Search results for: real-time rt-PCR assay

Authors: Gyati Shilakari Asthana, Abhay Asthana

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Purpose: The therapeutic potential of oligonucleotide (ODN) is primarily dependent upon its safe and efficient delivery to specific cells overcoming degradation and maximizing cellular uptake in vivo. The present study is focused to design low molecular weight chitosan nanoconstructs to meet the requirements of safe and effectual delivery of ODNs. LMW-chitosan is a biodegradable, water soluble, biocompatible polymer and is useful as a non-viral vector for gene delivery due to its better stability in water. Methods: LMW chitosan ODN nanoparticles (CHODN NPs) were formulated by self assembled method using various N/P ratios (moles ratio of amine groups of CH to phosphate moieties of ODNs; 0.5:1, 1:1, 3:1, 5:1 and 7:1) of CH to ODN. The developed CHODN NPs were evaluated with respect to gel retardation assay, particle size, zeta potential and cytotoxicity and transfection efficiency. Results: Complete complexation of CH/ODN was achieved at the charge ratio of 0.5:1 or above and CHODN NPs displayed resistance against DNase I. On increasing the N/P ratio of CH/ODN, particle size of the NPs decreased whereas zeta potential (ZV) value increased. No significant toxicity was observed at all CH concentrations. The transfection efficiency was increased on increasing N/P ratio from 1:1 to 3:1, whereas it was decreased with further increment in N/P ratio upto 7:1. Maximum transfection of CHODN NPs with both the cell lines (Raw 267.4 cells and Hela cells) was achieved at N/P ratio of 3:1. The results suggest that transfection efficiency of CHODN NPs is dependent on N/P ratio. Conclusion: Thus the present study states that LMW chitosan nanoparticulate carriers would be acceptable choice to improve transfection efficiency in vitro as well as in vivo delivery of oligonucleotide.

Keywords: LMW-chitosan, chitosan nanoparticles, biocompatibility, cytotoxicity study, transfection efficiency, oligonucleotide

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Authors: Tsige Reda

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Essential oil of Thymus vulgaris was extracted by means of hydro-distillation. This study was done to investigate the chemical composition, antibacterial and antioxidant activities. The chemical composition of the essential oils was determined using gas chromatography coupled to mass spectroscopy (GC-MS). Using disc diffusion assay the antibacterial activity was assessed on one Gram-positive bacteria and one Gram-negative bacteria. The percentage oil yield of the essential oil was found to be 0.97 ± 0.08% (w/w) with yellow color. The physicochemical constants of the oil were also noted. The phytochemical screening of the plant extract revealed the presence of tannins, saponins, phenol, flavonoids, terpenoids, steroids and alkaloids. A total of 18 chemical constituents were identified by Gas Chromatography-Mass Spectroscopy analysis representing 100% of the total essential oil of Thymus vulgaris, with thymol (31.977%), o-cymene (29.992%), and carvacrol (14.541%). Previous studies have revealed that the thymol, o-cymen and carvacrol components of Thymus vulgaris are responsible for their biological activities. Thymus vulgaris have been used traditionally to treat a wide variety of infections. Based on the extensive use and lack of scientific evidence, a study was embarked upon to determine its bioactivity. The essential oil of Thymus vulgaris leaves exhibited higher activity towards the Gram-positive bacteria (Staphylococcus aurous) than the Gram-negative bacteria (Escherichia coli) and also has good antioxidant activity, and can be used medicinal and therapeutic applications. This activity may be due to the high amount of thymol, o-cymen and carvacrol.

Keywords: hydro-distillation, Thymus vulgaris, essential oil composition, phytochemical screening, physicochemical constants, antioxidant activity, antibacterial activity

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Authors: Muhammad Imran Nisar, Fyezah Jehan, Tauseef Akhund, Sadia Shakoor, Kanwal Nayani, Furqan Kabir, Asad Ali, Anita Zaidi

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Pneumococcal Vaccine -10 (PCV 10) was included in the Expanded Program of immunization (EPI) in Sindh, Pakistan in February 2013. This study was carried out immediately before the introduction of PCV 10 to establish baseline pneumococcal carriage and prevalent serotypes in naso-pharynx of children 3-11 months of age in an urban and rural community in Sindh, Pakistan. An additional sample of children aged 12 to 59 months was drawn from the urban community. Nasopharyngeal specimens were collected from a random sample of children. Samples were processed in a central laboratory in Karachi. Pneumococci were cultured on 5% Sheep Blood Agar and serotyping was performed using CDC standardized sequential multiplex PCR assay on bacterial colonies. Serotypes were then categorized into vaccine (PCV-10 and PCV-13) type and non-vaccine types. A total of 670 children were enrolled. Carriage rate for pneumococcus based on culture positivity was 74% and 79.5 % in the infant group in Karachi and Matiari respectively. Carriage rate was 78.2% for children aged 12 to 59 months in Karachi. Proportion of PCV 10 serotypes in infants was 38.8% and 33.5% in Karachi and Matiari respectively. In the older age group in Karachi, the proportion was 30.6%. Most common serotypes were 6A, 6B, 23F, 19A and 18C. This survey establishes vaccine and non-vaccine serotype carriage rate in a vaccine-naïve pediatric population among rural and urban communities in Sindh province. Annually planned surveys in the same communities will inform change in carriage rate after the introduction and uptake of PCV 10 in these communities.

Keywords: Naso-Pharyngeal carriage, Pakistan, PCV10, Pneumococcus

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Authors: Benchouala Amira, Bojic Clement, Poupin Pascal, Cossu Leguille-carole

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The objective of this study is to evaluate in vitro the cytotoxic and androgenic potential of several antifungal molecules (amphotericin B, econazole, ketoconazole and miconazole) on MDA-Kb2 cell lines. This biological model is an effective tool for the detection of endocrine disruptors because it responds well to the main agonist of the androgen receptor (testosterone) and also to an antagonist: flutamide. The cytotoxicity of each chemical compound tested was measured using an MTT assay (tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) which measures the activity of the reductase function of mitochondrial succinate dehydrogenase enzymes of cultured cells. This complementary cytotoxicity test is essential to ensure that the effects of reduction in luminescence intensity observed during androgenic tests are only attributable to the anti-androgenic action of the compounds tested and not to their possible cytotoxic properties. Tests of the androgenic activity of antifungals show that these compounds do not have the capacity to induce transcription of the luciferase gene. These compounds do not exert an androgenic effect on MDA-Kb2 cells in culture for the environmental concentrations tested. The addition of flutamide for the same tested concentrations of antifungal molecules reduces the luminescence induced by amphotericin B, econazole and miconazole, which is explained by a strong interaction of these molecules with flutamide which may have a greater toxic effect than when tested alone. The cytotoxicity test shows that econazole and ketoconazole can cause cell death at certain concentrations tested. This cell mortality is perhaps induced by a direct or indirect action on deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or proteins necessary for cell division.

Keywords: cytotoxicity, androgenic potential, antifungals, MDA-Kb2

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Authors: Simona Pileviciene

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On 24th January 2014, Lithuania notified two primary cases of African swine fever (ASF) in wild boars. The animals were tested positive for ASF virus (ASFV) genome by real-time PCR at the National Reference Laboratory for ASF in Lithuania (NRL), results were confirmed by the European Union Reference Laboratory for African swine fever (CISA-INIA). Intensive wild and domestic animal monitoring program was started. During the period of 2014-2017 ASF was confirmed in two large commercial pig holding with the highest biosecurity. Pigs were killed and destroyed. Since 2014 ASF outbreak territory from east and south has expanded to the middle of Lithuania. Diagnosis by PCR is one of the highly recommended diagnostic methods by World Organization for Animal Health (OIE) for diagnosis of ASF. The aim of the present study was to compare singleplex real-time PCR assays to a duplex assay allowing the identification of ASF and internal control in a single PCR tube and to compare primers, that target the p72 gene (ASF 250 bp and ASF 75 bp) effectivity. Multiplex real-time PCR assays prove to be less time consuming and cost-efficient and therefore have a high potential to be applied in the routine analysis. It is important to have effective and fast method that allows virus detection at the beginning of disease for wild boar population and in outbreaks for domestic pigs. For experiments, we used reference samples (INIA, Spain), and positive samples from infected animals in Lithuania. Results show 100% sensitivity and specificity.

Keywords: African swine fewer, real-time PCR, wild boar, domestic pig

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Authors: Ahmad Ali Shahid, M Shakil Shaukat

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Agriculture is the backbone of economy of Pakistan and Cotton is the major agricultural export and supreme source of raw fiber for our textile industry. To combat against the developing resistance in the target insects and combating these challenges wholesomely, a novel combination of pyramided/stacked genes was conceptualized and later realized, through the means of biotechnology i.e., transformation of three genes namely, Cry1Ac, Cry2A, and EPSP synthase (glyphosate tolerant) genes in the locally cultivated cotton variety. The progenies of the transformed plants were successfully raised and screened under the tunnel conditions for two generations and the present study focused on the screening of plants which were confirmed for containing all of these three genes and their expressions. Initially, the screening was done through glyphosate spray assay and the plants which were healthy and showed no damage on leaves were selected after 07 days of spray. In the laboratory, the DNA of these plants were isolated and subjected to amplification of the three genes. Thus, seventeen out of twenty were confirmed positive for Cry1Ac gene and ten out of twenty were positive for Cry2A gene and all twenty were positive for presence of EPSP synthase gene. Then, the ten plant samples which were confirmed with presence of all three genes were subjected to expression analysis of these proteins through ELISA. The results showed that eight out of ten plants were actively expressing the three transgenes. Real-time PCR was also done to quantify the expression levels of the EPSP synthase gene. Finally, eight plants were confirmed for the presence and active expression of all three genes in T3 generation of the triple gene transformed cotton. These plants may be subjected to T4 generation to develop a new stable variety in due course of time.

Keywords: agriculture, cotton, transformation, cry genes, ELISA, PCR

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Authors: H. Aldewachi, M. Hines, M. McCulloch, N. Woodroofe, P. Gardiner

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DPP-IV is an enzyme whose expression is affected in a variety of diseases, therefore, has been identified as possible diagnostic or prognostic marker for various tumours, immunological, inflammatory, neuroendocrine, and viral diseases. Recently, DPP-IV enzyme has been identified as a novel target for type II diabetes treatment where the enzyme is involved. There is, therefore, a need to develop sensitive and specific methods that can be easily deployed for the screening of the enzyme either as a tool for drug screening or disease marker in biological samples. A variety of assays have been introduced for the determination of DPP-IV enzyme activity using chromogenic and fluorogenic substrates, nevertheless these assays either lack the required sensitivity especially in inhibited enzyme samples or displays low water solubility implying difficulty for use in vivo samples in addition to labour and time-consuming sample preparation. In this study, novel strategies based on exploiting the high extinction coefficient of gold nanoparticles (GNPs) are investigated in order to develop fast, specific and reliable enzymatic assay by investigating synthetic peptide sequences containing a DPP IV cleavage site and coupling them to GNPs. The DPP IV could be detected by colorimetric response of peptide capped GNPs (P-GNPS) that could be monitored by a UV-visible spectrophotometer or even naked eyes, and the detection limit could reach 0.01 unit/ml. The P-GNPs, when subjected to DPP IV, showed excellent selectivity compared to other proteins (thrombin and human serum albumin) , which led to prominent colour change. This provided a simple and effective colorimetric sensor for on-site and real-time detection of DPP IV.

Keywords: gold nanoparticles, synthetic peptides, colorimetric detection, DPP-IV enzyme

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Authors: Juan Huang, Nanqu Huang, Jingshan Shi, Yu Qiu

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Objectives: There are pathophysiological connections between type 2 diabetes mellitus (T2DM) and Alzheimer's disease (AD), and research on drugs with hypoglycemic and beta-amyloid (Aβ)-clearing effects have great therapeutic potential for AD. Dendrobium nobile Lindl. Alkaloids (DNLA) as one of the active compounds of Dendrobium nobile Lindl. In this study, we attempted to verify the hypoglycemic effect and investigate the effects of DNLA on the amyloid precursor protein (APP) metabolic pathway of the hippocampus in db/db mice. Methods: 4-weeks-old male C57BL/KsJ mice were the control group. And the same age and sexuality db/db mice were: model, DNLA-L (20 mg/kg), DNLA-M (40 mg/kg), and DNLA-H (80 mg/kg). After, mice were treated with different concentrations of DNLA for 17 weeks. The fasting blood glucose (FBG) was detected by glucose oxidase assay every week from the 4th to last week. The protein expression of β-amyloid 1-42 (Aβ1-42), β-site amyloid precursor protein-cleaving enzyme 1 (BACE1), and APP were examined by Western blotting. Results: The concentration of FBG and the protein expression of Aβ1-42, BACE1, and APP were increased in the hippocampus of the model group. Moreover, DNLA not only significantly decreased the concentration of FBG but also reduced the protein expressions of Aβ1-42, BACE1 and APP in the hippocampus of db/db mice in a dose-dependent manner. Conclusions: DNLA can decrease the protein expressions of Aβ1-42 in the hippocampus of db/db mice, and the mechanism may be involved in the APP metabolic pathway.

Keywords: Alzheimer's disease, type 2 diabetes mellitus, β-site amyloid precursor protein-cleaving enzyme 1, traditional Chinese medicines, beta-amyloid

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Authors: Sahar Essa, Hussain A. Safar, Raj Raghupathy

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Human cytomegalovirus (CMV) continues to be a source of severe complications in immunologically immature and immunocompromised hosts. Effective CMV vaccines that help diminish CMV disease in transplant patients and avoid congenital infection are of great importance. Though the exact roles of defense mechanisms are unidentified, viral-specific antibodies and cytokine responses are known to be involved in controlling CMV infections. CMV envelope glycoprotein B (UL55/gB), matrix proteins (UL83/pp65, UL99/pp28, UL32/pp150), and assembly protein UL80a/pp38 are known to be targets of antiviral immune responses. We immunized mice intraperitoneally with these five CMV-related proteins (commercial) for their ability to induce specific antibody responses (in-house immunoassay) and cytokine production (commercial assay) in a mouse model. We observed a significant CMV-antigen-specific antibody response to pp38 and pp65 (E/C ˃2.0, p˂0.001). Mice immunized with pp38 had significantly higher concentrations of GM-CSF, IFN-α, IL-2 IL-4, IL-5, and IL-17A (p˂0.05). Mice immunized with pp65 showed significantly higher concentrations of GM-CSF, IFN-γ, IL-2 IL-4, IL-10, IL-12, IL-17A, and TNF-α. Th1 to Th2 cytokines ratios revealed a Th1 cytokine bias in mice immunized with pp38, pp65, pp150, and gB. We suggest that stimulation with multiple CMV-related proteins, which include pp38, pp65, and gB antigens, will allow both humoral and cellular immune responses to be efficiently activated, thus serving as appropriate CMV antigens for future vaccines.

Keywords: cytomegalovirus, UL99/pp28, UL80a/pp38, UL83/pp65, UL32/pp150, UL55/gB, CMV-antigen-specific antibody, CMV antigen-specific cytokine responses

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Authors: Rajandeep Kaur, Rajeev Gupta

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Background: Chronic myeloid leukemia (CML) is the most common leukaemia in India. The annual incidence of chronic myeloid leukemia in India was originally reported to be 0.8 to 2.2 per 1,00,000 population. CML is a clonal disorder that is usually easily diagnosed because the leukemic cells of more than 95% of patients have a distinctive cytogenetic abnormality, the Philadelphia chromosome (Ph1). The approval of tyrosine kinase inhibitors (TKIs), which target BCR-ABL1 kinase activity, has significantly reduced the mortality rate associated with chronic myeloid leukemia (CML) and revolutionized treatment. Material and Methods: 80 diagnosed cases of CML were taken. Investigations were done. Bone marrow and molecular studies were also done and with EUTOS, patients were stratified into low and high-risk groups and then treatment with Imatinib was given to all patients and the molecular response was evaluated at 6 months and 12 months follow up with BCR-ABL by RT-PCR quantitative assay. Results: In the study population, out of 80 patients in the study population, 40 were females and 40 were males, with M: F is 1:1. Out of total 80 patients’ maximum patients (54) were in 31-60 years age group. Our study showed a most common symptom of presentation is abdominal discomfort followed by fever. Out of the total 80 patients, 25 (31.3%) patients had high EUTOS scores and 55 (68.8%) patients had low EUTOS scores. On 6 months follow up 36.3% of patients had Complete Molecular Response, 16.3% of patients had Major Molecular Response and 47.5% of patients had No Molecular Response but on 12 months follow up 71.3% of patients had Complete Molecular Response, 16.25% of patients had Major Molecular Response and 12.5% patients had No Molecular Response. Conclusion: In this study, we found a significant correlation between EUTOS score and Molecular response at 6 months and 12 months follow up after Imatinib therapy.

Keywords: chronic myeloid leukemia, European treatment and outcome study score, hematological response, molecular response, tyrosine kinase inhibitor

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Authors: Siriporn Okonogi, Pimpak Phumat, Sakornrat Khongkhunthian

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Piper betle has been extensively reported for various pharmacological effects including antimicrobial activity. 4-Allylpyrocatechol (AC) is a principle active compound found in P. betle. However, AC has a problem of solubility in water. The aims of the present study were to prepare AC loaded polymeric micelles for enhancing its water solubility and to evaluate its anti-biofilm activity against oral phathogenic bacteria. AC was loaded in polymeric micelles (PM) of Pluronic F127 by using thin film hydration method to obtain AC loaded PM (PMAC). The results revealed that AC in the form of PMAC possessed high water solubility. PMAC particles were characterized using a transmission electron microscope and photon correlation spectroscopy. Determination of entrapment efficiency (EE) and loading capacity (LC) of PMAC was done by using high-performance liquid chromatography. The highest EE (86.33 ± 14.27 %) and LC (19.25 ± 3.18 %) of PMAC were found when the weight ratio of polymer to AC was 4 to 1. At this ratio, the particles showed spherical in shape with the size of 38.83 ± 1.36 nm and polydispersity index of 0.28 ± 0.10. Zeta potential of the particles is negative with the value of 16.43 ± 0.55 mV. Crystal violet assay and confocal microscopy were applied to evaluate the effects of PMAC on Streptococcus mutans biofilms using chlorhexidine (CHX) as a positive control. PMAC contained 1.5 mg/mL AC could potentially inhibit (102.01 ± 9.18%) and significantly eradicate (85.05 ± 2.03 %) these biofilms (p < 0.05). Comparison with CHX, PMAC showed slightly similar biofilm inhibition but significantly stronger biofilm eradication (p < 0.05) than CHX. It is concluded that PMAC can enhance water solubility and anti-biofilm activity of AC.

Keywords: pluronic, polymeric micelles, solubility, 4-allylpyrocathecol, Streptococcus mutans, anti-biofilm

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Authors: Asma Ansari, Afsheen Aman, Shah Ali Ul Qader

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In the past few years, numerous concerns have been raised against increased bacterial resistance towards effective drugs and become a debated issue all over the world. With the emergence of drug resistant pathogens, the interaction of natural antimicrobial compounds and antibacterial nanoparticles has emerged as a potential candidate for combating infectious diseases. Microbial diversity in the biome provides an opportunity to screen new species which are capable of producing large number of antimicrobial compounds. Among these antimicrobial compounds, bacteriocins are highly specific and efficient antagonists. A combination of bacteriocin along with nanoparticles could prove to be more potent due to broadened antibacterial spectrum with possibly lower doses. In the current study, silver nanoparticles were synthesized through biological reduction using various isolated bacterial, fungal and yeast strains. Spectroscopy and scanning electron microscopy (SEM) was performed for the confirmation of nanoparticles. Bacteriocin was characterized and purified to homogeneity through gel permeation chromatography. The estimated molecular weight of bacteriocin was 10 kDa. Amino acid analysis and N-terminal sequencing revealed the novelty of the protein. Then antibacterial potential of silver nanoparticles and broad inhibitory spectrum bacteriocin was determined through agar well diffusion assay. These synthesized bacteriocin-Nanoparticles exhibit a good potential for clinical applications as compared to bacteriocin alone. This combination of bacteriocin with nanoparticles will be used as a new sort of biocide in the field of nano-proteomics. The advancement of nanoparticles-mediated drug delivery system will open a new age for rapid eradication of pathogens from biological systems.

Keywords: BAC-IB17, multidrug resistance, purification, silver nanoparticles

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Authors: Alaa E. El-sisi, Sherin Zakaria

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Rebamipide is an antiulcer drug with unique properties such as anti-inflammatory action. It induces endogenous prostaglandin e2 (PGE2). PGE2 is considered as a potent physiological suppressor of liver fibrosis. Aim of study: This study investigated the effect of rebamipide on hepatic fibrosis. Material and Method: Hepatic fibrosis was induced by intraperitoneal injections (IP) injection of CCl4 (0.45 mL/kg) in corn oil 1:5 twice a week for 4 weeks. Rats were divided into four groups as follow: Group 1 treated with CCL4 only, group 2 and 3 treated with CCL4 and rebamipide 60 mg/kg/day (group2) or 100 mg/kg/day (group3), and the fourth group was considered as control group and treated with vehicles. ALT, AST, and Bilirubin were assayed in serum. Antioxidant markers such as malondialdhyde (MDA) and superoxide dismutase (SOD) and fibrotic markers such as hyaluronic acid (HA) and procollagen-III (procol-III) were evaluated in liver tissues. IL-10 as well as PGE2 were also assayed in liver tissues. Pathologic changes in the liver were detected by hematoxylin and eosin staining. Collagen precipitation in liver tissues was visualized using masson trichrom stain. Results: Rebamipide inhibit CCL4 induced increase in ALT and AST significantly (p < 0.05). Rebamipide exerted an antioxidant effect as it inhibits CCL4 induced increased MDA level and decreased SOD activity. Fibrotic markers assay revealed that repamipide (60 or 100 mg/kg/day) decreased the level of procol-III and HA compared to CCl4 (p < 0.05). Oral administration of Rebamipide was associated with a significant increase (p < 0.05) of PGE2 and IL-10. Rebamipide especially at the dose of (100 mg/kg/day) restores liver histology structure and abolish collagen precipitation in liver tissues. Conclusion: Rebamipide retards hepatic fibrosis induced by CCL4 may be through the induction of PGE2 level.

Keywords: fibrotic markers, hepatic fibrosis, PGE2, rebamipide

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Authors: Amir Saber Gharamaleki, Beitollah Alipour, Zeinab Faghfoori, Ahmad YariKhosroushahi

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Colorectal cancer (CRC) is one of the most prevalent cancers and intestinal microbial community plays an important role in colorectal tumorigenesis. Probiotics have recently been assessed as effective anti-proliferative agents and thus this study was performed to examine whether CRC undergo apoptosis by treating with isolated Iranian native dairy yeast, Kluyveromyces marxianus YAS, secretion metabolites. The cytotoxicity assessments on cells (HT-29, Caco-2) were accomplished through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as well as qualitative DAPI (4',6-diamidino-2-phenylindole staining) and quantitative (flow cytometry assessments) evaluations of apoptosis. To evaluate the main mechanism of apoptosis, Real time PCR method was applied. Kluyveromyces marxianus YAS secretions (IC50) showed significant cytotoxicity against HT-29 and Caco-2 cancer cell lines (66.57 % and 66.34 % apoptosis) similar to 5-Fluorouracil (5-FU) while apoptosis only was developed in 27.57 % of KDR normal cells. The prophylactic effects of Kluyveromyces marxianus (PTCC 5195), as a reference yeast, was not similar to Kluyveromyces marxianus YAS indicating strain dependency of bioactivities on CRC disease prevention. Based on real time PCR results, the main cytotoxicity is related to apoptosis phenomenon and the core related mechanism is depended on the overexpression of BAX, CASP 9, CASP 8 and CASP 3 inducing apoptosis genes. However, several investigations should be conducted to precisely determine the effective compounds to be used as anticancer therapeutics in the future.

Keywords: anticancer, anti-proliferative, apoptosis, cytotoxicity, yeast

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Authors: Amita Jain, Shantanu Prakash, Suruchi Shukla

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Introduction: Acute encephalitis syndrome (AES) is a complex multi etiology syndrome posing a great public health problem in the northern part of India. Japanese encephalitis (JE) virus is an established etiology of AES in this region. Recently, Scrub typhus (ST) is being recognized as an emerging aetiology of AES in JE endemic belt. This study was conducted to establish the direct evidence of Central nervous system invasion by Orientia tsutsugamushi leading to AES. Methodology: A total of 849 cases with clinical diagnosis of AES were enrolled from six districts (Deoria and its adjoining area) of the traditional north Indian Japanese encephalitis (JE) belt. Serum and Cerebrospinal fluid samples were collected and tested for major agent causing acute encephalitis. AES cases either positive for anti-ST IgM antibodies or negative for all tested etiologies were investigated for ST-DNA by real-time PCR. Results: Of these 505 cases, 250 patients were laboratory confirmed for O. tsutsugamushi infection either by anti-ST IgM antibodies positivity (n=206) on serum sample or by ST-DNA detection by real-time PCR assay on CSF sample (n=2) or by both (n=42).Total 29 isolate could be sequenced for 56KDa gene. Conclusion: All the strains were found to cluster with Gilliam strains. The majority of the isolates showed a 97–99% sequence similarity with Thailand and Cambodian strains. Gilliam strain of O.tsusugamushi is an emerging as one of the major aetiologies leading to AES in northern part of India.

Keywords: acute encephalitis syndrome, O. tsutsugamushi, Gilliam strain, North India, cerebrospinal fluid

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Authors: Ji Won Kim, Moo Yeol Lee, Keon Wook Kang

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GV-1001, 16 amino acid fragment of human telomerase reverse transcriptase catalytic subunit (hTERT), has been developed as an injectable cancer vaccine for many types of solid tumors showing high-level of telomerase activity. In the present study, we evaluated the anti-cancer effect of GV-1001 on androgen-receptor-positive prostate cancer. Two signaling pathways, Gs-adenylate cyclase-cAMP and Gq-IP3-Ca2+ pathways play a central role in GnRH receptor (GnRHR)-mediated activities. We found that leuprolide acetate (LA) mainly acted on Gq-mediated Ca2+ signaling, while GV-1001 preferentially acted on cAMP signaling; and both the effects were counteracted by cetrorelix, a GnRHR antagonist. We further tested whether GV-1001 affects tumor growth of human prostate cancer cells in vivo. Prostate tumor xenografts were established using LNCap, androgen receptor-positive prostate cancer cells, and the nude mice bearing tumors were subcutaneously injected with GV-1001 (0.01, 0.1, 1, 10 microg/kg/day) and LA (0.01 microg/kg/day) for 2 weeks. GV-1001 (1 and 10 microg/kg/day) significantly inhibited tumor growth of LNCap xenografts. Interestingly, mRNA expression of MMP2 and MMP9 was significantly suppressed by GV-1001 injection, but not by LA administration. Boyden chamber assay revealed that GV-1001 potently inhibited cell migration of LNCap. Our finding suggests that GV-1001 as a novel GnRHR ligand, has anti-proliferative and anti-migratory effects on androgen receptor-positive prostate cancer cells.

Keywords: GV-1001, GnRH, hTERT, prostate cancer

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Authors: Mona T. Kashef, Omneya M. Helmy

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Multidrug resistant (MDR) bacteria have become a significant public health threat. The prevalence rates, of Gram negative MDR bacteria, are in continuous increase. However, few data are available about these resistant strains. Since, third generation cephalosporins are one of the most commonly used antimicrobials, we set out to investigate the prevalence, different mechanisms and clonal relatedness of multidrug resistance among third generation resistant Gram negative clinical isolates. A total of 114 Gram negative clinical isolates, previously characterized as being resistant to at least one of 3rd generation cephalosporins, were included in this study. Each isolate was tested, using Kirby Bauer disk diffusion method, against its assigned categories of antimicrobials. The role of efflux pump in resistance development was tested by the efflux pump inhibitor-based microplate assay using chloropromazine as an inhibitor. Detecting different aminoglycosides, β-lactams and quinolones resistance genes was done using polymerase chain reaction. The genetic diversity of MDR isolates was investigated using Random Amplification of Polymorphic DNA technique. MDR phenotype was detected in 101 isolates (89%). Efflux pump mediated resistance was detected in 49/101 isolates. Aminoglycosides resistance genes; armA and aac(6)-Ib were detected in one and 53 isolates, respectively. The aac(6)-Ib-cr allele, that also confers resistance to floroquinolones, was detected in 28/53 isolates. β-lactam resistance genes; blaTEM, blaSHV, blaCTX-M group 1 and group 9 were detected in 52, 29, 61 and 35 isolates, respectively. Quinolone resistance genes; qnrA, qnrB and qnrS were detectable in 2, 14, 8 isolates respectively, while qepA was not detectable at all. High diversity was observed among tested MDR isolates. MDR is common among 3rd generation cephalosporins resistant Gram negative bacteria, in Egypt. In most cases, resistance was caused by different mechanisms. Therefore, new treatment strategies should be implemented.

Keywords: gram negative, multidrug resistance, RAPD typing, resistance genes

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Authors: R. Posia, J. Mistry, K. Kamani

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The health screening of laboratory rodents is essential for ensuring animal health and the validity of biomedical research data. Routine health monitoring is necessary to verify the effectiveness of biosecurity and the specific pathogen free (SPF) status of the colony. The present screening was performed in barrier maintained rat (Rattus norvegicus) colony. Rats were maintained under a controlled environment and strict biosecurity in the facility. The screening was performed on quarterly bases from randomly selected animals from breeding and or maintenance colonies. Selected animals were subject to blood collection under isoflurane anaesthesia. Serum was separated from the collected blood and stored samples at -60 ± 10 °C until further use. A total of 88 samples were collected quarterly bases from animals in a year. In the serological test, enzyme-linked immunosorbent assay (ELISA) was used for screening of serum samples against sialodacryoadenitis virus (SDAV), Sendai virus (SV), and Kilham’s rat virus (KRV). ELISA kits were procured from XpressBio, USA. Test serum samples were run along with positive control, negative control serum in 96 well ELISA plates as per the procedure recommended by the vendor. Test ELISA plate reading was taken in the microplate reader. This screening observed that none of the samples was observed positive for the sialodacryoadenitis virus (SDAV), Sendai virus (SV), and Kilham’s rat virus (KRV), indicating that effectiveness of biosecurity practices followed in the rodent colony. The result of serological screening helps us to declare that our rodent colony is specifically pathogen free for these pathogens.

Keywords: biosecurity, ELISA, specific pathogen free, serological screening, serum

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Authors: Bernard L. Middleton, Susan P. Cosgrove

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There is an immediate need for alternative anti-herpetic treatment options effective for both primary infections and reoccurring reactivations of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2). Alternatives currently approved for the purposes of clinical administration includes antivirals and a reduced set of nucleoside analogues. The present article tests a treatment based on a systemic understanding of how the herpes virus affects cell inhibition and breakdown and targets different phases of the viral cycle, including the entry stage, reproductive cross mutation, and cell-to-cell infection. The treatment consisted of five immunotherapeutic core compounds (5CC), which were hypothesized to be capable of neutralizing human monoclonal antibodies. The tested 5CC were noted as being functional in the application of eliminating the DNA synthesis of herpes viral interferon (IFN) - induced cellular antiviral response. They were here found to neutralize antiviral reproduction by blocking cell-to-cell infection. The activity of the 5CC was tested on RC-37 in vitro using an assay plaque reduction and in vivo against HSV-1 and HSV-2. The 50% inhibitory concentration (IC50) of 5CC was 0.0009% for HSV-1 plaque formation and 0.0008% for HSV-2 plaque formation. Further tests were performed to evaluate the susceptibility of HSV-1 and HSV-2 to anti-herpetic drugs in Vero cells after virus entry. There were high-level markers of the 5CC virucidal activity in the viral suspension of HSV-1 and HSV-2. These concentrations of the 5CC are nontoxic and reduced plaque formation by 98.2% for HSV-1 and 93.0% for HSV-2. Virus HSV-1 and HSV-2 titers were reduced significantly by 5CC to the point of being negative, ranging 0.01–0.09 in 72%. The results concluded the 5CC as being an effective treatment option for the herpes simplex virus.

Keywords: synergy pharmaceuticals, herpes treatment, herpes cure, synergy pharmaceuticals treatment

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Authors: Sandy Van Vuuren, Lerato Kose, Annah Moteetee

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Lesotho, a country surrounded by South Africa has one of the highest rates of sexually transmitted infections (STI’s) in the world. In fact, the country ranks third highest with respect to infections related to the human immunodeficiency virus (HIV). Despite the high prevalence of STI’s, treatment has been a challenge due to limited accessibility to health facilities. An estimated 77% of the population lives in rural areas and more than 60% of the country is mountainous. Therefore, many villages remain accessible only by foot or horse-back. Thus, the Basotho (indigenous people from Lesotho) have a rich cultural heritage of plant use. The aim of this study was to determine what plant species are used for the treatment of STI’s and which of these have in vitro efficacy against pathogens such as Candida albicans, Gardnerella vaginalis, Oligella ureolytica, and Neisseria gonorrhoeae. A total of 34 medicinal plants were reported by traditional practitioners for the treatment of STI’s. Sixty extracts, both aqueous and organic (mixture of methanol and dichloromethane), from 24 of the recorded plant species were assessed for antimicrobial activity using the minimum inhibition concentration (MIC) micro-titre plate dilution assay. Neisseria gonorrhoeae (ATCC 19424) was found to be the most susceptible among the test pathogens, with the majority of the extracts (21) displaying noteworthy activity (MIC values ≤ 1 mg/ml). Helichrysum caespititium was found to be the most antimicrobially active species (MIC value of 0.01 mg/ml). The results of this study support, to some extent, the traditional medicinal uses of the evaluated plants for the treatment of STI’s, particularly infections related to gonorrhoea.

Keywords: Africa, Candida albicans, Gardnerella vaginalis, Neisseria gonorrhoeae, Oligella urealytica

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Authors: Sadegh Lotfieblisofla, Arash Khodabakhshi

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Tissue plasminogen activator (tPA) as a serine protease plays an important role in the fibrinolytic system and the dissolution of fibrin clots in human body. The production of this drug in plants such as tobacco could reduce its production costs. In this study, expression of tPA gene and protein targeting to different plant cell compartments, using various signal peptides has been investigated. For high level of expression, Kozak sequence was used after CaMV35S in the beginning of the gene. In order to design the final construction, Extensin, KDEL (amino acid sequence including Lys-Asp-Glu-Leu) and SP (γ-zein signal peptide coding sequence) were used as leader signals to conduct this protein into apoplast, endoplasmic reticulum and cytosol spaces, respectively. Cloned human tPA gene under the CaMV (Cauliflower mosaic virus) 35S promoter and NOS (Nopaline Synthase) terminator into pBI121 plasmid was transferred into tobacco explants by Agrobacterium tumefaciens strain LBA₄₄₀₄. The presence and copy number of genes in transgenic tobacco was proved by Southern blotting. Enzymatic activity of the rt-PA protein in transgenic plants compared to non-transgenic plants was confirmed by Zymography assay. The presence and amount of rt-PA recombinant protein in plants was estimated by ELISA analysis on crude protein extract of transgenic tobacco using a specific antibody. The yield of recombinant tPA in transgenic tobacco for SP, KDEL, Extensin signals were counted 0.50, 0.68, 0.69 microgram per milligram of total soluble proteins.

Keywords: tPA, recombinant, transgenic, tobacco

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Authors: Jie Gao

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The combination of chemotherapy with gene therapy is highly effective in cancer therapy. To achieve combined therapeutic effects in human gastric cancer over expressing EGFR, we developed targeted LPD (liposome-polycation-DNA complex) conjugated with anti-EGFR (epidermal growth factor receptor) Fab’ for co-delivery of doxorubicin (DOX) and ribonucleotide reductase M2 (RRM2) siRNA (DOX-RRM2-TLPD). The results showed that EGFR was over expressed in several gastric cancer cell lines and gastric cancer tissues. Gene Expression Omnibus (GEO) results showed that RRM2 expression was significantly higher in gastric cancer than in non-gastric cancer tissue, and RRM2 siRNA inhibited the proliferation of several gastric cancer cells, indicating that RRM2 is a candidate target for gastric cancer therapy. Confocal studies and flow cytometry showed that DOX-RRM2-TLPD delivered DOX and RRM2 siRNA to EGFR over expressing gastric cancer cells specifically and efficiently both in vitro and in vivo, resulting in enhanced therapeutic effects (cytotoxicity and apoptosis) compared with single-drug loaded or non-targeted controls, including DOX-NC-TLPD (targeted LPD co-delivering DOX and negative control siRNA), RRM2-TLPD (targeted LPD delivering RRM2 siRNA) and DOX-RRM2-NTLPD (non-targeted LPD co-delivering DOX and RRM2 siRNA). The in vivo antitumor assay showed that the average weight of the gastric cancer in mice treated with DOX-RRM2-TLPD was significantly lighter than that of mice treated with other controls. DOX-RRM2-TLPD represents an effective approach for combined therapy of gastric cancer over expressing EGFR.

Keywords: gene therapy, chemotherapy, immunoliposomes, gastric cancer

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Authors: Bhawna Chandravanshi, Ramesh Bhonde

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In the present study, we focused on the improvisation of islet survival in hypoxia. The Islet-like cell aggregates (ICAs) derived from Wharton's jelly mesenchymal stem cells (WJ-MSC) were cultured with and without WJ-MSC for 48h in hypoxia and normoxia and tested for their direct trophic effect on β cell survival. The WJ MSCs themselves secreted insulin upon glucose challenge and expressed the pancreatic markers at both transcription and translational level (C-peptide, Insulin, Glucagon and Glut 2). Direct contact of MSCs with ICAs facilitate the highest viability under hypoxia as evidenced by fluorescein diacetate/propidium iodide and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cytokine analysis of the co-cultured ICAs revealed amplification of anti-inflammatory cytokine-like TGFβ and TNFα accompanied by depletion of pro-inflammatory cytokines. The increment in VEGF and PDGFa was also seen showing their ability to vascularize upon transplantation. This was further accompanied by reduction in total reactive oxygen species, nitric oxide, and super oxide ions and down-regulation of Caspase3, Caspase8, p53 and up regulation of Bcl2 confirming prevention of apoptosis in ICAs. There was a significant reduction in the expression of p38 protein in the presence of MSCs making the ICAs responsive to glucose. Taken together our data demonstrate for the first time that the WJ-MSC expressed pancreatic markers and their supplementation protected engineered islets against hypoxia, oxidative stress, and inflammatory cytokines by inhibiting p38 MAPK protein.

Keywords: hypoxia, islet-like cell aggregates, inflammatory cytokines, oxidative stress

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Authors: Dian Muzdalifah, Anastasia F. Devi, Zatil A. Athaillah, Linar Z. Udin

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Tempe is a functional food prepared from soybeans through Rhizopus spp fermentation. It is well known as functional food, originated from Indonesia. Most studies on tempe functionalities refer to ripe (48 h fermentation) tempe and only limited studies discuss overripe tempe while longer fermentation time possibly increased tempe health benefit. Hence, the present study was performed to investigate the cytotoxic activity againts MCF-7 breast cancer cells and antioxidant property of tempe prepared from 0–156 h of fermentation. Tempe samples were dried and extracted with acetone and ethanol, respectively. Their extracts were used for subsequent analysis. The cytotoxic activity was assessed on MCF 7 breast cancer cells using Alamar Blue method. The antioxidant activity was determined by DPPH free radical scavenging assay. The results indicated that acetone extracts of 108 h tempe had a potent cytotoxic activity against MCF-7 breast cancer cells (IC50 = 2.54 ± 0,30 μg/mL). Ethanol extracts of 108 h tempe also showed the potency, but at slightly higher IC50 (5.20 ± 1.01 μg/mL). Both acetone and ethanol extracts of 108 and 120 h tempe showed high antioxidant activity expressed as percent inhibition with no significant difference. However, acetone extracts of 120 h tempe (81.31 ± 3.70 %) had better ability to inhibit oxidation reaction than that of ethanol extracts (75.77 ± 6.00 %). It can be concluded that the cytotoxic activity of tempe from 0–156 h of fermentation is positively correlated to their corresponding antioxidant property. Longer fermentation time, up to 108 h, increased the ability of tempe to inhibit the growth of MCF-7 breast cancer cells and oxidative reaction. But extended fermentation time, up to 156 h, tends to decrease its ability. Further studies are encouraged to identify the active components contained in each extract.

Keywords: antioxidant property, cytotoxic activity, extracts, overripe tempeh

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Authors: Dwitya Elvira, Eryati Darwin

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Background: Graves’ disease (GD) is an autoimmune thyroid disease. Imbalance of Th1/Th2 cells and T-regulatory (Treg)/Th17 cells was thought to play pivotal role in the pathogenesis of GD. Treg FoxP3 produced TGF-β to maintain regulatory function, and Th17 cells produced IL-17 as cytokines that were thought in mediating several autoimmune diseases. The aim of this study is to assess the role of IL-17 and TGF-β in the pathogenesis of GD and to investigate its correlation with Thyroid Stimulating Hormone Receptor Antibody (TRAb) and Treg FoxP3 expression. Method: 30 GD patients and 27 age and sex-matched controls were enrolled in this study. Diagnosis of GD was based on clinical and biochemical of GD. Serum IL-17, TGF-β, TRAb, and FoxP3 were measured by enzyme-linked immunosorbent assay (ELISA). Data were analyzed by using SPSS 21.0 (SPSS Inc.). Spearman rank correlation test was used for assessment of correlation. The statistical significance was accepted as P<0.05. Result: There was no significant correlation between IL-17 and TGF-β serum with expression of FoxP3 level in GD, but there was significant correlation between TGF-β and TRAb serum level (P<0.05). Serum levels of IL-17 and TGF-β were found to be elevated in patient group compared to control, where mean values of IL-17 were 14.43±2.15 pg/mL and TGF-β were 10.44±3.19 pg/mL in patients group; and in control group, level of IL-17 were 7.1±1.45 pg/mL and TGF-β were 4.95±1.35 pg/mL. Conclusion: Serum Il-17 and TGF-β were elevated in GD patients that reflect the role of inflammatory and regulatory cytokines activation in pathogenesis of GD. There was significant correlation between TGF-β and TRAb, revealing that Treg cytokines may play a role in pathogenesis of GD.

Keywords: IL-17, TGF-B, FoxP3, TRAb, Graves’ disease

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Authors: Huong M. T. Nguyen, Hoa A. P. Nguyen, Mai P. T. Nguyen, Ngoc D. Ngo, Van T. Ta, Hai T. Le, Chi V. Phan

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Wilson’s disease (WD) is an autosomal recessive disorder of the copper metabolism, which is caused by a mutation in the copper-transporting P-type ATPase (ATP7B). The mechanism of this disease is the failure of hepatic excretion of copper to bile, and leads to copper deposits in the liver and other organs. The ATP7B gene is located on the long arm of chromosome 13 (13q14.3). This study aimed to investigate the gene mutation in the Vietnamese patients with WD, and make a presymptomatic diagnosis for their familial members. Forty-three WD patients and their 65 siblings were identified as having ATP7B gene mutations. Genomic DNA was extracted from peripheral blood samples; 21 exons and exon-intron boundaries of the ATP7B gene were analyzed by direct sequencing. We recognized four mutations ([R723=; H724Tfs*34], V1042Cfs*79, D1027H, and IVS6+3A>G) in the sum of 20 detectable mutations, accounting for 87.2% of the total. Mutation S105* was determined to have a high rate (32.6%) in this study. The hotspot regions of ATP7B were found at exons 2, 16, and 8, and intron 14, in 39.6 %, 11.6 %, 9.3%, and 7 % of patients, respectively. Among nine homozygote/compound heterozygote siblings of the patients with WD, three individuals were determined as asymptomatic by screening mutations of the probands. They would begin treatment after diagnosis. In conclusion, 20 different mutations were detected in 43 WD patients. Of this number, four novel mutations were explored, including [R723=; H724Tfs*34], V1042Cfs*79, D1027H, and IVS6+3A>G. The mutation S105* is the most prevalent and has been considered as a biomarker that can be used in a rapid detection assay for diagnosis of WD patients. Exons 2, 8, and 16, and intron 14 should be screened initially for WD patients in Vietnam. Based on risk profile for WD, genetic testing for presymptomatic patients is also useful in diagnosis and treatment.

Keywords: ATP7B gene, mutation detection, presymptomatic diagnosis, Vietnamese Wilson’s disease

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Authors: Mudawi M. Nour

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Background: Now a days, the multidisciplinary scientific research conception in the field of nanotechnology has witnessed development with regard to the numerous applications and synthesis of nanomaterials. Objective: The current investigation has been conducted with the main focus on the green synthesis of silver nanoparticles from the leaves of Salvadora persica and its antibacterial activity against MDR pathogens E. coli and S. aureus. Methodology: Silver nanoparticles (AgNPs) were prepared after addition of aqueous extract of Salvadora persica leaves. The UV-Vis spectrophotometer, Transmission Electron Microscopy (TEM), zeta potential and Scanning Electron Microscopy (SEM) were employed to detect the particle size and morphology, besides Fourier transform infra-red spectrometer (FTIR) analysis was performed to determine the capping and stabilizing agents in the extract. Antibacterial assay for the biogenic AgNPs was conducted against E. coli and S. aureus. Results: Color change of the mixture from yellow to dark brown is the first indication to AgNPs formation. Furthermore, 420 nm was the peak value for UV-Vis spectroscopy absorption of the mixture. Besides, TEM and SEM micrographs showed wide variability in the diameter of smaller NPs aggregated together with spherical shapes, and zeta sizer showed about 153.3 nm as an average size of nanoparticles. Microbial suppression was noticed for the tested microorganisms. Furthermore, with the help of FTIR analysis, the biomolecules that act as capping and stabilizing agents of AgNPs are proteins and phenols present in the plant extract. Conclusion: Salvadora persica leaves extract act as a reducing and stabilizing agent for the synthesis of AgNPs, keeping its ability to suppress the MDR pathogen.

Keywords: green synthesis, FTIR, MDR pathogen, salvadora persica

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Authors: Maia Kukhaleishvili, Ekaterine Bulauri, Iveta Megrelishvili, Tamar Shamatava, Tamar Chipashvili

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Plant viruses can cause loss of yield and quality in a lot of important crops. Symptoms of pathogens are variable depending on the cultivars and virus strain. Selection of resistant potato varieties would reduce the risk of virus transmission and significant economic impact. Other way to avoid reduced harvest yields is regular potato seed production sampling and testing for viral infection. The aim of this study was to determine the occurrence and distribution of viral diseases according potato cultivars for further selection of virus-free material in Georgia. During the summer 2015- 2016, 5 potato cultivars (Sante, Laura, Jelly, Red Sonia, Anushka) at 5 different farms located in Akhalkalaki were tested for 6 different potato viruses: Potato virus A (PVA), Potato virus M (PVM), Potato virus S (PVS), Potato virus X (PVX), Potato virus Y (PVY) and potato leaf roll virus (PLRV). A serological method, Double Antibody Sandwich-Enzyme linked Immunosorbent Assay (DASELISA) was used at the laboratory to analyze the results. The result showed that PVY (21.4%) and PLRV (19.7%) virus presence in collected samples was relatively high compared to others. Researched potato cultivars except Jelly and Laura were infected by PVY with different concentrations. PLRV was found only in three potato cultivars (Sante, Jelly, Red Sonia) and PVM virus (3.12%) was characterized with low prevalence. PVX, PVA and PVS virus infection was not reported. It would be noted that 7.9% of samples were containing PVY/PLRV mix infection. Based on the results it can be concluded that PVY and PLRV infections are dominant in all research cultivars. Therefore significant yield losses are expected. Systematic, long-term control of potato viral infection, especially seed-potatoes, must be regarded as the most important factor to increase seed productivity.

Keywords: virus, potato, infection, diseases

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Authors: Gyati Shilakari Asthana, Abhay Asthana, Dharm Veer Kohli, Suresh Prasad Vyas

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Purpose: The therapeutic potential of oligonucleotide (ODN) is primarily dependent upon its safe and efficient delivery to specific cells overcoming degradation and maximizing cellular uptake in vivo. The present study is focused to design low molecular weight chitosan nanoconstructs to meet the requirements of safe and effectual delivery of ODNs. LMW-chitosan is a biodegradable, water soluble, biocompatible polymer and is useful as a non-viral vector for gene delivery due to its better stability in water. Methods: LMW chitosan ODN nanoparticles (CHODN NPs) were formulated by self-assembled method using various N/P ratios (moles ratio of amine groups of CH to phosphate moieties of ODNs; 0.5:1, 1:1, 3:1, 5:1, and 7:1) of CH to ODN. The developed CHODN NPs were evaluated with respect to gel retardation assay, particle size, zeta potential and cytotoxicity and transfection efficiency. Results: Complete complexation of CH/ODN was achieved at the charge ratio of 0.5:1 or above and CHODN NPs displayed resistance against DNase I. On increasing the N/P ratio of CH/ODN, the particle size of the NPs decreased whereas zeta potential (ZV) value increased. No significant toxicity was observed at all CH concentrations. The transfection efficiency was increased on increasing N/P ratio from 1:1 to 3:1, whereas it was decreased with further increment in N/P ratio upto 7:1. Maximum transfection of CHODN NPs with both the cell lines (Raw 267.4 cells and Hela cells) was achieved at N/P ratio of 3:1. The results suggest that transfection efficiency of CHODN NPs is dependent on N/P ratio. Conclusion: Thus the present study states that LMW chitosan nanoparticulate carriers would be acceptable choice to improve transfection efficiency in vitro as well as in vivo delivery of oligonucleotide.

Keywords: LMW-chitosan, chitosan nanoparticles, biocompatibility, cytotoxicity study, transfection efficiency, oligonucleotide

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Authors: Tannaz Alimardani, Amirhossein Moghanian, Morteza Elsa

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Bioactive glasses (BGs) are a group of surface-reactive biomaterials used in clinical applications as implants or filler materials in the human body to repair and replace diseased or damaged bone. Sol-gel technique was employed to prepare a SiO₂-CaO-P₂O₅ glass with a nominal composition of 58S BG with the addition of Sr and Li modifiers which imparts special properties to the BG. The effect of simultaneous addition of Sr and Li on bioactivity and biocompatibility, proliferation, alkaline phosphatase (ALP) activity of osteoblast cell line MC3T3-E1 and antibacterial property against methicillin-resistant Staphylococcus aureus (MRSA) bacteria were examined. BGs were characterized by X-ray diffraction, Fourier transform infrared spectroscopy and scanning electron microscopy before and after soaking the samples in the simulated body fluid (SBF) for different time intervals to characterize the formation of hydroxyapatite (HA) formed on the surface of BGs. Structural characterization indicated that the simultaneous presence of 5% Sr and 5% Li in 58S-BG composition not only did not retard HA formation because of the opposite effect of Sr and Li of the dissolution of BG in the SBF, but also stimulated the differentiation and proliferation of MC3T3-E1s. Moreover, the presence of Sr and Li on the dissolution of the ions resulted in an increase in the mean number of DAPI-labeled nuclei which was in good agreement with the live/dead assay. The result of antibacterial tests revealed that Sr and Li-substituted 58S bioactive glass exhibited a potential antibacterial effect against MRSA bacteria. Because of optimal proliferation and ALP activity of MC3T3-E1cells, proper bioactivity and high antibacterial potential against MRSA, BG-5/5 is suggested as a multifunctional candidate for bone tissue engineering.

Keywords: alkaline, alkaline earth, bioglass, co-doping, ion release

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