Search results for: Inhibition

Authors: R. P. Hewawasam, A. F. Dulhunty

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The ryanodine receptor (RyR) is an intracellular ion channel that releases Ca2+ from the sarcoplasmic reticulum and is essential for the excitation-contraction coupling and contraction in striated muscle. Human muscle specific glutathione transferase M2-2 (GSTM2-2) is a highly specific inhibitor of cardiac ryanodine receptor (RyR2) activity. Single channel-lipid bilayer studies and Ca2+ release assays performed using the C-terminal half of the GSTM2-2 and its mutants F157A and Y160A confirmed the ability of the C terminal domain of GSTM2-2 to specifically inhibit the cardiac ryanodine receptor activity. Objective of the present study is to determine the effect of C terminal domain of GSTM2-2 (GSTM2-2C) and the mutants, F157A and Y160A on the Ca2+ transients of neonatal ventricular cardiomyocytes. Primary cardiomyocytes were cultured from neonatal rats. They were treated with GSTM2-2C and the two mutants F157A and Y160A at 15µM and incubated for 2 hours. Then the cells were led with Fluo-4AM, fluorescent Ca2+ indicator, and the field stimulated (1 Hz, 3V and 2ms) cells were excited using the 488 nm argon laser. Contractility of the cells were measured and the Ca2+ transients in the stained cells were imaged using Leica SP5 confocal microscope. Peak amplitude of the Ca2+ transient, rise time and decay time from the peak were measured for each transient. In contrast to GSTM2C which significantly reduced the % shortening (42.8%) in the field stimulated cells, F157A and Y160A failed to reduce the % shortening.Analysis revealed that the average amplitude of the Ca2+ transient was significantly reduced (P<0.001) in cells treated with the wild type GSTM2-2C compared to that of untreated cells. Cells treated with the mutants F157A and Y160A didn’t change the Ca2+ transient significantly compared to the control. A significant increase in the rise time (P< 0.001) and a significant reduction in the decay time (P< 0.001) were observed in cardiomyocytes treated with GSTM2-2C compared to the control but not with F157A and Y160A. These results are consistent with the observation that GSTM2-2C reduced the Ca2+ release from the cardiac SR significantly whereas the mutants, F157A and Y160A didn’t show any effect compared to the control. GSTM2-2C has an isoform-specific effect on the cardiac ryanodine receptor activity and also it inhibits RyR2 channel activity only during diastole. Selective inhibition of RyR2 by GSTM2-2C has significant clinical potential in the treatment of cardiac arrhythmias and heart failure. Since GSTM2-2C-terminal construct has no GST enzyme activity, its introduction to the cardiomyocyte would not exert any unwanted side effects that may alter its enzymatic action. The present study further confirms that GSTM2-2C is capable of decreasing the Ca2+ release from the cardiac SR during diastole. These results raise the future possibility of using GSTM2-2C as a template for therapeutics that can depress RyR2 function when the channel is hyperactive in cardiac arrhythmias and heart failure.

Keywords: arrhythmia, cardiac muscle, cardiac ryanodine receptor, GSTM2-2

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Authors: Andrey A. Tyuftin, Rachael Reid, Paula Bourke, Patrick J. Cullen, Seamus Fanning, Paul Whyte, Declan Bolton , Joe P. Kerry

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One of the primary spoilage issues associated with vacuum-packed beef products is blown pack spoilage (BPS) caused by the psychrophilic spore-forming strain of Clostridium spp. Spores derived from this organism can be activated after heat-shrinking (eg. 90°C for 3 seconds). To date, research into the control of Clostridium spp in beef packaging is limited. Active packaging in the form of antimicrobially-active coatings may be one approach to its control. Antimicrobial compounds may be incorporated into packaging films or coated onto the internal surfaces of packaging films using a carrier matrix. Three naturally-sourced, commercially-available antimicrobials, namely; Auranta FV (AFV) (bitter oranges extract) from Envirotech Innovative Products Ltd, Ireland; Inbac-MDA (IMDA) from Chemital LLC, Spain, mixture of different organic acids and sodium octanoate (SO) from Sigma-Aldrich, UK, were added into gelatin solutions at 2 concentrations: 2.5 and 3.5 times their minimum inhibition concentration (MIC) against Clostridium estertheticum (DSMZ 8809). These gelatin solutions were coated onto the internal polyethylene layer of cold plasma treated, heat-shrinkable laminates conventionally used for meat packaging applications. Atmospheric plasma was used in order to enhance adhesion between packaging films and gelatin coatings. Pouches were formed from these coated packaging materials, and beef cuts which had been inoculated with C. estertheticum were vacuum packaged. Inoculated beef was vacuum packaged without employing active films and this treatment served as the control. All pouches were heat-sealed and then heat-shrunk at 90°C for 3 seconds and incubated at 2°C for 100 days. During this storage period, packs were monitored for the indicators of blown pack spoilage as follows; gas bubbles in drip, loss of vacuum (onset of BPS), blown, the presence of sufficient gas inside the packs to produce pack distension and tightly stretched, “overblown” packs/ packs leaking. Following storage and assessment of indicator date, it was concluded that AFV- and SO-containing packaging inhibited the growth of C. estertheticum, significantly delaying the blown pack spoilage of beef primals. IMDA did not inhibit the growth of C. estertheticum. This may be attributed to differences in release rates and possible reactions with gelatin. Overall, active films were successfully produced following plasma surface treatment, and experimental data demonstrated clearly that the use of antimicrobially-active films could significantly prolong the storage stability of beef primals through the effective control of BPS.

Keywords: active packaging, blown pack spoilage, Clostridium, antimicrobials, edible coatings, food packaging, gelatin films, meat science

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Authors: Navid Mosallaei, Mahmoud Reza Jaafari, Mohammad Yahya Hanafi-Bojd, Shiva Golmohammadzadeh, Bizhan Malaekeh-Nikouei

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Background: Docetaxel (DTX), a potent anticancer drug derived from the European yew tree, is effective against various human cancers by inhibiting microtubule depolymerization. Solid lipid nanoparticles (SLNs) have gained attention as drug carriers for enhancing drug effectiveness and safety. SLNs, submicron-sized lipid-based particles, can passively target tumors through the "enhanced permeability and retention" (EPR) effect, providing stability, drug protection, and controlled release while being biocompatible. Methods: The SLN formulation included biodegradable lipids (Compritol and Precirol), hydrogenated soy phosphatidylcholine (H-SPC) as a lipophilic co-surfactant, and Poloxamer 188 as a non-ionic polymeric stabilizer. Two SLN preparation techniques, probe sonication and microemulsion, were assessed. Characterization encompassed SLNs' morphology, particle size, zeta potential, matrix, and encapsulation efficacy. In-vitro cytotoxicity and cellular uptake studies were conducted using mouse colorectal (C-26) and human malignant melanoma (A-375) cell lines, comparing SLN-DTX with Taxotere®. In-vivo studies evaluated tumor inhibitory efficacy and survival in mice with colorectal (C-26) tumors, comparing SLNDTX withTaxotere®. Results: SLN-DTX demonstrated stability, with an average size of 180 nm and a low polydispersity index (PDI) of 0.2 and encapsulation efficacy of 98.0 ± 0.1%. Differential scanning calorimetry (DSC) suggested amorphous encapsulation of DTX within SLNs. In vitro studies revealed that SLN-DTX exhibited nearly equivalent cytotoxicity to Taxotere®, depending on concentration and exposure time. Cellular uptake studies demonstrated superior intracellular DTX accumulation with SLN-DTX. In a C-26 mouse model, SLN-DTX at 10 mg/kg outperformed Taxotere® at 10 and 20 mg/kg, with no significant differences in body weight changes and a remarkably high survival rate of 60%. Conclusion: This study concludes that SLN-DTX, prepared using the probe sonication, offers stability and enhanced therapeutic effects. It displayed almost same in vitro cytotoxicity to Taxotere® but showed superior cellular uptake. In a mouse model, SLN-DTX effectively inhibited tumor growth, with 10 mg/kg outperforming even 20 mg/kg of Taxotere®, without adverse body weight changes and with higher survival rates. This suggests that SLN-DTX has the potential to reduce adverse effects while maintaining or enhancing docetaxel's therapeutic profile, making it a promising drug delivery strategy suitable for industrialization.

Keywords: docetaxel, Taxotere®, solid lipid nanoparticles, enhanced permeability and retention effect, drug delivery, cancer chemotherapy, cytotoxicity, cellular uptake, tumor inhibition

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Authors: M. Cardenas-Garcia, P. P. Gonzalez-Perez

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Intercellular communication is a necessary condition for cellular functions and it allows a group of cells to survive as a population. Throughout this interaction, the cells work in a coordinated and collaborative way which facilitates their survival. In the case of cancerous cells, these take advantage of intercellular communication to preserve their malignancy, since through these physical unions they can send signs of malignancy. The Wnt/β-catenin signaling pathway plays an important role in the formation of intercellular communications, being also involved in a large number of cellular processes such as proliferation, differentiation, adhesion, cell survival, and cell death. The modeling and simulation of cellular signaling systems have found valuable support in a wide range of modeling approaches, which cover a wide spectrum ranging from mathematical models; e.g., ordinary differential equations, statistical methods, and numerical methods– to computational models; e.g., process algebra for modeling behavior and variation in molecular systems. Based on these models, different simulation tools have been developed from mathematical ones to computational ones. Regarding cellular and molecular processes in cancer, its study has also found a valuable support in different simulation tools that, covering a spectrum as mentioned above, have allowed the in silico experimentation of this phenomenon at the cellular and molecular level. In this work, we simulate and explore the complex interaction patterns of intercellular communication in cancer cells using the Cellulat bioinformatics tool, a computational simulation tool developed by us and motivated by two key elements: 1) a biochemically inspired model of self-organizing coordination in tuple spaces, and 2) the Gillespie’s algorithm, a stochastic simulation algorithm typically used to mimic systems of chemical/biochemical reactions in an efficient and accurate way. The main idea behind the Cellulat simulation tool is to provide an in silico experimentation environment that complements and guides in vitro experimentation in intra and intercellular signaling networks. Unlike most of the cell signaling simulation tools, such as E-Cell, BetaWB and Cell Illustrator which provides abstractions to model only intracellular behavior, Cellulat is appropriate for modeling both intracellular signaling and intercellular communication, providing the abstractions required to model –and as a result, simulate– the interaction mechanisms that involve two or more cells, that is essential in the scenario discussed in this work. During the development of this work we made evident the application of our computational simulation tool (Cellulat) for the modeling and simulation of intercellular communication between normal and cancerous cells, and in this way, propose key molecules that may prevent the arrival of malignant signals to the cells that surround the tumor cells. In this manner, we could identify the significant role that has the Wnt/β-catenin signaling pathway in cellular communication, and therefore, in the dissemination of cancer cells. We verified, using in silico experiments, how the inhibition of this signaling pathway prevents that the cells that surround a cancerous cell are transformed.

Keywords: cancer cells, in silico approach, intercellular communication, key molecules, modeling and simulation

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Authors: Jan Masak, Eva Kvasnickova, Vladimir Scholtz, Olga Matatkova, Marketa Valkova, Alena Cejkova

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Microbial colonization of medical instruments, catheters, implants, etc. is a serious problem in the spread of nosocomial infections. Biofilms exhibit enormous resistance to environment. The resistance of biofilm populations to antibiotic or biocides often increases by two to three orders of magnitude in comparison with suspension populations. Subjects of interests are substances or physical processes that primarily cause the destruction of biofilm, while the released cells can be killed by existing antibiotics. In addition, agents that do not have a strong lethal effect do not cause such a significant selection pressure to further enhance resistance. Non-thermal plasma (NTP) is defined as neutral, ionized gas composed of particles (photons, electrons, positive and negative ions, free radicals and excited or non-excited molecules) which are in permanent interaction. In this work, the effect of NTP generated by the cometary corona with a metallic grid on the formation and stability of biofilm and metabolic activity of cells in biofilm was studied. NTP was applied on biofilm populations of Staphylococcus epidermidis DBM 3179, Pseudomonas aeruginosa DBM 3081, DBM 3777, ATCC 15442 and ATCC 10145, Escherichia coli DBM 3125 and Candida albicans DBM 2164 grown on solid media on Petri dishes and on the titanium alloy (Ti6Al4V) surface used for the production joint replacements. Erythromycin (for S. epidermidis), polymyxin B (for E. coli and P. aeruginosa), amphotericin B (for C. albicans) and ceftazidime (for P. aeruginosa) were used to study the combined effect of NTP and antibiotics. Biofilms were quantified by crystal violet assay. Metabolic activity of the cells in biofilm was measured using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) colorimetric test based on the reduction of MTT into formazan by the dehydrogenase system of living cells. Fluorescence microscopy was applied to visualize the biofilm on the surface of the titanium alloy; SYTO 13 was used as a fluorescence probe to stain cells in the biofilm. It has been shown that biofilm populations of all studied microorganisms are very sensitive to the type of used NTP. The inhibition zone of biofilm recorded after 60 minutes exposure to NTP exceeded 20 cm², except P. aeruginosa DBM 3777 and ATCC 10145, where it was about 9 cm². Also metabolic activity of cells in biofilm differed for individual microbial strains. High sensitivity to NTP was observed in S. epidermidis, in which the metabolic activity of biofilm decreased after 30 minutes of NTP exposure to 15% and after 60 minutes to 1%. Conversely, the metabolic activity of cells of C. albicans decreased to 53% after 30 minutes of NTP exposure. Nevertheless, this result can be considered very good. Suitable combinations of exposure time of NTP and the concentration of antibiotic achieved in most cases a remarkable synergic effect on the reduction of the metabolic activity of the cells of the biofilm. For example, in the case of P. aeruginosa DBM 3777, a combination of 30 minutes of NTP with 1 mg/l of ceftazidime resulted in a decrease metabolic activity below 4%.

Keywords: anti-biofilm activity, antibiotic, non-thermal plasma, opportunistic pathogens

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Authors: Maia Kharadze, Indira Djaparidze, Maia Vanidze, Aleko Kalandia

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Modern scientific world studies chemical components and antioxidant activity of different kinds of vines according to their breed purity and location. To our knowledge, this kind of research has not been conducted in Georgia yet. The object of our research was to study Chkhaveri vine, which is included in the oldest varieties of the Black Sea basin vine. We have studied different-altitude Chkaveri grapes, juice, and wine (half dry rose-colored produced with European technologies) and their technical markers, qualitative and quantitive composition of their biologically active compounds and their antioxidant activity. We were determining the amount of phenols using Folin-Ciocalteu reagent, Flavonoids, Catechins and Anthocyanins using Spectral method and antioxidant activity using DPPH method. Several compounds were identified using –HPLC-UV-Vis, UPLC-MS methods. Six samples of Chkhaveri species– 5, 300, 360, 380, 400, 780 meter altitudes were taken and analyzed. The sample taken from 360 m altitude is distinguished by its cluster mass (383.6 grams) and high amount of sugar (20.1%). The sample taken from the five-meter altitude is distinguished by having high acidity (0.95%). Unlike other grapes varieties, such concentration of sugar and relatively low levels of citric acid ultimately leads to Chkhaveri wine individuality. Biologically active compounds of Chkhaveri were researched in 2014, 2015, 2016. The amount of total phenols in samples of 2016 fruit varies from 976.7 to 1767.0 mg/kg. Amount of Anthocians is 721.2-1630.2 mg/kg, and the amount of Flavanoids varies from 300.6 to 825.5 mg/kg. Relatively high amount of anthocyanins was found in the Chkhaveri at 780-meter altitude - 1630.2 mg/kg. Accordingly, the amount of Phenols and Flavanoids is high- 1767.9 mg/kg and 825.5 mg/kg. These characteristics are low in samples gathered from 5 meters above sea level, Anthocyanins-721.2 mg/ kg, total Phenols-976.7 mg/ kg, and Flavanoids-300.6 mg/kg. The highest amount of bioactive compounds can be found in the Chkhaveri samples of high altitudes because with rising height environment becomes harsh, the plant has to develop a better immune system using Phenolic compounds. The technology that is used for the production of wine also plays a huge role in the composition of the final product. Optimal techniques of maceration and ageing were worked out. While squeezing Chkhaveri, there are no anthocyanins in the juice. However, the amount of Anthocyanins rises during maceration. After the fermentation of dregs, the amount of anthocyanins is 55%, 521.3 gm/l, total Phenols 80% 1057.7 mg/l and Flavanoids 23.5 mg/l. Antioxidant activity of samples was also determined using the effect of 50% inhibition of the samples. All samples have high antioxidant activity. For instance, in samples at 780 meters above the sea-level antioxidant activity was 53.5%. It is relatively high compared to the sample at 5 m above sea-level with the antioxidant activity of 30.5%. Thus, there is a correlation between the amount Anthocyanins and antioxidant activity. The designated project has been fulfilled by financial support of the Georgia National Science Foundation (Grant AP/96/13, Grant 216816), Any idea in this publication is possessed by the author and may not represent the opinion of the Georgia National Science Foundation.

Keywords: antioxidants, bioactive content, wine, chkhaveri

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Authors: Sundru Manjulata Devi, Prakash M. Halami

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The immemorial use of fermented foods from vegetables, dairy and other biological sources are of great demand in India because of their health benefits. However, the diversity of Lactobacillus plantarum group (LPG) of vegetable origin has not been revealed yet, particularly with reference to their probiotic functionalities. In the present study, the different species of probiotic Lactobacillus plantarum group (LPG) i.e., L. plantarum subsp. plantarum MTCC 5422 (from fermented cereals), L. plantarum subsp. argentoratensis FG16 (from fermented bamboo shoot) and L. paraplantarum MTCC 9483 (from fermented gundruk) (as characterized by multiplex recA PCR assay) were considered to investigate their relative expression of MUB domains of mub gene (mucin binding protein) by Real time PCR. Initially, the allelic variation in the mub gene was assessed and found to encode three different variants (Type I, II and III). All the three types had 8, 9 and 10 MUB domains respectively (as analysed by Pfam database) and were found to be responsible for adhesion of bacteria to the host intestinal epithelial cells. These domains either get inserted or deleted during speciation or evolutionary events and lead to divergence. The reverse transcriptase qPCR analysis with mubLPF1+R1 primer pair supported variation in amplicon sizes with 300, 500 and 700 bp among different LPG strains. The relative expression of these MUB domains significantly unregulated in the presence of 1% mucin in overnight grown cultures. Simultaneously, the mub gene expressed efficiently by 7 fold in the culture L. paraplantarum MTCC 9483 with 10 MUB domains. An increase in the expression levels for L. plantarum subsp. plantarum MTCC 5422 and L. plantarum subsp. argentoratensis FG16 (MCC 2974) with 9 and 8 repetitive domains was around 4 and 2 fold, respectively. The detection and expression of an integrase (int) gene in the upstream region of mub gene reveals the excision and integration of these repetitive domains. Concurrently, an in vitro adhesion assay to mucin and exclusion of pathogens (such as Listeria monocytogenes and Micrococcus leuteus) was investigated and observed that the L. paraplantarum MTCC 9483 with more adhesion domains has more ability to adhere to mucin and inhibited the growth of pathogens. The production and expression of plantaricin-like bacteriocin (plnNC8 type) in MTCC 9483 suggests the pathogen inhibition. Hence, the expression of MUB domains can act as potential biomarkers in the screening of a novel probiotic LPG strain with adherence property. The present study provides a platform for an easy, rapid, less time consuming, low-cost methodology for the detection of potential probiotic bacteria. It was known that the traditional practices followed in the preparation of fermented bamboo shoots/gundruk/cereals of Indian foods contain different kinds of neutraceuticals for functional food and novel compounds with health promoting factors. In future, a detailed study of these food products can add more nutritive value, consumption and suitable for commercialization.

Keywords: adhesion gene, fermented foods, MUB domains, probiotics

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Authors: Hannah Dean

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The growth ideology, and its association with progress, is an important construct in the narrative of modernity. This ideology is embedded in neoclassical economic growth theory which conceptualises growth as linear and predictable, and the entrepreneur as a rational economic manager. This conceptualisation has been critiqued for reinforcing the managerial discourse in entrepreneurship studies. Despite these critiques, both the neoclassical growth theory and its adjacent managerial discourse dominate entrepreneurship studies notably the literature on female entrepreneurs. The latter is the focus of this paper. Given this emphasis on growth, female entrepreneurs are portrayed as problematic because their growth lags behind their male counterparts. This image which ignores the complexity and diversity of female entrepreneurs’ experience persists in the literature due to the lack of studies that analyse the process and contextual factors surrounding female entrepreneurs’ experience. This study aims to address the subordination of female entrepreneurs by questioning the hegemonic logic of economic growth and the managerial discourse as a true representation for the entrepreneurial experience. This objective is achieved by drawing on Schumpeter’s theorising and narrative inquiry. This exploratory study undertakes in depth interviews to gain insights into female entrepreneurs’ experience and the impact of the economic growth model and the managerial discourse on their performance. The narratives challenge a number of assumptions about female entrepreneurs. The participants occupied senior positions in the corporate world before setting up their businesses. This is at odds with much writing which assumes that women underperform because they leave their career without gaining managerial experience to achieve work-life balance. In line with Schumpeter, who distinguishes the entrepreneur from the manager, the participants’ main function was innovation. They did not believe that the managerial paradigm governing their corporate careers was applicable to their entrepreneurial experience. Formal planning and managerial rationality can hinder their decision making process. The narratives point to the gap between the two worlds which makes stepping into entrepreneurship a scary move. Schumpeter argues that the entrepreneurial process is evolutionary and that failure is an integral part of it. The participants’ entrepreneurial process was in fact irregular. The performance of new combinations was not always predictable. They therefore relied on their initiative. The inhibition to deploy these traits had an adverse effect on business growth. The narratives also indicate that over-reliance on growth threaten the business survival as it faces competing pressures. The study offers theoretical and empirical contributions to (female) entrepreneurship studies by presenting Schumpeter’s theorising as an alternative theoretical framework to the neoclassical economic growth theory. The study also reduces entrepreneurs’ vulnerability by making them aware of the negative influence that the linear growth model and the managerial discourse hold upon their performance. The study has implications for policy makers as it generates new knowledge that incorporates the current social and economic changes in the context of entrepreneurs that can no longer be sustained by the linear growth models especially in the current economic climate.

Keywords: economic growth, female entrepreneurs, managerial discourse, Schumpeter

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Authors: Alexandra Sargent, Sarah Ferris, Ioannis Theofanous

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The Abbott RealTime High Risk HPV test (RealTime HPV) is one of five assays clinically validated and approved by the English NHS Cervical Screening Programme (CSP) for HPV triage of low grade dyskaryosis and test-of-cure of treated Cervical Intraepithelial Neoplasia. The assay is a highly automated multiplex real-time PCR test for detecting 14 high risk (hr) HPV types, with simultaneous differentiation of HPV 16 and HPV 18 versus non-HPV 16/18 hrHPV. An endogenous internal control ensures sample cellularity, controls extraction efficiency and PCR inhibition. The original cervical specimen collected in SurePath (SP) liquid-based cytology (LBC) medium (BD Diagnostics) and the SP post-gradient cell pellets (SPG) after cytological processing are both CE marked for testing with the RealTime HPV test. During the 2011 NHSCSP validation of new tests only the original aliquot of SP LBC medium was investigated. Residual sample volume left after cytology slide preparation is low and may not always have sufficient volume for repeat HPV testing or for testing of other biomarkers that may be implemented in testing algorithms in the future. The SPG samples, however, have sufficient volumes to carry out additional testing and necessary laboratory validation procedures. This study investigates the correlation of RealTime HPV results of cervical specimens collected in SP LBC medium from women with low grade cytological abnormalities observed with matched pairs of original SP LBC medium and SP post-gradient cell pellets (SPG) after cytology processing. Matched pairs of SP and SPG samples from 750 women with borderline (N = 392) and mild (N = 351) cytology were available for this study. Both specimen types were processed and parallel tested for the presence of hrHPV with RealTime HPV according to the manufacturer´s instructions. HrHPV detection rates and concordance between test results from matched SP and SPGCP pairs were calculated. A total of 743 matched pairs with valid test results on both sample types were available for analysis. An overall-agreement of hrHPV test results of 97.5% (k: 0.95) was found with matched SP/SPG pairs and slightly lower concordance (96.9%; k: 0.94) was observed on 392 pairs from women with borderline cytology compared to 351 pairs from women with mild cytology (98.0%; k: 0.95). Partial typing results were highly concordant in matched SP/SPG pairs for HPV 16 (99.1%), HPV 18 (99.7%) and non-HPV16/18 hrHPV (97.0%), respectively. 19 matched pairs were found with discrepant results: 9 from women with borderline cytology and 4 from women with mild cytology were negative on SPG and positive on SP; 3 from women with borderline cytology and 3 from women with mild cytology were negative on SP and positive on SPG. Excellent correlation of hrHPV DNA test results was found between matched pairs of SP original fluid and post-gradient cell pellets from women with low grade cytological abnormalities tested with the Abbott RealTime High-Risk HPV assay, demonstrating robust performance of the test with both specimen types and reassuring the utility of the assay for cytology triage with both specimen types.

Keywords: Abbott realtime test, HPV, SurePath liquid based cytology, surepath post-gradient cell pellet

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Authors: N. G. Klivleyeva, T. I. Glebova, G. V. Lukmanova, S. B. Bayseit, S. Z. Taubaeva, M. K. Kalkozhaeva

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The role of viral diseases in the general infectious disease incidence increases every year and requires special attention to the problem of interpreting the etiology of infectious agents. Influenza and acute respiratory viral infections are one of the most pressing public health issues. In the period 2012-2014, collection of 419 nasal swabs and 150 blood sera has been carried out in the patient care institutions of the various Kazakhstan regions from patients with symptoms of ARVI and pneumonia. Primary identification of biosamples for the presence of influenza viral antigens in enzyme immunoassay on nitrocellulose membrane gave positive results in 125 swabs (29.8%). Biosample screening in immunofluorescence test revealed the presence of influenza viral antigens against A/H1 in 63 samples (15.0%), A/H3 – in 70 samples (16.7%) and type B – in 9 samples (2.1%). As a result of primary infection, and successive passages in chick embryos and MDCK cell cultures, 38 HAAg were isolated from 419 samples with a clear cytopathic effect and hemagglutination titre in MDCK cell culture within 1:2-1:4, in CE - 1:8-1:256. The infectivity of isolates in chicken embryos were 3.5-6.5 lg EID50/0.2, in MDCK cell culture – 2.5-6.5 lg PFU/ml. Identification of 28 isolates was carried out in inhibition reactions of hemagglutinating activity and neuraminidase activity, showed their belonging to the influenza virus: 26 strains to A/H1N1, one - to A/H3N2, and one - to type B. Serological examination of blood sera for the presence of specific antibodies being an indirect evidence of the performed isolation and contributing to the timely interpretation of the disease etiology in the epidemics takes an important place in the comprehensive study of influenza viruses circulating among people. Serological analyzes were carried out in HAI assay using a kit consisting of 12 reference strains obtained from the WHO centre for reference and research on Influenza (CDC, Atlanta, USA) and three Kazakhstan (A/Almaty/347/09 (H1N1v), A/Almaty/462/11 (H3N2) and B/Almaty/414/10) human influenza viruses that are stored in the laboratory collection. The results of serological analysis of 150 blood sera showed that antihaemagglutinins against the A/H3N2 virus serosubtype were found in 46 samples (49.4%) out of 93 sera collected in 2012-2013. The antibody titres were within 1:160-1:320. 19 sera (20.4%) were seropositive against influenza A/H1N1 virus, the antibodies were observed in titres of 1:20-1:40. Six sera (6.4%) were positive against the influenza A/H1N1+A/H3N2 virus (mixed infection); the antibodies were recorded in titres of 1:20-1:40. Antihaemagglutinins against influenza type B virus were detected only in five sera (5.4%). The results of analysis of 57 sera collected in 2014 showed that antihaemagglutinins against A/H3N2 virus subtype were detected in 32 blood sera (56.1%) in titres of 1:160-1:640. Ten sera (17.5%) were seropositive against A/H1N1 virus; antihaemagglutinins against influenza type B virus were not detected. Therefore, virological and serological studies have shown that in Kazakhstan, as well as in the world, the influenza viruses A/H1N1, A/H3N2 and influenza B viruses were actively circulating during the epidemic seasons in 2012-2014.

Keywords: influenza, MDCK cell, serological analysis, virus

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Authors: Karen L. Rincon-Granados, América R. Vázquez-Olmos, Adriana-Patricia Rodríguez-Hernández, Gina Prado-Prone, Margarita Rivera, Roberto Y. Sato-Berrú

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In this work, a hybrid film based on poly-3-hydroxybutyrate (P3HB) and nickel ferrite (NiFe₂O₄) nanoparticles (NPs) was obtained by a simple and reproducible methodology in order to study its antibacterial and cytotoxic properties. The motivation for this research is the current antimicrobial resistance (RAM). This is a threat to human health and development worldwide. RAM is caused by the emergence of bacterial strains resistant to traditional antibiotics that were used as treatment. Due to this, the need to investigate new alternatives for preventing and treating bacterial infections emerges. In this sense, metal oxide NPs have aroused great interest due to their unique physicochemical properties. However, their use is limited by the nanostructured nature, commonly obtained by chemical and physical synthesis methods, as powders or colloidal dispersions. Therefore, the incorporation of nanostructured materials in polymer matrices to obtain hybrid materials that allow disinfecting and preventing the spread of bacteria on various surfaces. Accordingly, this work presents the synthesis and study of the antibacterial properties of the P3HB@NiFe₂O₄ hybrid film as a potential material to inhibit bacterial growth. The NiFe₂O₄ NPs were previously synthesized by a mechanochemical method. The P3HB and P3HB@NiFe₂O₄ films were obtained by the solvent casting method. The films were characterized by X-ray diffraction (XRD), Raman scattering, and scanning electron microscopy (SEM). The XRD pattern showed that the NiFe₂O₄ NPs were incorporated into the P3HB polymer matrix and retained their nanometric sizes. By energy dispersive X-ray spectroscopy (EDS), it was observed that the NPs are homogeneously distributed in the film. The bactericidal effect of the films obtained was evaluated in vitro using the broth surface method against two opportunistic and nosocomial pathogens, Staphylococcus aureus and Pseudomonas aeruginosa. The bacterial growth results showed that the P3HB@NiFe₂O₄ hybrid film was inhibited by 97% and 96% for S. aureus and P. aeruginosa, respectively. Surprisingly, the P3HB film inhibited both bacterial strains by around 90%. The cytotoxicity of the NiFe₂O₄ NPs, P3HB@NiFe₂O₄ hybrid film, and the P3HB film was evaluated using human skin cells, keratinocytes, and fibroblasts, finding that the NPs are biocompatible. The P3HB film and hybrids are cytotoxic, which demonstrated that although P3HB is known and reported as a biocompatible polymer, under our work conditions, P3HB was cytotoxic. Its bactericidal effect could be related to this activity. Its films are bactericidal and cytotoxic to keratinocytes and fibroblasts, the first barrier of human skin. Despite this, the hybrid film of P3HB@NiFe₂O₄ presents synergy with the bactericidal effect between P3HB and NPs, increasing bacterial inhibition. In addition, NPs decrease the cytotoxicity of P3HB to keratinocytes. The methodology used in this work was successful in producing hybrid films with antibacterial activity. However, future challenges are generated to find relationships between NPs and P3HB that allow taking advantage of their bactericidal properties and do not compromise biocompatibility.

Keywords: poly-3-hydroxybutyrate, nanoparticles, hybrid film, antibacterial

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Authors: Poojan Gharat, Haridas Pal, Sharmistha Dutta Choudhury

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Tuning photophysical properties of guest dyes through host-guest interactions involving macrocyclic hosts are the attractive research areas since past few decades, as these changes can directly be implemented in chemical sensing, molecular recognition, fluorescence imaging and dye laser applications. Excited state intramolecular proton transfer (ESIPT) is an intramolecular prototautomerization process display by some specific dyes. The process is quite amenable to tunability by the presence of different macrocyclic hosts. The present study explores the interesting effect of p-sulfonatocalix[n]arene (SCXn) and cyclodextrin (CD) hosts on the excited-state prototautomeric equilibrium of Chrysazine (CZ), a model antitumour drug. CZ exists exclusively in its normal form (N) in the ground state. However, in the excited state, the excited N* form undergoes ESIPT along with its pre-existing intramolecular hydrogen bonds, giving the excited state prototautomer (T*). Accordingly, CZ shows a single absorption band due to N form, but two emission bands due to N* and T* forms. Facile prototautomerization of CZ is considerably inhibited when the dye gets bound to SCXn hosts. However, in spite of lower binding affinity, the inhibition is more profound with SCX6 host as compared to SCX4 host. For CD-CZ system, while prototautomerization process is hindered by the presence of β-CD, it remains unaffected in the presence of γCD. Reduction in the prototautomerization process of CZ by SCXn and βCD hosts is unusual, because T* form is less dipolar in nature than the N*, hence binding of CZ within relatively hydrophobic hosts cavities should have enhanced the prototautomerization process. At the same time, considering the similar chemical nature of two CD hosts, their effect on prototautomerization process of CZ would have also been similar. The atypical effects on the prototautomerization process of CZ by the studied hosts are suggested to arise due to the partial inclusion or external binding of CZ with the hosts. As a result, there is a strong possibility of intermolecular H-bonding interaction between CZ dye and the functional groups present at the portals of SCXn and βCD hosts. Formation of these intermolecular H-bonds effectively causes the pre-existing intramolecular H-bonding network within CZ molecule to become weak, and this consequently reduces the prototautomerization process for the dye. Our results suggest that rather than the binding affinity between the dye and host, it is the orientation of CZ in the case of SCXn-CZ complexes and the binding stoichiometry in the case of CD-CZ complexes that play the predominant role in influencing the prototautomeric equilibrium of the dye CZ. In the case of SCXn-CZ complexes, the results obtained through experimental findings are well supported by quantum chemical calculations. Similarly for CD-CZ systems, binding stoichiometries obtained through geometry optimization studies on the complexes between CZ and CD hosts correlate nicely with the experimental results. Formation of βCD-CZ complexes with 1:1 stoichiometry while formation of γCD-CZ complexes with 1:1, 1:2 and 2:2 stoichiometries are revealed from geometry optimization studies and these results are in good accordance with the observed effects by the βCD and γCD hosts on the ESIPT process of CZ dye.

Keywords: intermolecular proton transfer, macrocyclic hosts, quantum chemical studies, photophysical studies

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Authors: Heba Kamal Mohamed

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Introduction: Tramadol is a weak -opioid receptor agonist with an analgesic effect because of the inhibition of uptake of norepinephrine and serotonin. Nowadays, tramadol hydrochloride is frequently used as a pain reliever. Tramadol is recommended for the management of acute and chronic pain of moderate to severe intensity associated with a variety of diseases or problems, including osteoarthritis, diabetic neuropathy, neuropathic pain, and even perioperative pain in human patients. In obstetrics and gynecology, tramadol is used extensively to treat postoperative pain. Aim of the study: This study was undertaken to investigate the histological (light and electron microscopic) and immunohistochemical effects of long term tramadol treatment on the ovary of adult rats and the possible recovery after tramadol withdrawal. Design: Experimental study. Materials and methods: Thirty adult female albino rats were used in this study. They were classified into three main groups (10 rats each). Group I served as the control group. Group II, rats were subcutaneously injected with tramadol 40 mg/kg three times per week for 8 weeks. Group III, rats were subcutaneously injected with tramadol 40 mg/kg three times per week for 8 weeks then were kept for another 8 weeks without treatment for recovery. At the end of the experiment rats were sacrificed and bilateral oophorectomy was carried out; the ovaries were processed for histological study (light and electron microscopic) and immunohistochemical reaction for caspase-3 (apoptotic protein). Results: Examination of the ovary of tramadol-treated rats (group II) revealed many atretic ovarian follicles, some follicles showed detachment of the oocyte from surrounding granulosa cells and others showed loss of the oocyte. Many follicles revealed degenerated vacuolated oocytes and vacuolated theca folliculi cells. Granulosa cells appeared shrunken, disrupted and loosely attached with vacuolated cytoplasm and pyknotic nuclei. Some follicles showed separation of granulosa cells from the theca folliculi layer. The ultrastructural study revealed the presence of granulosa cells with electron dense indented nuclei, damaged mitochondria and granular vacuolated cytoplasm. Other cells showed accumulation of large amount of lipid droplets in their cytoplasm. Some follicles revealed rarifaction of the cytoplasm of oocytes and absent zona pellucida. Moreover, apoptotic changes were detected by immunohistochemical staining in the form of increased staining intensity to caspase-3 (apoptotic protein). With Masson's Trichrome stain, there was an increased collagen fibre deposition in the ovarian cortical stroma. The wall of blood vessels appeared thickened. In the withdrawal group (group III), there was a little improvement in the histological and immunohistochemical changes. Conclusion: Tramadol had serious deleterious effects on ovarian structure. Thus, it should be used with caution, especially when a long term treatment is indicated. Withdrawal of tramadol led to a little improvement in the structural impairment of the ovary.

Keywords: tramadol, ovary, withdrawal, rats

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Authors: Yuvraj Singh Dangi, Brajesh Kumar Tiwari, Ashok Kumar Jain, Kamta Prasad Namdeo

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The potential use of liposomes as drug carriers by i.v. injection is limited by their low stability in blood stream. Firstly, phospholipid exchange and transfer to lipoproteins, mainly HDL destabilizes and disintegrates liposomes with subsequent loss of content. To avoid the pain associated with injection and to obtain better patient compliance studies concerning various dosage forms, have been developed. Conventional liposomes (unilamellar and multilamellar) have certain drawbacks like low entrapment efficiency, stability and release of drug after single breach in external membrane, have led to the new type of liposomal systems. The challenge has been successfully met in the form of Double Liposomes (DL). DL is a recently developed type of liposome, consisting of smaller liposomes enveloped in lipid bilayers. The outer lipid layer of DL can protect inner liposomes against various enzymes, therefore DL was thought to be more effective than ordinary liposomes. This concept was also supported by in vitro release characteristics i.e. DL formation inhibited the release of drugs encapsulated in inner liposomes. DL consists of several small liposomes encapsulated in large liposomes, i.e., multivesicular vesicles (MVV), therefore, DL should be discriminated from ordinary classification of multilamellar vesicles (MLV), large unilamellar vesicles (LUV), small unilamellar vesicles (SUV). However, for these liposomes, the volume of inner phase is small and loading volume of water-soluble drugs is low. In the present study, the potential of phosphatidylethanolamine (PE) lipid anchored double liposomes (DL) to incorporate two drugs in a single system is exploited as a tool to augment the H. pylori eradication rate. Preparation of DL involves two steps, first formation of primary (inner) liposomes by thin film hydration method containing one drug, then addition of suspension of inner liposomes on thin film of lipid containing the other drug. The success of formation of DL was characterized by optical and transmission electron microscopy. Quantitation of DL-bacterial interaction was evaluated in terms of percent growth inhibition (%GI) on reference strain of H. pylori ATCC 26695. To confirm specific binding efficacy of DL to H. pylori PE surface receptor we performed an agglutination assay. Agglutination in DL treated H. pylori suspension suggested selectivity of DL towards the PE surface receptor of H. pylori. Monotherapy is generally not recommended for treatment of a H. pylori infection due to the danger of development of resistance and unacceptably low eradication rates. Therefore, combination therapy with amoxicillin trihydrate (AMOX) as anti-H. pylori agent and ranitidine bismuth citrate (RBC) as antisecretory agent were selected for the study with an expectation that this dual-drug delivery approach will exert acceptable anti-H. pylori activity.

Keywords: Helicobacter pylorI, amoxicillin trihydrate, Ranitidine Bismuth citrate, phosphatidylethanolamine, multi vesicular systems

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Authors: Anupalli Roja Rani, Pavithra Dasari

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Background: Among several, breast cancer has emerged as the most common female cancer in developing countries. It is the most common cause of cancer-related deaths worldwide among women. It is a molecularly and clinically heterogeneous disease. Moreover, it is a hormone–dependent tumor in which estrogens can regulate the growth of breast cells by binding with estrogen receptors (ERs). Moreover, the use of natural products in cancer therapeutics is due to their properties of biocompatibility and less toxicity. Plants are the vast reservoirs for various bioactive compounds. Coleus barbatus (Lamiaceae) contains anticancer properties against several cancer cell lines. Method: In the present study, an attempt is being made to enrich the knowledge of the anticancer activity of pure compounds extracted from Coleus barbatus (Andrew). On human breast cancer cell lines MCF-7. Here in, we are assessing the antiproliferative activity of Coleus barbatus (Andrew) plant extracts against MCF 7 and also evaluating their toxicity in normal human mammary cell lines such as Human Mammary Epithelial Cells (HMEC). The active fraction of plant extract was further purified with the help of Flash chromatography, Medium Pressure Liquid Chromatography (MPLC) and preparative High-Performance Liquid Chromatography (HPLC). The structure of pure compounds will be elucidated by using modern spectroscopic methods like Nuclear magnetic resonance (NMR), Electrospray Ionisation Mass Spectrometry (ESI-MS) methods. Later, the growth inhibition morphological assessment of cancer cells and cell cycle analysis of purified compounds were assessed using FACS. The growth and progression of signaling molecules HER2, GRP78 was studied by secretion assay using ELISA and expression analysis by flow cytometry. Result: Cytotoxic effect against MCF-7 with IC50 values were derived from dose response curves, using six concentrations of twofold serially diluted samples, by SOFTMax Pro software (Molecular device) and respectively Ellipticine and 0.5% DMSO were used as a positive and negative control. Conclusion: The present study shows the significance of various bioactive compounds extracted from Coleus barbatus (Andrew) root material. It acts as an anti-proliferative and shows cytotoxic effects on human breast cancer cell lines MCF7. The plant extracts play an important role pharmacologically. The whole plant has been used in traditional medicine for decades and the studies done have authenticated the practice. Earlier, as described, the plant has been used in the ayurveda and homeopathy medicine. However, more clinical and pathological studies must be conducted to investigate the unexploited potential of the plant. These studies will be very useful for drug designing in the future.

Keywords: coleus barbatus, HPLC, MPLC, NMR, MCF7, flash chromatograph, ESI-MS, FACS, ELISA.

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Authors: Wei Xiao, Gangzhi Cai, Xingliang Qin, Hongyan Ren, Zaidong Hua, Zhe Zhu, Hongwei Xiao, Ximin Zheng, Jie Yao, Yanzhen Bi

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Myostatin (MSTN) is the master regulator of double muscling phenotype with overgrown muscle and decreased fatness in animals, but its action mode to regulate fat deposition remains to be elucidated. In this study a swin-specific pathway through which MSTN acts to regulate the fat deposition was deciphered. Deep sequenincing of the mRNA and miRNA of fat tissues of MSTN knockout (KO) and wildtype (WT) pigs discovered the positive correlation of myocyte enhancer factor 2C (MEF2C) and fat-inhibiting miR222 expression, and the inverse correlation of miR222 and stearoyl-CoA desaturase 5 (SCD5) expression. SCD5 is rodent-absent and expressed only in pig, sheep and cattle. Fatty acid spectrum of fat tissues revealed a lower percentage of oleoyl-CoA (18:1) and palmitoleyl CoA (16:1) in MSTN KO pigs, which are the catalyzing products of SCD5-mediated desaturation of steroyl CoA (18:0) and palmitoyl CoA (16:0). Blood metrics demonstrated a 45% decline of triglyceride (TG) content in MSTN KO pigs. In light of these observations we hypothesized that MSTN might act through MEF2C/miR222/SCD5 pathway to regulate desaturation of fatty acid as well as triglyceride synthesis in pigs. To this end, real-time PCR and Western blotting were carried out to detect the expression of the three genes stated above. These experiments showed that MEF2C expression was up-regulated by nearly 2-fold, miR222 up-regulated by nearly 3-fold and SCD5 down-regulated by nearly 50% in MSTN KO pigs. These data were consistent with the expression change in deep sequencing analysis. Dual luciferase reporter was then used to confirm the regulation of MEF2C upon the promoter of miR222. Ecotopic expression of MEF2C in preadipocyte cells enhanced miR222 expression by 3.48-fold. CHIP-PCR identified a putative binding site of MEF2C on -2077 to -2066 region of miR222 promoter. Electrophoretic mobility shift assay (EMSA) demonstrated the interaction of MEF2C and miR222 promoter in vitro. These data indicated that MEF2C transcriptionally regulates the expression of miR222. Next, the regulation of miR222 on SCD5 mRNA as well as its physiological consequences were examined. Dual luciferase reporter testing revealed the translational inhibition of miR222 upon the 3´ UTR (untranslated region) of SCD5 in preadipocyte cells. Transfection of miR222 mimics and inhibitors resulted in the down-regulation and up-regulation of SCD5 in preadipocyte cells respectively, consistent with the results from reporter testing. RNA interference of SCD5 in preadipocyte cells caused 26.2% reduction of TG, in agreement with the results of TG content in MSTN KO pigs. In summary, the results above supported the existence of a molecular pathway that MSTN signals through MEF2C/miR222/SCD5 to regulate the fat deposition in pigs. This swine-specific pathway offers potential molecular markers for the development and breeding of a new pig line with optimised fatty acid composition. This would benefit human health by decreasing the takeup of saturated fatty acid.

Keywords: fat deposition, MEF2C, miR222, myostatin, SCD5, pig

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Authors: Anil Saini, Jatinder Kumar Ratan

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Now a day, the key issue for the scientific community is to devise the innovative technologies for sustainable control of urban pollution. In urban cities, a large surface area of the masonry structures, buildings, and pavements is exposed to the open environment, which may be utilized for the control of air pollution, if it is built from the photocatalytically active cement-based constructional materials such as concrete, mortars, paints, and blocks, etc. The photocatalytically active cement is formulated by incorporating a photocatalyst in the cement matrix, and such cement is generally known as self-cleaning cement In the literature, self-cleaning cement has been synthesized by incorporating nanosized-TiO₂ (n-TiO₂) as a photocatalyst in the formulation of the cement. However, the utilization of n-TiO₂ for the formulation of self-cleaning cement has the drawbacks of nano-toxicity, higher cost, and agglomeration as far as the commercial production and applications are concerned. The use of microsized-TiO₂ (m-TiO₂) in place of n-TiO₂ for the commercial manufacture of self-cleaning cement could avoid the above-mentioned problems. However, m-TiO₂ is less photocatalytically active as compared to n- TiO₂ due to smaller surface area, higher band gap, and increased recombination rate. As such, the use of m-TiO₂ in the formulation of self-cleaning cement may lead to a reduction in photocatalytic activity, thus, reducing the self-cleaning, depolluting, and antimicrobial abilities of the resultant cement material. So improvement in the photoactivity of m-TiO₂ based self-cleaning cement is the key issue for its practical applications in the present scenario. The current work proposes the use of surface-fluorinated m-TiO₂ for the formulation of self-cleaning cement to enhance its photocatalytic activity. The calcined dolomite, a constructional material, has also been utilized as co-adsorbent along with the surface-fluorinated m-TiO₂ in the formulation of self-cleaning cement to enhance the photocatalytic performance. The surface-fluorinated m-TiO₂, calcined dolomite, and the formulated self-cleaning cement were characterized using diffuse reflectance spectroscopy (DRS), X-ray diffraction analysis (XRD), field emission-scanning electron microscopy (FE-SEM), energy dispersive x-ray spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), BET (Brunauer–Emmett–Teller) surface area, and energy dispersive X-ray fluorescence spectrometry (EDXRF). The self-cleaning property of the as-prepared self-cleaning cement was evaluated using the methylene blue (MB) test. The depolluting ability of the formulated self-cleaning cement was assessed through a continuous NOX removal test. The antimicrobial activity of the self-cleaning cement was appraised using the method of the zone of inhibition. The as-prepared self-cleaning cement obtained by uniform mixing of 87% clinker, 10% calcined dolomite, and 3% surface-fluorinated m-TiO₂ showed a remarkable self-cleaning property by providing 53.9% degradation of the coated MB dye. The self-cleaning cement also depicted a noteworthy depolluting ability by removing 5.5% of NOx from the air. The inactivation of B. subtiltis bacteria in the presence of light confirmed the significant antimicrobial property of the formulated self-cleaning cement. The self-cleaning, depolluting, and antimicrobial results are attributed to the synergetic effect of surface-fluorinated m-TiO₂ and calcined dolomite in the cement matrix. The present study opens an idea and route for further research for acile and economical formulation of self-cleaning cement.

Keywords: microsized-titanium dioxide (m-TiO₂), self-cleaning cement, photocatalysis, surface-fluorination

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Authors: Douglas N. Simões, Michele Pittol, Vanda F. Ribeiro, Daiane Tomacheski, Ruth M. C. Santana

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The increasing demand of thermoplastic elastomers is related to the wide range of applications, such as automotive, footwear, wire and cable industries, adhesives and medical devices, cell phones, sporting goods, toys and others. These materials are susceptible to microbial attack. Moisture and organic matter present in some areas (such as shower area and sink), provide favorable conditions for microbial proliferation, which contributes to the spread of diseases and reduces the product life cycle. Compounds based on SEBS copolymers, poly(styrene-b-(ethylene-co-butylene)-b-styrene, are a class of thermoplastic elastomers (TPE), fully recyclable and largely used in domestic appliances like bath mats and tooth brushes (soft touch). Zinc oxide and zinc ions loaded in personal and home care products have become common in the last years due to its biocidal effect. In that sense, the aim of this study was to evaluate the effect of zinc as antimicrobial agent in compounds based on SEBS/polypropylene/oil/ calcite for use as refrigerator seals (gaskets), bath mats and sink squeegee. Two zinc oxides from different suppliers (ZnO-Pe and ZnO-WR) and one masterbatch of zinc ions (M-Zn-ion) were used in proportions of 0%, 1%, 3% and 5%. The compounds were prepared using a co-rotating double screw extruder (L/D ratio of 40/1 and 16 mm screw diameter). The extrusion parameters were kept constant for all materials. Tests specimens were prepared using the injection molding machine. A compound with no antimicrobial additive (standard) was also tested. Compounds were characterized by physical (density), mechanical (hardness and tensile properties) and rheological properties (melt flow rate - MFR). The Japan Industrial Standard (JIS) Z 2801:2010 was applied to evaluate antibacterial properties against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). The Brazilian Association of Technical Standards (ABNT) NBR 15275:2014 were used to evaluate antifungal properties against Aspergillus niger (A. niger), Aureobasidium pullulans (A. pullulans), Candida albicans (C. albicans), and Penicillium chrysogenum (P. chrysogenum). The microbiological assay showed a reduction over 42% in E. coli and over 49% in S. aureus population. The tests with fungi showed inconclusive results because the sample without zinc also demonstrated an inhibition of fungal development when tested against A. pullulans, C. albicans and P. chrysogenum. In addition, the zinc loaded samples showed worse results than the standard sample when tested against A. niger. The zinc addition did not show significant variation in mechanical properties. However, the density values increased with the rise in ZnO additives concentration, and had a little decrease in M-Zn-ion samples. Also, there were differences in the MFR results in all compounds compared to the standard.

Keywords: antimicrobial, home device, SEBS, zinc

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Authors: Gabriela Wyszogrodzka, Przemyslaw Dorozynski, Barbara Gil, Maciej Strzempek, Bartosz Marszalek, Piotr Kulinowski, Wladyslaw Piotr Weglarz, Elzbieta Menaszek

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MOFs (Metal-Organic Frameworks) belong to a new group of porous materials with a hybrid organic-inorganic construction. Their structure is a network consisting of metal cations or clusters (acting as metallic centers, nodes) and the organic linkers between nodes. The interest in MOFs is primarily associated with the use of their well-developed surface and large porous. Possibility to build MOFs of biocompatible components let to use them as potential drug carriers. Furthermore, forming MOFs structure from cations possessing paramagnetic properties (e.g. iron cations) allows to use them as MRI (Magnetic Resonance Imaging) contrast agents. The concept of formation of particles that combine the ability to transfer active substance with imaging properties has been called theranostic (from words combination therapy and diagnostics). By building MOF structure from iron cations it is possible to use them as theranostic agents and monitoring the distribution of the active substance after administration in real time. In the study iron-MOF: Fe-MIL-101-NH2 was chosen, consisting of iron cluster in nodes of the structure and amino-terephthalic acid as a linker. The aim of the study was to investigate the possibility of applying Fe-MIL-101-NH2 as inhalable theranostic particulate system for the first-line anti-tuberculosis antibiotic – isoniazid. The drug content incorporated into Fe-MIL-101-NH2 was evaluated by dissolution study using spectrophotometric method. Results showed isoniazid encapsulation efficiency – ca. 12.5% wt. Possibility of Fe-MIL-101-NH2 application as the MRI contrast agent was demonstrated by magnetic resonance tomography. FeMIL-101-NH2 effectively shortening T1 and T2 relaxation times (increasing R1 and R2 relaxation rates) linearly with the concentrations of suspended material. Images obtained using multi-echo magnetic resonance imaging sequence revealed possibility to use FeMIL-101-NH2 as positive and negative contrasts depending on applied repetition time. MOFs micronization via ultrasound was evaluated by XRD, nitrogen adsorption, FTIR, SEM imaging and did not influence their crystal shape and size. Ultrasonication let to break the aggregates and achieve very homogeneously looking SEM images. MOFs cytotoxicity was evaluated in in vitro test with a highly sensitive resazurin based reagent PrestoBlue™ on L929 fibroblast cell line. After 24h no inhibition of cell proliferation was observed. All results proved potential possibility of application of ironMOFs as an isoniazid carrier and as MRI contrast agent in inhalatory treatment of tuberculosis. Acknowledgments: Authors gratefully acknowledge the National Science Center Poland for providing financial support, grant no 2014/15/B/ST5/04498.

Keywords: imaging agents, metal-organic frameworks, theranostics, tuberculosis

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Authors: Akram Abi, Mohammad Hossein Sarrafzadeh

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Because of their several advantages over chemical surfactants, biosurfactants have given rise to a growing interest in the past decades. Advantages such as lower toxicity, higher biodegradability, higher selectivity and applicable at extreme temperature and pH which enables them to be used in a variety of applications such as: enhanced oil recovery, environmental and pharmaceutical applications, etc. Bacillus subtilis produces a cyclic lipopeptide, called surfactin, which is one of the most powerful biosurfactants with ability to decrease surface tension of water from 72 mN/m to 27 mN/m. In addition to its biosurfactant character, surfactin exhibits interesting biological activities such as: inhibition of fibrin clot formation, lyses of erythrocytes and several bacterial spheroplasts, antiviral, anti-tumoral and antibacterial properties. Surfactin is an antibiotic substance and has been shown recently to possess anti-HIV activity. However, application of biosurfactants is limited by their high production cost. The cost can be reduced by optimizing biosurfactant production using cheap feed stock. Utilization of inexpensive substrates and unconventional carbon sources like urban or agro-industrial wastes is a promising strategy to decrease the production cost of biosurfactants. With suitable engineering optimization and microbiological modifications, these wastes can be used as substrates for large-scale production of biosurfactants. As an effort to fulfill this purpose, in this work we have tried to utilize olive oil as second carbon source and also yeast extract as second nitrogen source to investigate the effect on both biomass and biosurfactant production improvement in Bacillus subtilis cultures. Since the turbidity of the culture was affected by presence of the oil, optical density was compromised and no longer could be used as an index of growth and biomass concentration. Therefore, cell Dry Weight measurements with applying necessary tactics for removing oil drops to prevent interference with biomass weight were carried out to monitor biomass concentration during the growth of the bacterium. The surface tension and critical micelle dilutions (CMD-1, CMD-2) were considered as an indirect measurement of biosurfactant production. Distinctive and promising results were obtained in the cultures containing olive oil compared to cultures without it: more than two fold increase in biomass production (from 2 g/l to 5 g/l) and considerable reduction in surface tension, down to 40 mN/m at surprisingly early hours of culture time (only 5hr after inoculation). This early onset of biosurfactant production in this culture is specially interesting when compared to the conventional cultures at which this reduction in surface tension is not obtained until 30 hour of culture time. Reducing the production time is a very prominent result to be considered for large scale process development. Furthermore, these results can be used to develop strategies for utilization of agro-industrial wastes (such as olive oil mill residue, molasses, etc.) as cheap and easily accessible feed stocks to decrease the high costs of biosurfactant production.

Keywords: agro-industrial waste, bacillus subtilis, biosurfactant, fermentation, second carbon and nitrogen source, surfactin

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Authors: Alec Chabwinja, Cannan Tawonezvi, Jerneja Vidmar, Constance Chingwaru, Walter Chingwaru

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Objective: To evaluate the potential of commercial fermented / probiotic products available in Zimbabwe or internationally, and strains of Lactobacillus plantarum (L. plantarum) as prophylaxis and therapy against diarrhoeal and sexually transmitted infections. Methods: The antimicrobial potential of cultures of lactobacilli enriched from 4 Zimbabwean commercial food/beverage products, namely Dairibord Lacto sour milk (DLSM), Probrand sour milk (PSM), Kefalos Vuka cheese (KVC) and Chibuku opaque beer (COB); three probiotic products obtainable in Europe and internationally; and four strains of L. plantarum obtained from Balkan traditional cheeses and Zimbabwean foods against clinical strains of Escherichia coli (E. coli) and non-clinical strains of Candida albicans and Rhodotorula spp. was assayed using the well diffusion method. Three commercial Agar diffusion assay and a competitive exclusion assay were carried out on Mueller-Hinton agar. Results: Crude cultures of putative lactobacillus strains obtained from Zimbabwean dairy products (Probrand sour milk, Kefalos Vuka vuka cheese and Chibuku opaque beer) exhibited significantly greater antimicrobial activities against clinical strains of E. coli than strains of L. plantarum isolated from Balkan cheeses (CLP1, CLP2 or CLP3) or crude microbial cultures from commercial paediatric probiotic products (BG, PJ and PL) of a culture of Lactobacillus rhamnosus LGG (p < 0.05). Furthermore, the following has high antifungal activities against the two yeasts: supernatant-free microbial pellet (SFMP) from an extract of M. azedarach leaves (27mm ± 2.5) > cell-free culture supernatants (CFCS) from Maaz Dairy sour milk and Mnandi sour milk (approximately 26mm ± 1.8) > CFCS and SFMP from Amansi hodzeko (25mm ± 1.5) > CFCS from Parinari curatellifolia fruit (24mm ± 1.5), SFMP from P. curatellifolia fruit (24mm ± 1.4) and SFMP from mahewu (20mm ± 1.5). These cultures also showed high tolerance to acidic conditions (~pH4). Conclusions: The putative lactobacilli from four commercial Zimbabwean dairy products (Probrand sour milk, Kefalos Vuka vuka cheese and Chibuku opaque beer), and three strains of L. plantarum from Balkan cheeses (CLP1, CLP2 or CLP3) exhibited high antibacterial activities, while Maaz Dairy sour-, Mnandi sour- and Amansi hodzeko milk products had high antifungal activities. Our selection of Zimbabwean probiotic products has potential for further development into probiotic products for use in the control diarrhea caused by pathogenic strains of E. coli or yeast infections. Studies to characterise the probiotic potential of the live cultures in the products are underway.

Keywords: lactic acid bacteria, Staphylococcus aureus, Streptococcus spp, yeast, inhibition, acid tolerance

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Authors: Hanan M. Hassan, Laila Abouzeid, Lamya H. Al-Wahaibi, George S. G. Shehatou, Ali A. El-Emam

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Cancer is a major public health problem and the second leading cause of death worldwide. In 2020, cancer diagnosis and treatment have been negatively affected by the coronavirus 2019 (COVID-19) pandemic. During the quarantine, because of the limited access to healthcare and avoiding exposure to COVID-19 as a contagious disease; patients of cancer suffered deferments in follow-up and treatment regimens leading to substantial worsening of disease, death, and increased healthcare costs. Thus, this study is designed to investigate the molecular mechanisms by which adamantne derivatives attenuate hepatocllular carcinoma experimentally and theoretically. There is a close association between increased resistance to anticancer drugs and defective apoptosis that considered a causative factor for oncogenesis. Cancer cells use different molecular pathways to inhibit apoptosis, BAX and Bcl-2 proteins have essential roles in the progression or inhibition of intrinsic apoptotic pathways triggered by mitochondrial dysfunction. Therefore, their balance ratio can promote the cellular apoptotic fate. In this study, the in vitro cytotoxic effects of seven synthetic adamantyl isothiorea derivatives were evaluated against five human tumor cell lines by MTT assay. Compounds 5 and 6 showed the best results, mostly against hepatocellular carcinoma (HCC). Hence, in vivo studies were performed in male Sprague-Dawley (SD) rats in which experimental hepatocellular carcinoma was induced with thioacetamide (TAA) (200 mg/kg, i.p., twice weekly) for 16 weeks. The most promising compounds, 5 and 6, were administered to treat liver cancer rats at a dose of 10 mg/kg/day for an additional two weeks, and the effects were compared with doxorubicin (DR), the anticancer drug. Hepatocellular carcinoma was evidenced by a dramatic increase in liver indices, oxidative stress markers, and immunohistochemical studies that were accompanied by a plethora of inflammatory mediators and alterations in the apoptotic cascade. Our results showed that treatment with adamantane derivatives 5 and 6 significantly suppressed fibrosis, inflammation, and other histopathological insults resulting in the diminished formation of hepatocyte tumorigenesis. Moreover, administration of the tested compounds resulted in amelioration of EGFR protein expression, upregulation of BAX, and lessening down of Bcl-2 levels that prove their role as apoptosis inducers. Also, the docking simulations performed for adamantane showed good fit and binding to the EGFR protein through hydrogen bond formation with conservative amino acids, which gives a shred of strong evidence for its hepatoprotective effect. In most analyses, the effects of compound 6 were more comparable to DR than compound 5. Our findings suggest that adamantane derivatives 5 and 6 are shown to have cytotoxic activity against HCC in vitro and in vivo, by more than one mechanism, possibly by inhibiting the TLR4-MyD88-NF-κB pathway and targeting EGFR signaling.

Keywords: adamantane, EGFR, HCC, apoptosis

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Authors: Tiantian Min, Chuanxiang Cheng, Jin Yue

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The growth and reproduction of microorganisms in food packaging can cause food decay and foodborne diseases, which pose a serious threat to the health of consumers and even cause serious economic losses. Active food packaging containing antibacterial bioactive compounds is a promising strategy for extending the shelf life of products and maintaining the food quality, as well as reducing the food waste. However, most active packaging can only act as slow-release effect for antimicrobials, which causes the release rate of antimicrobials not match the growth rate of microorganisms. Stimuli-responsive active packaging materials based on biopolymeric substrates and bioactive substances that respond to some biological and non-biological trigger factors provide more opportunities for fresh food preservation. The biological stimuli factors such as relative humidity, pH and enzyme existed in the exudate secreted by microorganisms have been expected to design food packaging materials. These stimuli-responsive materials achieved accurate release or delivery of bioactive substances at specific time and appropriate dose. Recently, metal-organic-frameworks (MOFs) nanoparticles become attractive carriers to enhance the efficiency of bioactive compounds or drugs. Cellulose nanofibrils have been widely applied for film substrates due to their biodegradability and biocompatibility. The abundant hydroxyl groups in cellulose can be oxidized to carboxyl groups by TEMPO, making it easier to anchoring MOFs and to be further modification. In this study, a pH and enzyme dual-responsive CAR@ZIF-8/TOCNF/PE film was fabricated by in-situ growth of ZIF-8 nanoparticles onto TEMPO-oxidized cellulose (TOCNF) film and further coated with pectin (PE) for stabilization and controlled release of carvacrol (CAR). The enzyme triggered release of CAR was achieved owing to the degradation of pectin by pectinase secreted by microorganisms. Similarly, the pH-responsive release of CAR was attributed to the unique skeleton degradation of ZIF-8, further accelerating the release of CAR from the topological structure of ZIF-8. The composite film performed excellent crystallinity and adsorb ability confirmed by X-ray diffraction and BET analysis, and the inhibition efficiency against Escherichia coli, Staphylococcus aureus and Aspergillus niger reached more than 99%. The composite film was capable of releasing CAR when exposure to dose-dependent enzyme (0.1, 0.2, and 0.3 mg/mL) and acidic condition (pH = 5). When inoculated 10 μL of Aspergillus niger spore suspension on the equatorial position of mango and raspberries, this composite film acted as packaging pads effectively inhibited the mycelial growth and prolonged the shelf life of mango and raspberries to 7 days. Such MOF-TOCNF based film provided a targeted, controlled and sustained release of bioactive compounds for long-term antibacterial activity and preservation effect, which can also avoid the cross-contamination of fruits.

Keywords: active food packaging, controlled release, fruit preservation, in-situ growth, stimuli-responsive

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Authors: Jin Yue

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Fresh produces especially fruits, vegetables, meats and aquatic products have limited shelf life and are highly susceptible to deterioration. Essential oils (EOs) extracted from plants have excellent antioxidant and broad-spectrum antibacterial activities, and they can play as natural food preservatives. But EOs are volatile, water insoluble, pungent, and easily decomposing under light and heat. Many approaches have been developed to improve the solubility and stability of EOs such as polymeric film, coating, nanoparticles, nano-emulsions and nanofibers. Construction of active packaging film which can incorporate EOs with high loading efficiency and controlled release of EOs has received great attention. It is still difficult to achieve accurate release of antibacterial compounds at specific target locations in active packaging. In this research, a relative humidity-responsive packaging material was designed, employing the electrospinning technique to fabricate a nanofibrous film loaded with a 4-terpineol/β-cyclodextrin inclusion complexes (4-TA/β-CD ICs). Functioning as an innovative food packaging material, the film demonstrated commendable attributes including pleasing appearance, thermal stability, mechanical properties, and effective barrier properties. The incorporation of inclusion complexes greatly enhanced the antioxidant and antibacterial activity of the film, particularly against Shewanella putrefaciens, with an inhibitory efficiency of up to 65%. Crucially, the film realized controlled release of 4-TA under 98% high relative humidity conditions by inducing the plasticization of polymers caused by water molecules, swelling of polymer chains, and destruction of hydrogen bonds within the cyclodextrin inclusion complex. This film with a long-term antimicrobial effect successfully extended the shelf life of Litopenaeus vannamei shrimp to 7 days at 4 °C. To further improve the loading efficiency and long-acting release of EOs, we synthesized the γ-cyclodextrin-metal organic frameworks (γ-CD-MOFs), and then efficiently anchored γ-CD-MOFs on chitosan-cellulose (CS-CEL) composite film by in situ growth method for controlled releasing of carvacrol (CAR). We found that the growth efficiency of γ-CD-MOFs was the highest when the concentration of CEL dispersion was 5%. The anchoring of γ-CD-MOFs on CS-CEL film significantly improved the surface area of CS-CEL film from 1.0294 m2/g to 43.3458 m2/g. The molecular docking and 1H NMR spectra indicated that γ-CD-MOF has better complexing and stabilizing ability for CAR molecules than γ-CD. In addition, the release of CAR reached 99.71±0.22% on the 10th day, while under 22% RH, the release pattern of CAR was a plateau with 14.71 ± 4.46%. The inhibition rate of this film against E. coli, S. aureus and B. cinerea was more than 99%, and extended the shelf life of strawberries to 7 days. By incorporating the merits of natural biopolymers and MOFs, this active packaging offers great potential as a substitute for traditional packaging materials.

Keywords: active packaging, antibacterial activity, controlled release, essential oils, food quality control

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Authors: Yuli Hou, Jianping Sun, Mengdan Gao, Hui Liu, Ling Qin, Ang Li, Dongfu Li, Yonghong Zhang, Yan Zhao

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Background and Aims: Interferon-induced transmembrane protein 3 (IFITM3) is a component of ISG (Interferon-Stimulated Gene) family. IFITM3 has been recognized as a key signal molecule regulating cell growth in some tumors. However, the function of IFITM3 rs12252-CC genotype in the hepatocellular carcinoma (HCC) remains unknown to author’s best knowledge. A cohort study was employed to clarify the relationship between IFITM3 rs12252-CC genotype and HCC progression, and cellular experiments were used to investigate the correlation of function of IFITM3 and the progress of HCC. Methods: 336 candidates were enrolled in study, including 156 with HBV related HCC and 180 with chronic Hepatitis B infections or liver cirrhosis. Polymerase chain reaction (PCR) was employed to determine the gene polymorphism of IFITM3. The functions of IFITM3 were detected in PLC/PRF/5 cell with different treated:LV-IFITM3 transfected with lentivirus to knockdown the expression of IFITM3 and LV-NC transfected with empty lentivirus as negative control. The IFITM3 expression, proliferation and migration were detected by Quantitative reverse transcription polymerase chain reaction (qRT-PCR), QuantiGene Plex 2.0 assay, western blotting, immunohistochemistry, Cell Counting Kit(CCK)-8 and wound healing respectively. Six samples (three infected with empty lentiviral as control; three infected with LV-IFITM3 vector lentiviral as experimental group ) of PLC/PRF/5 were sequenced at BGI (Beijing Genomics Institute, Shenzhen,China) using RNA-seq technology to identify the IFITM3-related signaling pathways and chose PI3K/AKT pathway as related signaling to verify. Results: The patients with HCC had a significantly higher proportion of IFITM3 rs12252-CC compared with the patients with chronic HBV infection or liver cirrhosis. The distribution of CC genotype in HCC patients with low differentiation was significantly higher than that in those with high differentiation. Patients with CC genotype found with bigger tumor size, higher percentage of vascular thrombosis, higher distribution of low differentiation and higher 5-year relapse rate than those with CT/TT genotypes. The expression of IFITM3 was higher in HCC tissues than adjacent normal tissues, and the level of IFITM3 was higher in HCC tissues with low differentiation and metastatic than high/medium differentiation and without metastatic. Higher RNA level of IFITM3 was found in CC genotype than TT genotype. In PLC/PRF/5 cell with knockdown, the ability of cell proliferation and migration was inhibited. Analysis RNA sequencing and verification of RT-PCR found out the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR) pathway was associated with knockdown IFITM3.With the inhibition of IFITM3, the expression of PI3K/AKT/mTOR signaling pathway was blocked and the expression of vimentin was decreased. Conclusions: IFITM3 rs12252-CC with the higher expression plays a vital role in the progress of HCC by regulating HCC cell proliferation and migration. These effects are associated with PI3K/AKT/mTOR signaling pathway.

Keywords: IFITM3, interferon-induced transmembrane protein 3, HCC, hepatocellular carcinoma, PI3K/ AKT/mTOR, phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin

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Authors: S. Mamoucha, N. Tsafantakis, Α. Ioannidis, S. Chatzipanagiotou, C. Nikolaou, L. Skaltsounis, N. Fokialakis, N. Christodoulakis

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Introduction: Plant secondary metabolites (SM) can be grouped into three chemically distinct groups: terpenes, phenolics, and nitrogen-containing compounds. For many years the adaptive significance of SM was unknown. They were thought to be functionless end-products. Currently it is accepted that many secondary metabolites (also known as natural products) have important ecological roles in plants. For instance, they serve as attractants (odor, color, taste) for pollinators and seed-dispersing animals. Moreover, they protect plants from herbivores, microbial pathogens and from environmental stress (high and low temperatures, drought, alkalinity, salinity, radiation etc). It is well known that both biotic and abiotic stress often increase the accumulation of SM. The local climatic conditions, seasonal changes, external factors such as light, temperature, humidity affect the biosynthesis and composition of secondary metabolites. A well known dioecious evergreen plant, Pistacia lentiscus L. (mastic tree), was selected in order to study the metabolic variations occur in response to the different climate conditions, due to the seasonal variation and its effect on the biosynthesis of bioactive compounds. Materials-methods: Young and mature leaves were collected in January and July 2014, dried and extracted by accelerated solvent extraction (Dionex ASE™ 350) using solvents of increased polarity (DCM, MeOH, and H2O). GC-MS and UHPLC-HRMS analysis were carried out in order to define the nature and the relative abundance of SM. The antibacterial activity was evaluated by using the Agar Disc Diffusion Assay against ATCC and clinical isolates strains: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Streptococcus mutans and Klebsiella pneumoniae. All tests were carried out in duplicate and the average radii of the inhibition zones were calculated for each extract. Results: According to the phytochemical profile obtained from each extract, the biosynthesis of SM varied both qualitatively and quantitatively under the two different types of seasonal stress. With exception of the biologically inactive nonpolar DCM extract of July, all extracts inhibited the growth of most of the investigated microorganisms. A clear positive correlation has been observed between the relative abundance of SM and the bioactivity of the DCM extracts of January and July. Observed changes during phytochemical analysis were mainly focused on the triterpenoid content. On the other hand, the bioactivity of the polar extracts (MeOH and H2O) of January and July resulted practically invariable against most of the microorganisms, besides the significant variation of the SM content due to the seasonal variation. Conclusion: Our results clearly confirmed the hypothesis of abiotic stress as an important regulating factor that significantly affects the biosynthesis of secondary metabolites and thus the presence of bioactive compounds. Acknowledgment: This work was supported by IKY - State Scholarship Foundation, Athens, Greece.

Keywords: antibacterial screening, phytochemical profile, Pistacia lentiscus, abiotic stress

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Authors: S. F. Afzali, W. L. Wong

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The epizootic ulcerative syndrome (EUS) causes by the oomycete fungus, Aphanomyces invadans; known to be one of the infectious fish diseases for farmed and wild fishes in fresh and brackish-water from the Asia-pacific region, America and Africa. Although, EUS had been documented by the Office International des Epizooties (OIE) since 1995, hitherto, there is neither standard chemical agents that can be used for successful treatment of this destructive infection in the time of outbreak; nor available vaccine for prevention. Plant-based remedies in controlling fish diseases are gaining much attention recently as an alternative to chemical treatments, which possess negative effects to the environment and human. In present study, Sonneratia alba, a mangrove plant belongs to the Sonneratiaceae family, was screened in vitro and in vivo for its antifungal activity against A. invadans mycelium growth and its effects on fish innate immune system and disease resistant. The in vitro tests was performed using the disc diffusion methods with measurements of minimum inhibitory concentration (MIC) and inhibition zone. For in vivo study, the S. alba extract supplemented diets were administrated at 0.0, 1.0%, 3.0%, and 5.0% on healthy goldfish, Carassius auratus, which challenged with A. invadans zoospores (100 spores/ml). To compare the significant differences in the hematological and immunological parameters obtained from the experiments, the data were analysed using the SPSS. The methanol extract of S. alba effectively inhibited the mycelial growth of A. invadans at a minimum concentration of 1000 ppm for agar and filter paper diffusion experiments. In the agar diffusion test, 500 ppm of the extract inhibited the fungus mycelial growth up to 96 hours after exposure. The mycelial growth from the edge of the pre-inoculated A. invadans agar discs treated with S. alba extracts at concentrations of 100, 500 and 1000 ppm were 15, 8 and 0 mm respectively. The results of the filter paper disc test showed that the S. alba extract at its minimal inhibitory concentration (1000 ppm) has similar qualitative inhibitory effect as malachite green at 1 ppm and formalin at 250 ppm. According to the in vivo tests findings, in the infected fish fed with 3.0% and 5.0% supplementation diet, the numbers of white blood cell and myeloperoxidase activity significantly increased after the second week of treatment. Whilst the numbers of red blood cell significantly decreased in the infected fish fed with 0.0 and 1.0% supplementation diet. After the third week of feeding, significant increases in the total protein, albumin level, lysozyme activity were recorded in the infected fish fed with 3.0% and 5.0% supplementation diet. Also, the enriched diets increased the survival rate as compared to the untreated group that suffered from 90% mortality. The present study indicated that S. alba extract may inhibit the mycelial growth of A. invadans effectively, suggesting an alternative to other chemotherapeutic agents, which brought much environmental and health concerns to the public, for EUS treatment.

Keywords: fungal pathogen, goldfish, organic extract, treatment

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Authors: Hsiang-Yu Yuan, Marc Baguelin, Kin O. Kwok, Nimalan Arinaminpathy, Edwin Leeuwen, Steven Riley

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Traditional syndromic surveillance for influenza has substantial public health value in characterizing epidemics. Because the relationship between syndromic incidence and the true infection events can vary from one population to another and from one year to another, recent studies rely on combining serological test results with syndromic data from traditional surveillance into epidemic models to make inference on epidemiological processes of influenza. However, despite the widespread availability of serological data, epidemic models have thus far not explicitly represented antibody titre levels and their correspondence with immunity. Most studies use dichotomized data with a threshold (Typically, a titre of 1:40 was used) to define individuals as likely recently infected and likely immune and further estimate the cumulative incidence. Underestimation of Influenza attack rate could be resulted from the dichotomized data. In order to improve the use of serosurveillance data, here, a refinement of the concept of the stratified immunity within an epidemic model for influenza transmission was proposed, such that all individual antibody titre levels were enumerated explicitly and mapped onto a variable scale of susceptibility in different age groups. Haemagglutination inhibition titres from 523 individuals and 465 individuals during pre- and post-pandemic phase of the 2009 pandemic in Hong Kong were collected. The model was fitted to serological data in age-structured population using Bayesian framework and was able to reproduce key features of the epidemics. The effects of age-specific antibody boosting and protection were explored in greater detail. RB was defined to be the effective reproductive number in the presence of stratified immunity and its temporal dynamics was compared to the traditional epidemic model using use dichotomized seropositivity data. Deviance Information Criterion (DIC) was used to measure the fitness of the model to serological data with different mechanisms of the serological response. The results demonstrated that the differential antibody response with age was present (ΔDIC = -7.0). The age-specific mixing patterns with children specific transmissibility, rather than pre-existing immunity, was most likely to explain the high serological attack rates in children and low serological attack rates in elderly (ΔDIC = -38.5). Our results suggested that the disease dynamics and herd immunity of a population could be described more accurately for influenza when the distribution of immunity was explicitly represented, rather than relying only on the dichotomous states 'susceptible' and 'immune' defined by the threshold titre (1:40) (ΔDIC = -11.5). During the outbreak, RB declined slowly from 1.22[1.16-1.28] in the first four months after 1st May. RB dropped rapidly below to 1 during September and October, which was consistent to the observed epidemic peak time in the late September. One of the most important challenges for infectious disease control is to monitor disease transmissibility in real time with statistics such as the effective reproduction number. Once early estimates of antibody boosting and protection are obtained, disease dynamics can be reconstructed, which are valuable for infectious disease prevention and control.

Keywords: effective reproductive number, epidemic model, influenza epidemic dynamics, stratified immunity

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Authors: Andrey A. Tyuftin, David Clarke, Malco C. Cruz-Romero, Declan Bolton, Seamus Fanning, Shashi K. Pankaj, Carmen Bueno-Ferrer, Patrick J. Cullen, Joe P. Kerry

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Prolonging of shelf-life is essential in order to address issues such as; supplier demands across continents, economical profit, customer satisfaction, and reduction of food wastage. Smart packaging solutions presented in the form of naturally occurred antimicrobially-active packaging may be a solution to these and other issues. Gelatin film forming solution with adding of natural sourced antimicrobials is a promising tool for the active smart packaging. The objective of this study was to coat conventional plastic hydrophobic packaging material with hydrophilic antimicrobial active beef gelatin coating and conduct shelf life trials on beef sub-primal cuts. Minimal inhibition concentration (MIC) of Caprylic acid sodium salt (SO) and commercially available Auranta FV (AFV) (bitter oranges extract with mixture of nutritive organic acids) were found of 1 and 1.5 % respectively against bacterial strains Bacillus cereus, Pseudomonas fluorescens, Escherichia coli, Staphylococcus aureus and aerobic and anaerobic beef microflora. Therefore SO or AFV were incorporated in beef gelatin film forming solution in concentration of two times of MIC which was coated on a conventional plastic LDPE/PA film on the inner cold plasma treated polyethylene surface. Beef samples were vacuum packed in this material and stored under chilling conditions, sampled at weekly intervals during 42 days shelf life study. No significant differences (p < 0.05) in the cook loss was observed among the different treatments compared to control samples until the day 29. Only for AFV coated beef sample it was 3% higher (37.3%) than the control (34.4 %) on the day 36. It was found antimicrobial films did not protect beef against discoloration. SO containing packages significantly (p < 0.05) reduced Total viable bacterial counts (TVC) compared to the control and AFV samples until the day 35. No significant reduction in TVC was observed between SO and AFV films on the day 42 but a significant difference was observed compared to control samples with a 1.40 log of bacteria reduction on the day 42. AFV films significantly (p < 0.05) reduced TVC compared to control samples from the day 14 until the day 42. Control samples reached the set value of 7 log CFU/g on day 27 of testing, AFV films did not reach this set limit until day 35 and SO films until day 42 of testing. The antimicrobial AFV and SO coated films significantly prolonged the shelf-life of beef steaks by 33 or 55% (on 7 and 14 days respectively) compared to control film samples. It is concluded antimicrobial coated films were successfully developed by coating the inner polyethylene layer of conventional LDPE/PA laminated films after plasma surface treatment. The results indicated that the use of antimicrobial active packaging coated with SO or AFV increased significantly (p < 0.05) the shelf life of the beef sub-primal. Overall, AFV or SO containing gelatin coatings have the potential of being used as effective antimicrobials for active packaging applications for muscle-based food products.

Keywords: active packaging, antimicrobials, edible coatings, food packaging, gelatin films, meat science

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Authors: Vladimir Berezin, Aizhan Turmagambetova, Andrey Bogoyavlenskiy, Pavel Alexyuk, Madina Alexyuk, Irina Zaitceva, Nadezhda Sokolova

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Introduction: Influenza viruses could infect approximately 5% to 10% of the global human population annually, resulting in serious social and economic damage. Vaccination and etiotropic antiviral drugs are used for the prevention and treatment of influenza. Vaccination is important; however, antiviral drugs represent the second line of defense against new emerging influenza virus strains for which vaccines may be unsuccessful. However, the significant drawback of commercial synthetic anti-flu drugs is the appearance of drug-resistant influenza virus strains. Therefore, the search and development of new anti-flu drugs efficient against drug-resistant strains is an important medical problem for today. The aim of this work was a study of four phenolic acids of plant origin (Gallic, Syringic, Vanillic, and Protocatechuic acids) as a possible tool for treatment against influenza virus. Methods: Phenolic acids; gallic, syringic, vanillic, and protocatechuic have been prepared by extraction from plant tissues and purified using high-performance liquid chromatography fractionation. Avian influenza virus, strain A/Tern/South Africa/1/1961 (H5N3) and human epidemic influenza virus, strain A/Almaty/8/98 (H3N2) resistant to commercial anti-flu drugs (Rimantadine, Oseltamivir) were used for testing antiviral activity. Viruses were grown in the allantoic cavity of 10 days old chicken embryos. The chemotherapeutic index (CTI), determined as the ratio of an average toxic concentration of the tested compound (TC₅₀) to the average effective virus-inhibition concentration (EC₅₀), has been used as a criteria of specific antiviral action. Results: The results of study have shown that the structure of phenolic acids significantly affected their ability to suppress the reproduction of tested influenza virus strains. The highest antiviral activity among tested phenolic acids was detected for gallic acid, which contains three hydroxyl groups in the molecule at C3, C4, and C5 positions. Antiviral activity of gallic acid against A/H5N3 and A/H3N2 influenza virus strains was higher than antiviral activity of Oseltamivir and Rimantadine. gallic acid inhibited almost 100% of the infection activity of both tested viruses. Protocatechuic acid, which possesses 2 hydroxyl groups (C3 and C4) have shown weaker antiviral activity in comparison with gallic acid and inhibited less than 10% of virus infection activity. Syringic acid, which contains two hydroxyl groups (C3 and C5), was able to suppress up to 12% of infection activity. Substitution of two hydroxyl groups by methoxy groups resulted in the complete loss of antiviral activity. Vanillic acid, which is different from protocatechuic acid by replacing of C3 hydroxyl group to methoxy group, was able to suppress about 30% of infection activity of tested influenza viruses. Conclusion: For pronounced antiviral activity, the molecular of phenolic acid must have at least two hydroxyl groups. Replacement of hydroxyl groups to methoxy group leads to a reduction of antiviral properties. Gallic acid demonstrated high antiviral activity against influenza viruses, including Rimantadine and Oseltamivir resistant strains, and could be used as a potential candidate for the development of antiviral drug against influenza virus.

Keywords: antiviral activity, influenza virus, drug resistance, phenolic acids

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