Search results for: cellular uptake

Authors: Siddhant Sethi, Yasuharu Takashima, Shigetaka Nakamura, Kenzo Fujimoto

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Site-directed mutagenesis is a renowned technique to introduce specific mutations in the genome. To achieve site-directed mutagenesis, many chemical and enzymatic approaches have been reported in the past like disulphite induced genome editing, CRISPR-Cas9, TALEN etc. The chemical methods are invasive whereas the enzymatic approaches are time-consuming and expensive. Most of these techniques are unusable in the cellular application due to their toxicity and other limitations. Photo-chemical cytosine deamination, introduced in 2010, is one of the major technique for enzyme-free single-point mutation of cytosine to uracil in DNA and RNA, wherein, 3-cyanovinylcarbazole nucleoside (CNVK) containing oligodeoxyribonucleotide (ODN) having CNVK at -1 position to that of target cytosine is reversibly crosslinked to target DNA strand using 366 nm and then incubated at 90ºC to accommodate deamination. This technique is superior to enzymatic methods of site-directed mutagenesis but has a disadvantage that it requires the use of high temperature for the deamination step which restricts its applicability in the in vivo applications. This study has been focused on improving the technique by reducing the temperature required for deamination. Firstly, the photo-cross-linker, CNVK has been modified by replacing cyano group attached to vinyl group with methyl ester (OMeVK), amide (NH2VK), and carboxylic acid (OHVK) to observe the acceleration in the deamination of target cytosine cross-linked to vinylcarbazole derivative. Among the derivatives, OHVK has shown 2 times acceleration in deamination reaction as compared to CNVK, while the other two derivatives have shown deceleration towards deamination reaction. The trend of rate of deamination reaction follows the same order as that of hydrophilicity of the vinylcarbazole derivatives. OHVK being most hydrophilic has shown highest acceleration while OMeVK is least hydrophilic has proven to be least active for deamination. Secondly, in the related study, the counter-base of the target cytosine, guanine has been replaced by inosine, 2-aminopurine, nebularine, and 5-nitroindole having distinct hydrogen bonding patterns with target cytosine. Among the ODNs with these counter bases, ODN with inosine has shown 12 fold acceleration towards deamination of cytosine cross-linked to CNVK at physiological conditions as compared to guanosine. Whereas, when 2-aminopurine, nebularine, and 5-nitroindole were used, no deamination reaction took place. It can be concluded that inosine has potential to be used as the counter base of target cytosine for the CNVK mediated photo-cross-linking induced deamination of cytosine. The increase in rate of deamination reaction has been attributed to pattern and number of hydrogen bonding between the cytosine and counter base. One of the important factor is presence of hydrogen bond between exo-cyclic amino group of cytosine and the counter base. These results will be useful for development of more efficient technique for site-directed mutagenesis for C → U transformations in the DNA/RNA which might be used in the living system for treatment of various genetic disorders and genome engineering for making designer and non-native proteins.

Keywords: C to U transformation, DNA editing, genome engineering, ultra-fast photo-cross-linking

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Authors: Farwa Rizvi, Joanne Williams, Humaira Maheen, Elizabeth Hoban

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There is an increase in risky sexual behavior and unsafe sex in adolescent girls and young women aged 15 to 24 years in Cambodia, which negatively affects their reproductive health by increasing the risk of contracting sexually transmitted infections and unintended pregnancies. Risky sexual behavior includes ‘having sex at an early age, having multiple sexual partners, having sex while under the influence of alcohol or drugs, and unprotected sexual behaviors’. A systematic review of quantitative research conducted in Cambodia was undertaken, using the theoretical framework of the Social Ecological Model to identify the personal, social and cultural factors associated with risky sexual behavior and unsafe sex in young Cambodian women. PRISMA guidelines were used to search databases including Medline Complete, PsycINFO, CINAHL Complete, Academic Search Complete, Global Health, and Social Work Abstracts. Additional searches were conducted in Science Direct, Google Scholar and in the grey literature sources. A risk-of-bias tool developed explicitly for the systematic review of cross-sectional studies was used. Summary item on the overall risk of study bias after the inter-rater response showed that the risk-of-bias was high in two studies, moderate in one study and low in one study. The search strategy included a combination of subject terms and free text terms. The medical subject headings (MeSH) terms included were; contracept* or ‘birth control’ or ‘family planning’ or pregnan* or ‘safe sex’ or ‘protected intercourse’ or ‘unprotected intercourse’ or ‘protected sex’ or ‘unprotected sex’ or ‘risky sexual behaviour*’ or ‘abort*’ or ‘planned parenthood’ or ‘unplanned pregnancy’ AND ( barrier* or obstacle* or challenge* or knowledge or attitude* or factor* or determinant* or choic* or uptake or discontinu* or acceptance or satisfaction or ‘needs assessment’ or ‘non-use’ or ‘unmet need’ or ‘decision making’ ) AND Cambodia*. Initially, 300 studies were identified by using key words and finally, four quantitative studies were selected based on the inclusion criteria. The four studies were published between 2010 and 2016. The study participants ranged in age from 10-24 years, single or married, with 3 to 10 completed years of education. The mean age at sexual debut was reported to be 18 years. Using the perspective of the Social Ecological Model, risky sexual behavior was associated with individual-level factors including young age at sexual debut, low education, unsafe sex under the influence of alcohol and substance abuse, multiple sexual partners or transactional sex. Family level factors included living away from parents, orphan status and low levels of family support. Peer and partner level factors included peer delinquency and lack of condom use. Low socioeconomic status at the society level was also associated with risky sexual behaviour. There is scant research on sexual and reproductive health of adolescent girls and young women in Cambodia. Individual, family and social factors were significantly associated with risky sexual behaviour. More research is required to inform potential preventive strategies and policies that address young women’s sexual and reproductive health.

Keywords: adolescents, high-risk sex, sexual activity, unplanned pregnancies

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Authors: Iryna Atamaniuk, Hannah Boysen, Nils Wieczorek, Natalia Politaeva, Iuliia Bazarnova, Kerstin Kuchta

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Over the last decade due to climate change and a strategy of natural resources preservation, the interest for the aquatic biomass has dramatically increased. Along with mitigation of the environmental pressure and connection of waste streams (including CO₂ and heat emissions), microalgae bioeconomy can supply food, feed, as well as the pharmaceutical and power industry with number of value-added products. Furthermore, in comparison to conventional biomass, microalgae can be cultivated in wide range of conditions without compromising food and feed production, thus addressing issues associated with negative social and the environmental impacts. This paper presents the state-of-the art technology for microalgae bioeconomy from cultivation process to production of valuable components and by-streams. Microalgae Chlorella sorokiniana were cultivated in the pilot-scale innovation concept in Hamburg (Germany) using different systems such as race way pond (5000 L) and flat panel reactors (8 x 180 L). In order to achieve the optimum growth conditions along with suitable cellular composition for the further extraction of the value-added components, process parameters such as light intensity, temperature and pH are continuously being monitored. On the other hand, metabolic needs in nutrients were provided by addition of micro- and macro-nutrients into a medium to ensure autotrophic growth conditions of microalgae. The cultivation was further followed by downstream process and extraction of lipids, proteins and saccharides. Lipids extraction is conducted in repeated-batch semi-automatic mode using hot extraction method according to Randall. As solvents hexane and ethanol are used at different ratio of 9:1 and 1:9, respectively. Depending on cell disruption method along with solvents ratio, the total lipids content showed significant variations between 8.1% and 13.9 %. The highest percentage of extracted biomass was reached with a sample pretreated with microwave digestion using 90% of hexane and 10% of ethanol as solvents. Proteins content in microalgae was determined by two different methods, namely: Total Kejadahl Nitrogen (TKN), which further was converted to protein content, as well as Bradford method using Brilliant Blue G-250 dye. Obtained results, showed a good correlation between both methods with protein content being in the range of 39.8–47.1%. Characterization of neutral and acid saccharides from microalgae was conducted by phenol-sulfuric acid method at two wavelengths of 480 nm and 490 nm. The average concentration of neutral and acid saccharides under the optimal cultivation conditions was 19.5% and 26.1%, respectively. Subsequently, biomass residues are used as substrate for anaerobic digestion on the laboratory-scale. The methane concentration, which was measured on the daily bases, showed some variations for different samples after extraction steps but was in the range between 48% and 55%. CO₂ which is formed during the fermentation process and after the combustion in the Combined Heat and Power unit can potentially be used within the cultivation process as a carbon source for the photoautotrophic synthesis of biomass.

Keywords: bioeconomy, lipids, microalgae, proteins, saccharides

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Authors: Jessica M. Black

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Although much of natural and social science research aims to enhance human flourishing and address social problems, the training within the two fields is significantly different across theory, methodology, and implementation of results. Social scientists are trained in social, psychological, and to the extent that it is relevant to their discipline, spiritual development, theory, and accompanying methodologies. They tend not to receive training or learn about accompanying methodology related to interrogating human development and social problems from a biological perspective. On the other hand, those in the natural sciences, and for the purpose of this work, human biological sciences specifically – biology, neuroscience, genetics, epigenetics, and physiology – are often trained first to consider cellular development and related methodologies, and may not have opportunity to receive formal training in many of the foundational principles that guide human development, such as systems theory or person-in-environment framework, methodology related to tapping both proximal and distal psycho-social-spiritual influences on human development, and foundational principles of equity, justice and inclusion in research design. There is a need for disciplines heretofore siloed to know one another, to receive streamlined, easy to access training in theory and methods from one another and to learn how to build interdisciplinary teams that can speak and act upon a shared research language. Team science is more essential than ever, as are transdisciplinary approaches to training and research design. This study explores the use of a methodological toolbox that natural and social scientists can use by employing a decision-making tree regarding project aims, costs, and participants, among other important study variables. The decision tree begins with a decision about whether the researcher wants to learn more about social sciences approaches or biological approaches to study design. The toolbox and platform are flexible, such that users could also choose among modules, for instance, reviewing epigenetics or community-based participatory research even if those are aspects already a part of their home field. To start, both natural and social scientists would receive training on systems science, team science, transdisciplinary approaches, and translational science. Next, social scientists would receive training on grounding biological theory and the following methodological approaches and tools: physiology, (epi)genetics, non-invasive neuroimaging, invasive neuroimaging, endocrinology, and the gut-brain connection. Natural scientists would receive training on grounding social science theory, and measurement including variables, assessment and surveys on human development as related to the developing person (e.g., temperament and identity), microsystems (e.g., systems that directly interact with the person such as family and peers), mesosystems (e.g., systems that interact with one another but do not directly interact with the individual person, such as parent and teacher relationships with one another), exosystems (e.g., spaces and settings that may come back to affect the individual person, such as a parent’s work environment, but within which the individual does not directly interact, macrosystems (e.g., wider culture and policy), and the chronosystem (e.g., historical time, such as the generational impact of trauma). Participants will be able to engage with the toolbox and one another to foster increased transdisciplinary work

Keywords: methodology, natural science, social science, transdisciplinary

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Authors: Anna Höfer, Johannes Herrmann, Patrick Meybohm, Christopher Lotz

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Mitochondria are pivotal for energy supply and regulation of cellular functions. Deficiencies of mitochondrial metabolism have been implicated in diverse stressful conditions including infections. Platelets are key mediators for thrombo-inflammation during development and resolution of acute respiratory distress syndrome (ARDS). Previous data point to an exhausted platelet phenotype in critically-ill patients with coronavirus 19 disease (COVID-19) impacting the course of disease. The objective of this work was to characterize platelet mitochondrial metabolism in patients suffering from COVID-19 ARDSA longitudinal analysis of platelet mitochondrial metabolism in 24 patients with COVID-19 induced ARDS compared to 35 healthy controls (ctrl) was performed. Blood samples were analyzed at two time points (t1=day 1; t2=day 5-7 after study inclusion). The activity of mitochondrial citrate synthase was photometrically measured. The impact of oxidative stress on mitochondrial permeability was assessed by a photometric calcium-induced swelling assay and the activity of superoxide dismutase (SOD) by a SOD assay kit. The amount of protein carbonylation and the activity of mitochondria complexes I-IV were photometrically determined. Levels of interleukins (IL)-1α, IL-1β and tumor necrosis factor (TNF-) α were measured by a Multiplex assay kit. Median age was 54 years, 63 % were male and BMI was 29.8 kg/m2. SOFA (12; IQR: 10-15) and APACHE II (27; IQR: 24-30) indicated critical illness. Median Murray Score was 3.4 (IQR: 2.8-3.4), 21/24 (88%) required mechanical ventilation and V-V ECMO support in 14/24 (58%). Platelet counts in ARDS did not change during ICU stay (t1: 212 vs. t2: 209 x109/L). However, mean platelet volume (MPV) significantly increased (t1: 10.6 vs. t2: 11.9 fL; p<0.0001). Citrate synthase activity showed no significant differences between ctrl and ARDS patients. Calcium induced swelling was more pronounced in patients at t1 compared to t2 and to ctrl (50µM; t1: 0.006 vs. ctrl: 0.016 ΔOD; p=0.001). The amount of protein carbonylation as marker for irreversible proteomic modification constantly increased during ICU stay and compared to ctrl., without reaching significance. In parallel, superoxid dismutase activity gradually declined during ICU treatment vs. ctrl (t2: - 29 vs. ctrl.: - 17 %; p=0.0464). Complex I analysis revealed significantly stronger activity in ARDS vs. ctrl. (t1: 0.633 vs. ctrl.: 0.415 ΔOD; p=0.0086). There were no significant differences in complex II, III or IV activity in platelets from ARDS patients compared to ctrl. IL-18 constantly increased during the observation period without reaching significance. IL-1α and TNF-α did not differ from ctrl. However, IL-1β levels were significantly elevated in ARDS (t1: 16.8; t2: 16.6 vs. ctrl.: 12.4 pg/mL; p1=0.0335, p2=0.0032). This study reveals new insights in platelet mitochondrial metabolism during COVID-19 caused ARDS. it data point towards enhanced platelet activity with a pronounced turnover rate. We found increased activity of mitochondria complex I and evidence for enhanced oxidative stress. In parallel, protective mechanisms against oxidative stress were narrowed with elevated levels of IL-1β likely causing a pro-apoptotic environment. These mechanisms may contribute to platelet exhaustion in ARDS.

Keywords: acute respiratory distress syndrome (ARDS), coronavirus 19 disease (COVID-19), oxidative stress, platelet mitochondrial metabolism

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Authors: Marcin Dębowski, Marcin Zieliński, Mirosław Krzemieniewski

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As methane fermentation is a complex series of successive biochemical transformations, its subsequent stages are determined, to a various extent, by physical and chemical factors. A specific state of equilibrium is being settled in the functioning fermentation system between environmental conditions and the rate of biochemical reactions and products of successive transformations. In the case of physical factors that influence the effectiveness of methane fermentation transformations, the key significance is ascribed to temperature and intensity of biomass agitation. Among the chemical factors, significant are pH value, type, and availability of the culture medium (to put it simply: the C/N ratio) as well as the presence of toxic substances. One of the important elements which influence the effectiveness of methane fermentation is the pre-treatment of organic substrates and the mode in which the organic matter is made available to anaerobes. Out of all known and described methods for organic substrate pre-treatment before methane fermentation process, the ultrasound disintegration is one of the most interesting technologies. Investigations undertaken on the ultrasound field and the use of installations operating on the existing systems result principally from very wide and universal technological possibilities offered by the sonication process. This physical factor may induce deep physicochemical changes in ultrasonicated substrates that are highly beneficial from the viewpoint of methane fermentation processes. In this case, special role is ascribed to disintegration of biomass that is further subjected to methane fermentation. Once cell walls are damaged, cytoplasm and cellular enzymes are released. The released substances – either in dissolved or colloidal form – are immediately available to anaerobic bacteria for biodegradation. To ensure the maximal release of organic matter from dead biomass cells, disintegration processes are aimed to achieve particle size below 50 μm. It has been demonstrated in many research works and in systems operating in the technical scale that immediately after substrate supersonication the content of organic matter (characterized by COD, BOD5 and TOC indices) was increasing in the dissolved phase of sedimentation water. This phenomenon points to the immediate sonolysis of solid substances contained in the biomass and to the release of cell material, and consequently to the intensification of the hydrolytic phase of fermentation. It results in a significant reduction of fermentation time and increased effectiveness of production of gaseous metabolites of anaerobic bacteria. Because disintegration of Virginia fanpetals biomass via ultrasounds applied in order to intensify its conversion is a novel technique, it is often underestimated by exploiters of agri-biogas works. It has, however, many advantages that have a direct impact on its technological and economical superiority over thus far applied methods of biomass conversion. As for now, ultrasound disintegrators for biomass conversion are not produced on the mass-scale, but by specialized groups in scientific or R&D centers. Therefore, their quality and effectiveness are to a large extent determined by their manufacturers’ knowledge and skills in the fields of acoustics and electronic engineering.

Keywords: ultrasound disintegration, biomass, methane fermentation, biogas, Virginia fanpetals

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Authors: G. M. Vlasceanu, H. Iovu, E. Vasile, M. Ionita

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Herein, the synthesis and characterization of chitosan-gelatin highly porous scaffold reinforced with graphene oxide, and hydroxyapatite (HAp), crosslinked with genipin was targeted. In tissue engineering, chitosan and gelatin are two of the most robust biopolymers with wide applicability due to intrinsic biocompatibility, biodegradability, low antigenicity properties, affordability, and ease of processing. HAp, per its exceptional activity in tuning cell-matrix interactions, is acknowledged for its capability of sustaining cellular proliferation by promoting bone-like native micro-media for cell adjustment. Genipin is regarded as a top class cross-linker, while graphene oxide (GO) is viewed as one of the most performant and versatile fillers. The composites with natural bone HAp/biopolymer ratio were obtained by cascading sonochemical treatments, followed by uncomplicated casting methods and by freeze-drying. Their structure was characterized by Fourier Transform Infrared Spectroscopy and X-ray Diffraction, while overall morphology was investigated by Scanning Electron Microscopy (SEM) and micro-Computer Tomography (µ-CT). Ensuing that, in vitro enzyme degradation was performed to detect the most promising compositions for the development of in vivo assays. Suitable GO dispersion was ascertained within the biopolymer mix as nanolayers specific signals lack in both FTIR and XRD spectra, and the specific spectral features of the polymers persisted with GO load enhancement. Overall, correlations between the GO induced material structuration, crystallinity variations, and chemical interaction of the compounds can be correlated with the physical features and bioactivity of each composite formulation. Moreover, the HAp distribution within follows an auspicious density gradient tuned for hybrid osseous/cartilage matter architectures, which were mirrored in the mice model tests. Hence, the synthesis route of a natural polymer blend/hydroxyapatite-graphene oxide composite material is anticipated to emerge as influential formulation in bone tissue engineering. Acknowledgement: This work was supported by the project 'Work-based learning systems using entrepreneurship grants for doctoral and post-doctoral students' (Sisteme de invatare bazate pe munca prin burse antreprenor pentru doctoranzi si postdoctoranzi) - SIMBA, SMIS code 124705 and by a grant of the National Authority for Scientific Research and Innovation, Operational Program Competitiveness Axis 1 - Section E, Program co-financed from European Regional Development Fund 'Investments for your future' under the project number 154/25.11.2016, P_37_221/2015. The nano-CT experiments were possible due to European Regional Development Fund through Competitiveness Operational Program 2014-2020, Priority axis 1, ID P_36_611, MySMIS code 107066, INOVABIOMED.

Keywords: biopolymer blend, ectopic osteoinduction, graphene oxide composite, hydroxyapatite

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Authors: Diego R. Boiarski, Camila M. Toigo, Thais M. Sobjak, Andrey F. P. Santos, Silvia Romao, Ana T. B. Guimaraes

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Anthropogenic activities promote changes in the community’s structures and decrease the species abundance of amphibians. Biological communities of fluvial systems are assemblies of organisms that have adapted to regional conditions, including the physical environment and food resources, and are further refined through interactions with other species. The aim of this study was to assess neurotoxic alterations and in the antioxidant system on tadpoles of Lithobates catesbeianus exposed to waters from Cascavel River, in the south of Brazil. A total of 420 L of water was collected from the Cascavel River, 140 L from each of the three different locations: Site 1 – headwater; Site 2 – stretch of the stream that runs through an urbanized area; Site 3 – a stretch from the rural area. Twelve tadpoles were acclimated in each aquarium (100 L of water) for seven days. The water from each aquarium was replaced with the ones sampled from the river, except the one from the control aquarium. After seven days, a portion of the liver was removed and conditioned for ChE, SOD, CAT and LPO analysis; other part of the tissue was conditioned for histological analysis. The statistical analysis performed was one-way ANOVA, followed by post-hoc Tukey-HSD test, and the multivariate principal components analysis. It was not observed any neurotoxic effect, but a slight increase in SOD activity and elevation of CAT activity in both urban and rural environment. A decrease in LPO reaction was detected, mainly among the tadpoles exposed to the waters from the rural area. The results of the present study demonstrate the alteration of the antioxidant system, as well as liver histopathologies in tadpoles exposed mainly to waters collected in urban and rural environments. These alterations may cause the reduction in the velocity of the metamorphosis process from the tadpoles. Further, were observed histological alterations, highlighting necrotic areas mainly among the animals exposed to urban waters. Those damages can lead to metabolic dysfunction, interfering with survival capacity, diminishing not only individual fitness but for the whole population. In the interpretation synthesis of all biomarkers, the cellular damage gradient is perceptible, characterized by the variables related to the antioxidant system, due to the flow direction of the stream. This result is indicative that along the course of the creek occurs dumping of organic material, which promoted an acute response upon tadpoles of L. catesbeianus. and it was also observed the difference in tissue damage between the experimental groups and the control group, the latter presenting histological alterations, but to a lesser degree than the animals exposed to the waters of the Cascavel river. These damages, caused by reactive oxygen species possibly resulting from the contamination by organic compounds, can lead the animals to a series of metabolic dysfunctions, interfering with its metamorphosis capacity. Interruption of metamorphosis may affect survival, which may impair its growth, development and reproduction, diminishing not only the fitness of each individual but in a long-term, to the entire population.

Keywords: American bullfrog, histopathology, oxidative stress, urban creeks pollution

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Authors: Iman Farasat, Howard M. Salis

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Engineered genetic circuits reprogram cellular behavior to act as living computers with applications in detecting cancer, creating self-controlling artificial tissues, and dynamically regulating metabolic pathways. Phenemenological models are often used to simulate and design genetic circuit behavior towards a desired behavior. While such models assume that each circuit component’s function is modular and independent, even small changes in a circuit (e.g. a new promoter, a change in transcription factor expression level, or even a new media) can have significant effects on the circuit’s function. Here, we use statistical thermodynamics to account for the several factors that control transcriptional regulation in bacteria, and experimentally demonstrate the model’s accuracy across 825 measurements in several genetic contexts and hosts. We then employ our first principles model to design, experimentally construct, and characterize a family of signal amplifying genetic circuits (genetic OpAmps) that expand the dynamic range of cell sensors. To develop these models, we needed a new approach to measuring the in vivo binding free energies of transcription factors (TFs), a key ingredient of statistical thermodynamic models of gene regulation. We developed a new high-throughput assay to measure RNA polymerase and TF binding free energies, requiring the construction and characterization of only a few constructs and data analysis (Figure 1A). We experimentally verified the assay on 6 TetR-homolog repressors and a CRISPR/dCas9 guide RNA. We found that our binding free energy measurements quantitatively explains why changing TF expression levels alters circuit function. Altogether, by combining these measurements with our biophysical model of translation (the RBS Calculator) as well as other measurements (Figure 1B), our model can account for changes in TF binding sites, TF expression levels, circuit copy number, host genome size, and host growth rate (Figure 1C). Model predictions correctly accounted for how these 8 factors control a promoter’s transcription rate (Figure 1D). Using the model, we developed a design framework for engineering multi-promoter genetic circuits that greatly reduces the number of degrees of freedom (8 factors per promoter) to a single dimensionless unit. We propose the Ptashne (Pt) number to encapsulate the 8 co-dependent factors that control transcriptional regulation into a single number. Therefore, a single number controls a promoter’s output rather than these 8 co-dependent factors, and designing a genetic circuit with N promoters requires specification of only N Pt numbers. We demonstrate how to design genetic circuits in Pt number space by constructing and characterizing 15 2-repressor OpAmp circuits that act as signal amplifiers when within an optimal Pt region. We experimentally show that OpAmp circuits using different TFs and TF expression levels will only amplify the dynamic range of input signals when their corresponding Pt numbers are within the optimal region. Thus, the use of the Pt number greatly simplifies the genetic circuit design, particularly important as circuits employ more TFs to perform increasingly complex functions.

Keywords: transcription factor, synthetic biology, genetic circuit, biophysical model, binding energy measurement

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Authors: Adriana Muñoz-Talavera, Ana Rosa Rincón-Sánchez, Abraham Escobedo-Moratilla, María Cristina Islas-Carbajal, Miguel Ángel Gómez-Lim

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The highest rates of diabetes incidence and prevalence worldwide will increase the number of diabetic patients requiring insulin or insulin analogues. Then, current production systems would not be sufficient to meet the future market demands. Therefore, developing efficient expression systems for insulin and insulin analogues are needed. In addition, insulin analogues with better pharmacokinetics and pharmacodynamics properties and without mitogenic potential will be required. SCI-57 (single chain insulin-57) is an insulin analogue having 10 times greater affinity to the insulin receptor, higher resistance to thermal degradation than insulin, native mitogenicity and biological effect. Plants as expression platforms have been used to produce recombinant proteins because of their advantages such as cost-effectiveness, posttranslational modifications, absence of human pathogens and high quality. Immunoglobulin production with a yield of 50% has been achieved by transient expression in Nicotiana benthamiana (Nb). The aim of this study is to produce SCI-57 by transient expression in Nb. Methodology: DNA sequence encoding SCI-57 was cloned in pICH31070. This construction was introduced into Agrobacterium tumefaciens by electroporation. The resulting strain was used to infiltrate leaves of Nb. In order to isolate SCI-57, leaves from transformed plants were incubated 3 hours with the extraction buffer therefore filtrated to remove solid material. The resultant protein solution was subjected to anion exchange chromatography on an FPLC system and ultrafiltration to purify SCI-57. Detection of SCI-57 was made by electrophoresis pattern (SDS-PAGE). Protein band was digested with trypsin and the peptides were analyzed by Liquid chromatography tandem-mass spectrometry (LC-MS/MS). A purified protein sample (20µM) was analyzed by ESI-Q-TOF-MS to obtain the ionization pattern and the exact molecular weight determination. Chromatography pattern and impurities detection were performed using RP-HPLC using recombinant insulin as standard. The identity of the SCI-57 was confirmed by anti-insulin ELISA. The total soluble protein concentration was quantified by Bradford assay. Results: The expression cassette was verified by restriction mapping (5393 bp fragment). The SDS-PAGE of crude leaf extract (CLE) of transformed plants, revealed a protein of about 6.4 kDa, non-present in CLE of untransformed plants. The LC-MS/MS results displayed one peptide with a high score that matches SCI-57 amino acid sequence in the sample, confirming the identity of SCI-57. From the purified SCI-57 sample (PSCI-57) the most intense charge state was 1069 m/z (+6) on the displayed ionization pattern corresponding to the molecular weight of SCI-57 (6412.6554 Da). The RP-HPLC of the PSCI-57 shows the presence of a peak with similar retention time (rt) and UV spectroscopic profile to the insulin standard (SCI-57 rt=12.96 and insulin rt=12.70 min). The collected SCI-57 peak had ELISA signal. The total protein amount in CLE from transformed plants was higher compared to untransformed plants. Conclusions: Our results suggest the feasibility to produce insulin analogue SCI-57 by transient expression in Nicotiana benthamiana. Further work is being undertaken to evaluate the biological activity by glucose uptake by insulin-sensitive and insulin-resistant murine and human cultured adipocytes.

Keywords: insulin analogue, mass spectrometry, Nicotiana benthamiana, transient expression

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Authors: Tesfaye Dagne, Dagmawit Solomon, Firmaye Bogale, Yosef Gebreyohannes, Samson Mideksa, Mamuye Hadis, Desalegn Ararso, Ermias Woldie, Tsegaye Getachew, Sabit Ababor, Zelalem Kebede

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Globally, road traffic accident (RTA) is causing millions of deaths and injuries every year. It is one of the leading causes of death among people of all age groups and the problem is worse among young reproductive age group. Moreover the problem is increasing with an increasing number of vehicles. The majority of the problem happen in low and middle income countries (LMIC), even if the number of vehicles in these countries is low compared to their population. So, the objective of this paper is to summarize the best available evidence on interventions that can reduce road traffic accidents in low and middle income countries (LMIC). Method: A rapid evidence synthesis approach adapted from the SURE Rapid Response Service was applied to search, appraise and summarize the best available evidence on effective intervention in reducing road traffic injury. To answer the question under review, we searched for relevant studies from databases including PubMed, the Cochrane Library, TRANSPORT, Health system evidence, Epistemonikos, and SUPPORT summary. The following key terms were used for searching: Road traffic accident, RTA, Injury, Reduc*, Prevent*, Minimiz*, “Low and middle-income country”, LMIC. We found 18 articles through a search of different databases mentioned above. After screening for the titles and abstracts of the articles, four of them which satisfy the inclusion criteria were included in the final review. Then we appraised and graded the methodological quality of systematic reviews that are deemed to be highly relevant using AMSTAR. Finding: The identified interventions to reduce road traffic accidents were legislation and enforcement, public awareness/education, speed control/ rumble strips, road improvement, mandatory motorcycle helmet, graduated driver license, street lighting. Legislation and Enforcement: Legislation focusing on mandatory motorcycle helmet usage, banning cellular phone usage when driving, seat belt laws, decreasing the legal blood alcohol content (BAC) level from 0.06 g/L to 0.02 g/L bring the best result where enforcement is there. Public Awareness/Education: focusing on seat belt use, child restraint use, educational training in health centers and schools/universities, and public awareness with media through the distribution of videos, posters/souvenirs, and pamphlets are effective in the short run. Speed Control: through traffic calming bumps, or speed bumps, rumbled strips are effective in reducing accidents and fatality. Mandatory Motorcycle Helmet: is associated with reduction in mortality. Graduated driver’s license (GDL): reduce road traffic injury by 19%. Street lighting: is a low-cost intervention which may reduce road traffic accidents.

Keywords: evidence synthesis, injury, rapid review, reducing, road traffic accident

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Authors: Marcin Zieliński, Marcin Dębowski, Mirosław Krzemieniewski

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The anaerobic degradation of substrates is limited especially by the rate and effectiveness of the first (hydrolytic) stage of fermentation. This stage may be intensified through pre-treatment of substrate aimed at disintegration of the solid phase and destruction of substrate tissues and cells. The most frequently applied criterion of disintegration outcomes evaluation is the increase in biogas recovery owing to the possibility of its use for energetic purposes and, simultaneously, recovery of input energy consumed for the pre-treatment of substrate before fermentation. Hydrodynamic cavitation is one of the methods for organic substrate disintegration that has a high implementation potential. Cavitation is explained as the phenomenon of the formation of discontinuity cavities filled with vapor or gas in a liquid induced by pressure drop to the critical value. It is induced by a varying field of pressures. A void needs to occur in the flow in which the pressure first drops to the value close to the pressure of saturated vapor and then increases. The process of cavitation conducted under controlled conditions was found to significantly improve the effectiveness of anaerobic conversion of organic substrates having various characteristics. This phenomenon allows effective damage and disintegration of cellular and tissue structures. Disintegration of structures and release of organic compounds to the dissolved phase has a direct effect on the intensification of biogas production in the process of anaerobic fermentation, on reduced dry matter content in the post-fermentation sludge as well as a high degree of its hygienization and its increased susceptibility to dehydration. A device the efficiency of which was confirmed both in laboratory conditions and in systems operating in the technical scale is a hydrodynamic generator of cavitation. Cavitators, agitators and emulsifiers constructed and tested worldwide so far have been characterized by low efficiency and high energy demand. Many of them proved effective under laboratory conditions but failed under industrial ones. The only task successfully realized by these appliances and utilized on a wider scale is the heating of liquids. For this reason, their usability was limited to the function of heating installations. Design of the presented cavitation generator allows achieving satisfactory energy efficiency and enables its use under industrial conditions in depolymerization processes of biomass with various characteristics. Investigations conducted on the laboratory and industrial scale confirmed the effectiveness of applying cavitation in the process of biomass destruction. The use of the cavitation generator in laboratory studies for disintegration of sewage sludge allowed increasing biogas production by ca. 30% and shortening the treatment process by ca. 20 - 25%. The shortening of the technological process and increase of wastewater treatment plant effectiveness may delay investments aimed at increasing system output. The use of a mechanical cavitator and application of repeated cavitation process (4-6 times) enables significant acceleration of the biogassing process. In addition, mechanical cavitation accelerates increases in COD and VFA levels.

Keywords: hydrodynamic cavitation, pretreatment, biomass, methane fermentation, Virginia fanpetals

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Authors: Vrushali Guhe, Shailza Singh

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Cutaneous leishmaniasis is neglected tropical disease present worldwide caused by the protozoan parasite Leishmania major, the therapeutic armamentarium for leishmaniasis are showing several limitations as drugs are showing toxic effects with increasing resistance by a parasite. Thus identification of novel therapeutic targets is of paramount importance. Previous studies have shown that autophagy, a cellular process, can either facilitate infection or aid in the elimination of the parasite, depending on the specific parasite species and host background in leishmaniasis. In the present study, our objective was to target the essential autophagy protein ATG8, which plays a crucial role in the survival, infection dynamics, and differentiation of the Leishmania parasite. ATG8 in Leishmania major and its homologue, LC3, in Homo sapiens, act as autophagic markers. Present study manifested the crucial role of ATG8 protein as a potential target for combating Leishmania major infection. Through bioinformatics analysis, we identified non-conserved motifs within the ATG8 protein of Leishmania major, which are not present in LC3 of Homo sapiens. Against these two non-conserved motifs, we generated a peptide library of 60 peptides on the basis of physicochemical properties. These peptides underwent a filtering process based on various parameters, including feasibility of synthesis and purification, compatibility with Selective Reaction Monitoring (SRM)/Multiple reaction monitoring (MRM), hydrophobicity, hydropathy index, average molecular weight (Mw average), monoisotopic molecular weight (Mw monoisotopic), theoretical isoelectric point (pI), and half-life. Further filtering criterion shortlisted three peptides by using molecular docking and molecular dynamics simulations. The direct interaction between ATG8 and the shortlisted peptides was confirmed through Surface Plasmon Resonance (SPR) experiments. Notably, these peptides exhibited the remarkable ability to penetrate the parasite membrane and exert profound effects on Leishmania major. The treatment with these peptides significantly impacted parasite survival, leading to alterations in the cell cycle and morphology. Furthermore, the peptides were found to modulate autophagosome formation, particularly under starved conditions, suggesting their involvement in disrupting the regulation of autophagy within Leishmania major. In vitro, studies demonstrated that the selected peptides effectively reduced the parasite load within infected host cells. Encouragingly, these findings were corroborated by in vivo experiments, which showed a reduction in parasite burden upon peptide administration. Additionally, the peptides were observed to affect the levels of LC3II within host cells. In conclusion, our findings highlight the efficacy of these novel peptides in targeting Leishmania major’s ATG8 and disrupting parasite survival. These results provide valuable insights into the development of innovative therapeutic strategies against leishmaniasis via targeting autophagy protein ATG8 of Leishmania major.

Keywords: ATG8, leishmaniasis, surface plasmon resonance, MD simulation, molecular docking, peptide designing, therapeutics

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Authors: Eduardo Rodrigues, Gisela Mendes, José M. Torres, José E. Sousa

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In the sequence of the liberalisation of the electricity sector a progressive engagement of consumers has been considered and targeted by sector regulatory policies. With the objective of promoting market competition while protecting consumers interests, by transferring some of the upstream benefits to the end users while reaching a fair distribution of system costs, different market models to value consumers’ demand flexibility at the energy community level are envisioned. Local Energy and Flexibility Markets (LEFM) involve stakeholders interested in providing or procure local flexibility for community, services and markets’ value. Under the scope of DOMINOES, a European research project supported by Horizon 2020, the local market concept developed is expected to: • Enable consumers/prosumers empowerment, by allowing them to value their demand flexibility and Distributed Energy Resources (DER); • Value local liquid flexibility to support innovative distribution grid management, e.g., local balancing and congestion management, voltage control and grid restoration; • Ease the wholesale market uptake of DER, namely small-scale flexible loads aggregation as Virtual Power Plants (VPPs), facilitating Demand Response (DR) service provision; • Optimise the management and local sharing of Renewable Energy Sources (RES) in Medium Voltage (MV) and Low Voltage (LV) grids, trough energy transactions within an energy community; • Enhance the development of energy markets through innovative business models, compatible with ongoing policy developments, that promote the easy access of retailers and other service providers to the local markets, allowing them to take advantage of communities’ flexibility to optimise their portfolio and subsequently their participation in external markets. The general concept proposed foresees a flow of market actions, technical validations, subsequent deliveries of energy and/or flexibility and balance settlements. Since the market operation should be dynamic and capable of addressing different requests, either prioritising balancing and prosumer services or system’s operation, direct procurement of flexibility within the local market must also be considered. This paper aims to highlight the research on the definition of suitable DR models to be used by the Distribution System Operator (DSO), in case of technical needs, and by the retailer, mainly for portfolio optimisation and solve unbalances. The models to be proposed and implemented within relevant smart distribution grid and microgrid validation environments, are focused on day-ahead and intraday operation scenarios, for predictive management and near-real-time control respectively under the DSO’s perspective. At local level, the DSO will be able to procure flexibility in advance to tackle different grid constrains (e.g., demand peaks, forecasted voltage and current problems and maintenance works), or during the operating day-to-day, to answer unpredictable constraints (e.g., outages, frequency deviations and voltage problems). Due to the inherent risks of their active market participation retailers may resort to DR models to manage their portfolio, by optimising their market actions and solve unbalances. The interaction among the market actors involved in the DR activation and in flexibility exchange is explained by a set of sequence diagrams for the DR modes of use from the DSO and the energy provider perspectives. • DR for DSO’s predictive management – before the operating day; • DR for DSO’s real-time control – during the operating day; • DR for retailer’s day-ahead operation; • DR for retailer’s intraday operation.

Keywords: demand response, energy communities, flexible demand, local energy and flexibility markets

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Authors: Luisa Barreira, Viana Castaneda, Maria J. Rodrigues, Florinda Gama, Tamara Santos, Marta Oliveira, Catarina Pereira, Maribela Pestana, Pedro Correia, Miguel Salazar, Carla Nunes, Luisa Custodio, Joao Varela

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In the last century, the world witnessed an exponential demographic increase that has put an enormous pressure on agriculture and food production. Associated also with climate changes, there has been a decrease in the amount of available freshwater and an increased salinization of soils which can affect the production of most food crops. Halophytes, however, are plants able to withstand high salinities while maintaining a good growth productivity. To cope with the excess salt, they produce secondary metabolites (e.g. vitamins and phenolic compounds) which, along with the natural presence of some minerals, makes them not only nutritionally rich but also functional foods. Some halophytes, as quinoa or salicornia, are already used in some countries, mostly as gourmet food. Hydroponic cultivation of halophytes using seawater or diluted seawater for watering can decrease the pressure on freshwater resources while producing a nutritional and functional food. The XtremeGourmet project funded by the EU aims to develop and optimize the production of different halophytes by hydroponics. One of the more specific objectives of this project is the study of halophytes’ productivity and chemical composition under different abiotic conditions, e.g. salt and nutrient concentration and light intensity. Three species of halophytes commonly occurring in saltmarshes of the South of Portugal (Inula chrithmoides, Salicornia ramosissima and Mesembryanthemum nodiflorum) were cultivated using hydroponics under different salinities, ranging from 5 to 45 dS/m. For each condition, several parameters were assessed namely: total and commercial productivity, electrical conductivity, total soluble solids, proximal composition, mineral profile, total phenolics, flavonoids and condensed tannins content and antioxidant activity. Results show that productivity was significantly reduced for all plants with increasing salinity up to salinity 29 dS/m and remained low onwards. Oppositely, the electrical conductivity and the total soluble solids content of the produced plants increased with salinity, reaching a plateau at 29 dS/m. It seems that plants reflect the salt concentration of the water up to some point, being able to regulate their salt content for higher salinities. The same tendency was observed for the ash content of these plants, which is related to the mineral uptake from the cultivating media and the plants’ capacity to both accumulate and regulate ions’ concentration in their tissues. Nonetheless, this comes with a metabolic cost which is observed by a decrease in productivity. The mineral profile of these plants shows high concentrations of sodium but also high amounts of potassium. In what concerns the microelements, these plants appear to be a good source of manganese and iron and the low amounts of toxic metals account for their safe consumption in moderate amounts. Concerning the phenolics composition, plants presented moderate concentrations of phenolics but high amounts of condensed tannins, particularly I. crithmoides which accounts for its characteristic sour and spicy taste. Contrary to some studies in which higher amounts of phenolics were found in plants cultivated under higher salinities, in this study, the highest amount of phenolic compounds were found in plants grown at the lowest or intermediate salinities. Nonetheless, there was a positive correlation between the concentration of these compounds and the antioxidant capacity of the plants’ extracts.

Keywords: functional properties, halophytes, hydroponics, nutritional composition, salinity effect

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Authors: E. Carraro, Sa. Bonetta, Si. Bonetta, E. Ceretti, G. C. V. Viola, C. Pignata, S. Levorato, T. Salvatori, S. Vannini, V. Romanazzi, A. Carducci, G. Donzelli, T. Schilirò, A. De Donno, T. Grassi, S. Bonizzoni, A. Bonetti, G. Gilli, U. Gelatti

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In 2013, air pollution and particulate matter were classified as carcinogenic to human by the IARC. At present, PM is Europe's most problematic pollutant in terms of harm to health, as reported by European Environmental Agency (EEA) in the EEA Technical Report on Air quality in Europe, 2015. A percentage between 17-30 of the EU urban population lives in areas where the EU air quality 24-hour limit value for PM10 is exceeded. Many studies have found a consistent association between exposure to PM and the incidence and mortality for some chronic diseases (i.e. lung cancer, cardiovascular diseases). Among the mechanisms responsible for these adverse effects, genotoxic damage is of particular concern. Children are a high-risk group in terms of the health effects of air pollution and early exposure during childhood can increase the risk of developing chronic diseases in adulthood. The MAPEC_LIFE (Monitoring Air Pollution Effects on Children for supporting public health policy) is a project founded by EU Life+ Programme (LIFE12 ENV/IT/000614) which intends to evaluate the associations between air pollution and early biological effects in children and to propose a model for estimating the global risk of early biological effects due to air pollutants and other factors in children. This work is focused on the micronuclei frequency in child buccal cells in association with airborne PM levels taking into account the influence of other factors associated with the lifestyle of children. The micronucleus test was performed in exfoliated buccal cells of 6–8 years old children from 5 Italian towns with different air pollution levels. Data on air quality during the study period were obtained from the Regional Agency for Environmental Protection. A questionnaire administered to children’s parents was used to obtain details on family socio-economic status, children health condition, exposures to other indoor and outdoor pollutants (i.e. passive smoke) and life-style, with particular reference to eating habits. During the first sampling campaign (winter 2014-15) 1315 children were recruited and sampled for Micronuclei test in buccal cells. In the sampling period the levels of the main pollutants and PM10 were, as expected, higher in the North of Italy (PM10 mean values 62 μg/m3 in Torino and 40 μg/m3 in Brescia) than in the other towns (Pisa, Perugia, Lecce). A higher Micronucleus frequency in buccal cells of children was found in Brescia (0.6/1000 cells) than in the other towns (range 0.3-0.5/1000 cells). The statistical analysis underlines a relation of the micronuclei frequency with PM concentrations, traffic level near child residence, and level of education of parents. The results suggest that, in addition to air pollution exposure, some other factors, related to lifestyle or further exposures, may influence micronucleus frequency and cellular response to air pollutants.

Keywords: air pollution, buccal cells, children, micronucleus cytome assay

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Authors: Wellemans Isabelle, Remmelink Myriam, Foucart Annick, Rusu Stefan, Compère Christophe

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Primary pulmonary meningioma (PPM) is a very rare tumor, and its occurrence has been reported only sporadically. Multiple PPMs are even more exceptional, and herein, we report, to the best of our knowledge, the fourth case, focusing on the clinicopathological features of the tumor. Moreover, the possible relationship between the use of progesterone–only contraceptives and the development of these neoplasms will be discussed. Case Report: We report a case of a 51-year-old female presenting three solid pulmonary nodules, with the following localizations: right upper lobe, middle lobe, and left lower lobe, described as incidental findings on computed tomography (CT) during a pre-bariatric surgery check-up. The patient revealed no drinking or smoking history. The physical exam was unremarkable except for the obesity. The lesions ranged in size between 6 and 24 mm and presented as solid nodules with lobulated contours. The largest lesion situated in the middle lobe had mild fluorodeoxyglucose (FDG) uptake on F-18 FDG positron emission tomography (PET)/CT, highly suggestive of primary lung neoplasm. For pathological assessment, video-assisted thoracoscopic middle lobectomy and wedge resection of the right upper nodule was performed. Histological examination revealed relatively well-circumscribed solid proliferation of bland meningothelial cells growing in whorls and lobular nests, presenting intranuclear pseudo-inclusions and psammoma bodies. No signs of anaplasia were observed. The meningothelial cells expressed diffusely Vimentin, focally Progesterone receptors and were negative for epithelial (cytokeratin (CK) AE1/AE3, CK7, CK20, Epithelial Membrane Antigen (EMA)), neuroendocrine markers (Synaptophysin, Chromogranin, CD56) and Estrogenic receptors. The proliferation labelling index Ki-67 was low (<5%). Metastatic meningioma was ruled out by brain and spine magnetic resonance imaging (MRI) scans. The third lesion localized in the left lower lobe was followed-up and resected three years later because of its slow but significant growth (14 mm to 16 mm), alongside two new infra centimetric lesions. Those three lesions showed a morphological and immunohistochemical profile similar to previously resected lesions. The patient was disease-free one year post-last surgery. Discussion: Although PPMs are mostly benign and slow-growing tumors with an excellent prognosis, they do not present specific radiological characteristics, and it is difficult to differentiate it from other lung tumors, histopathologic examination being essential. Aggressive behavior is associated with atypical or anaplastic features (WHO grades II–III) The etiology is still uncertain and different mechanisms have been proposed. A causal connection between sexual hormones and meningothelial proliferation has long been suspected and few studies examining progesterone only contraception and meningioma risk have all suggested an association. In line with this, our patient was treated with Levonorgestrel, a progesterone agonist, intra-uterine device (IUD). Conclusions: PPM, defined by the typical histological and immunohistochemical features of meningioma in the lungs and the absence of central nervous system lesions, is an extremely rare neoplasm, mainly solitary and associating, and indolent growth. Because of the unspecific radiologic findings, it should always be considered in the differential diagnosis of lung neoplasms. Regarding multiple PPM, only three cases are reported in the literature, and this is the first described in a woman treated by a progesterone-only IUD to the best of our knowledge.

Keywords: pulmonary meningioma, multiple meningioma, meningioma, pulmonary nodules

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Authors: Panagiotis Gkekas, Christos Sougles, Dionysios Kehagias, Dimitrios Tzovaras

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This paper presents the architecture of the “Intelligent cooperative integrated system for road safety and road infrastructure maintenance towards 2020” (ODOS2020) advanced infrastructure, which implements a number of cooperative ITS applications based on Internet of Things and Infrastructure-to-Vehicle (V2I) technologies with the purpose to enhance the active road safety level of vehicles through the provision of a fully automated V2I environment. The primary objective of the ODOS2020 project is to contribute to increased road safety but also to the optimization of time for maintenance of road infrastructure. The integrated technological solution presented in this paper addresses all types of vehicles and requires minimum vehicle equipment. Thus, the ODOS2020 comprises a low-cost solution, which is one of its main benefits. The system architecture includes an integrated notification system to transmit personalized information on road, traffic, and environmental conditions, in order for the drivers to receive real-time and reliable alerts concerning upcoming critical situations. The latter include potential dangers on the road, such as obstacles or road works ahead, extreme environmental conditions, etc., but also informative messages, such as information on upcoming tolls and their charging policies. At the core of the system architecture lies an integrated sensorial network embedded in special road infrastructures (strips) that constantly collect and transmit wirelessly information about passing vehicles’ identification, type, speed, moving direction and other traffic information in combination with environmental conditions and road wear monitoring and predictive maintenance data. Data collected from sensors is transmitted by roadside infrastructure, which supports a variety of communication technologies such as ITS-G5 (IEEE-802.11p) wireless network and Internet connectivity through cellular networks (3G, LTE). All information could be forwarded to both vehicles and Traffic Management Centers (TMC) operators, either directly through the ITS-G5 network, or to smart devices with Internet connectivity, through cloud-based services. Therefore, through its functionality, the system could send personalized notifications/information/warnings and recommendations for upcoming events to both road users and TMC operators. In the course of the ODOS2020 project pilot operation has been conducted to allow drivers of both C-ITS equipped and non-equipped vehicles to experience the provided added value services. For non-equipped vehicles, the provided information is transmitted to a smartphone application. Finally, the ODOS2020 system and infrastructure is appropriate for installation on both urban, rural, and highway environments. The paper presents the various parts of the system architecture and concludes by outlining the various challenges that had to be overcome during its design, development, and deployment in a real operational environment. Acknowledgments: Work presented in this paper was co-financed by the European Regional Development Fund of the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation (call RESEARCH–CREATE–INNOVATE) under contract no. Τ1EDK-03081 (project ODOS2020).

Keywords: infrastructure to vehicle, intelligent transportation systems, internet of things, road safety

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Authors: Inna Kornienko, Svetlana Guryeva, Anatoly Shekhter, Elena Petersen

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Transplantation is an effective treatment option for patients suffering from different end-stage diseases. However, it is plagued by a constant shortage of donor organs and the subsequent need of a lifelong immunosuppressive therapy for the patient. Currently some researchers look towards using of pig organs to replace human organs for transplantation since the matrix derived from porcine organs is a convenient substitute for the human matrix. As an initial step to create a new ex vivo tissue engineered model, optimized protocols have been created to obtain organ-specific acellular matrices and evaluated their potential as tissue engineered scaffolds for culture of normal cells and tumor cell lines. These protocols include decellularization by perfusion in a bioreactor system and immersion-agitation on an orbital shaker with use of various detergents (SDS, Triton X-100) and freezing. Complete decellularization – in terms of residual DNA amount – is an important predictor of probability of immune rejection of materials of natural origin. However, the signs of cellular material may still remain within the matrix even after harsh decellularization protocols. In this regard, the matrices obtained from tissues of low-immunogenic pigs with α3Galactosyl-tranferase gene knock out (GalT-KO) may be a promising alternative to native animal sources. The research included a study of induced effect of frozen and fresh fragments of GalT-KO skin on healing of full-thickness plane wounds in 80 rats. Commercially available wound dressings (Ksenoderm, Hyamatrix and Alloderm) as well as allogenic skin were used as a positive control and untreated wounds were analyzed as a negative control. The results were evaluated on the 4th day after grafting, which corresponds to the time of start of normal wound epithelization. It has been shown that a non-specific immune response in models treated with GalT-Ko pig skin was milder than in all the control groups. Research has been performed to measure technical skin characteristics: stiffness and elasticity properties, corneometry, tevametry, and cutometry. These metrics enabled the evaluation of hydratation level, corneous layer husking level, as well as skin elasticity and micro- and macro-landscape. These preliminary data may contribute to development of personalized transplantable organs from GalT-Ko pigs with significantly limited potential of immune rejection. By applying growth factors to a decellularized skin sample it is possible to achieve various regenerative effects based on the particular situation. In this particular research BMP2 and Heparin-binding EGF-like growth factor have been used. Ideally, a bioengineered organ must be biocompatible, non-immunogenic and support cell growth. Porcine organs are attractive for xenotransplantation if severe immunologic concerns can be bypassed. The results indicate that genetically modified pig tissues with knock-outed α3Galactosyl-tranferase gene may be used for production of low-immunogenic matrix suitable for transplantation.

Keywords: decellularization, low-immunogenic, matrix, scaffolds, transplants

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Authors: Sophio Kobauri, Temur Kantaria, Nina Kulikova, David Tugushi, Ramaz Katsarava

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Polymeric nanoparticles-based drug delivery systems and therapeutics have a great potential in the treatment of a numerous diseases, due to they are characterizing the flexible properties which is giving possibility to modify their structures with a complex definition over their structures, compositions and properties. Important characteristics of the polymeric nanoparticles (PNPs) used as drug carriers are high particle’s stability, high carrier capacity, feasibility of encapsulation of both hydrophilic and hydrophobic drugs, and feasibility of variable routes of administration, including oral application and inhalation; NPs are especially effective for intracellular drug delivery since they penetrate into the cells’ interior though endocytosis. A variety of PNPs based drug delivery systems including charged and neutral, degradable and non-degradable polymers of both natural and synthetic origin have been developed. Among these huge varieties the biodegradable PNPs which can be cleared from the body after the fulfillment of their function could be considered as one of the most promising. For intracellular uptake it is highly desirable to have positively charged PNPs since they can penetrate deep into cell membranes. For long-lasting circulation of PNPs in the body it is important they have so called “stealth coatings” to protect them from the attack of immune system of the organism. One of the effective ways to render the PNPs “invisible” for immune system is their PEGylation which represent the process of pretreatment of polyethylene glycol (PEG) on the surface of PNPs. The present work deals with constructing PNPs from amino acid based biodegradable polymers – regular poly(ester amide) (PEA) composed of sebacic acid, leucine and 1,6-hexandiol (labeled as 8L6), cationic PEA composed of sebacic acid, arginine and 1,6-hexandiol (labeled as 8R6), and comb-like co-PEA composed of sebacic acid, malic acid, leucine and 1,6-hexandiol (labeled as PEG-PEA). The PNPs were fabricated using the polymer deposition/solvent displacement (nanoprecipitation) method. The regular PEA 8L6 form stable negatively charged (zeta-potential within 2-12 mV) PNPs of desired size (within 150-200 nm) in the presence of various surfactants (Tween 20, Tween 80, Brij 010, etc.). Blending the PEAs 8L6 and 8R6 gave the 130-140 nm sized positively charged PNPs having zeta-potential within +20 ÷ +28 mV depending 8L6/8R6 ratio. The PEGylating PEA PEG-PEA was synthesized by interaction of epoxy-co-PEA [8L6]0,5-[tES-L6]0,5 with mPEG-amine-2000 The stable and positively charged PNPs were fabricated using pure PEG-PEA as a surfactant. A firm anchoring of the PEG-PEA with 8L6/8R6 based PNPs (owing to a high afinity of the backbones of all three PEAs) provided good stabilization of the NPs. In vitro biocompatibility study of the new PNPs with four different stable cell lines: A549 (human), U-937 (human), RAW264.7 (murine), Hepa 1-6 (murine) showed they are biocompatible. Considering high stability and cell compatibility of the elaborated PNPs one can conclude that they are promising for subsequent therapeutic applications. This work was supported by the joint grant from the Science and Technology Center in Ukraine and Shota Rustaveli National Science Foundation of Georgia #6298 “New biodegradable cationic polymers composed of arginine and spermine-versatile biomaterials for various biomedical applications”.

Keywords: biodegradable poly(ester amide)s, cationic poly(ester amide), pegylating poly(ester amide), nanoparticles

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Authors: Oluwaseun Adeleke, Samuel O. Ikani, Fidelis Edet, Anthony Nwala, Mopelola Raji, Simeon Christian Chukwu

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Background: The DISC project collaborated with partners to implement demand creation and service delivery interventions, including the MoT (Moment of Truth) innovation, in over 500 health facilities across 15 states. This has increased the voluntary conversion rate to self-injection among women who opt for injectable contraception. While some facilities recorded an increasing trend in key performance indicators, few others persistently performed sub-optimally due to provider and system-related barriers. Methodology: Twenty-two facilities performing sub-optimally were selected purposively from three Nigerian states. Low productivity was appraised using low reporting rates and poor SI conversion rates as indicators. Interviews were conducted with health providers across these health facilities using a rapid diagnosis tool. The project also conducted a data quality assessment that evaluated the veracity of data elements reported across the three major sources of family planning data in the facility. Findings: The inability and sometimes refusal of providers to support clients to self-inject effectively was associated with the misunderstanding of its value to their work experience. It was also observed that providers still held a strong influence over clients’ method choices. Furthermore, providers held biases and misconceptions about DMPA-SC that restricted the access of obese clients and new acceptors to services – a clear departure from the recommendations of the national guidelines. Additionally, quality of care standards was compromised because job aids were not used to inform service delivery. Facilities performing sub-optimally often under-reported DMPA-SC utilization data, and there were multiple uncoordinated responsibilities for recording and reporting. Additionally, data validation meetings were not regularly convened, and these meetings were ineffective in authenticating data received from health facilities. Other reasons for sub-optimal performance included poor documentation and tracking of stock inventory resulting in commodity stockouts, low client flow because of poor positioning of health facilities, and ineffective messaging. Some facilities lacked adequate human and material resources to provide services effectively and received very few supportive supervision visits. Supportive supervision visits and Data Quality Audits have been useful to address the aforementioned performance barriers. The project has deployed digital DMPA-SC self-injection checklists that have been aligned with nationally approved templates. During visits, each provider and community mobilizer is accorded special attention by the supervisor until he/she can perform procedures in line with best practice (protocol). Conclusion: This narrative provides a summary of a range of factors that identify health facilities performing sub-optimally in their provision of DMPA-SC services. Findings from this assessment will be useful during project design to inform effective strategies. As the project enters its final stages of implementation, it is transitioning high-impact activities to state institutions in the quest to sustain the quality of service beyond the tenure of the project. The project has flagged activities, as well as created protocols and tools aimed at placing state-level stakeholders at the forefront of improving productivity in health facilities.

Keywords: family planning, contraception, DMPA-SC, self-care, self-injection, barriers, opportunities, performance

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Authors: Aberdam Edith, Sangari Linda, Petit Isabelle, Aberdam Daniel

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Background and Rationale: Transparent avascularised cornea is essential for normal vision and depends on limbal stem cells (LSC) that reside between the cornea and the conjunctiva. Ocular burns or injuries may destroy the limbus, causing limbal stem cell deficiency (LSCD). The cornea becomes vascularised by invaded conjunctival cells, the stroma is scarring, resulting in corneal opacity and loss of vision. Grafted autologous limbus or cultivated autologous LCS can restore the vision, unless the two eyes are affected. Alternative cellular sources have been tested in the last decades, including oral mucosa or hair follicle epithelial cells. However, only partial success has been achieved by the use of these cells since they were not able to uniformly commit into corneal epithelial cells. Human pluripotent stem cells (iPSC) display both unlimited growth capacity and ability to differentiate into any cell type. Our goal was to design a standardized and reproducible protocol to produce transplantable autologous LSC from patients through cell reprogramming technology. Methodology: First, keratinocyte primary culture was established from a small number of plucked hair follicles of healthy donors. The resulting epithelial cells were reprogrammed into induced pluripotent stem cells (iPSCs) and further differentiate into corneal epithelial cells (CEC), according to a robust protocol that recapitulates the main step of corneal embryonic development. qRT-PCR analysis and immunofluorescent staining during the course of differentiation confirm the expression of stage specific markers of corneal embryonic lineage. First appear ectodermal progenitor-specific cytokeratins K8/K18, followed at day 7 by limbal-specific PAX6, TP63 and cytokeratins K5/K14. At day 15, K3/K12+-corneal cells are present. To amplify the iPSC-derived LSC (named COiPSC), intact small epithelial colonies were detached and cultivated in limbal cell-specific medium. In that culture conditions, the COiPSC can be frozen and thaw at any passage, while retaining their corneal characteristics for at least eight passages. To evaluate the potential of COiPSC as an alternative ocular toxicity model, COiPSC were treated at passage P0 to P4 with increasing amounts of SDS and Benzalkonium. Cell proliferation and apoptosis of treated cells was compared to LSC and the SV40-immortalized human corneal epithelial cell line (HCE) routinely used by cosmetological industrials. Of note, HCE are more resistant to toxicity than LSC. At P0, COiPSC were systematically more resistant to chemical toxicity than LSC and even to HCE. Remarkably, this behavior changed with passage since COiPSC at P2 became identical to LSC and thus closer to physiology than HCE. Comparative transcriptome analysis confirmed that COiPSC from P2 are similar to a mixture of LSC and CEC. Finally, by organotypic reconstitution assay, we demonstrated the ability of COiPSC to produce a 3D corneal epithelium on a stromal equivalent made of keratocytes. Conclusion: COiPSC could become valuable for two main applications: (1) an alternative robust tool to perform, in a reproducible and physiological manner, toxicity assays for cosmetic products and pharmacological tests of drugs. (2). COiPSC could become an alternative autologous source for cornea transplantation for LSCD.

Keywords: Limbal stem cell deficiency, iPSC, cornea, limbal stem cells

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Authors: Mohammadjavad Sotoudeheian

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Heart failure (HF) prevalence, as a global cardiovascular problem, is increasing gradually. A variety of molecular mechanisms contribute to HF. Proteins involved in cardiac contractility regulation, such as ion channels and calcium handling proteins, are altered. Additionally, epigenetic modifications and gene expression can lead to altered cardiac function. Moreover, inflammation and oxidative stress contribute to HF. The progression of HF can be attributed to mitochondrial dysfunction that impairs energy production and increases apoptosis. Molecular mechanisms such as these contribute to the development of cardiomyocyte defects and HF and can be therapeutically targeted. The heart's contractile function is controlled by cardiomyocytes. Calpain, and its related molecules, including Bax, VEGF, and AMPK, are among the proteins involved in regulating cardiomyocyte function. Apoptosis is facilitated by Bax. Cardiomyocyte apoptosis is regulated by this protein. Furthermore, cardiomyocyte survival, contractility, wound healing, and proliferation are all regulated by VEGF, which is produced by cardiomyocytes during inflammation and cytokine stress. Cardiomyocyte proliferation and survival are also influenced by AMPK, an enzyme that plays an active role in energy metabolism. They all play key roles in apoptosis, angiogenesis, hypertrophy, and metabolism during myocardial inflammation. The role of calpains has been linked to several molecular pathways. The calpain pathway plays an important role in signal transduction and apoptosis, as well as autophagy, endocytosis, and exocytosis. Cell death and survival are regulated by these calcium-dependent cysteine proteases that cleave proteins. As a result, protein fragments can be used for various cellular functions. By cleaving adhesion and motility proteins, calcium proteins also contribute to cell migration. HF may be brought about by calpain-mediated pathways. Many physiological processes are mediated by the calpain molecular pathways. Signal transduction, cell death, and cell migration are all regulated by these molecular pathways. Calpain is activated by calcium binding to calmodulin. In the presence of calcium, calmodulin activates calpain. Calpains are stimulated by calcium, which increases matrix metalloproteinases (MMPs). In order to develop novel treatments for these diseases, we must understand how this pathway works. A variety of myocardial remodeling processes involve calpains, including remodeling of the extracellular matrix and hypertrophy of cardiomyocytes. Calpains also play a role in maintaining cardiac homeostasis through apoptosis and autophagy. The development of HF may be in part due to calpain-mediated pathways promoting cardiomyocyte death. Numerous studies have suggested the importance of the Ca2+ -dependent protease calpain in cardiac physiology and pathology. Therefore, it is important to consider this pathway to develop and test therapeutic options in humans that targets calpain in HF. Apoptosis, autophagy, endocytosis, exocytosis, signal transduction, and disease progression all involve calpain molecular pathways. Therefore, it is conceivable that calpain inhibitors might have therapeutic potential as they have been investigated in preclinical models of several conditions in which the enzyme has been implicated that might be treated with them. Ca 2+ - dependent proteases and calpains contribute to adverse ventricular remodeling and HF in multiple experimental models. In this manuscript, we will discuss the calpain molecular pathway's important roles in HF development.

Keywords: calpain, heart failure, autophagy, apoptosis, cardiomyocyte

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Authors: Ekaterina Zvorykina

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Nowadays, Arctic and Antarctic regions are distinguished to be one of the most important strategic resources for global development. Nonetheless, living conditions in Arctic regions still demand certain improvements. As soon as the region is rarely populated, one of the main points of interest is health accommodation of the people, who migrate to Arctic region for permanent and shift work. At Arctic and Antarctic latitudes, personnel face polar day and polar night conditions during the time of the year. It means that they are deprived of natural sunlight in winter season and have continuous daylight in summer. Firstly, the change in light intensity during 24-hours period due to migration affects circadian rhythms. Moreover, the controlled artificial light in winter is also an issue. The results of the recent studies on night shift medical professionals, who were exposed to permanent artificial light, have already demonstrated higher risks in cancer, depression, Alzheimer disease. Moreover, people exposed to frequent time zones change are also subjected to higher risks of heart attack and cancer. Thus, our main goals are to understand how high latitude work and living conditions can affect human health and how it can be prevented. In our study, we analyze molecular and cellular factors, which play important role in circadian rhythm change and distinguish main risk groups in people, migrating to high latitudes. The main well-studied index of circadian timing is melatonin or its metabolite 6-sulfatoxymelatonin. In low light intensity melatonin synthesis is disturbed and as a result human organism requires more time for sleep, which is still disregarded when it comes to working time organization. Lack of melatonin also causes shortage in serotonin production, which leads to higher depression risk. Melatonin is also known to inhibit oncogenes and increase apoptosis level in cells, the main factors for tumor growth, as well as circadian clock genes (for example Per2). Thus, people who work in high latitudes can be distinguished as a risk group for cancer diseases and demand more attention. Clock/Clock genes, known to be one of the main circadian clock regulators, decrease sensitivity of hypothalamus to estrogen and decrease glucose sensibility, which leads to premature aging and oestrous cycle disruption. Permanent light exposure also leads to accumulation superoxide dismutase and oxidative stress, which is one of the main factors for early dementia and Alzheimer disease. We propose a new screening system adjusted for people, migrating from middle to high latitudes and accommodation therapy. Screening is focused on melatonin and estrogen levels, sleep deprivation and neural disorders, depression level, cancer risks and heart and vascular disorders. Accommodation therapy includes different types artificial light exposure, additional melatonin and neuroprotectors. Preventive procedures can lead to increase of migration intensity to high latitudes and, as a result, the prosperity of Arctic region.

Keywords: circadian rhythm, high latitudes, melatonin, neuroprotectors

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Authors: Ricardo Alberto Chiong Zevallos, Eduardo Moraes Rego Reis

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Pancreatic adenocarcinoma (PDAC) patients have a less than 10% 5-year survival rate. PDAC has no defined diagnostic and prognostic biomarkers. Gemcitabine is the first-line drug in PDAC and several other cancers. Long non-coding RNAs (lncRNAs) contribute to the tumorigenesis and are potential biomarkers for PDAC. Although lncRNAs aren’t translated into proteins, they have important functions. LncRNAs can decoy or recruit proteins from the epigenetic machinery, act as microRNA sponges, participate in protein translocation through different cellular compartments, and even promote chemoresistance. The chromatin remodeling enzyme EZH2 is a histone methyltransferase that catalyzes the methylation of histone 3 at lysine 27, silencing local expression. EZH2 is ambivalent, it can also activate gene expression independently of its histone methyltransferase activity. EZH2 is overexpressed in several cancers and interacts with lncRNAs, being recruited to a specific locus. EZH2 can be recruited to activate an oncogene or silence a tumor suppressor. The lncRNAs misregulation in cancer can result in the differential recruitment of EZH2 and in a distinct epigenetic landscape, promoting chemoresistance. The relevance of the EZH2-lncRNAs interaction to chemoresistant PDAC was assessed by Real Time quantitative PCR (RT-qPCR) and RNA Immunoprecipitation (RIP) experiments with naïve and gemcitabine-resistant PDAC cells. The expression of several lncRNAs and EZH2 gene targets was evaluated contrasting naïve and resistant cells. Selection of candidate genes was made by bioinformatic analysis and literature curation. Indeed, the resistant cell line showed higher expression of chemoresistant-associated lncRNAs and protein coding genes. RIP detected lncRNAs interacting with EZH2 with varying intensity levels in the cell lines. During RIP, the nuclear fraction of the cells was incubated with an antibody for EZH2 and with magnetic beads. The RNA precipitated with the beads-antibody-EZH2 complex was isolated and reverse transcribed. The presence of candidate lncRNAs was detected by RT-qPCR, and the enrichment was calculated relative to INPUT (total lysate control sample collected before RIP). The enrichment levels varied across the several lncRNAs and cell lines. The EZH2-lncRNA interaction might be responsible for the regulation of chemoresistance-associated genes in multiple cancers. The relevance of the lncRNA-EZH2 interaction to PDAC was assessed by siRNA knockdown of a lncRNA, followed by the analysis of the EZH2 target expression by RT-qPCR. The chromatin immunoprecipitation (ChIP) of EZH2 and H3K27me3 followed by RT-qPCR with primers for EZH2 targets also assess the specificity of the EZH2 recruitment by the lncRNA. This is the first report of the interaction of EZH2 and lncRNAs HOTTIP and PVT1 in chemoresistant PDAC. HOTTIP and PVT1 were described as promoting chemoresistance in several cancers, but the role of EZH2 is not clarified. For the first time, the lncRNA LINC01133 was detected in a chemoresistant cancer. The interaction of EZH2 with LINC02577, LINC00920, LINC00941, and LINC01559 have never been reported in any context. The novel lncRNAs-EZH2 interactions regulate chemoresistant-associated genes in PDAC and might be relevant to other cancers. Therapies targeting EZH2 alone weren’t successful, and a combinatorial approach also targeting the lncRNAs interacting with it might be key to overcome chemoresistance in several cancers.

Keywords: epigenetics, chemoresistance, long non-coding RNAs, pancreatic cancer, histone modification

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Authors: Zuzana Tatarkova, Ivana Pilchova, Michal Cibulka, Martin Kolisek, Peter Racay, Peter Kaplan

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Introduction: Normal functioning of mitochondria is crucial for cardiac performance. Mitochondria undergo mitophagy and biogenesis, and mitochondrial proteins are subject to extensive post-translational modifications. The state of mitochondrial homeostasis reflects overall cellular fitness and longevity. Perturbed mitochondria produce less ATP, release greater amounts of reactive molecules, and are more prone to apoptosis. Therefore mitochondrial turnover is an integral aspect of quality control in which dysfunctional mitochondria are selectively eliminated through mitophagy. Currently, the progressive deterioration of physiological functions is seen as accumulation of modified/damaged proteins with limiting regenerative ability and disturbance of such affected protein-protein communication throughout aging in myocardial cells. Methodologies: For our study was used immunohistochemistry, biochemical methods: spectrophotometry, western blotting, immunodetection as well as more sophisticated 2D electrophoresis and mass spectrometry for evaluation protein-protein interactions and specific post-translational modification. Results and Discussion: Mitochondrial stress response to reactive species was evaluated as electron transport chain (ETC) complexes, redox-active molecules, and their possible communication. Protein-protein interactions revealed a strong linkage between age and ETC protein subunits. Redox state was strongly affected in senescent mitochondria with shift in favor of more pro-oxidizing condition within cardiomyocytes. Acute myocardial ischemia and ischemia-reperfusion (IR) injury affected ETC complexes I, II and IV with no change in complex III. Ischemia induced decrease in total antioxidant capacity, MnSOD, GSH and catalase activity with recovery in some extent during reperfusion. While MnSOD protein content was higher in IR group, activity returned to 95% of control. Nitric oxide is one of the biological molecules that can out compete MnSOD for superoxide and produce peroxynitrite. This process is faster than dismutation and led to the 10-fold higher production of nitrotyrosine after IR injury in adult with higher protection in senescent ones. 2D protein profiling revealed 140 mitochondrial proteins, 12 of them with significant changes after IR injury and 36 individual nitrotyrosine-modified proteins further identified by mass spectrometry. Linking these two groups, 5 proteins were altered after IR as well as nitrated, but only one showed massive nitration per lowering content of protein after IR injury in adult. Conclusions: Senescent cells have greater proportion of protein content, which might be modulated by several post-translational modifications. If these protein modifications are connected to functional consequences and protein-protein interactions are revealed, link may lead to the solution. Assume all together, dysfunctional proteostasis can play a causative role and restoration of protein homeostasis machinery is protective against aging and possibly age-related disorders. This work was supported by the project VEGA 1/0018/18 and by project 'Competence Center for Research and Development in the field of Diagnostics and Therapy of Oncological diseases', ITMS: 26220220153, co-financed from EU sources.

Keywords: aging heart, mitochondria, proteomics, redox state

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Authors: Sami El Khatib, Agnès Leroux, Jean-Louis Merlin, François Guillemin, Marie-Ange D’Hallewin

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Photodynamic therapy (PDT) is a treatment modality based on the cytotoxic effect occurring on the target tissues by interaction of a photosensitizer with light in the presence of oxygen. One of the major advances in PDT can be attributed to the use of topical aminolevulinic (ALA) to induce Protoporphyrin IX (PpIX) for the treatment of early stage cancers as well as diagnosis. ALA is a precursor of the heme synthesis pathway. Locally delivered to the target tissue ALA overcomes the negative feedback exerted by heme and promotes the transient formation of PpIX in situ to reach critical effective levels in cells and tissue. Whereas early steps of the heme pathway occur in the cytosol, PpIX synthesis is shown to be held in the mitochondrial membranes and PpIX fluorescence is expected to accumulate in close vicinity of the initial building site and to progressively diffuse to the neighboring cytoplasmic compartment or other lipophylic organelles. PpIX is known to be highly reactive and will be degraded when irradiated with light. PpIX photobleaching is believed to be governed by a singlet oxygen mediated mechanism in the presence of oxidized amino acids and proteins. PpIX photobleaching and subsequent spectral phototransformation were described widely in tumor cells incubated in vitro with ALA solution, or ex vivo in human and porcine mucosa superfused with hexylaminolevulinate (hALA). PpIX photobleaching was also studied in vivo, using animal models such as normal or tumor mice skin and orthotopic rat bladder model. Hexyl aminolevulinate a more potent lipophilic derivative of ALA was proposed as an adjunct to standard cystoscopy in the fluorescence diagnosis of bladder cancer and other malignancies. We have previously reported the effectiveness of hALA mediated PDT of rat bladder cancer. Although normal and tumor bladder epithelium exhibit similar fluorescence intensities after intravesical instillation of two hALA concentrations (8 and 16 mM), the therapeutic response at 8mM and 20J/cm2 was completely different from the one observed at 16mM irradiated with the same light dose. Where the tumor is destroyed, leaving the underlying submucosa and muscle intact after an 8 mM instillation, 16mM sensitization and subsequent illumination results in the complete destruction of the underlying bladder wall but leaves the tumor undamaged. The object of the current study is to try to unravel the underlying mechanism for this apparent contradiction. PpIX extraction showed identical amounts of photosensitizer in tumor bearing bladders at both concentrations. Photobleaching experiments revealed mono-exponential decay curves in both situations but with a two times faster decay constant in case of 16mM bladders. Fluorescence microscopy shows an identical fluorescence pattern for normal bladders at both concentrations and tumor bladders at 8mM with bright spots. Tumor bladders at 16 mM exhibit a more diffuse cytoplasmic fluorescence distribution. The different response to PDT with regard to the initial pro-drug concentration can thus be attributed to the different cellular localization.

Keywords: bladder cancer, hexyl-aminolevulinate, photobleaching, confocal fluorescence microscopy

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Authors: Iñaki Iturria, Pasquale Russo, Montserrat Nacher-Vázquez, Giuseppe Spano, Paloma López, Miguel Angel Pardo

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Introduction: It is known that some Lactic Acid Bacteria (LAB) present an interesting probiotic effect. Probiotic bacteria stimulate host resistance to microbial pathogens and thereby aid in immune response, and modulate the host's immune responses to antigens with a potential to down-regulate hypersensitivity reactions. Therefore, probiotic therapy is valuable against intestinal infections and may be beneficial in the treatment of Inflammatory Bowel Disease (IBD). Several in vitro tests are available to evaluate the probiotic potential of a LAB strain. However, an in vivo model is required to understand the interaction between the host immune system and the bacteria. During the last few years, zebrafish (Danio rerio) has gained interest as a promising vertebrate model in this field. This organism has been extensively used to study the interaction between the host and the microbiota, as well as the host immune response under several microbial infections. In this work, we report on the use of the zebrafish model to investigate in vivo the colonizing ability and the immunomodulatory effect of probiotic LAB. Methods: Lactobacillus strains belonging to different LAB species were fluorescently tagged and used to colonize germ-free zebrafish larvae gastrointestinal tract (GIT). Some of the strains had a well-documented probiotic effect (L. acidophilus LA5); while others presented an exopolysaccharide (EPS) producing phenotype, thus allowing evaluating the influence of EPS in the colonization and immunomodulatory effect. Bacteria colonization was monitored for 72 h by direct observation in real time using fluorescent microscopy. CFU count per larva was also evaluated at different times. The immunomodulatory effect was assessed analysing the differential expression of several innate immune system genes (MyD88, NF-κB, Tlr4, Il1β and Il10) by qRT- PCR. The anti-inflammatory effect was evaluated using a chemical enterocolitis zebrafish model. The protective effect against a pathogen was also studied. To that end, a challenge test was developed using a fluorescently tagged pathogen (Vibrio anguillarum-GFP+). The progression of the infection was monitored up to 3 days using a fluorescent stereomicroscope. Mortality rates and CFU counts were also registered. Results and conclusions: Larvae exposed to EPS-producing bacteria showed a higher fluorescence and CFU count than those colonized with no-EPS phenotype LAB. In the same way, qRT-PCR results revealed an immunomodulatory effect on the host after the administration of the strains with probiotic activity. A downregulation of proinflammatory cytoquines as well as other cellular mediators of inflammation was observed. The anti-inflammatory effect was found to be particularly marked following exposure to LA% strain, as well as EPS producing strains. Furthermore, the challenge test revealed a protective effect of probiotic administration. As a matter of fact, larvae fed with probiotics showed a decrease in the mortality rate ranging from 20 to 35%. Discussion: In this work, we developed a promising model, based on the use of gnotobiotic zebrafish coupled with a bacterial fluorescent tagging in order to evaluate the probiotic potential of different LAB strains. We have successfully used this system to monitor in real time the colonization and persistence of exogenous LAB within the gut of zebrafish larvae, to evaluate their immunomodulatory effect and for in vivo competition assays. This approach could bring further insights into the complex microbial-host interactions at intestinal level.

Keywords: gnotobiotic, immune system, lactic acid bacteria, probiotics, zebrafish

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Authors: Şeyma Dombaycıoğlu, Hilal Köse, Ali Osman Aydın, Hatem Akbulut

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Rechargeable lithium ion batteries (LIBs) have been considered as one of the most attractive energy storage choices for laptop computers, electric vehicles and cellular phones owing to their high energy and power density. Compared with conventional carbonaceous materials, transition metal oxides (TMOs) have attracted great interests and stand out among versatile novel anode materials due to their high theoretical specific capacity, wide availability and good safety performance. ZnO, as an anode material for LIBs, has a high theoretical capacity of 978 mAh g-1, much higher than that of the conventional graphite anode (∼370 mAhg-1). However, several major problems such as poor cycleability, resulting from the severe volume expansion and contraction during the alloying-dealloying cycles with Li+ ions and the associated charge transfer process, the pulverization and the agglomeration of individual particles, which drastically reduces the total entrance/exit sites available for Li+ ions still hinder the practical use of ZnO powders as an anode material for LIBs. Therefore, a great deal of effort has been devoted to overcome these problems, and many methods have been developed. In most of these methods, it is claimed that carbon nanotubes (CNTs) will radically improve the performance of batteries, because their unique structure may especially enhance the kinetic properties of the electrodes and result in an extremely high specific charge compared with the theoretical limits of graphitic carbon. Due to outstanding properties of CNTs, MWCNT buckypaper substrate is considered a buffer material to prevent mechanical disintegration of anode material during the battery applications. As the bridge connecting the positive and negative electrodes, the electrolyte plays a critical role affecting the overall electrochemical performance of the cell including rate, capacity, durability and safety. Commercial electrolytes for Li-ion batteries normally consist of certain lithium salts and mixed organic linear and cyclic carbonate solvents. Most commonly, LiPF6 is attributed to its remarkable features including high solubility, good ionic conductivity, high dissociation constant and satisfactory electrochemical stability for commercial fabrication. Besides LiPF6, LiBF4 is well known as a conducting salt for LIBs. LiBF4 shows a better temperature stability in organic carbonate based solutions and less moisture sensitivity compared to LiPF6. In this work, free standing zinc oxide (ZnO) and multiwalled carbon nanotube (MWCNT) nanocomposite materials were prepared by a sol gel technique giving a high capacity anode material for lithium ion batteries. Electrolyte solutions (including 1 m Li+ ion) were prepared with different Li salts in glove box. For this purpose, LiPF6 and LiBF4 salts and also mixed of these salts were solved in EC:DMC solvents (1:1, w/w). CR2016 cells were assembled by using these prepared electrolyte solutions, the ZnO/MWCNT buckypaper nanocomposites as working electrodes, metallic lithium as cathode and polypropylene (PP) as separator. For investigating the effect of different Li salts on the electrochemical performance of ZnO/MWCNT nanocomposite anode material electrochemical tests were performed at room temperature.

Keywords: anode, electrolyte, Li-ion battery, ZnO/MWCNT

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Authors: Turky Al-Qahtani, Roustem Miftahof

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Introduction: Under common assumptions of excitabi-lity, morphological (cellular) homogeneity, and spatial structural anomalies added as required, it has been shown that biological systems are able to display travelling wave dynamics. Being not self-sustainable, existence depends on the electrophysiological state of transmembrane ion channels and it requires an extrinsic/intrinsic periodic source. However, organs in the body are highly multicellular, heterogeneous, and their functionality is the outcome of electro-mechanical conjugation, rather than excitability only. Thus, peristalsis in the gut relies on spatiotemporal myoelectrical pattern formations between the mechanical, represented by smooth muscle cells (SM), and the control, comprised of a chain of primary sensory and motor neurones, components. Synaptically linked through the afferent and efferent pathways, they form a functional unit (FU) of the gut. Aims: These are: i) to study numerically the complex dynamics, and ii) to investigate the possibility of self-sustained myoelectrical activity in the FU. Methods: The FU recreates the following sequence of physiological events: deformation of mechanoreceptors of located in SM; generation and propagation of electrical waves of depolarisation - spikes - along the axon to the soma of the primary neurone; discharge of the primary neurone and spike propagation towards the motor neurone; burst of the motor neurone and transduction of spikes to SM, subsequently producing forces of contraction. These are governed by a system of nonlinear partial and ordinary differential equations being a modified version of the Hodgkin-Huxley model and SM fibre mechanics. In numerical experiments; the source of excitation is mechanical stretches of SM at a fixed amplitude and variable frequencies. Results: Low frequency (0.5 < v < 2 Hz) stimuli cause the propagation of spikes in the neuronal chain and, finally, the generation of active forces by SM. However, induced contractions are not sufficient to initiate travelling wave dynamics in the control system. At frequencies, 2 < v < 4 Hz, multiple low amplitude and short-lasting contractions are observed in SM after the termination of stretching. For frequencies (0.5 < v < 4 Hz), primary and sensory neurones demonstrate strong connectivity and coherent electrical activity. Significant qualitative and quantitative changes in dynamics of myoelectical patterns with a transition to a self-organised mode are recorded with the high degree of stretches at v = 4.5 Hz. Increased rates of deformation lead to the production of high amplitude signals at the mechanoreceptors with subsequent self-sustained excitation within the neuronal chain. Remarkably, the connection between neurones weakens resulting in incoherent firing. Further increase in a frequency of stimulation (v > 4.5 Hz) has a detrimental effect on the system. The mechanical and control systems become disconnected and exhibit uncoordinated electromechanical activity. Conclusion: To our knowledge, the existence of periodic activity in a multicellular, functionally heterogeneous biological system with mechano-electrical dynamics, such as the FU, has been demonstrated for the first time. These findings support the notion of possible peristalsis in the gut even in the absence of intrinsic sources - pacemaker cells. Results could be implicated in the pathogenesis of intestinal dysrythmia, a medical condition associated with motor dysfunction.

Keywords: complex dynamics, functional unit, the gut, dysrythmia

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