Search results for: pseudo-random sequence

Authors: Bello Gonzalez T. D. J., Setten Van M., Essen Van A., Brouwer M., Veldman K. T.

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Resistance to fluoroquinolones (FQ) has evolved increasingly over the years, posing a significant challenge for the treatment of human infections, particularly gastrointestinal tract infections caused by zoonotic bacteria transmitted through the food chain and environment. In broiler chickens, a relatively high proportion of FQ resistance has been observed in Escherichia coli indicator, Salmonella and Campylobacter isolates. We hypothesize that flumequine (Flu), used as a secondary choice for the treatment of poultry infections, could potentially be associated with a high proportion of FQ resistance. To evaluate this hypothesis, we used an in vitro fermentation chicken caeca model. Two continuous single-stage fermenters were used to simulate in real time the physiological conditions of the chicken caeca microbial content (temperature, pH, caecal content mixing, and anoxic environment). A pool of chicken caecal content containing FQ-resistant E. coli obtained from chickens at slaughter age was used as inoculum along with a spiked FQ-susceptible Campylobacter jejuni strain isolated from broilers. Flu was added to one of the fermenters (Flu-fermenter) every 24 hours for two days to evaluate the selection and maintenance of FQ resistance over time, while the other served as a control (C-Fermenter). The experiment duration was 5 days. Samples were collected at three different time points: before, during and after Flu administration. Serial dilutions were plated on Butzler culture media with and without Flu (8mg/L) and enrofloxacin (4mg/L) and on MacConkey culture media with and without Flu (4mg/L) and enrofloxacin (1mg/L) to determine the proportion of resistant strains over time. Positive cultures were identified by mass spectrometry and matrix-assisted laser desorption/ionization (MALDI). A subset of the obtained isolates were used for Whole Genome Sequencing analysis. Over time, E. coli exhibited positive growth in both fermenters, while C. jejuni growth was detected up to day 3. The proportion of Flu-resistant E. coli strains recovered remained consistent over time after antibiotic selective pressure, while in the C-fermenter, a decrease was observed at day 5; a similar pattern was observed in the enrofloxacin-resistant E. coli strains. This suggests that Flu might play a role in the selection and persistence of enrofloxacin resistance, compared to C-fermenter, where enrofloxacin-resistant E. coli strains appear at a later time. Furthermore, positive growth was detected from both fermenters only on Butzler plates without antibiotics. A subset of C. jejuni strains from the Flu-fermenter revealed that those strains were susceptible to ciprofloxacin (MIC < 0.12 μg/mL). A selection of E. coli strains from both fermenters revealed the presence of plasmid-mediated quinolone resistance (PMQR) (qnr-B19) in only one strain from the C-fermenter belonging to sequence type (ST) 48, and in all from Flu-fermenter belonged to ST189. Our results showed that Flu selective impact on PMQR-positive E. coli strains, while no effect was observed in C. jejuni. Maintenance of Flu-resistance was correlated with antibiotic selective pressure. Further studies into antibiotic resistance gene transfer among commensal and zoonotic bacteria in the chicken caeca content may help to elucidate the resistance spread mechanisms.

Keywords: fluoroquinolone-resistance, escherichia coli, campylobacter jejuni, in vitro model

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Authors: Zeina Nasr, Bashar Merheb

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The honey bee is a social insect that had driven the human interest much more than any other organism. Beekeeping practices dated the appearance of Man on earth and now it provides a hobby or a secondary work that contributes to the family revenue and requires a little time engagement and money investment. Honey production is not the only contribution of honey bees to the economy, since honey bees play an important role in the pollination. Bee keeping in Lebanon is an important part of the agricultural economy. However, a growing concern about bees is spreading globally, due to an accelerated decline of bees colonies. This raises the alert to preserve and protect local bee strains against uncontrolled introduction of foreign strains and invasive parasitic species. Mitochondrial DNA (mtDNA) markers are commonly used in studying genetic variation in the Apis mellifera species. The DraI-COI-COII test is based on the analysis of the intergenic region between the two genes COI and COII. The different honey bee strains differ in the presence or absence of the p sequence and the number of Q sequences present. A. m. syriaca belonging to the lineage Z, is the native honey bee subspecies in Lebanon, Syria, Jordan, and Palestine. A. m. syriaca is known for its high defensiveness, even though it has many important advantages. However, commercial breeder strains, such as the Italian (A. m. ligustica), and Carniolan (A. m. carnica) strains, have been introduced by beekeepers and regularly used for honey production. This raises worries about the disappearance of the local subspecies. It is obvious that identifying A. m. syriaca colonies and protecting them against uncontrolled mating with other bee strains is a crucial step to protect and improve the original local strain. This study aims to reveal the existing sub-species of honey bee in Akkar – Lebanon and to assess the influence of introgression on the hybridization of the local strain. This will help to identify areas of pure A.m. syriaca population over this district to be considered in choosing syriaca reserves. We collected samples of bees from different regions of Akkar district in order to perform mtDNA analysis. We determined the restriction fragments length of the intergenic region COI-COII, using the restriction enzyme DraI. The results showed both the C and the Z lineages. Four restriction patterns were identified among the restriction maps of the studied samples. The most abundant mitochondrial lineage is the Z lineage constituting about 60% of the identified samples. Al-Dreib region reported the lowest introgression with foreign mtDNA of 21% making it the most suitable area for a genetic reserve of syriaca in Akkar based on its lowest introgression and suitable environment in addition to the attitude of local beekeepers to conserve the local strain. Finally, this study is the first step in constructing conservation programs for the preservation of the local strain and should be generalized to the whole Lebanese population, consistent with the effort done in neighboring countries.

Keywords: Akkar Lebanon, Apis millefera syriaca, DraI-COI-COII test, mitochondrial DNA

Procedia PDF Downloads 276

Authors: Vikas Kumar, Kailash Chandra, Kaomud Tyagi

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The insect order Thysanoptera includes small insects commonly called thrips. As insect vectors, only thrips are capable of Tospoviruses transmission (genus Tospovirus, family Bunyaviridae) affecting various crops. Currently, fifteen species of subfamily Thripinae (Thripidae) have been reported as vectors for tospoviruses. Frankliniella schultzei, which is reported as act as a vector for at least five tospovirses, have been suspected to be a species complex with more than one species. It is one of the historical unresolved issues where, two species namely, F. schultzei Trybom and F. sulphurea Schmutz were erected from South Africa and Srilanaka respectively. These two species were considered to be valid until 1968 when sulphurea was treated as colour morph (pale form) and synonymised under schultzei (dark form) However, these two have been considered as valid species by some of the thrips workers. Parallel studies have indicated that brown form of schultzei is a vector for tospoviruses while yellow form is a non-vector. However, recent studies have shown that yellow populations have also been documented as vectors. In view of all these facts, it is highly important to have a clear understanding whether these colour forms represent true species or merely different populations with different vector carrying capacities and whether there is some hidden diversity in 'Frankliniella schultzei species complex'. In this study, we aim to study the 'Frankliniella schultzei species complex' with molecular spectacles with DNA data from India and Australia and Africa. A total of fifty-five specimens was collected from diverse locations in India and Australia. We generated molecular data using partial fragments of mitochondrial cytochrome c oxidase I gene (mtCOI) and 28S rRNA gene. For COI dataset, there were seventy-four sequences, out of which data on fifty-five was generated in the current study and others were retrieved from NCBI. All the four different tree construction methods: neighbor-joining, maximum parsimony, maximum likelihood and Bayesian analysis, yielded the same tree topology and produced five cryptic species with high genetic divergence. For, rDNA, there were forty-five sequences, out of which data on thirty-nine was generated in the current study and others were retrieved from NCBI. The four tree building methods yielded four cryptic species with high bootstrap support value/posterior probability. Here we could not retrieve one cryptic species from South Africa as we could not generate data on rDNA from South Africa and sequence for rDNA from African region were not available in the database. The results of multiple species delimitation methods (barcode index numbers, automatic barcode gap discovery, general mixed Yule-coalescent, and Poisson-tree-processes) also supported the phylogenetic data and produced 5 and 4 Molecular Operational Taxonomic Units (MOTUs) for mtCOI and 28S dataset respectively. These results of our study indicate the likelihood that F. sulphurea may be a valid species, however, more morphological and molecular data is required on specimens from type localities of these two species and comparison with type specimens.

Keywords: DNA barcoding, species complex, thrips, species delimitation

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Authors: Sukran Acikel, Mehmet Ekmekci, Otgonbayar Namkhai

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Lignite coal fields in western Turkey generally occurs in tensional Neogene basins bordered by major faults. Karacahisar coal field in Mugla province of western Turkey is a large Neogene basin filled with alternation of silisic and calcerous layers. The basement of the basin is composed of mainly karstified carbonate rocks of Mesozoic and schists of Paleozoic age. The basement rocks are exposed at highlands surrounding the basin. The basin fill deposits forms shallow, low yield and local aquifers whereas karstic carbonate rock masses forms the major aquifer in the region. The karstic aquifer discharges through a spring zone issuing at intersection of two major faults. Municipal water demand in Bodrum city, a touristic attraction area is almost totally supplied by boreholes tapping the karstic aquifer. A well field has been constructed on the eastern edge of the coal basin, which forms a ridge separating two Neogene basins. A major concern was raised about the plausible impact of mining activities on groundwater system in general and on water supply well field in particular. The hydrogeological studies carried out in the area revealed that the coal seam is located below the groundwater level. Mining operations will be affected by groundwater inflow to the pits, which will require dewatering measures. Dewatering activities in mine sites have two-sided effects: a) lowers the groundwater level at and around the pit for a safe and effective mining operation, b) continuous dewatering causes expansion of cone of depression to reach a spring, stream and/or well being utilized by local people, capturing their water. Plausible effect of mining operations on the flow of the spring zone was another issue of concern. Therefore, a detailed representative hydrogeological conceptual model of the site was developed on the basis of available data and field work. According to the hydrogeological conceptual model, dewatering of Neogene layers will not hydraulically affect the water supply wells, however, the ultimate perimeter of the open pit will expand to intersect the well field. According to the conceptual model, the coal seam is separated from the bottom by a thick impervious clay layer sitting on the carbonate basement. Therefore, the hydrostratigraphy does not allow a hydraulic interaction between the mine pit and the karstic carbonate rock aquifer. However, the structural setting in the basin suggests that deep faults intersecting the basement and the Neogene sequence will most probably carry the deep groundwater up to a level above the bottom of the pit. This will require taking necessary measure to lower the piezometric level of the carbonate rock aquifer along the faults. Dewatering the carbonate rock aquifer will reduce the flow to the spring zone. All findings were put together to recommend a strategy for safe and effective mining operation.

Keywords: conceptual model, dewatering, groundwater, mining operation

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Authors: Ruth Goldschmidt, Vyacheslav Kalchenko, Lilah Agemy, Rachel Elmoalem, Avigdor Scherz

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Vascular Targeted Photodynamic therapy (VTP) is a new modality for selective cancer treatment that leads to the complete tumor ablation. A photosensitizer, a bacteriochlorophyll derivative in our case, is first administered to the patient and followed by the illumination of the tumor area, by a near-IR laser for its photoactivation. The photoactivated drug releases reactive oxygen species (ROS) in the circulation, which reacts with blood cells and the endothelium leading to the occlusion of the blood vasculature. If the blood vessels are only partially closed, the tumor may recover, and cancer cells could survive. On the other hand, excessive treatment may lead to toxicity of healthy tissues nearby. Simultaneous VTP monitoring and image processing independent of the photoexcitation laser has not yet been reported, to our knowledge. Here we present a method for blood flow monitoring, using a real-time laser speckle imaging (RTLSI) in the tumor during VTP. We have synthesized over the years a library of bacteriochlorophyll derivatives, among them WST11 and STL-6014. Both are water soluble derivatives that are retained in the blood vasculature through their partial binding to HSA. WST11 has been approved in Mexico for VTP treatment of prostate cancer at a certain drug dose, and time/intensity of illumination. Application to other bacteriochlorophyll derivatives or other cancers may require different treatment parameters (such as light/drug administration). VTP parameters for STL-6014 are still under study. This new derivative mainly differs from WST11 by its lack of the central Palladium, and its conjugation to an Arg-Gly-Asp (RGD) sequence. RGD is a tumor-specific ligand that is used for targeting the necrotic tumor domains through its affinity to αVβ3 integrin receptors. This enables the study of cell-targeted VTP. We developed a special RTLSI module, based on Labview software environment for data processing. The new module enables to acquire raw laser speckle images and calculate the values of the laser temporal statistics of time-integrated speckles in real time, without additional off-line processing. Using RTLSI, we could monitor the tumor’s blood flow following VTP in a CT26 colon carcinoma ear model. VTP with WST11 induced an immediate slow down of the blood flow within the tumor and a complete final flow arrest, after some sporadic reperfusions. If the irradiation continued further, the blood flow stopped also in the blood vessels of the surrounding healthy tissue. This emphasizes the significance of light dose control. Using our RTLSI system, we could prevent any additional healthy tissue damage by controlling the illumination time and restrict blood flow arrest within the tumor only. In addition, we found that VTP with STL-6014 was the most effective when the photoactivation was conducted 4h post-injection, in terms of tumor ablation success in-vivo and blood vessel flow arrest. In conclusion, RTSLI application should allow to optimize VTP efficacy vs. toxicity in both the preclinical and clinical arenas.

Keywords: blood vessel occlusion, cancer treatment, photodynamic therapy, real time imaging

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Authors: Mohsina Aktar, Bishawjit Mallick

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Bangladesh faces the multidimensional manifestations of climate change e.g. flood, cyclone, sea level rise, drainage congestion, salinity, etc. This study aimed at to find out the community’s perception of the perceived impact of climate change on their wetland resource based livelihood, to analyze their present livelihood scenario and to find out required institutional setup to strengthen present livelihood scenario. Therefore, this study required both quantitative analysis like quantification of wetland resources, occupation, etc. and also exploratory information like policy and institutional reform. For quantitative information 200 questionnaire survey and in some cases observation survey and for socially shareable qualitative and quantitative issues case study and focus group discussion were conducted. In-Depth interview was conducted for socially non-shareable qualitative issues. The overall findings of this study have been presented maintaining a sequence- perception about climate change effect, livelihood scenario and required institutional support of the wetland community. Flood has been ranked where cyclone effect is comparatively less disastrous in this area. Heavy rainfall comes after the cyclone. Female members responded almost same about the ranking and effects of frequently occurred and devastating effects of climate change. People are much more aware of the impact of climate change. Training of Care in RVCC project helps to increase their knowledge level. If the level of education can be increased, people can fight against calamity and poverty with more confidence. People seem to overcome the problems of water logging and thus besides involving in Hydroponics (33.3%) as prime occupation in monsoon; they are also engaged in other business related activities. January to May is the low-income season for the farmers. But some people don’t want to change their traditional occupation and their age is above 45. The young earning member wants to utilize their lean income period by alternative occupation. People who do not have own land and performing water transportation or other types of occupation are now interested about Hydroponics. People who give their land on rent are now thinking about renting their land in monsoon as through that they can earn a sound amount rather than get nothing. What they require is just seed, training, and capital. Present marketing system faces the problem of communication. So this sector needed to be developed. Involvement of women in income earning activity is very low (5.1%), and 100% women are housewives. They became inferior due to their educational level and dominance of their husband. Only one NGO named BCAS (Bangladesh Center for Advanced Studies) has been found engage training facilities and advocacy for this purpose. Upazilla agricultural extension office like other GO remains inactive to give support the community for extension and improvement of Hydroponics agriculture. If the community gets proper support and inspiration, they can fight against crisis of low-income and climate change, with the Hydroponics cultivation system successfully.

Keywords: wetland community, hydroponics, climate change adaptation, livelihood

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Authors: Davit Pipoyan, Meline Beglaryan, Nicolò Merendino

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Mining industry is one of the priority sectors of Armenian economy. Along with the solution of some socio-economic development, it brings about numerous environmental problems, especially toxic element pollution, which largely influences the safety of agricultural products. In addition, accumulation of toxic elements in agricultural products, mainly in edible parts of plants represents a direct pathway for their penetration into the human food chain. In Armenia, the share of plant origin food in overall diet is significantly high, so estimation of dietary intakes of toxic trace elements via consumption of selected fruits and vegetables are of great importance for observing the underlying health risks. Therefore, the present study was aimed to assess dietary exposure of potentially toxic trace elements through the intake of locally grown fruits and vegetables in Akhtala community (Armenia), where not only mining industry is developed, but also cultivation of fruits and vegetables. Moreover, this investigation represents one of the very first attempts to estimate human dietary exposure of potentially toxic trace elements in the study area. Samples of some commonly grown fruits and vegetables (fig, cornel, raspberry, grape, apple, plum, maize, bean, potato, cucumber, onion, greens) were randomly collected from several home gardens located near mining areas in Akhtala community. The concentration of Cu, Mo, Ni, Cr, Pb, Zn, Hg, As and Cd in samples were determined by using an atomic absorption spectrophotometer (AAS). Precision and accuracy of analyses were guaranteed by repeated analysis of samples against NIST Standard Reference Materials. For a diet study, individual-based approach was used, so the consumption of selected fruits and vegetables was investigated through food frequency questionnaire (FFQ). Combining concentration data with contamination data, the estimated daily intakes (EDI) and cumulative daily intakes were assessed and compared with health-based guidance values (HBGVs). According to the determined concentrations of the studied trace elements in fruits and vegetables, it can be stressed that some trace elements (Cu, Ni, Pb, Zn) among the majority of samples exceeded maximum allowable limits set by international organizations. Meanwhile, others (Cr, Hg, As, Cd, Mo) either did not exceed these limits or still do not have established allowable limits. The obtained results indicated that only for Cu the EDI values exceeded dietary reference intake (0.01 mg/kg/Bw/day) for some investigated fruits and vegetables in decreasing order of potato > grape > bean > raspberry > fig > greens. In contrast to this, for combined consumption of selected fruits and vegetables estimated cumulative daily intakes exceeded reference doses in the following sequence: Zn > Cu > Ni > Mo > Pb. It may be concluded that habitual and combined consumption of the above mentioned fruits and vegetables can pose a health risk to the local population. Hence, further detailed studies are needed for the overall assessment of potential health implications taking into consideration adverse health effects posed by more than one toxic trace element.

Keywords: daily intake, dietary exposure, fruits, trace elements, vegetables

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Authors: Larysa Novak-Kalyayeva, Aleksander Kuczabski, Orystlava Sydorchuk, Nataliia Fersman, Tatyana Zemlinskaia

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The article presents the results of the creative analysis and comparison of trends in the development of the theory of public administration during the period from the second half of the 20^th to the beginning of the 21^st century. The process of conceptualization of the priorities of public administration in the dynamics of reforming was held under the influence of such factors as globalization, integration, information and technological changes and human rights is examined. The priorities of the social state in the concepts of the second half of the 20^th century are studied. Peculiar approaches to determining the priorities of public administration in the countries of "Soviet dictatorship" in Central and Eastern Europe in the same period are outlined. Particular attention is paid to the priorities of public administration regarding the interaction between public power and society and the development of conceptual foundations for the modern managerial process. There is a thought that the dynamics of the formation of concepts of the European governance is characterized by the sequence of priorities: from socio-economic and moral-ethical to organizational-procedural and non-hierarchical ones. The priorities of the "welfare state" were focused on the decent level of material wellbeing of population. At the same time, the conception of "minimal state" emphasized priorities of human responsibility for their own fate under the conditions of minimal state protection. Later on, the emphasis was placed on horizontal ties and redistribution of powers and competences of "effective state" with its developed procedures and limits of responsibility at all levels of government and in close cooperation with the civil society. The priorities of the contemporary period are concentrated on human rights in the concepts of "good governance" and all the following ones, which recognize the absolute priority of public administration with compliance, provision and protection of human rights. There is a proved point of view that civilizational changes taking place under the influence of information and technological imperatives also stipulate changes in priorities, redistribution of emphases and update principles of managerial concepts on the basis of publicity, transparency, departure from traditional forms of hierarchy and control in favor of interactivity and inter-sectoral interaction, decentralization and humanization of managerial processes. The necessity to permanently carry out the reorganization, by establishing the interaction between different participants of public power and social relations, to establish a balance between political forces and social interests on the basis of mutual trust and mutual understanding determines changes of social, political, economic and humanitarian paradigms of public administration and their theoretical comprehension. The further studies of theoretical foundations of modern public administration in interdisciplinary discourse in the context of ambiguous consequences of the globalizational and integrational processes of modern European state-building would be advisable. This is especially true during the period of political transformations and economic crises which are the characteristic of the contemporary Europe, especially for democratic transition countries.

Keywords: concepts of public administration, democratic transition countries, human rights, the priorities of public administration, theory of public administration

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Authors: Su Mon Saw, Joe Rothnagel

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Short open reading frames (sORFs) located in 5’UTR of mRNAs are known as uORFs. Characterization of uORF-encoded peptides (uPEPs) i.e., a subset of short open reading frame encoded peptides (sPEPs) and their translation regulation lead to understanding of causes of genetic disease, proteome complexity and development of treatments. Existence of uORFs within cellular proteome could be detected by LC-MS/MS. The ability of uORF to be translated into uPEP and achievement of uPEP identification will allow uPEP’s characterization, structures, functions, subcellular localization, evolutionary maintenance (conservation in human and other species) and abundance in cells. It is hypothesized that a subset of sORFs are translatable and that their encoded sPEPs are functional and are endogenously expressed contributing to the eukaryotic cellular proteome complexity. This project aimed to investigate whether sORFs encode functional peptides. Liquid chromatography-mass spectrometry (LC-MS) and bioinformatics were thus employed. Due to probable low abundance of sPEPs and small in sizes, the need for efficient peptide enrichment strategies for enriching small proteins and depleting the sub-proteome of large and abundant proteins is crucial for identifying sPEPs. Low molecular weight proteins were extracted using SDS-PAGE from Human Embryonic Kidney (HEK293) cells and Strong Cation Exchange Chromatography (SCX) from secreted HEK293 cells. Extracted proteins were digested by trypsin to peptides, which were detected by LC-MS/MS. The MS/MS data obtained was searched against Swiss-Prot using MASCOT version 2.4 to filter out known proteins, and all unmatched spectra were re-searched against human RefSeq database. ProteinPilot v5.0.1 was used to identify sPEPs by searching against human RefSeq, Vanderperre and Human Alternative Open Reading Frame (HaltORF) databases. Potential sPEPs were analyzed by bioinformatics. Since SDS PAGE electrophoresis could not separate proteins <20kDa, this could not identify sPEPs. All MASCOT-identified peptide fragments were parts of main open reading frame (mORF) by ORF Finder search and blastp search. No sPEP was detected and existence of sPEPs could not be identified in this study. 13 translated sORFs in HEK293 cells by mass spectrometry in previous studies were characterized by bioinformatics. Identified sPEPs from previous studies were <100 amino acids and <15 kDa. Bioinformatics results showed that sORFs are translated to sPEPs and contribute to proteome complexity. uPEP translated from uORF of SLC35A4 was strongly conserved in human and mouse while uPEP translated from uORF of MKKS was strongly conserved in human and Rhesus monkey. Cross-species conserved uORFs in association with protein translation strongly suggest evolutionary maintenance of coding sequence and indicate probable functional expression of peptides encoded within these uORFs. Translation of sORFs was confirmed by mass spectrometry and sPEPs were characterized with bioinformatics.

Keywords: bioinformatics, HEK293 cells, liquid chromatography-mass spectrometry, ProteinPilot, Strong Cation Exchange Chromatography, SDS-PAGE, sPEPs

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Authors: M. A. C. S. Sampath Fernando, James M. Curran, Renate Meyer

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Model assessment, in the Bayesian context, involves evaluation of the goodness-of-fit and the comparison of several alternative candidate models for predictive accuracy and improvements. In posterior predictive checks, the data simulated under the fitted model is compared with the actual data. Predictive model accuracy is estimated using information criteria such as the Akaike information criterion (AIC), the Bayesian information criterion (BIC), the Deviance information criterion (DIC), and the Watanabe-Akaike information criterion (WAIC). The goal of an information criterion is to obtain an unbiased measure of out-of-sample prediction error. Since posterior checks use the data twice; once for model estimation and once for testing, a bias correction which penalises the model complexity is incorporated in these criteria. Cross-validation (CV) is another method used for examining out-of-sample prediction accuracy. Leave-one-out cross-validation (LOO-CV) is the most computationally expensive variant among the other CV methods, as it fits as many models as the number of observations. Importance sampling (IS), truncated importance sampling (TIS) and Pareto-smoothed importance sampling (PSIS) are generally used as approximations to the exact LOO-CV and utilise the existing MCMC results avoiding expensive computational issues. The reciprocals of the predictive densities calculated over posterior draws for each observation are treated as the raw importance weights. These are in turn used to calculate the approximate LOO-CV of the observation as a weighted average of posterior densities. In IS-LOO, the raw weights are directly used. In contrast, the larger weights are replaced by their modified truncated weights in calculating TIS-LOO and PSIS-LOO. Although, information criteria and LOO-CV are unable to reflect the goodness-of-fit in absolute sense, the differences can be used to measure the relative performance of the models of interest. However, the use of these measures is only valid under specific circumstances. This study has developed 11 models using normal, log-normal, gamma, and student’s t distributions to improve the PCR stutter prediction with forensic data. These models are comprised of four with profile-wide variances, four with locus specific variances, and three which are two-component mixture models. The mean stutter ratio in each model is modeled as a locus specific simple linear regression against a feature of the alleles under study known as the longest uninterrupted sequence (LUS). The use of AIC, BIC, DIC, and WAIC in model comparison has some practical limitations. Even though, IS-LOO, TIS-LOO, and PSIS-LOO are considered to be approximations of the exact LOO-CV, the study observed some drastic deviations in the results. However, there are some interesting relationships among the logarithms of pointwise predictive densities (lppd) calculated under WAIC and the LOO approximation methods. The estimated overall lppd is a relative measure that reflects the overall goodness-of-fit of the model. Parallel log-likelihood profiles for the models conditional on equal posterior variances in lppds were observed. This study illustrates the limitations of the information criteria in practical model comparison problems. In addition, the relationships among LOO-CV approximation methods and WAIC with their limitations are discussed. Finally, useful recommendations that may help in practical model comparisons with these methods are provided.

Keywords: cross-validation, importance sampling, information criteria, predictive accuracy

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Authors: Lara Bettinelli, Bernhard Glatz, Josef Fink

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Planning engineers and researchers use various calculation models with different levels of complexity, calculation efficiency and accuracy in dynamic calculations of railway bridges under high-speed traffic. When choosing a vehicle model to depict the dynamic loading on the bridge structure caused by passing high-speed trains, different goals are pursued: On the one hand, the selected vehicle models should allow the calculation of a bridge’s vibrations as realistic as possible. On the other hand, the computational efficiency and manageability of the models should be preferably high to enable a wide range of applications. The commonly adopted and straightforward vehicle model is the moving load model (MLM), which simplifies the train to a sequence of static axle loads moving at a constant speed over the structure. However, the MLM can significantly overestimate the structure vibrations, especially when resonance events occur. More complex vehicle models, which depict the train as a system of oscillating and coupled masses, can reproduce the interaction dynamics between the vehicle and the bridge superstructure to some extent and enable the calculation of more realistic bridge accelerations. At the same time, such multi-body models require significantly greater processing capacities and precise knowledge of various vehicle properties. The European standards allow for applying the so-called additional damping method when simple load models, such as the MLM, are used in dynamic calculations. An additional damping factor depending on the bridge span, which should take into account the vibration-reducing benefits of the vehicle-bridge interaction, is assigned to the supporting structure in the calculations. However, numerous studies show that when the current standard specifications are applied, the calculation results for the bridge accelerations are in many cases still too high compared to the measured bridge accelerations, while in other cases, they are not on the safe side. A proposal to calculate the additional damping based on extensive dynamic calculations for a parametric field of simply supported bridges with a ballasted track was developed to address this issue. In this contribution, several different approaches to determine the additional damping of the supporting structure considering the vehicle-bridge interaction when using the MLM are compared with one another. Besides the standard specifications, this includes the approach mentioned above and two additional recently published alternative formulations derived from analytical approaches. For a bridge catalogue of 65 existing bridges in Austria in steel, concrete or composite construction, calculations are carried out with the MLM for two different high-speed trains and the different approaches for additional damping. The results are compared with the calculation results obtained by applying a more sophisticated multi-body model of the trains used. The evaluation and comparison of the results allow assessing the benefits of different calculation concepts for the additional damping regarding their accuracy and possible applications. The evaluation shows that by applying one of the recently published redesigned additional damping methods, the calculation results can reflect the influence of the vehicle-bridge interaction on the design-relevant structural accelerations considerably more reliable than by using normative specifications.

Keywords: Additional Damping Method, Bridge Dynamics, High-Speed Railway Traffic, Vehicle-Bridge-Interaction

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Authors: Weili Jiang, Simon Cadavid Lopera, Klaus Drechsler

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Fuel cell vehicle has become the most competitive solution for the transportation sector in the hydrogen economy. Type IV pressure vessel is currently the most popular and widely developed technology for the on-board storage, based on their high reliability and relatively low cost. Due to the stringent requirement on mechanical performance, the pressure vessel is subject to great amount of composite material, a major cost driver for the hydrogen tanks. Evidently, the optimization of composite layup design shows great potential in reducing the overall material usage, yet requires comprehensive understanding on underlying mechanisms as well as the influence of different design parameters on mechanical performance. Given the type of materials and manufacturing processes by which the type IV pressure vessels are manufactured, the design and optimization are a nuanced subject. The manifold of stacking sequence and fiber orientation variation possibilities have an out-standing effect on vessel strength due to the anisotropic property of carbon fiber composites, which make the design space high dimensional. Each variation of design parameters requires computational resources. Using finite element analysis to evaluate different designs is the most common method, however, the model-ing, setup and simulation process can be very time consuming and result in high computational cost. For this reason, it is necessary to build a reliable automation scheme to set up and analyze the di-verse composite layups. In this research, the simulation process of different tank designs regarding various parameters is conducted and automatized in a commercial finite element analysis framework Abaqus. Worth mentioning, the modeling of the composite overwrap is automatically generated using an Abaqus-Python scripting interface. The prediction of the winding angle of each layer and corresponding thickness variation on dome region is the most crucial step of the modeling, which is calculated and implemented using analytical methods. Subsequently, these different composites layups are simulated as axisymmetric models to facilitate the computational complexity and reduce the calculation time. Finally, the results are evaluated and compared regarding the ultimate tank strength. By automatically modeling, evaluating and comparing various composites layups, this system is applicable for the optimization of the tanks structures. As mentioned above, the mechanical property of the pressure vessel is highly dependent on composites layup, which requires big amount of simulations. Consequently, to automatize the simulation process gains a rapid way to compare the various designs and provide an indication of the optimum one. Moreover, this automation process can also be operated for creating a data bank of layups and corresponding mechanical properties with few preliminary configuration steps for the further case analysis. Subsequently, using e.g. machine learning to gather the optimum by the data pool directly without the simulation process.

Keywords: type IV pressure vessels, carbon composites, finite element analy-sis, automation of simulation process

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Authors: Pamela Chia, Tay Yoong Chuan

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Introduction: Severe pulmonary hypertension (PH) patients requiring non-cardiac surgery risk have increased mortality rates ranging. These patients are plagued with cardiorespiratory failure, dysrhythmias and anticoagulation potentially with concurrent sepsis and renal insufficiency, perioperative morbidity. We present a deaf-mute patient with severe idiopathic PH emergently prepared for ruptured ectopic laparotomy. Case Report: A 20 year-old female, 62kg (BMI 25 kg/m2) with severe idiopathic PH (2DE Ejection Fraction was 41%, Pulmonary Artery Systolic Pressure (PASP) 105 mmHg, Right ventricle strain and hypertrophy) and selective mutism was rushed in for emergency laparotomy after presenting to the emergency department for abdominal pain. The patient had an NYHA Class II with room air SpO2 93-95%. While awaiting lung transplant, the patient takes warfarin, Sildanefil, Macitentan and even Selexipag for rising PASP. At presentation, vital signs: BP 95/63, HR 119 SpO2 88% (room air). Despite decreasing haemoglobin 14 to 10g/dL, INR 2.59 was reversed with prothrombin concentrate, and Vitamin K. ECG revealed Right Bundle Branch Block with right ventricular strain and x-ray showed cardiomegaly, dilated Right Ventricle, Pulmonary Arteries, basal atelectasis. Arterial blood gas showed compensated metabolic acidosis pH 7.4 pCO2 32 pO2 53 HCO3 20 BE -4 SaO2 88%. The cardiothoracic surgeon concluded no role for Extracorporeal Membrane Oxygenation (ECMO). We inserted invasive arterial and central venous lines with blood transfusion via an 18G cannula before the patient underwent a midline laparotomy, haemostasis of ruptured ovarian cyst with 2.4L of clots under general anesthesia and FloTrac cardiac output monitoring. Rapid sequence induction was done with Midazolam/Propofol, remifentanil infusion, and rocuronium. The patient was maintained on Desflurane. Blood products and colloids were transfused for further 1.5L blood loss. Postoperatively, the patient was transferred to the intensive care unit and was extubated uneventfully 7hours later. The patient went home a week later. Discussion: Emergency hemostasis laparotomy in anticoagulated WHO Class I PH patient awaiting lung transplant with no ECMO backup poses tremendous stress on the deaf-mute patient and the anesthesiologist. Balancing hemodynamics avoiding hypotension while awaiting hemostasis in the presence of pulmonary arterial dilators and anticoagulation requires close titration of volatiles, which decreases RV contractility. We review the contraindicated anesthetic agents (ketamine, N2O), choice of vasopressors in hypotension to maintain Aortic-right ventricular pressure gradients and nitric oxide use perioperatively. Conclusion: Interdisciplinary communication with a deaf-mute moribund patient and anesthesia considerations pose many rare challenges worth sharing.

Keywords: pulmonary hypertension, case report, warfarin reversal, emergency surgery

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Authors: Niklas Ravn-Boess, Nainita Bhowmick, Takamitsu Hattori, Shohei Koide, Christopher Park, Dimitris Placantonakis

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Background: Glioblastoma (GBM) is the most common and deadly primary brain malignancy in adults. Tumor propagation, brain invasion, and resistance to therapy critically depend on GBM stem-like cells (GSCs); however, the mechanisms that regulate GSC self-renewal are incompletely understood. Given the aggressiveness and poor prognosis of GBM, it is imperative to find biomarkers that could also translate into novel drug targets. Along these lines, we have identified a cell surface antigen, CD97 (ADGRE5), an adhesion G protein-coupled receptor (GPCR), that is expressed on GBM cells but is absent from non-neoplastic brain tissue. CD97 has been shown to promote invasiveness, angiogenesis, and migration in several human cancers, but its frequency of expression and functional role in regulating GBM growth and survival, and its potential as a therapeutic target has not been investigated. Design: We assessed CD97 mRNA and protein expression in patient derived GBM samples and cell lines using publicly available RNA-sequencing datasets and flow cytometry, respectively. To assess CD97 function, we generated shRNA lentiviral constructs that target a sequence in the CD97 extracellular domain (ECD). A scrambled shRNA (scr) with no predicted targets in the genome was used as a control. We evaluated CD97 shRNA lentivirally transduced GBM cells for Ki67, Annexin V, and DAPI. We also tested CD97 KD cells for their ability to self-renew using clonogenic tumorsphere formation assays. Further, we utilized synthetic Abs (sAbs) generated against the ECD of CD97 to test for potential antitumor effects using patient-derived GBM cell lines. Results: CD97 mRNA expression was expressed at high levels in all GBM samples available in the TCGA cohort. We found high levels of surface CD97 protein expression in 6/6 patient-derived GBM cell cultures, but not human neural stem cells. Flow cytometry confirmed downregulation of CD97 in CD97 shRNA lentivirally transduced cells. CD97 KD induced a significant reduction in cell growth in 3 independent GBM cell lines representing mesenchymal and proneural subtypes, which was accompanied by reduced (~20%) Ki67 staining and increased (~30%) apoptosis. Incubation of GBM cells with sAbs (20 ug/ ml) against the ECD of CD97 for 3 days induced GSC differentiation, as determined by the expression of GFAP and Tubulin. Using three unique GBM patient derived cultures, we found that CD97 KD attenuated the ability of GBM cells to initiate sphere formation by over 300 fold, consistent with an impairment in GSC self-renewal. Conclusion: Loss of CD97 expression in patient-derived GBM cells markedly decreases proliferation, induces cell death, and reduces tumorsphere formation. sAbs against the ECD of CD97 reduce tumorsphere formation, recapitulating the phenotype of CD97 KD, suggesting that sAbs that inhibit CD97 function exhibit anti-tumor activity. Collectively, these findings indicate that CD97 is necessary for the proliferation and survival of human GBM cells and identify CD97 as a promising therapeutically targetable vulnerability in GBM.

Keywords: adhesion GPCR, CD97, GBM stem cell, glioblastoma

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Authors: Franciska Weise, Ralph Bruder

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Due to the demographic change in Germany along with the declining birth rate and the increasing age of population, the share of older people in society is rising. This development is also reflected in the work force of German companies. Therefore companies should focus on improving ergonomics, especially in the area of age-related work design. Literature shows that studies on age-related work design have been carried out in the past, some of whose results have been put into practice. However, there is still a need for further research. One of the most important methods for taking into account the needs of an aging population is job rotation. This method aims at preventing or reducing health risks and inappropriate physical strain. It is conceived as a systematic change of workplaces within a group. Existing literature does not cover any methods for the investigation of the attitudes of employees towards job rotation. However, in order to evaluate job rotation, it is essential to have knowledge of the views of people towards rotation. In addition to an investigation of attitudes, the design of rotation plays a crucial role. The sequence of activities and the rotation frequency influence the worker and as well the work result. The evaluation of preliminary talks on the shop floor showed that team speakers and foremen share a common understanding of job rotation. In practice, different varieties of job rotation exist. One important aspect is the frequency of rotation. It is possible to rotate never, more than one time or even during every break, or more often than every break. It depends on the opportunity or possibility to rotate whenever workers want to rotate. From the preliminary talks some challenges can be derived. For example a rotation in the whole team is not possible, if a team member requires to be trained for a new task. In order to be able to determine the relation of the design and the attitude towards job rotation, a questionnaire is carried out in the vehicle manufacturing. The questionnaire will be employed to determine the different varieties of job rotation that exist in production, as well as the attitudes of workers towards those different frequencies of job rotation. In addition, younger and older employees will be compared with regard to their rotation frequency and their attitudes towards rotation. There are three kinds of age groups. Three questions are under examination. The first question is whether older employees rotate less frequently than younger employees. Also it is investigated to know whether the frequency of job rotation and the attitude towards the frequency of job rotation are interconnected. Moreover, the attitudes of the different age groups towards the frequency of rotation will be examined. Up to now 144 employees, all working in production, took part in the survey. 36.8 % were younger than thirty, 37.5 % were between thirty und forty-four and 25.7 % were above forty-five years old. The data shows no difference between the three age groups in relation to the frequency of job rotation (N=139, median=4, Chi²=.859, df=2, p=.651). Most employees rotate between six and seven workplaces per day. In addition there is a statistically significant correlation between the frequency of job rotation and the attitude towards the frequency (Spearman-Rho: 2-sided=.008, correlation coefficient=.223). Less than four workplaces per day are not enough for the employees. The third question, which differences can be found between older and younger people who rotate in a different way and with different attitudes towards job rotation, cannot be possible answered. Till now the data shows that younger people would like to rotate very often. Regarding to older people no correlation can be found with acceptable significance. The results of the survey will be used to improve the current practice of job rotation. In addition, the discussions during the survey are expected to help sensitize the employees with respect to rotation issues, and to contribute to optimizing rotation by means of qualification and an improved design of job rotation. Together with the employees and the results of the survey there must be found standards which show how to rotate in an ergonomic way while consider the attitude towards job rotation.

Keywords: job rotation, age-related work design, questionnaire, automotive industry

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Authors: Rauan Sakenov, Peter Bukovics, Peter Gaszler, Veronika Tokacs-Kollar, Beata Bugyi

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FH2 (formin homology-2) domains of several proteins, collectively known as formins, including DAAM, DAAM1 and mDia1, promote G-actin nucleation and elongation. FH2 domains of these formins exist as oligomers. Chain dimerization by ring structure formation serves as a structural basis for actin polymerization function of FH2 domain. Proper single chain configuration and specific interactions between its various regions are necessary for individual chains to form a dimer functional in G-actin nucleation and elongation. FH1 and WH2 domain-containing formins were shown to behave as intrinsically disordered proteins. Thus, the aim of this research was to study structural dynamics of FH2 domain of DAAM. To investigate structural features of FH2 domain of DAAM, molecular dynamics simulation of chain A of FH2 domain of DAAM solvated in water box in 50 mM NaCl was conducted at temperatures from 293.15 to 353.15K, with VMD 1.9.2, NAMD 2.14 and Amber Tools 21 using 2z6e and 1v9d PDB structures of DAAM was obtained on I-TASSER webserver. Calcium and ATP bound G-actin 3hbt PDB structure was used as a reference protein with well-described structural dynamics of denaturation. Topology and parameter information of CHARMM 2012 additive all-atom force fields for proteins, carbohydrate derivatives, water and ions were used in NAMD 2.14 and ff19SB force field for proteins in Amber Tools 21. The systems were energy minimized for the first 1000 steps, equilibrated and produced in NPT ensemble for 1ns using stochastic Langevin dynamics and the particle mesh Ewald method. Our root-mean square deviation (RMSD) analysis of molecular dynamics of chain A of FH2 domains of DAAM revealed similar insignificant changes of total molecular average RMSD values of FH2 domain of these formins at temperatures from 293.15 to 353.15K. In contrast, total molecular average RMSD values of G-actin showed considerable increase at 328K, which corresponds to the denaturation of G-actin molecule at this temperature and its transition from native, ordered, to denatured, disordered, state which is well-described in the literature. RMSD values of lasso and tail regions of chain A of FH2 domain of DAAM exhibited higher than total molecular average RMSD at temperatures from 293.15 to 353.15K. These regions are functional in intra- and interchain interactions and contain highly conserved tryptophan residues of lasso region, highly conserved GNYMN sequence of post region and amino acids of the shell of hydrophobic pocket of the salt bridge between Arg171 and Asp321, which are important for structural stability and ordered state of FH2 domain of DAAM and its functions in FH2 domain dimerization. In conclusion, higher than total molecular average RMSD values of lasso and post regions of chain A of FH2 domain of DAAM may explain disordered state of FH2 domain of DAAM at temperatures from 293.15 to 353.15K. Finally, absence of marked transition, in terms of significant changes in average molecular RMSD values between native and denatured states of FH2 domain of DAAM at temperatures from 293.15 to 353.15K, can make it possible to attribute these formins to the group of intrinsically disordered proteins rather than to the group of intrinsically ordered proteins such as G-actin.

Keywords: FH2 domain, DAAM, formins, molecular modelling, computational biophysics

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Authors: Olga A. Momot, Boris I. Synzynys, Alla A. Oudalova

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Obninsk is situated in the Kaluga region 100 km southwest of Moscow on the left bank of the Protva River. Several enterprises utilizing nuclear energy are operating in the town. A special attention in the region where radiation-hazardous facilities are located has traditionally been paid to radioactive gas and aerosol releases into the atmosphere; liquid waste discharges into the Protva river and groundwater pollution. Municipal intakes involve 34 wells arranged 15 km apart in a sequence north-south along the foot of the left slope of the Protva river valley. Northern and southern water intakes are upstream and downstream of the town, respectively. They belong to river valley intakes with mixed feeding, i.e. precipitation infiltration is responsible for a smaller part of groundwater, and a greater amount is being formed by overflowing from Protva. Water intakes are maintained by the Protva river runoff, the volume of which depends on the precipitation fallen out and watershed area. Groundwater contamination with tritium was first detected in a sanitary-protective zone of the Institute of Physics and Power Engineering (SRC-IPPE) by Roshydromet researchers when realizing the “Program of radiological monitoring in the territory of nuclear industry enterprises”. A comprehensive survey of the SRC-IPPE’s industrial site and adjacent territories has revealed that research nuclear reactors and accelerators where tritium targets are applied as well as radioactive waste storages could be considered as potential sources of technogenic tritium. All the above sources are located within the sanitary controlled area of intakes. Tritium activity in water of springs and wells near the SRC-IPPE is about 17.4 – 3200 Bq/l. The observed values of tritium activity are below the intervention levels (7600 Bq/l for inorganic compounds and 3300 Bq/l for organically bound tritium). The risk has being assessed to estimate possible effect of considered tritium concentrations on human health. Data on tritium concentrations in pipe-line drinking water were used for calculations. The activity of 3H amounted to 10.6 Bq/l and corresponded to the risk of such water consumption of ~ 3·10-7 year-1. The risk value given in magnitude is close to the individual annual death risk for population living near a NPP – 1.6·10-8 year-1 and at the same time corresponds to the level of tolerable risk (10-6) and falls within “risk optimization”, i.e. in the sphere for planning the economically sound measures on exposure risk reduction. To estimate the chemical risk, physical and chemical analysis was made of waters from all springs and wells near the SRC-IPPE. Chemical risk from groundwater contamination was estimated according to the EPA US guidance. The risk of carcinogenic diseases at a drinking water consumption amounts to 5·10-5. According to the classification accepted the health risk in case of spring water consumption is inadmissible. The compared assessments of risk associated with tritium exposure, on the one hand, and the dangerous chemical (e.g. heavy metals) contamination of Obninsk drinking water, on the other hand, have confirmed that just these chemical pollutants are responsible for health risk.

Keywords: radiation-hazardous facilities, water intakes, tritium, heavy metal, health risk

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Authors: Asma Ben Hmidene, Mizuho Hanaki, Kazuma Murakami, Kazuhiro Irie, Hiroko Isoda, Hideyuki Shigemori

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Type 2 diabetes mellitus (T2DM) and Alzheimer’s disease (AD) are characterized as a peripheral metabolic disorder and a degenerative disease of the central nervous system, respectively. It is now widely recognized that T2DM and AD share many pathophysiological features including glucose metabolism, increased oxidative stress and amyloid aggregation. Amyloid beta (Aβ) is the components of the amyloid deposits in the AD brain and while the component of the amyloidogenic peptide deposit in the pancreatic islets of Langerhans is identified as human islet amyloid polypeptide (hIAPP). These two proteins are originated from the amyloid precursor protein and have a high sequence similarity. Although the amino acid sequences of amyloidogenic proteins are diverse, they all adopt a similar structure in aggregates called cross-beta-spine. Add at that, extensive studies in the past years have found that like Aβ1-42, IAPP forms early intermediate assemblies as spherical oligomers, implicating that these oligomers possess a common folding pattern or conformation. These similarities can be used in the search for effective pharmacotherapy for DM, since potent therapeutic agents such as antioxidants with a catechol moiety, proved to inhibit Aβ aggregation, may play a key role in the inhibit the aggregation of hIAPP treatment of patients with DM. Tamarix gallica is one of the halophyte species having a powerful antioxidant system. Although it was traditionally used for the treatment of various liver metabolic disorders, there is no report about the use of this plant for the treatment or prevention of T2DM and AD. Therefore, the aim of this work is to investigate their protective effect towards T2DM and AD by isolation and identification of α-glucosidase inhibitors, with antioxidant potential, that play an important role in the glucose metabolism in diabetic patient, as well as, the polymerization of hIAPP and Aβ aggregation inhibitors. Structure-activity relationship study was conducted for both assays. And as for α-glucosidase inhibitors, their mechanism of action and their synergistic potential when applied with a very low concentration of acarbose were also suggesting that they can be used not only as α-glucosidase inhibitors but also be combined with established α-glucosidase inhibitors to reduce their adverse effect. The antioxidant potential of the purified substances was evaluated by DPPH and SOD assays. Th-T assay using 42-mer amyloid β-protein (Aβ42) for AD and hIAPP which is a 37-residue peptide secreted by the pancreatic β –cells for T2DM and Transmission electronic microscopy (TEM) were conducted to evaluate the amyloid aggragation of the actives substances. For α-glucosidase, p-NPG and glucose oxidase assays were performed for determining the inhibition potential and structure-activity relationship study. The Enzyme kinetic protocol was used to study the mechanism of action. From this research, it was concluded that polyphenols playing a role in the glucose metabolism and oxidative stress can also inhibit the amyloid aggregation, and that substances with a catechol and glucuronide moieties inhibiting amyloid-β aggregation, might be used to inhibit the aggregation of hIAPP.

Keywords: α-glucosidase inhibitors, amyloid aggregation inhibition, mechanism of action, polyphenols, structure activity relationship, synergistic potential, tamarix gallica

Procedia PDF Downloads 279

Authors: Sachin Mulmi Shrestha, Xin Fang, Hui Ye, Lihua Ren, Qinghua Ji, Ruihua Shi

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Background: Esophageal squamous cell carcinoma (ESCC) is a tumor arising from esophageal epithelial cells and is one of the major disease subtype in Asian countries, including China. Esophageal cancer is the 7th highest incidence based on the 2020 data of GLOBOCAN. The pathogenesis of cancer is still not well understood as many molecular and genetic basis of esophageal carcinogenesis has yet to be clearly elucidated. Circular RNAs are RNA molecules that are formed by back-splicing covalently joined 3′- and 5′-endsrather than canonical splicing, and recent data suggest circular RNAs could sponge miRNAs and are enriched with functional miRNA binding sites. Hence, we studied the mechanism of circular RNA, its biological function, and the relationship between microRNA in the carcinogenesis of ESCC. Methods: 4 pairs of normal and esophageal cancer tissues were collected in Zhongda hospital, affiliated to Southeast University, and high-throughput RNA sequencing was done. The result revealed that circ_0032746 was upregulated, and thus we selected circ_0032746 for further study. The backsplice junction of circRNA was validated by sanger sequence, and stability was determined by RNASE R assay. The binding site of circRNA and microRNA was predicted by circinteractome,mirandaand RNAhybrid database. Furthermore, circRNA was silenced by siRNA and then by lentivirus. The regulatory axis of circ0032746/miR4270 was validated by shRNA, mimic, and inhibitor transfection. Then, in vitro experiments were performed to assess the role of circ0032746 on proliferation (CCK-8 assay and colon formation assay), migration and invasion (Transewell assay), and apoptosis of ESCC. Results: The upregulated circ0032746 was validated in 9 pairs of tissues and 5 types of cell lines by qPCR, which showed high expression and was statistically significant (P<0.005) ). Upregulated circ0032746 was silenced by shRNA, which showed significant knockdown in KYSE 30 and TE-1 cell lines expression compared to control. Nuclear and cytoplasmic mRNA fraction experiment displayed the cytoplasmic location of circ0032746. The sponging of miR4270 was validated by co-transfection of sh-circ0032746 and mimic or inhibitor. Transfection with mimic showed the decreased expression of circ_0032746, whereas inhibitor inhibited the result. In vitro experiments showed that silencing of circ_0032746 inhibited the proliferation, migration, and invasion compared to the negative control group. The apoptosis was seen higher in a knockdown group than in the control group. Furthermore, 11 common mircoRNA target mRNAs were predicted by Targetscan, MirTarbase, and miRanda database, which may further play role in the pathogenesis. Conclusion: Our results showed that novel circ_0032746 is upregulated in ESCC and plays role in itsoncogenicity. Silencing of circ_0032746 inhibits the proliferation and migration of ESCC whereas increases the apoptosis of cancer cells. Hence, circ0032746 acts as an oncogene in ESCC by sponging with miR4270 and could be a potential biomarker in the diagnosis of ESCC in the future.

Keywords: circRNA, esophageal squamous cell carcinoma, microRNA, upregulated

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Authors: Jean Pierre Kabue, Emma Meader, Afsatou Ndama Traore, Paul R. Hunter, Natasha Potgieter

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Norovirus is now considered the most common cause of outbreaks of nonbacterial gastroenteritis. Limited data are available for Norovirus strains in Africa, especially in rural and peri-urban areas. Despite the excessive burden of diarrhea disease in developing countries, Norovirus infections have been to date mostly reported in developed countries. There is a need to investigate intensively the role of viral agents associated with diarrhea in different settings in Africa continent. To determine the prevalence and genetic diversity of Norovirus strains circulating in the rural communities in the Limpopo Province, South Africa and investigate the genetic relationship between Norovirus strains, a cross-sectional study was performed on human stools collected from rural communities. Between July 2014 and April 2015, outpatient children under 5 years of age from rural communities of Vhembe District, South Africa, were recorded for the study. A total of 303 stool specimens were collected from those with diarrhea (n=253) and without (n=50) diarrhea. NoVs were identified using real-time one-step RT-PCR. Partial Sequence analyses were performed to genotype the strains. Phylogenetic analyses were performed to compare identified NoVs genotypes to the worldwide circulating strains. Norovirus detection rate was 41.1% (104/253) in children with diarrhea. There was no significant difference (OR=1.24; 95% CI 0.66-2.33) in Norovirus detection between symptomatic and asymptomatic children. Comparison of the median CT values for NoV in children with diarrhea and without diarrhea revealed significant statistical difference of estimated GII viral load from both groups, with a much higher viral burden in children with diarrhea. To our knowledge, this is the first study reporting on the differences in estimated viral load of GII and GI NoV positive cases and controls. GII.Pe (n=9) were the predominant genotypes followed by GII.Pe/GII.4 Sydney 2012 (n=8) suspected recombinant and GII.4 Sydney 2012 variants(n=7). Two unassigned GII.4 variants and an unusual RdRp genotype GII.P15 were found. With note, the rare GIIP15 identified in this study has a common ancestor with GIIP15 strain from Japan previously reported as GII/untypeable recombinant strain implicated in a gastroenteritis outbreak. To our knowledge, this is the first report of this unusual genotype in the African continent. Though not confirmed predictive of diarrhea disease in this study, the high detection rate of NoV is an indication of subsequent exposure of children from rural communities to enteric pathogens due to poor sanitation and hygiene practices. The results reveal that the difference between asymptomatic and symptomatic children with NoV may possibly be related to the NoV genogroups involved. The findings emphasize NoV genetic diversity and predominance of GII.Pe/GII.4 Sydney 2012, indicative of increased NoV activity. An uncommon GII.P15 and two unassigned GII.4 variants were also identified from rural settings of the Vhembe District/South Africa. NoV surveillance is required to help to inform investigations into NoV evolution, and to support vaccine development programmes in Africa.

Keywords: asymptomatic, common, outpatients, norovirus genetic diversity, sporadic gastroenteritis, South African rural communities, symptomatic

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Authors: L. Matthewman, J. Nowlan

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Background: Holistic or Integrative Psychology emphasizes the interdependence of physiological, spiritual and psychological dynamics. Studying “wholeness and well-being” from a systems perspective combines innovative psychological science interventions with Eastern orientated healing wisdoms and therapies. The literature surrounding holistic/integrative psychology focuses on multi-stage interventions in attempts to enhance the mind-body experiences of well-being for participants. This study proposes a new single stage model as an intervention for UG/PG students, time-constrained workplace employees and managers/leaders for improved well-being and life enhancement. The main research objective was to investigate participants’ experiences of holistic and mindfulness interventions for impact on emotional well-being. The main research question asked was if single stage holistic interventions could impact on psychological well-being. This is of consequence because many people report that a reason for not taking part in mind-body or wellness programmes is that they believe that they do not have sufficient time to engage in such pursuits. Experimental Approach: The study employed a mixed methods pre-test/post-test research design. Data was analyzed using descriptive statistics and interpretative phenomenological analysis. Purposive sampling methods were employed. An adapted mindfulness measurement questionnaire (MAAS) was administered to 20 volunteer final year UG student participants prior to the single stage intervention and following the intervention. A further post-test longitudinal follow-up took place one week later. Intervention: The single stage model intervention consisted of a half hour session of mindfulness, yoga stretches and head and neck massage in the following sequence: Mindful awareness of the breath, yoga stretches 1, mindfulness of the body, head and neck massage, mindfulness of sounds, yoga stretches 2 and finished with pure awareness mindfulness. Results: The findings on the pre-test indicated key themes concerning: “being largely unaware of feelings”, “overwhelmed with final year exams”, “juggling other priorities” , “not feeling in control”, “stress” and “negative emotional display episodes”. Themes indicated on the post-test included: ‘more aware of self’, ‘in more control’, ‘immediately more alive’ and ‘just happier’ compared to the pre-test. Themes from post-test 2 indicated similar findings to post-test 1 in terms of themes. but on a lesser scale when scored for intensity. Interestingly, the majority of participants reported that they would now seek other similar interventions in the future and would be likely to engage with a multi-stage intervention type on a longer-term basis. Overall, participants reported increased psychological well-being after the single stage intervention. Conclusion: A single stage one-off intervention model can be effective to help towards the wellbeing of final year UG students. There is little indication to suggest that this would not be generalizable to others in different areas of life and business. However this study must be taken with caution due to low participant numbers. Implications: Single stage one-off interventions can be used to enhance peoples’ lives who might not otherwise sign up for a longer multi-stage intervention. In addition, single stage interventions can be utilized to help participants progress onto longer multiple stage interventions. Finally, further research into one stage well-being interventions is encouraged.

Keywords: holistic/integrative psychology, mindfulness, well-being, yoga

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Authors: Luis F. Caballero-castro, Dirsa Feliciano, Daniela Novoa, Orlando Arroyo, Jesús D. Villalba-morales

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Colombia faces a critical challenge in seismic resilience due to the prevalence of informal housing, which constitutes approximately 70% of residential structures. More than 10 million Colombians (20% of the population), live in homes susceptible to collapse in the event of an earthquake. This, combined with the fact that 83% of the population is in intermediate and high seismic hazard areas, has brought serious consequences to the country. These consequences became evident during the 1999 Armenia earthquake, which affected nearly 100,000 properties and represented economic losses equivalent to 1.88% of that year's Gross Domestic Product (GDP). Despite previous efforts to reinforce informal housing through methods like externally reinforced masonry walls, alternatives related to seismic protection systems (SPDs), such as Buckling-Restrained Braces (BRB), have not yet been explored in the country. BRBs are reinforcement elements capable of withstanding both compression and tension, making them effective in enhancing the lateral stiffness of structures. In this study, the use of low-cost and easily installable BRBs for the retrofit of informal housing in Colombia was evaluated, considering the economic limitations of the communities. For this purpose, a case study was selected involving an informally constructed dwelling in the country, from which field information on its structural characteristics and construction materials was collected. Based on the gathered information, nonlinear models with and without BRBs were created, and their seismic performance was analyzed and compared through incremental static (pushover) and nonlinear dynamic analyses. In the first analysis, the capacity curve was identified, showcasing the sequence of failure events occurring from initial yielding to structural collapse. In the second case, the model underwent nonlinear dynamic analyses using a set of seismic records consistent with the country's seismic hazard. Based on the results, fragility curves were calculated to evaluate the probability of failure of the informal housings before and after the intervention with BRBs, providing essential information about their effectiveness in reducing seismic vulnerability. The results indicate that low-cost BRBs can significantly increase the capacity of informal housing to withstand earthquakes. The dynamic analysis revealed that retrofit structures experienced lower displacements and deformations, enhancing the safety of residents and the seismic performance of informally constructed houses. In other words, the use of low-cost BRBs in the retrofit of informal housing in Colombia is a promising strategy for improving structural safety in seismic-prone areas. This study emphasizes the importance of seeking affordable and practical solutions to address seismic risk in vulnerable communities in earthquake-prone regions in Colombia and serves as a model for addressing similar challenges of informal housing worldwide.

Keywords: buckling-restrained braces, fragility curves, informal housing, incremental dynamic analysis, seismic retrofit

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Authors: Massimiliano Biagini, Marco Spinsanti, Gabriella De Angelis, Sara Tomei, Ilaria Ferlenghi, Maria Scarselli, Alessia Biolchi, Alessandro Muzzi, Brunella Brunelli, Silvana Savino, Marzia M. Giuliani, Isabel Delany, Paolo Costantino, Rino Rappuoli, Vega Masignani, Nathalie Norais

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The gram-negative bacterium Neisseria meningitidis serogroup B (MenB) is an exclusively human pathogen representing the major cause of meningitides and severe sepsis in infants and children but also in young adults. This pathogen is usually present in the 30% of healthy population that act as a reservoir, spreading it through saliva and respiratory fluids during coughing, sneezing, kissing. Among surface-exposed protein components of this diplococcus, factor H binding protein is a lipoprotein proved to be a protective antigen used as a component of the recently licensed Bexsero vaccine. fHbp is a highly variable meningococcal protein: to reflect its remarkable sequence variability, it has been classified in three variants (or two subfamilies), and with poor cross-protection among the different variants. Furthermore, the level of fHbp expression varies significantly among strains, and this has also been considered an important factor for predicting MenB strain susceptibility to anti-fHbp antisera. Different methods have been used to assess fHbp expression on meningococcal strains, however, all these methods use anti-fHbp antibodies, and for this reason, the results are affected by the different affinity that antibodies can have to different antigenic variants. To overcome the limitations of an antibody-based quantification, we developed a quantitative Mass Spectrometry (MS) approach. Selected Reaction Monitoring (SRM) recently emerged as a powerful MS tool for detecting and quantifying proteins in complex mixtures. SRM is based on the targeted detection of ProteoTypicPeptides (PTPs), which are unique signatures of a protein that can be easily detected and quantified by MS. This approach, proven to be highly sensitive, quantitatively accurate and highly reproducible, was used to quantify the absolute amount of fHbp antigen in total extracts derived from 105 clinical isolates, evenly distributed among the three main variant groups and selected to be representative of the fHbp circulating subvariants around the world. We extended the study at the genetic level investigating the correlation between the differential level of expression and polymorphisms present within the genes and their promoter sequences. The implications of fHbp expression on the susceptibility of the strain to killing by anti-fHbp antisera are also presented. To date this is the first comprehensive fHbp expression profiling in a large panel of Neisseria meningitidis clinical isolates driven by an antibody-independent MS-based methodology, opening the door to new applications in vaccine coverage prediction and reinforcing the molecular understanding of released vaccines.

Keywords: quantitative mass spectrometry, Neisseria meningitidis, vaccines, bexsero, molecular epidemiology

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Authors: Kirsten Techmer, Jan-Erik Rybak, Simon Rudolph

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Natural formed pyrites (FeS2) are frequent sulfides in sedimentary and metamorphic rocks. Growth textures of idiomorphic pyrite assemblages reflect the conditions during their formation in the geologic sequence, furtheron the local texture analyses of the growth patterns of pyrite assemblages by EBSD reveal the possibility to resolve the growth conditions during the formation of pyrite at the micron scale. The spatial resolution of local texture measurements in the Scanning Electron Microscope used can be in the nanomete scale. Orientation contrasts resulting from domains of smaller misorientations within larger pyrite crystals can be resolved as well. The electron optical studies have been carried out in a Field-Emission Scanning Electron Microscope (FEI Quanta 200) equipped with a CCD camera to study the orientation contrasts along the surfaces of pyrite. Idiomorphic cubic single crystals of pyrite, polycrystalline assemblages of pyrite, spherically grown spheres of pyrite as well as pyrite-bearing ammonites have been studied by EBSD in the Scanning Electron Microscope. Samples were chosen to show no or minor secondary deformation and an idiomorphic 3D crystal habit, so the local textures of pyrite result mainly from growth and minor from deformation. The samples studied derived from Navajun (Spain), Chalchidiki (Greece), Thüringen (Germany) and Unterkliem (Austria). Chemical analyses by EDAX show pyrite with minor inhomogeneities e.g., single crystals of galena and chalcopyrite along the grain boundaries of larger pyrite crystals. Intergrowth between marcasite and pyrite can be detected in one sample. Pyrite may form intense growth twinning lamellae on {011}. Twinning, e.g., contact twinning is abundant within the crystals studied and the individual twinning lamellaes can be resolved by EBSD. The ammonites studied show a replacement of the shale by newly formed pyrite resulting in an intense intergrowth of calcite and pyrite. EBSD measurements indicate a polycrystalline microfabric of both minerals, still reflecting primary surface structures of the ammonites e.g, the Septen. Discs of pyrite (“pyrite dollar”) as well as pyrite framboids show growth patterns comprising a typical microfabric. EBSD studies reveal an equigranular matrix in the inner part of the discs of pyrite and a fiber growth with larger misorientations in the outer regions between the individual segments. This typical microfabric derived from a formation of pyrite crystals starting at a higher nucleation rate and followed by directional crystal growth. EBSD studies show, that the growth texture of pyrite in the samples studied reveals a correlation between nucleation rate and following growth rate of the pyrites, thus leading to the characteristic crystal habits. Preferential directional growth at lower nucleation rates may lead to the formation of 3D framboids of pyrite. Crystallographic misorientations between the individual fibers are similar. In ammonites studied, primary anisotropies of the substrates like e.g., ammonitic sutures, influence the nucleation, crystal growth and habit of the newly formed pyrites along the surfaces.

Keywords: Electron Back Scatter Diffraction (EBSD), growth pattern, Fe-sulfides (pyrite), texture analyses

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Authors: Raha Bahreini

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This paper discusses the roll-back of women’s sexual and reproductive rights in the Islamic Republic of Iran, which has come in the wake of a striking shift in the country’s official population policies. Since the late 1980s, Iran has won worldwide praise for its sexual and reproductive health and services, which have contributed to a steady decline in the country’s fertility rate–from 7.0 births per women in 1980 to 5.5 in 1988, 2.8 in 1996 and 1.85 in 2014. This is owed to a significant increase in the voluntary use of modern contraception in both rural and urban areas. In 1976, only 37 per cent of women were using at least one method of contraception; by 2014 this figure had reportedly risen to a high of nearly 79 per cent for married girls and women living in urban areas and 73.78 per cent for those living in rural areas. Such progress may soon be halted. In July 2012, Iran’s Supreme Leader Ayatollah Sayed Ali Khamenei denounced Iran’s family planning policies as an imitation of Western lifestyle. He exhorted the authorities to increase Iran’s population to 150 to 200 million (from around 78.5 million), including by cutting subsidies for contraceptive methods and dismantling the state’s Family and Population Planning Programme. Shortly thereafter, Iran’s Minister of Health and Medical Education announced the scrapping of the budget for the state-funded Family and Population Planning Programme. Iran’s Parliament subsequently introduced two bills; the Comprehensive Population and Exaltation of Family Bill (Bill 315), and the Bill to Increase Fertility Rates and Prevent Population Decline (Bill 446). Bill 446 outlaws voluntary tubectomies, which are believed to be the second most common method of modern contraception in Iran, and blocks access to information about contraception, denying women the opportunity to make informed decisions about the number and spacing of their children. Coupled with the elimination of state funding for Iran’s Family and Population Programme, the move would undoubtedly result in greater numbers of unwanted pregnancies, forcing more women to seek illegal and unsafe abortions. Bill 315 proposes various discriminatory measures in the areas of employment, divorce, and protection from domestic violence in order to promote a culture wherein wifedom and child-bearing is seen as women’s primary duty. The Bill, for example, instructs private and public entities to prioritize, in sequence, men with children, married men without children and married women with children when hiring for certain jobs. It also bans the recruitment of single individuals as family law lawyers, public and private school teachers and members of the academic boards of universities and higher education institutes. The paper discusses the consequences of these initiatives which would, if continued, set the human rights of women and girls in Iran back by decades, leaving them with a future shaped by increased inequality, discrimination, poor health, limited choices and restricted freedoms, in breach of Iran’s international human rights obligations.

Keywords: family planning and reproductive health, gender equality and empowerment of women, human rights, population growth

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Authors: T. C. C. Soo, S. Bhassu

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Unlike the common presence of both innate and adaptive immunity in vertebrates, crustaceans, in particular, shrimps, have been discovered to possess only innate immunity. This further emphasizes the importance of innate immunity within shrimps in pathogenic resistance. Under the study of pathogenic immune challenge, different shrimp species actually exhibit varying degrees of immune resistance towards the same pathogen. Furthermore, even within the same shrimp species, different batches of challenged shrimps can have different strengths of immune defence. Several important pathways are activated within shrimps during pathogenic infection. One of them is JAK-STAT pathway that is activated during bacterial, viral and fungal infections by which STAT(Signal Transducer and Activator of Transcription) gene is the core element of the pathway. Based on theory of Central Dogma, the genomic information is transmitted in the order of DNA, RNA and protein. This study is focused in uncovering the important evolutionary patterns present within the DNA (non-coding region) and RNA (coding region). The three shrimp species involved are Macrobrachium rosenbergii, Penaeus monodon and Litopenaeus vannamei which all possess commercial significance. The shrimp species were challenged with a famous penaeid shrimp virus called white spot syndrome virus (WSSV) which can cause serious lethality. Tissue samples were collected during time intervals of 0h, 3h, 6h, 12h, 24h, 36h and 48h. The DNA and RNA samples were then extracted using conventional kits from the hepatopancreas tissue samples. PCR technique together with designed STAT gene conserved primers were utilized for identification of the STAT coding sequences using RNA-converted cDNA samples and subsequent characterization using various bioinformatics approaches including Ramachandran plot, ProtParam and SWISS-MODEL. The varying levels of immune STAT gene activation for the three shrimp species during WSSV infection were confirmed using qRT-PCR technique. For one sample, three biological replicates with three technical replicates each were used for qRT-PCR. On the other hand, DNA samples were important for uncovering the structural variations within the genomic region of STAT gene which would greatly assist in understanding the STAT protein functional variations. The partially-overlapping primers technique was used for the genomic region sequencing. The evolutionary inferences and event predictions were then conducted through the Bayesian Inference method using all the acquired coding and non-coding sequences. This was supplemented by the construction of conventional phylogenetic trees using Maximum likelihood method. The results showed that adaptive evolution caused STAT gene sequence mutations between different shrimp species which led to evolutionary divergence event. Subsequently, the divergent sites were correlated to the differing expressions of STAT gene. Ultimately, this study assists in knowing the shrimp species innate immune variability and selection of disease resistant shrimps for breeding purpose. The deeper understanding of STAT gene evolution from the perspective of both purifying and adaptive approaches not only can provide better immunological insight among shrimp species, but also can be used as a good reference for immunological studies in humans or other model organisms.

Keywords: gene evolution, JAK-STAT pathway, immunology, STAT gene

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Authors: Wen-Yang Hu, Ranli Lu, Mark Maienschein-Cline, Danping Hu, Larisa Nonn, Toshi Shioda, Gail S. Prins

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Background: Genetic mutations are highly associated with increased prostate cancer risk. In addition to whole genome sequencing, somatic mutations can be identified by aligning transcriptome sequences to the human genome. Here we analyzed bulk RNAseq and single cell RNAseq data of human prostate cancer cells and their matched non-cancer cells in benign regions from 4 individual patients. Methods: Sequencing raw reads were aligned to the reference genome hg38 using STAR. Variants were annotated using Annovar with respect to overlap gene annotation information, effect on gene and protein sequence, and SIFT annotation of nonsynonymous variant effect. We determined cancer-specific novel alleles by comparing variant calls in cancer cells to matched benign cells from the same individual by selecting unique alleles that were only detected in the cancer samples. Results: In bulk RNAseq data from 3 patients, the most common variants were the noncoding mutations at UTR3/UTR5, and the major variant types were single-nucleotide polymorphisms (SNP) including frameshift mutations. C>T transversion is the most frequently presented substitution of SNP. A total of 222 genes carrying unique exonic or UTR variants were revealed in cancer cells across 3 patients but not in benign cells. Among them, transcriptome levels of 7 genes (CITED2, YOD1, MCM4, HNRNPA2B1, KIF20B, DPYSL2, NR4A1) were significantly up or down regulated in cancer stem cells. Out of the 222 commonly mutated genes in cancer, 19 have nonsynonymous variants and 11 are damaged genes with variants including SIFT, frameshifts, stop gain/loss, and insertions/deletions (indels). Two damaged genes, activating transcription factor 6 (ATF6) and histone demethylase KDM3A are of particular interest; the former is a survival factor for certain cancer cells while the later positively activates androgen receptor target genes in prostate cancer. Further, single cell RNAseq data of cancer cells and their matched non-cancer benign cells from both primary 2D and 3D tumoroid cultures were analyzed. Similar to the bulk RNAseq data, single cell RNAseq in cancer demonstrated that the exonic mutations are less common than noncoding variants, with SNPs including frameshift mutations the most frequently presented types in cancer. Compared to cancer stem cell enriched-3D tumoroids, 2D cancer cells carried 3-times higher variants, 8-times more coding mutations and 10-times more nonsynonymous SNP. Finally, in both 2D primary and 3D tumoroid cultures, cancer stem cells exhibited fewer coding mutations and noncoding SNP or insertions/deletions than non-stem cancer cells. Summary: Our study demonstrates the usefulness of bulk and single cell RNAseaq data in identifying somatic mutations in prostate cancer, providing an alternative method in screening candidate genes for prostate cancer diagnosis and potential therapeutic targets. Cancer stem cells carry fewer somatic mutations than non-stem cancer cells due to their inherited immortal stand DNA from parental stem cells that explains their long-lived characteristics.

Keywords: prostate cancer, stem cell, genomic mutation, RNAseq

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Authors: Yi H. Wong, Li L. Chan, Chee O. Leong, Stephen Ambu, Joon W. Mak, Priyadashi S. Sahu

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Background: Acanthamoeba is a free-living opportunistic protist which is ubiquitously distributed in the environment. Virulent Acanthamoeba can cause fatal encephalitis in immunocompromised patients and potential blinding keratitis in immunocompetent contact lens wearers. Approximately 24 species have been identified but only the A. castellanii, A. polyphaga and A. culbertsoni are commonly associated with human infections. Until to date, the precise molecular basis for Acanthamoeba pathogenesis remains unclear. Previous studies reported that Acanthamoeba virulence can be diminished through prolonged axenic culture but revived through serial mouse passages. As no clear explanation on this reversible pathogenesis is established, hereby, we postulate that the epigenetic regulators, DNA-methyltransferases (DNMT) and histone-deacetylases (HDAC), could possibly be involved in granting the virulence plasticity of Acanthamoeba spp. Methods: Four rounds of mouse passages were conducted to revive the virulence potential of the virulence-attenuated Acanthamoeba castellanii strain (ATCC 50492). Briefly, each mouse (n=6/group) was inoculated intraperitoneally with Acanthamoebae cells (2x 105 trophozoites/mouse) and incubated for 2 months. Acanthamoebae cells were isolated from infected mouse organs by culture method and subjected to subsequent mouse passage. In vitro cytopathic, encystment and gelatinolytic assays were conducted to evaluate the virulence characteristics of Acanthamoebae isolates for each passage. PCR primers which targeted on the 2 members (DNMT1 and DNMT2) and 5 members (HDAC1 to 5) of the DNMT and HDAC gene families respectively were custom designed. Quantitative real-time PCR (qPCR) was performed to detect and quantify the relative expression of the two gene families in each Acanthamoeba isolates. Beta-tubulin of A. castellanii (Genbank accession no: XP_004353728) was included as housekeeping gene for data normalisation. PCR mixtures were also analyzed by electrophoresis for amplicons detection. All statistical analyses were performed using the paired one-tailed Student’s t test. Results: Our pathogenicity tests showed that the virulence-reactivated Acanthamoeba had a higher degree of cytopathic effect on vero cells, a better resistance to encystment challenge and a higher gelatinolytic activity which was catalysed by serine protease. qPCR assay showed that DNMT1 expression was significantly higher in the virulence-reactivated compared to the virulence-attenuated Acanthamoeba strain (p ≤ 0.01). The specificity of primers which targeted on DNMT1 was confirmed by sequence analysis of PCR amplicons, which showed a 97% similarity to the published DNA-methyltransferase gene of A. castellanii (GenBank accession no: XM_004332804.1). Out of the five primer pairs which targeted on the HDAC family genes, only HDAC4 expression was significantly difference between the two variant strains. In contrast to DNMT1, HDAC4 expression was much higher in the virulence-attenuated Acanthamoeba strain. Conclusion: Our mouse passages had successfully restored the virulence of the attenuated strain. Our findings suggested that DNA-methyltransferase (DNMT1) and histone deacetylase (HDAC4) expressions are associated with virulence potential of Acanthamoeba spp.

Keywords: acanthamoeba, DNA-methyltransferase, histone deacetylase, virulence-associated proteins

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Authors: Ved Vrat Verma, Rani Gupta, Manisha Goel

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γ-glutamyltranspeptidase (GGT: EC 2.3.2.2) is an N-terminal nucleophile hydrolase conserved in all three domains of life. GGT plays a key role in glutathione metabolism where it catalyzes the breakage of the γ-glutamyl bonds and transfer of γ-glutamyl group to water (hydrolytic activity) or amino acids or short peptides (transpeptidase activity). GGTs from bacteria, archaea, and eukaryotes (human, rat and mouse) are homologous proteins sharing >50% sequence similarity and conserved four layered αββα sandwich like three dimensional structural fold. These proteins though similar in their structure to each other, are quite diverse in their enzyme activity: some GGTs are better at hydrolysis reactions but poor in transpeptidase activity, whereas many others may show opposite behaviour. GGT is known to be involved in various diseases like asthma, parkinson, arthritis, and gastric cancer. Its inhibition prior to chemotherapy treatments has been shown to sensitize tumours to the treatment. Microbial GGT is known to be a virulence factor too, important for the colonization of bacteria in host. However, all known inhibitors (mimics of its native substrate, glutamate) are highly toxic because they interfere with other enzyme pathways. However, a few successful efforts have been reported previously in designing species specific inhibitors. We aim to leverage the diversity seen in GGT family (pathogen vs. eukaryotes) for designing specific inhibitors. Thus, in the present study, we have used DIVERGE software to identify sites in GGT proteins, which are crucial for the functional and structural divergence of these proteins. Since, type II divergence sites vary in clade specific manner, so type II divergent sites were our focus of interest throughout the study. Type II divergent sites were identified for pathogen vs. eukaryotes clusters and sites were marked on clade specific representative structures HpGGT (2QM6) and HmGGT (4ZCG) of pathogen and eukaryotes clade respectively. The crucial divergent sites within 15 A radii of the binding cavity were highlighted, and in-silico mutations were performed on these sites to delineate the role of these sites on the mechanism of catalysis and protein folding. Further, the amino acid network (AAN) analysis was also performed by Cytoscape to delineate assortative mixing for cavity divergent sites which could strengthen our hypothesis. Additionally, molecular dynamics simulations were performed for wild complexes and mutant complexes close to physiological conditions (pH 7.0, 0.1 M ionic strength and 1 atm pressure) and the role of putative divergence sites and structural integrities of the homologous proteins have been analysed. The dynamics data were scrutinized in terms of RMSD, RMSF, non-native H-bonds and salt bridges. The RMSD, RMSF fluctuations of proteins complexes are compared, and the changes at protein ligand binding sites were highlighted. The outcomes of our study highlighted some crucial divergent sites which could be used for novel inhibitors designing in a species-specific manner. Since, for drug development, it is challenging to design novel drug by targeting similar protein which exists in eukaryotes, so this study could set up an initial platform to overcome this challenge and help to deduce the more effective targets for novel drug discovery.

Keywords: γ-glutamyltranspeptidase, divergence, species-specific, drug design

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Authors: Rama Munajat

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Discourse information levels may be viewed from either a background-foreground distinction or a multi-level perspective, and cross-linguistic studies on this area suggest that each information level is marked by a specific linguistic device. In this sense, Sundanese, spoken in Indonesia’s West Javanese Province, further differentiates the background and foreground information into ordinary and significant types. This paper will report an ongoing shift from particle-to-active voice marking in the way Sundanese signals foregrounding events. The shift relates to decades of contact with Bahasa Indonesia (Indonesia’s official language) and linguistic compatibility between the two surface marking strategies. Representative data analyzed include three groups of short stories in both Sundanese and Bahasa Indonesia (Indonesian) published in three periods: before 1945, 1965-2006, and 2016-2019. In the first group of Sundanese data, forward-moving events dominantly appear in particle KA (Kecap Anteuran, word-accompanying) constructions, where the KA represents different particles that co-occur with a special group of verbs. The second group, however, shows that the foregrounded events are more frequently described in active-voice forms with a subject-predicate (SP) order. Subsequently, the third offers stronger evidence for the use of the SP structure. As for the Indonesian data, the foregrounding events in the first group occur in verb-initial and passive-voice constructions, while in the second and third, the events more frequently appear in active-voice structures (subject-predicate sequence). The marking shift above suggests a structural influence from Indonesian, stemmed from generational differences among authors of the Sundanese short stories, particularly related to their education and language backgrounds. The first group of short stories – published before 1945 or before Indonesia's independence from Dutch – were written by native speakers of Sundanese who spoke Indonesian as a foreign language and went through the Dutch education system. The second group of authors, on the other hand, represents a generation of Sundanese native speakers who spoke Indonesian as a second language. Finally, the third group consists of authors who are bilingual speakers of both Sundanese and Indonesian. The data suggest that the last two groups of authors completed the Indonesian education system. With these, the use of subject-predicate sequences to denote foregrounding events began to appear more frequently in the second group and then became more dominant in those of the third. The coded data also signify that cohesion, coherence, and pragmatic purposes in Particle KA constructions are intact in their respective active-voice structure counterparts. For instance, the foregrounding events in Particle KA constructions occur in Sentence-initial KA and Pre-verbal KA forms, whereas those in the active-voice are described in Subject-Predicate (SP) and Zero-Subject active-voice patterns. Cross-language data further demonstrate that the Sentence-initial KA and the SP active-voice structures each contain an overt noun phrase (NP) co-referential with one of the entities introduced in a preceding context. Similarly, the pre-verbal KA and Zero-Subject active-voice patterns have a deleted noun phrase unambiguously referable to the only one entity previously mentioned. The presence and absence of an NP inform a pragmatic strategy to place prominence on topic/given and comment/new information, respectively.

Keywords: discourse analysis, foregrounding marking, pragmatics, language contact

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