed phase high-performance liquid chromatography (HPLC). This is the first report on the micropropagation, genetic integrity study and quantitative phytochemical analysis of in vitro regenerated plants of C. obcordata.

Keywords: Cephalantheropsis obcordata, genetic fidelity, ISSR markers, HPLC

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92 Pegylated Liposomes of Trans Resveratrol, an Anticancer Agent, for Enhancing Therapeutic Efficacy and Long Circulation

Authors: M. R. Vijayakumar, Sanjay Kumar Singh, Lakshmi, Hithesh Dewangan, Sanjay Singh

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Trans resveratrol (RES) is a natural molecule proved for cancer preventive and therapeutic activities devoid of any potential side effects. However, the therapeutic application of RES in disease management is limited because of its rapid elimination from blood circulation thereby low biological half life in mammals. Therefore, the main objective of this study is to enhance the circulation as well as therapeutic efficacy using PEGylated liposomes. D-α-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS) is applied as steric surface decorating agent to prepare RES liposomes by thin film hydration method. The prepared nanoparticles were evaluated by various state of the art techniques such as dynamic light scattering (DLS) technique for particle size and zeta potential, TEM for shape, differential scanning calorimetry (DSC) for interaction analysis and XRD for crystalline changes of drug. Encapsulation efficiency and invitro drug release were determined by dialysis bag method. Cancer cell viability studies were performed by MTT assay, respectively. Pharmacokinetic studies were performed in sprague dawley rats. The prepared liposomes were found to be spherical in shape. Particle size and zeta potential of prepared formulations varied from 64.5±3.16 to 262.3±7.45 nm and -2.1 to 1.76 mV, respectively. DSC study revealed absence of potential interaction. XRD study revealed presence of amorphous form in liposomes. Entrapment efficiency was found to be 87.45±2.14 % and the drug release was found to be controlled up to 24 hours. Minimized MEC in MTT assay and tremendous enhancement in circulation time of RES PEGylated liposomes than its pristine form revealed that the stearic stabilized PEGylated liposomes can be an alternative tool to commercialize this molecule for chemopreventive and therapeutic applications in cancer.

Keywords: trans resveratrol, cancer nanotechnology, long circulating liposomes, bioavailability enhancement, liposomes for cancer therapy, PEGylated liposomes

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91 The Comparison between bFGF and Small Molecules in Derivation of Chicken Primordial Germ Cells and Embryonic Germ Cells

Authors: Maryam Farzaneh, Seyyedeh Nafiseh Hassani, Hossein Baharvand

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Objective: Chicken gonadal tissue has a two population such primordial germ cells (PGCs) and stromal cells (somatic cells). PGCs and embryonic germ cells (EGCs) that is a pluripotent type of PGCs in long-term culture are suitable sources for the production of chicken pluripotent stem cell lines, transgenic birds, vaccine and recombinant protein production. In general, the effect of growth factors such bFGF and mouse LIF on derivation of PGCs in vitro are important and in this study we could see the unique effect of small molecules such PD032 and SB43 as a chemical, in comparison to growth factors. Materials and Methods: After incubation of fertilized chicken egg up to 6 days and isolation of primary gonadal tissues and culture of mixed cells like PGCs and stromal cells. PGCs proliferate in the present of fetal calf serum (FCS) and small molecules and in another group bFGF, that these factors are important for PGCs culture and derivation. Somatic cells produce a multilayer feeder under the PGCs in primary culture and PGCs make a small cluster under these cells. Results: In present of small molecules and high volume of FCS (15%), the present of EGCs as a pluripotent stem cells were clear four weeks, that they had a positive immune-staining and periodic acid-Schiff staining (PAS), but in present of growth factors like bFGF without any chemicals, the present of PGCs were clear but after 7 until 10 days, there were disappear. Conclusion: Until now we have seen many researches about derivation and maintenance of chicken PGCs, in the hope of understanding the mechanisms that occur during germline development and production of a therapeutic product by transgenic birds. There are still many unknowns in this area and this project will try to have efficient conditions for identification of suitable culture medium for long-term culture of PGCs in vitro without serum and feeder cells.

Keywords: chicken gonadal primordial germ cells, pluripotent stem cells, growth factors, small molecules, transgenic birds

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90 Functional Characterization of Rv1019, a Putative TetR Family Transcriptional Regulator of Mycobacterium Tuberculosis H37Rv

Authors: Akhil Raj Pushparajan, Ranjit Ramachandran, Jijimole Gopi Reji, Ajay Kumar Ramakrishnan

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Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of the leading causes of death by an infectious disease. In spite of the availability of effective drugs and a vaccine, TB is a major health concern and was declared a global emergency by the World Health Organization (WHO). The success of intracellular pathogens like Mtb depends on its ability to overcome the challenging environment in the host. Gene regulation controlled by transcriptional regulators (TRs) plays a crucial role for the bacteria to adapt to the host environment. In vitro studies on gene regulatory mechanisms during dormancy and reactivation have provided insights into the adaptations employed by Mtb to survive in the host. Here we present our efforts to functionally characterize Rv1019, a putative TR of Mtb H37Rv which was found to be present at significantly varying levels during dormancy and reactivation in vitro. The expression of this protein in the dormancy-reactivation model was validated by qRT-PCR and western blot. By DNA- protein interaction studies and reporter assays we found that under normal laboratory conditions of growth this protein behaves as an auto-repressor and tetracycline was found to abrogate this repression by interfering with its ability to bind DNA. Further, by cDNA analysis, we found that this TR is co-transcribed with its downstream genes Rv1020 (mfd) and Rv1021 (mazG) which are involved in DNA damage response in Mtb. Constitutive expression of this regulator in the surrogate host M. smegmatis showed downregulation of the orthologues of downstream genes suggested that Rv1019 could negatively regulate these genes. Our finds also show that M. smegmatis expressing Rv1019 is sensitive to DNA damage suggests the role of this protein in regulating DNA damage response induced by oxidative stress. Because of its role in regulating DNA damage response which may help in the persistence of Mtb, Rv1019 could be used as a prospective target for therapeutic intervention to fight TB.

Keywords: auto-repressor, DNA repair, mycobacterium smegmatis, mycobacterium tuberculosis, tuberculosis

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89 Cytotoxicity of 13 South African Macrofungal Species and Mechanism/s of Action against Cancer Cell Lines

Authors: Gerhardt Boukes, Maryna Van De Venter, Sharlene Govender

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Macrofungi have been used for the past two thousand years in Asian countries, and more recently in Western countries, for their medicinal properties. Biological activities include antimicrobial, antioxidant, anti-inflammatory, antidiabetic, anticancer and immunomodulatory to name a few. Several biologically active compounds have been identified and isolated. Macrofungal research in Africa is poorly documented and to the best of our knowledge non-existent. South Africa has a rich macrofungal biodiversity, which includes endemic and exotic macrofungal species. Ethanolic extracts of 13 macrofungal species, including mushrooms, bracket fungi and puffballs, were prepared and screened for cytotoxicity against a panel of seven cell lines, including A549 (human lung adenocarcinoma), HeLa (human cervical adenocarcinoma), HT-29 (human colorectal adenocarcinoma), MCF7 (human breast adenocarcinoma), MIA PaCa-2 (human pancreatic ductal adenocarcinoma), PC-3 (human prostate adenocarcinoma) and Vero (African green monkey kidney epithelial) cells using MTT. Cell lines were chosen according to the most prevalent cancer types affecting males and females in South Africa and globally, and the mutations they contain. Preliminary results have shown that three of the macrofungal genera, i.e. Fomitopsis, Gymnopilus and Pycnoporus, have shown cytotoxic activity, ranging between IC50 ~20 and 200 µg/mL. The molecular mechanism of action contributing to cell death investigated and being investigated include apoptosis (i.e. DNA cell cycle arrest, caspase-3 activation and mitochondrial membrane potential), autophagy (i.e. acridine orange and LC3B staining) and ER stress (i.e. thioflavin T staining and caspase-12) in the presence of melphalan, chloroquine and thapsigargin/tuncamycin as positive controls, respectively. The genus, Pycnoporus, has shown the best cytotoxicity of the three macrofungal genera. Future work will focus on the identification and isolation of novel active compounds and elucidating the mechanism/s of action.

Keywords: cancer, cytotoxicity, macrofungi, mechanism/s of action

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88 Longitudinal Profile of Antibody Response to SARS-CoV-2 in Patients with Covid-19 in a Setting from Sub–Saharan Africa: A Prospective Longitudinal Study

Authors: Teklay Gebrecherkos

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Background: Serological testing for SARS-CoV-2 plays an important role in epidemiological studies, in aiding the diagnosis of COVID-19 and assess vaccine responses. Little is known about the dynamics of SARS-CoV-2 serology in African settings. Here, we aimed to characterize the longitudinal antibody response profile to SARS-CoV-2 in Ethiopia. Methods: In this prospective study, a total of 102 PCR-confirmed COVID-19 patients were enrolled. We obtained 802 plasma samples collected serially. SARS-CoV-2 antibodies were determined using four lateral flow immune assays (LFIAs) and an electrochemiluminescent immunoassay. We determined longitudinal antibody response to SARS-CoV-2 as well as seroconversion dynamics. Results: Serological positivity rate ranged between 12%-91%, depending on timing after symptom onset. There was no difference in the positivity rate between severe and non-severe COVID-19 cases. The specificity ranged between 90%-97%. Agreement between different assays ranged between 84%-92%. The estimated positive predictive value (PPV) for IgM or IgG in a scenario with seroprevalence at 5% varies from 33% to 58%. Nonetheless, when the population seroprevalence increases to 25% and 50%, there is a corresponding increase in the estimated PPVs. The estimated negative-predictive value (NPV) in a low seroprevalence scenario (5%) is high (>99%). However, the estimated NPV in a high seroprevalence scenario (50%) for IgM or IgG is reduced significantly from 80% to 85%. Overall, 28/102 (27.5%) seroconverted by one or more assays tested within a median time of 11 (IQR: 9–15) days post symptom onset. The median seroconversion time among symptomatic cases tended to be shorter when compared to asymptomatic patients [9 (IQR: 6–11) vs. 15 (IQR: 13–21) days; p = 0.002]. Overall, seroconversion reached 100% 5.5 weeks after the onset of symptoms. Notably, of the remaining 74 COVID-19 patients included in the cohort, 64 (62.8%) were positive for antibodies at the time of enrollment, and 10 (9.8%) patients failed to mount a detectable antibody response by any of the assays tested during follow-up. Conclusions: Longitudinal assessment of antibody response in African COVID-19 patients revealed heterogeneous responses. This underscores the need for a comprehensive evaluation of serum assays before implementation. Factors associated with failure to seroconvert need further research.

Keywords: COVID-19, antibody, rapid diagnostic tests, ethiopia

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87 Raising Antibodies against Epoxyscillirosidine, the Toxic Principle Contained in Moraea pallida Bak. in Rabbits

Authors: Hamza I. Isa, Gezina C. H. Ferreira, Jan E. Crafford, Christoffel J. Botha

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Moraea pallida Bak. (yellow tulip) poisoning is the most important plant-induced cardiac glycoside toxicosis in South Africa. Cardiac glycoside poisonings collectively account for about 33 and 10 % mortalities due to plants, in large and small stock respectively, in South Africa. The toxic principle is 1α, 2α-epoxyscillirosidine, a bufadienolide. The aim of the study was to investigate the potential to develop a vaccine against epoxyscillirosidine. Epoxyscillirosidine and the related bufadienolides proscillaridin and bufalin, which are commercially available, were conjugated to the carrier proteins [Hen ovalbumin (OVA), bovine serum albumin (BSA) and keyhole limpet haemocyanin (KLH)], rendering them immunogenic. Adult male New Zealand White rabbits were immunized. In Trials 1 and 2, rabbits (n=6) were, each assigned to two groups. Experimental animals (n=3; n=4) were vaccinated with epoxyscillirosidine-OVA conjugate, while the control (n=3; n=2) were vaccinated with OVA, using Freund’s complete and incomplete and Montanide adjuvants, for Trials 1 and 2, respectively. In Trial 3, rabbits (n=15), randomly allocated to 5 equal groups (I, II, III, IV and V), were vaccinated with proscillaridin-BSA, bufalin-BSA, epoxyscillirosidine-KLH, epoxyscillirosidine-BSA conjugates, and BSA respectively, using Montanide as adjuvant. Vaccination was on Days 0, 21 and 42. Additional vaccinations were done on Day 56 and 63 for Trial 1. Vaccination was by intradermal injection of 0.4 ml of the immunogen (4 mg/ml [Trial 1] and 8 mg/ml for Trials 2 and Trial 3, respectively). Blood was collected pre-vaccination and at 3 week intervals following each vaccination. Antibody response was determined using an indirect ELISA. There was poor immune response associated with the dose (0.4 mg per rabbit) and adjuvant used in Trial 1. Antibodies were synthesized against the conjugate administered in Trial 2. For Trail 3, antibodies against the immunogens were successfully raised in rabbits with epoxyscillirosidine-KLH inducing the highest immune response. The antibodies raised against proscillaridin and bufalin cross-reacted with epoxyscillirosidine when used as antigen in the ELISA. The study successfully demonstrated the synthesis of antibodies against the bufadienolide conjugates administered. The cross-reactivity of proscillaridin and bufalin with epoxyscillirosidine could potentially be utilized as alternative to epoxyscillirosidine in future studies to prevent yellow tulp poisoning by vaccination.

Keywords: antibodies , bufadienolides, cross-reactivity, epoxyscillirosidine, Moraea pallida, poisoning

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86 Upconversion Nanoparticle-Mediated Carbon Monoxide Prodrug Delivery System for Cancer Therapy

Authors: Yaw Opoku-Damoah, Run Zhang, Hang Thu Ta, Zhi Ping Xu

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Gas therapy is still at an early stage of research and development. Even though most gasotransmitters have proven their therapeutic potential, their handling, delivery, and controlled release have been extremely challenging. This research work employs a versatile nanosystem that is capable of delivering a gasotransmitter in the form of a photo-responsive carbon monoxide-releasing molecule (CORM) for targeted cancer therapy. The therapeutic action was mediated by upconversion nanoparticles (UCNPs) designed to transfer bio-friendly low energy near-infrared (NIR) light to ultraviolet (UV) light capable of triggering carbon monoxide (CO) from a water-soluble amphiphilic manganese carbonyl complex CORM incorporated into a carefully designed lipid drug delivery system. Herein, gaseous CO that plays a role as a gasotransmitter with cytotoxic and homeostatic properties was investigated to instigate cellular apoptosis. After successfully synthesizing the drug delivery system, the ability of the system to encapsulate and mediate the sustained release of CO after light excitation was demonstrated. CO fluorescence probe (COFP) was successfully employed to determine the in vitro drug release profile upon NIR light irradiation. The uptake of nanoparticles enhanced by folates and its receptor interaction was also studied for cellular uptake purposes. The anticancer potential of the final lipid nanoparticle Lipid/UCNPs/CORM/FA (LUCF) was also determined by cell viability assay. Intracellular CO release and a subsequent therapeutic action involving ROS production, mitochondrial damage, and CO production was also evaluated. In all, this current project aims to use in vitro studies to determine the potency and efficiency of a NIR-mediated CORM prodrug delivery system.

Keywords: carbon monoxide-releasing molecule, upconversion nanoparticles, site-specific delivery, amphiphilic manganese carbonyl complex, prodrug delivery system.

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85 Investigating Anti-Tumourigenic and Anti-Angiogenic Effects of Resveratrol in Breast Carcinogenesis Using in-Silico Algorithms

Authors: Asma Zaib, Saeed Khan, Ayaz Ahmed Noonari, Sehrish Bint-e-Mohsin

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Breast cancer is the most common cancer among females worldwide and is estimated that more than 450,000 deaths are reported each year. It accounts for about 14% of all female cancer deaths. Angiogenesis plays an essential role in Breast cancer development, invasion, and metastasis. Breast cancer predominantly begins in luminal epithelial cells lining the normal breast ducts. Breast carcinoma likely requires coordinated efforts of both increased proliferation and increased motility to progress to metastatic stages.Resveratrol: a natural stilbenoid, has anti-inflammatory and anticancer effects that inhibits proliferation of variety of human cancer cell lines, including breast, prostate, stomach, colon, pancreatic, and thyroid cancers.The objective of this study is:To investigate anti-neoangiogenesis effects of Resveratrol in breast cancer and to analyze inhibitory effects of resveratrol on aromatase, Erα, HER2/neu, and VEGFR.Docking is the computational determination of binding affinity between molecule (protein structure and ligand).We performed molecular docking using Swiss-Dock and to determine docking effects of (1) Resveratrol with Aromatase, (2) Resveratrol with ERα (3) Resveratrol with HER2/neu and (4) Resveratrol with VEGFR2.Docking results of resveratrol determined inhibitory effects on aromatase with binding energy of -7.28 kcal/mol which shows anticancerous effects on estrogen dependent breast tumors. Resveratrol also show inhibitory effects on ERα and HER2/new with binging energy -8.02, and -6.74 respectively; which revealed anti-cytoproliferative effects upon breast cancer. On the other hand resveratrol v/s VEGFR showed potential inhibitory effects on neo-angiogenesis with binding energy -7.68 kcal/mol, angiogenesis is the important phenomenon that promote tumor development and metastasis. Resveratrol is an anti-breast cancer agent conformed by in silico studies, it has been identified that resveratrol can inhibit breast cancer cells proliferation by acting as competitive inhibitor of aromatase, ERα and HER2 neo, while neo-angiogemesis is restricted by binding to VEGFR which authenticates the anti-carcinogenic effects of resveratrol against breast cancer.

Keywords: angiogenesis, anti-cytoproliferative, molecular docking, resveratrol

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84 Autophagy Suppresses Bladder Tumor Formation in a Mouse Orthotopic Bladder Tumor Formation Model

Authors: Wan-Ting Kuo, Yi-Wen Liu, Hsiao-Sheng Liu

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Annual incidence of bladder cancer increases in the world and occurs frequently in the male. Most common type is transitional cell carcinoma (TCC) which is treated by transurethral resection followed by intravesical administration of agents. In clinical treatment of bladder cancer, chemotherapeutic drugs-induced apoptosis is always used in patients. However, cancers usually develop resistance to chemotherapeutic drugs and often lead to aggressive tumors with worse clinical outcomes. Approximate 70% TCC recurs and 30% recurrent tumors progress to high-grade invasive tumors, indicating that new therapeutic agents are urgently needed to improve the successful rate of overall treatment. Nonapoptotic program cell death may assist to overcome worse clinical outcomes. Autophagy which is one of the nonapoptotic pathways provides another option for bladder cancer patients. Autophagy is reported as a potent anticancer therapy in some cancers. First of all, we established a mouse orthotopic bladder tumor formation model in order to create a similar tumor microenvironment. IVIS system and micro-ultrasound were utilized to noninvasively monitor tumor formation. In addition, we carried out intravesical treatment in our animal model to be consistent with human clinical treatment. In our study, we carried out intravesical instillation of the autophagy inducer in mouse orthotopic bladder tumor to observe tumor formation by noninvasive IVIS system and micro-ultrasound. Our results showed that bladder tumor formation is suppressed by the autophagy inducer, and there are no significant side effects in the physiology of mice. Furthermore, the autophagy inducer upregulated autophagy in bladder tissues of the treated mice was confirmed by Western blot, immunohistochemistry, and immunofluorescence. In conclusion, we reveal that a novel autophagy inducer with low side effects suppresses bladder tumor formation in our mouse orthotopic bladder tumor model, and it provides another therapeutic approach in bladder cancer patients.

Keywords: bladder cancer, transitional cell carcinoma, orthotopic bladder tumor formation model, autophagy

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83 Risk Factors for Severe Typhoid Fever in Children: A French Retrospective Study about 78 Cases from 2000-2017 in Six Parisian Hospitals

Authors: Jonathan Soliman, Thomas Cavasino, Virginie Pommelet, Lahouari Amor, Pierre Mornand, Simon Escoda, Nina Droz, Soraya Matczak, Julie Toubiana, François Angoulvant, Etienne Carbonnelle, Albert Faye, Loic de Pontual, Luu-Ly Pham

Abstract:

Background: Typhoid and paratyphoid fever are systemic infections caused by Salmonella enterica serovar Typhi or paratyphi (A, B, C). Children traveling to tropical areas are at risk to contract these diseases which can be complicated. Methods: Clinical, biological and bacteriological data were collected from 78 pediatric cases reported between 2000 and 2017 in six Parisian hospitals. Children aged 0 to 18 years old, with a diagnosis of typhoid or paratyphoid fever confirmed by bacteriological exams, were included. Epidemiologic, clinical, biological features and presence of multidrug-resistant (MDR) bacteria or intermediate susceptibility to ciprofloxacin (nalidixic acid resistant) were examined by univariate analysis and by logistic regression analysis to identify risk factors of severe typhoid in children. Results: 84,6% of the children were imported cases of typhoid fever (n=66/78) and 15,4% were autochthonous cases (n=12/78). 89,7% were caused by S.typhi (n=70/78) and 12,8% by S.paratyphi (n=10/78) including 2 co-infections. 19,2% were intrafamilial cases (n=15/78). Median age at diagnosis was 6,4 years-old [6 months-17,9 years]. 28,2% of the cases were complicated forms (n=22/78): digestive (n=8; 10,3%), neurological (n=7; 9%), pulmonary complications (n=4; 5,1%) and hemophagocytic syndrome (n=4; 5,1%). Only 5% of the children had prior immunization with typhoid non-conjugated vaccine (n=4/78). 28% of the cases (n=22/78) were caused by resistant bacteria. Thrombocytopenia and diagnosis delay was significantly associated with severe infection (p= 0.029 and p=0,01). Complicated forms were more common with MDR (p=0,1) and not statistically associated with a young age or sex in this study. Conclusions: Typhoid and paratyphoid fever are not rare in children back from tropical areas. This multicentric pediatric study seems to show that thrombocytopenia, diagnosis delay, and multidrug resistant bacteria are associated with severe typhoid fever and complicated forms in children.

Keywords: antimicrobial resistance, children, Salmonella enterica typhi and paratyphi, severe typhoid

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82 Molecular Characterisation and Expression of Glutathione S-Transferase of Fasciola Gigantica

Authors: J. Adeppa, S. Samanta, O. K. Raina

Abstract:

Fasciolosis is a widespread economically important parasitic infection throughout the world caused by Fasciola hepatica and F. gigantica. In order to identify novel immunogen conferring significant protection against fasciolosis, currently, research has been focused on the defined antigens viz. glutathione S-transferase, fatty acid binding protein, cathepsin-L, fluke hemoglobin, paramyosin, myosin and F. hepatica- Kunitz Type Molecule. Among various antigens, GST which plays a crucial role in detoxification processes, i.e. phase II defense mechanism of this parasite, has a unique position as a novel vaccine candidate and a drug target in the control of this disease. For producing the antigens in large quantities and their purification to complete homogeneity, the recombinant DNA technology has become an important tool to achieve this milestone. RT- PCR was carried out using F. gigantica total RNA as template, and an amplicon of 657 bp GST gene was obtained. TA cloning vector was used for cloning of this gene, and the presence of insert was confirmed by blue-white selection for recombinant colonies. Sequence analysis of the present isolate showed 99.1% sequence homology with the published sequence of the F. gigantica GST gene of cattle origin (accession no. AF112657), with six nucleotide changes at 72, 74, 423, 513, 549 and 627th bp found in the present isolate, causing an overall change of 4 amino acids. The 657 bp GST gene was cloned at BamH1 and HindIII restriction sites of the prokaryotic expression vector pPROEXHTb in frame with six histidine residues and expressed in E. coli DH5α. Recombinant protein was purified from the bacterial lysate under non-denaturing conditions by the process of sonication after lysozyme treatment and subjecting the soluble fraction of the bacterial lysate to Ni-NTA affinity chromatography. Western blotting with rabbit hyper-immune serum showed immuno-reactivity with 25 kDa recombinant GST. Recombinant protein detected F. gigantica experimental as well as field infection in buffaloes by dot-ELISA. However, cross-reactivity studies on Fasciola gigantica GST antigen are needed to evaluate the utility of this protein in the serodiagnosis of fasciolosis.

Keywords: fasciola gigantic, fasciola hepatica, GST, RT- PCR

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81 Chikungunya Virus Infection among Patients with Febrile Illness Attending University of Maiduguri Teaching Hospital, Nigeria

Authors: Abdul-Dahiru El-Yuguda, Saka Saheed Baba, Tawa Monilade Adisa, Mustapha Bala Abubakar

Abstract:

Background: Chikungunya (CHIK) virus, a previously anecdotally described arbovirus, is now assuming a worldwide public health burden. The CHIK virus infection is characterized by potentially life threatening and debilitating arthritis in addition to the high fever, arthralgia, myalgia, headache and rash. Method: Three hundred and seventy (370) serum samples were collected from outpatients with febrile illness attending University of Maiduguri Teaching Hospital, Nigeria, and was used to detect for Chikungunya (CHIK) virus IgG and IgM antibodies using the Enzyme Linked Immunosorbent Assays (ELISAs). Result: Out of the 370 sera tested, 39 (10.5%) were positive for presence of CHIK virus antibodies. A total of 24 (6.5%) tested positive for CHIK virus IgM only while none (0.0%) was positive for presence of CHIK virus IgG only and 15 (4.1%) of the serum samples were positive for both IgG and IgM antibodies. A significant difference (p<0.0001) was observed in the distribution of CHIK virus antibodies in relation to gender. The males had prevalence of 8.5% IgM antibodies as against 4.6% observed in females. On the other hand 4.6% of the females were positive for concurrent CHIK virus IgG and IgM antibodies when compared to a prevalence of 3.4% observed in males. Only the age groups ≤ 60 years and the undisclosed age group were positive for presence of CHIK virus IgG and/or IgM antibodies. No significant difference (p>0.05) was observed in the seasonal prevalence of CHIK virus antibodies among the study subjects Analysis of the prevalence of CHIK virus antibodies in relation to clinical presentation (as observed by Clinicians) of the patients revealed that headache and fever were the most frequently encountered ailments. Conclusion: The CHIK virus IgM and concurrent IgM and IgG antibody prevalence rates of 6.5% and 4.1% observed in this study indicates a current infection and the lack of IgG antibody alone observed shows that the infection is not endemic but sporadic. Recommendation: Further studies should be carried to establish the seasonal prevalence of CHIK virus infection vis-à-vis vector dynamics in the study area. A comprehensive study need to be carried out on the molecular characterization of the CHIK virus circulating in Nigeria with a view to developing CHIK virus vaccine.

Keywords: Chikungunya virus, IgM and IgG antibodies, febrile patients, enzyme linked immunosorbent assay

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80 BRG1 and Ep300 as a Transcriptional Regulators of Breast Cancer Growth

Authors: Maciej Sobczak, Julita Pietrzak, Tomasz Płoszaj, Agnieszka Robaszkiewicz

Abstract:

Brg1, a member of SWI/SNF complex, plays a role in chromatin remodeling, therefore, regulates expression of many genes. Brg1 is an ATPase of SWI/SNF complex, thus its activity requires ATP. Through its bromodomain recognizes acetylated histone residues and evicts them, thus promoting transcriptionally active state of chromatin. One of the enzymes that is responsible for acetylation of histone residues is Ep300. It was previously shown in the literature that cooperation of Brg1 and Ep300 occurs at the promoter regions that have binding sites for E2F-family transcription factors as well as CpG islands. According to literature, approximately 20% of human cancer possess mutation in Brg1 or any other crucial SWI/SNF subunit. That phenomenon makes Brg1-Ep300 a very promising target for anti-cancer therapy. Therefore in our study, we investigated if physical interaction between Brg1 and Ep300 exists and what impact those two proteins have on key for breast cancer cells processes such as DNA damage repair and cell proliferation. Bioinformatical analysis pointed out, that genes involved in cell proliferation and DNA damage repair are overexpressed in MCF7 and MDA-MB-231 cells. Moreover, promoter regions of these genes are highly acetylated, which suggests high transcriptional activity of those sites. Notably, many of those gene possess within their promoters an E2F, Brg1 motives, as well as CpG islands and acetylated histones. Our data show that Brg1 physically interacts with Ep300, and together they regulate expression of genes involved in DNA damage repair and cell proliferation. Upon inhibiting Brg1 or Ep300, expression of vital for cancer cell survival genes such as CDK2/4, BRCA1/2, PCNA, and XRCC1 is decreased in MDA-MB-231 and MCF7 cells. Moreover, inhibition or silencing of either Brg1 or Ep300 leads to cell cycle arrest in G1. After inhibition of BRG1 or Ep300 on tested gene promoters, the repressor complex including Rb, HDAC1, and EZH2 is formed, which inhibits gene expression. These results highlight potentially significant target for targeted anticancer therapy to be introduced as a supportive therapy.

Keywords: brg1, ep300, breast cancer, epigenetics

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79 An Ecofriendly Approach for the Management of Aedes aegypti L (Diptera: Culicidae) by Ocimum sanctum

Authors: Mohd Shazad, Kamal Kumar Gupta

Abstract:

Aedes aegypti (Diptera: Culicidae), commonly known as tiger mosquito is the vector of dengue fever, yellow fever, chikungunya and zika virus. In the absence of any effective vaccine against these diseases, control the mosquito population is the only promising mean to prevent the diseases. Currently used chemical insecticides cause environmental contamination, high mammalian toxicity and hazards to non-target organisms, insecticide resistance and vector resurgence. Present research work aimed to explore the potentials of phytochemicals present in the Ocimum sanctum in management of mosquito population. The leaves of Ocimum were extracted with ethanol by ‘cold extraction method’. 0-24h old fourth instar larvae of Aedes aegypti were treated with the extract of concentrations 50ppm, 100ppm, 200ppm and 400ppm for 24h. Survival, growth and development of the treated larvae were evaluated. The adults emerged from the treated larvae were used for the reproductive fitness studies. Our results indicate 77.2% mortality in the larvae exposed to 400 ppm. At lower doses, although there was no significant reduction in the survival after 24h however, it decreased during subsequent days of observations. In control experiments, no mortality was observed. It was also observed that the larvae survived after treatment showed severe growth and developmental abnormalities. There was significant increase in larval duration. In control, fourth instar moulted into pupa after 3 days while larvae treated with 400 ppm extract were moulted after 4.6 days. Larva-pupa intermediates and the pupa-adult intermediates were observed in many cases. The adults emerged from the treated larvae showed impaired mating and oviposition behaviour. The females exhibited longer preoviposition period, reduced oviposition rate and decreased egg output. GCMS analysis of the ethanol extract revealed presence of JH mimics and intermediates of JH biosynthetic pathway. Potentials of Ocimum sanctum in integrated vector management programme of Aedes aegypti were discussed.

Keywords: Aedes aegypti, Ocimum sanctum, oviposition, survival

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78 Ficus Microcarpa Fruit Derived Iron Oxide Nanomaterials and Its Anti-bacterial, Antioxidant and Anticancer Efficacy

Authors: Fuad Abdullah Alatawi

Abstract:

Microbial infections-based diseases are a significant public health issue around the world, mainly when antibiotic-resistant bacterium types evolve. In this research, we explored the anti-bacterial and anti-cancer potency of iron-oxide (Fe₂O₃) nanoparticles prepared from F. macrocarpa fruit extract. The chemical composition of F. macrocarpa fruit extract was used as a reducing and capping agent for nanoparticles’ synthesis was examined by GC-MS/MS analysis. Then, the prepared nanoparticles were confirmed by various biophysical techniques, including X-ray powder diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), UV-Vis Spectroscopy, and Transmission Electron Microscopy (TEM) and Energy Dispersive Spectroscopy (EDAX), and Dynamic Light Scattering (DLS). Also, the antioxidant capacity of fruit extract was determined through 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), Fluorescence Recovery After Photobleaching (FRAP), Superoxide Dismutase (SOD) assays. Furthermore, the cytotoxicity activities of Fe₂O₃ NPs were determined using the (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) (MTT) test on MCF-7 cells. In the antibacterial assay, lethal doses of the Fe₂O₃NPs effectively inhibited the growth of gram-negative and gram-positive bacteria. The surface damage, ROS production, and protein leakage are the antibacterial mechanisms of Fe₂O₃NPs. Concerning antioxidant activity, the fruit extracts of F. macrocarpa had strong antioxidant properties, which were confirmed by DPPH, ABTS, FRAP, and SOD assays. In addition, the F. microcarpa-derived iron oxide nanomaterials greatly reduced the cell viability of (MCF-7). The GC-MS/MS analysis revealed the presence of 25 main bioactive compounds in the F. microcarpa extract. Overall, the finding of this research revealed that F. microcarpa-derived Fe₂O₃ nanoparticles could be employed as an alternative therapeutic agent to cure microbial infection and breast cancer in humans.

Keywords: ficus microcarpa, iron oxide, antibacterial activity, cytotoxicity

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77 Identification and Characterization of Polysaccharide Biosynthesis Protein (CAPD) of Enterococcus faecium

Authors: Liaqat Ali, Hubert E. Blum, Türkân Sakinc

Abstract:

Enterococcus faecium is an emerging multidrug-resistant nosocomial pathogen increased dramatically worldwide and causing bacteremia, endocarditis, urinary tract and surgical site infections in immunocomprised patients. The capsular polysaccharides that contribute to pathogenesis through evasion of the host innate immune system are also involved in hindering leukocyte killing of enterococci. The gene cluster (enterococcal polysaccharide antigen) of E. faecalis encoding homologues of many genes involved in polysaccharide biosynthesis. We identified two putative loci with 22 kb and 19 kb which contained 11 genes encoding for glycosyltransferases (GTFs); this was confirmed by using genome comparison of already sequenced strains that has no homology to known capsule genes and the epa-locus. The polysaccharide-conjugate vaccines have rapidly emerged as a suitable strategy to combat different pathogenic bacteria, therefore, we investigated a polysaccharide biosynthesis CapD protein in E. faecium contains 336 amino acids and had putative function for N-linked glycosylation. The deletion/knock-out capD mutant was constructed and complemented by homologues recombination method and confirmed by using PCR and sequencing. For further characterization and functional analysis, in-vitro cell culture and in-vivo a mouse infection models were used. Our ΔcapD mutant shows a strong hydrophobicity and all strains exhibited biofilm production. Subsequently, the opsonic activity was tested in an opsonophagocytic assay which shows increased in mutant compared complemented and wild type strains but more than two fold decreased in colonization and adherence was seen on surface of uroepithelial cells. However, a significant higher bacterial colonialization was observed in capD mutant during animal bacteremia infection. Unlike other polysaccharides biosynthesis proteins, CapD does not seems to be a major virulence factor in enterococci but further experiments and attention is needed to clarify its function, exact mechanism and involvement in pathogenesis of enteroccocal nosocomial infections eventually to develop a vaccine/ or targeted therapy.

Keywords: E. faecium, pathogenesis, polysaccharides, biofilm formation

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76 In Silico Screening, Identification and Validation of Cryptosporidium hominis Hypothetical Protein and Virtual Screening of Inhibitors as Therapeutics

Authors: Arpit Kumar Shrivastava, Subrat Kumar, Rajani Kanta Mohapatra, Priyadarshi Soumyaranjan Sahu

Abstract:

Computational approaches to predict structure, function and other biological characteristics of proteins are becoming more common in comparison to the traditional methods in drug discovery. Cryptosporidiosis is a major zoonotic diarrheal disease particularly in children, which is caused primarily by Cryptosporidium hominis and Cryptosporidium parvum. Currently, there are no vaccines for cryptosporidiosis and recommended drugs are not effective. With the availability of complete genome sequence of C. hominis, new targets have been recognized for the development of effective and better drugs and/or vaccines. We identified a unique hypothetical epitopic protein in C. hominis genome through BLASTP analysis. A 3D model of the hypothetical protein was generated using I-Tasser server through threading methodology. The quality of the model was validated through Ramachandran plot by PROCHECK server. The functional annotation of the hypothetical protein through DALI server revealed structural similarity with human Transportin 3. Phylogenetic analysis for this hypothetical protein also showed C. hominis hypothetical protein (CUV04613) was the closely related to human transportin 3 protein. The 3D protein model is further subjected to virtual screening study with inhibitors from the Zinc Database by using Dock Blaster software. Docking study reported N-(3-chlorobenzyl) ethane-1,2-diamine as the best inhibitor in terms of docking score. Docking analysis elucidated that Leu 525, Ile 526, Glu 528, Glu 529 are critical residues for ligand–receptor interactions. The molecular dynamic simulation was done to access the reliability of the binding pose of inhibitor and protein complex using GROMACS software at 10ns time point. Trajectories were analyzed at each 2.5 ns time interval, among which, H-bond with LEU-525 and GLY- 530 are significantly present in MD trajectories. Furthermore, antigenic determinants of the protein were determined with the help of DNA Star software. Our study findings showed a great potential in order to provide insights in the development of new drug(s) or vaccine(s) for control as well as prevention of cryptosporidiosis among humans and animals.

Keywords: cryptosporidium hominis, hypothetical protein, molecular docking, molecular dynamics simulation

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75 Profiling of Apoptotic Protein Expressions after Trabectedin Treatment in Human Prostate Cancer Cell Line PC-3 by Protein Array Technology

Authors: Harika Atmaca, Emir Bozkurt, Latife Merve Oktay, Selim Uzunoglu, Ruchan Uslu, Burçak Karaca

Abstract:

Microarrays have been developed for highly parallel enzyme-linked immunosorbent assay (ELISA) applications. The most common protein arrays are produced by using multiple monoclonal antibodies, since they are robust molecules which can be easily handled and immobilized by standard procedures without loss of activity. Protein expression profiling with protein array technology allows simultaneous analysis of the protein expression pattern of a large number of proteins. Trabectedin, a tetrahydroisoquinoline alkaloid derived from a Caribbean tunicate, Ecteinascidia turbinata, has been shown to have antitumor effects. Here, we used a novel proteomic approach to explore the mechanism of action of trabectedin in prostate cancer cell line PC-3 by apoptosis antibody microarray. XTT cell proliferation kit and Cell Death Detection Elisa Plus Kit (Roche) was used for measuring cytotoxicity and apoptosis. Human Apoptosis Protein Array (R&D Systems) which consists of 35 apoptosis related proteins was used to assess the omic protein expression pattern. Trabectedin induced cytotoxicity and apoptosis in prostate cancer cells in a time and concentration-dependent manner. The expression levels of the death receptor pathway molecules, TRAIL-R1/DR4, TRAIL R2/DR5, TNF R1/TNFRSF1A, FADD were significantly increased by 4.0-, 21.0-, 4.20- and 11.5-fold by trabectedin treatment in PC-3 cells. Moreover, mitochondrial pathway related pro-apoptotic proteins Bax, Bad, Cytochrome c, and Cleaved Caspase-3 expressions were induced by 2.68-, 2.07-, 2.8-, and 4.5-fold and the expression levels of anti-apoptotic proteins Bcl-2 and Bcl-XL were reduced by 3.5- and 5.2-fold in PC-3 cells. Proteomic (antibody microarray) analysis suggests that the mechanism of action of trabectedin may be exerted via the induction of both intrinsic and extrinsic apoptotic pathways. The antibody microarray platform can be utilised to explore the molecular mechanism of action of novel anticancer agents.

Keywords: trabectedin, prostate cancer, omic protein expression profile, apoptosis

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74 Quantitative and Fourier Transform Infrared Analysis of Saponins from Three Kenyan Ruellia Species: Ruellia prostrata, Ruellia lineari-bracteolata and Ruellia bignoniiflora

Authors: Christine O. Wangia, Jennifer A. Orwa, Francis W. Muregi, Patrick G. Kareru, Kipyegon Cheruiyot, Eric Guantai

Abstract:

Ruellia (syn. Dipteracanthus) species are wild perennial creepers belonging to the Acanthaceae family. These species are reported to possess anti-inflammatory, analgesic, antioxidant, gastroprotective, anticancer, and immuno-stimulant properties. Phytochemical screening of both aqueous and methanolic extracts of Ruellia species revealed the presence of saponins. Saponins have been reported to possess anti-inflammatory, antioxidant, immuno-stimulant, antihepatotoxic, antibacterial, anticarcinogenic, and antiulcerogenic activities. The objective of this study was to quantify and analyze the Fourier transform infrared (FTIR) spectra of saponins in crude extracts of three Kenyan Ruellia species namely Ruellia prostrata (RPM), Ruellia lineari-bracteolata (RLB) and Ruellia bignoniiflora (RBK). Sequential organic extraction of the ground whole plant material was done using petroleum ether (PE), chloroform, ethyl acetate (EtOAc), and absolute methanol by cold maceration, while aqueous extraction was by hot maceration. The plant powders and extracts were mixed with spectroscopic grade KBr and compressed into a pellet. The infrared spectra were recorded using a Shimadzu FTIR spectrophotometer of 8000 series in the range of 3500 cm-1 - 500 cm-1. Quantitative determination of the saponins was done using standard procedures. Quantitative analysis of saponins showed that RPM had the highest quantity of crude saponins (2.05% ± 0.03), followed by RLB (1.4% ± 0.15) and RBK (1.25% ± 0.11), respectively. FTIR spectra revealed the spectral peaks characteristic for saponins in RPM, RLB, and RBK plant powders, aqueous and methanol extracts; O-H absorption (3265 - 3393 cm-1), C-H absorption ranging from 2851 to 2924 cm-1, C=C absorbance (1628 - 1655 cm-1), oligosaccharide linkage (C-O-C) absorption due to sapogenins (1036 - 1042 cm-1). The crude saponins from RPM, RLB and RBK showed similar peaks to their respective extracts. The presence of the saponins in extracts of RPM, RLB and RBK may be responsible for some of the biological activities reported in the Ruellia species.1

Keywords: Ruellia bignoniiflora, Ruellia linearibracteolata, Ruellia prostrata, Saponins

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73 Synthesis, Characterization and Biological Properties of Half-Sandwich Complexes of Ruthenium(II), Rhodium(II) and Iridium(III)

Authors: A. Gilewska, J. Masternak, K. Kazimierczuk, L. Turlej, J. Wietrzyk, B. Barszcz

Abstract:

Platinum-based drugs are now widely used as chemotherapeutic agents. However the platinum complexes show the toxic side-effects: i) the development of platinum resistance; ii) the occurrence of severe side effects, such as nephro-, neuro- and ototoxicity; iii) the high toxicity towards human fibroblast. Therefore the development of new anticancer drugs containing different transition-metal ions, for example, ruthenium, rhodium, iridium is a valid strategy in cancer treatment. In this paper, we reported the synthesis, spectroscopic, structural and biological properties of complexes of ruthenium, rhodium, and iridium containing N,N-chelating ligand (2,2’-bisimidazole). These complexes were characterized by elemental analysis, UV-Vis and IR spectroscopy, X-ray diffraction analysis. These complexes exhibit a typical pseudotetrahedral three-legged piano-stool geometry, in which the aromatic arene ring forms the seat of the piano-stool, while the bidentate 2,2’-bisimidazole (ligand) and the one chlorido ligand form the three legs of the stool. The spectroscopy data (IR, UV-Vis) and elemental analysis correlate very well with molecular structures. Moreover, the cytotoxic activity of the complexes was carried out on human cancer cell lines: LoVo (colorectal adenoma), MV-4-11 (myelomonocytic leukaemia), MCF-7 (breast adenocarcinoma) and normal healthy mouse fibroblast BALB/3T3 cell lines. To predict a binding mode, a potential interaction of metal complexes with calf thymus DNA (CT-DNA) and protein (BSA) has been explored using UV absorption and circular dichroism (CD). It is interesting to note that the investigated complexes show no cytotoxic effect towards the normal BALB/3T3 cell line, compared to cisplatin, which IC₅₀ values was determined as 2.20 µM. Importantly, Ru(II) displayed the highest activity against HL-60 (IC₅₀ 4.35 µM). The biological studies (UV-Vis and circular dichroism) suggest that arene-complexes could interact with calf thymus DNA probably via an outside binding mode and interact with protein (BSA).

Keywords: ruthenium(II) complex, rhodium(III) complex, iridium(III) complex, biological activity

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72 A Retrospective Study on the Spectrum of Infection and Emerging Antimicrobial Resistance in Type 2 Diabetes Mellitus

Authors: Pampita Chakraborty, Sukumar Mukherjee

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People with diabetes mellitus are more susceptible to developing infections, as high blood sugar levels can weaken the patient's immune system defences. People with diabetes are more adversely affected when they get an infection than someone without the disease, because you have weakened immune defences in diabetes. People who have minimally elevated blood sugar levels experience worse outcomes with infections. Diabetic patients in hospitals do not necessarily have a higher mortality rate due to infections, but they do face longer hospitalisation and recovery times. A study was done in a tertiary care unit in eastern India. Patients with type 2 diabetes mellitus infection were recruited in the study. A total of 520 cases of Type 2 Diabetes Mellitus were recorded out of which 200 infectious cases was included in the study. All subjects underwent detailed history & clinical examination. Microbiological samples were collected from respective site of the infection for microbial culture and antibiotic sensitivity test. Out of the 200 infectious cases urinary tract infection(UTI) was found in majority of the cases followed by diabetic foot ulcer (DFU), respiratory tract infection(RTI) and sepsis. It was observed that Escherichia coli was the most commonest pathogen isolated from UTI cases and Staphylococcus aureus was predominant in foot ulcers followed by other organisms. Klebsiella pneumonia was the major organism isolated from RTI and Enterobacter aerogenes was commonly observed in patients with sepsis. Isolated bacteria showed differential sensitivity pattern against commonly used antibiotics. The majority of the isolates were resistant to several antibiotics that are usually prescribed on an empirical basis. These observations are important, especially for patient management and the development of antibiotic treatment guidelines. It is recommended that diabetic patients receive pneumococcal and influenza vaccine annually to reduce morbidity and mortality. Appropriate usage of antibiotics based on local antibiogram pattern can certainly help the clinician in reducing the burden of infections.

Keywords: antimicrobial resistance, diabetic foot ulcer, respiratory tract infection, urinary tract infection

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71 Antiproliferative and Apoptotic Effects of an Enantiomerically Pure β-Dipeptide Derivative through PI3K/Akt-Dependent and -Independent Pathways in Human Hormone-Refractory Prostate Cancer Cells

Authors: Mei-Ling Chan, Jin-Ming Wu, Konstantin V. Kudryavtsev, Jih-Hwa Guh

Abstract:

Prostate cancer is one of the most common malignant disease in men. KUD983 is an enantiomerically pure β-dipeptide derivative, which may have anti-cancer effects. In the present study, KUD983 exhibits powerful activity against hormone-refractory prostate cancer (HRPC) PC-3 and DU145 cells. The IC50 values of KUD983 in PC-3 and DU145 cells are 0.56±0.07M and 0.50±0.04 M respectively. KUD983 induced G1 arrest of the cell cycle and subsequent apoptosis associated with the down-regulation of several related proteins including cyclin D1, cyclin E and Cdk4, and the de-phosphorylation of RB. The protein expressions of nuclear and total c-Myc protein, which was able to regulate the expression of both cyclin D1 and cyclin E, were significantly suppressed by KUD983. Phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) is an important signaling pathway that influences the energy metabolism, cell cycle, proliferation, survival and apoptosis of cells, and is associated with numerous other signaling pathways. The Western Blot data revealed that KUD983 inhibited PI3K/Akt and mTOR/p70S6K/4E-BP1 pathways. The transient transfection of constitutively active myristylated Akt (myr-Akt) cDNA significantly reversed KUD983-induced caspase activation but did not abolish the suppression of mTOR/p70S6K/4E-BP1 signaling cascade indicating the presence of both Akt-dependent and -independent pathways. Moreover, KUD983-induced effect was collaborated with the down-regulation of anti-apoptotic Bcl-2 members (e.g., Bcl-2, and Mcl-1) and IAP family members (e.g., survivin). Furthermore, KUD983 induced autophagic cell death using confocal microscopic examination, investigating the level of conversion of LC3-I to LC3-II and flow cytometric detection of AVO-positive cells. Taken together, the data suggest that KUD983 is an anticancer β-dipeptide against HRPCs through the inhibition of cell proliferation and induction of apoptotic and autophagic cell death. The suppression of signaling pathways mediated by c-Myc, PI3K/Akt and mTOR/p70S6K/4E-BP1 and the collaboration with down-regulation of Mcl-1 and survivin may indicate the mechanism of KUD983 against HRPC.

Keywords: β-dipeptide, hormone-refractory prostate cancer, mTOR, PI3K/Akt

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70 Interrelationship of Socio-Demographic Factors, Health Belief Dimensions and Compliance to Measles Vaccination among Filipino Mothers

Authors: Beryl Rene R. Lopez, Lesley Anne M. Lipat, Rhogene Barbette C. Lirio, Laurice Joy H. Llanes, Karl Philippe M. Llapitan, Einstein James R. Lopez, Socorro S. GuanHing

Abstract:

Background: Measles remain as one of the most common childhood diseases despite the availability of the vaccine that is safe and cost-effective. Because of morbidity and mortality associated with the recent measles outbreak in the Philippines, there is an increasing concern from the health care professionals. Objective: The purpose of this study is to determine the relationship between the compliance of Filipino mothers to measles vaccination and their health beliefs when grouped according to the given socio-demographic factors using a researcher-made questionnaire. Research Methodology: This research utilized the descriptive-correlational research design. With the use of purposive sampling technique, the study involved 200 Filipino mothers aged 18 years old and above excluding those who are healthcare professionals with children aged 2-3 years old with either urban or rural as their settlements. Pre-testing was done prior to the actual data gathering. A questionnaire composed of 26 items involving socio-demographic, compliance, and health beliefs was distributed to the sample population. Statistical analysis was done with the use of Exploratory Factor Analysis (EFA) for the first research question and Structural Equation Model (SEM) for the second research question. Results: Four dimensions were generated with the use of EFA namely: Vulnerability-Oriented Beliefs (VOB), Knowledge-Oriented Beliefs (KOB), Accessibility-Oriented Beliefs (AOB), and Outcomes-Oriented Beliefs (OOB). These were then correlated with the mothers’ socio-demographic factors (age, educational attainment, the area of residence, the number of children, and family income) and their compliance to the measles vaccination schedule. Results showed significant and direct relationships between area of residence and compliance, family income and compliance, KOB and compliance, education and KOB, KOB and VOB, KOB and OOB, AOB and KOB, AOB and OOB, AOB and VOB, and lastly, OOB and VOB. Conclusion: The Knowledge – Oriented Belief dimension greatly influence compliance to measles vaccination. Other determinants of compliance like the area of residence, educational attainment, and family income significantly increase the Filipino mothers’ likelihood of compliance to measles vaccination, which have implications to health education.

Keywords: socio-demographic, health beliefs, compliance, measles vaccination

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69 Exploring Polypnenolics Content and Antioxidant Activity of R. damascena Dry Extract by Spectroscopic and Chromatographic Techniques

Authors: Daniela Nedeltcheva-Antonova, Kamelia Getchovska, Vera Deneva, Stanislav Bozhanov, Liudmil Antonov

Abstract:

Rosa damascena Mill. (Damask rose) is one of the most important plants belonging to the Rosaceae family, with a long historical use in traditional medicine and as a valuable oil-bearing plant. Many pharmacological effects have been reported from this plant, including anti-inflammatory, hypnotic, analgesic, anticonvulsant, anti-depressant, antianxiety, antitussive, antidiabetic, relaxant effects on tracheal chains, laxative, prokinetic and hepatoprotective activities. Pharmacological studies have shown that the various health effects of R. damascena flowers can mainly be attributed to its large amount of polyphenolic components. Phenolics possess a wide range of pharmacological activities, such as antioxidants, free-radical scavengers, anticancer, anti-inflammatory, antimutagenic, and antidepressant, with flavonoids being the most numerous group of natural polyphenolic compounds. According to the technological process in the production of rose concrete (solvent extraction with non-polar solvents of fresh rose flowers), it can be assumed that the resulting plant residue would be as rich of polyphenolics, as the plant itself, and could be used for the development of novel products with promising health-promoting effect. Therefore, an optimisation of the extraction procedure of the by-product from the rose concrete production was carried out. An assay of the extracts in respect of their total polyphenols and total flavonoids content was performed. HPLC analysis of quercetin and kaempferol, the two main flavonoids found in R. damascena, was also carried out. The preliminary results have shown that the flavonoid content in the rose extracts is comparable to that of the green tea or Gingko biloba, and they could be used for the development of various products (food supplements, natural cosmetics and phyto-pharmaceutical formulation, etc.). The fact that they are derived from the by-product of industrial plant processing could add the marketing value of the final products in addition to the well-known reputation of the products obtained from Bulgarian roses (R. damascena Mill.).

Keywords: gas chromatography-mass-spectromrtry, dry extract, flavonoids, Rosa damascena Mill

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