Search results for: endogenous reference gene

Authors: H. Boulahyaoui, S. Alaoui Amine, C. Loutfi, H. El Annaz, N. Touil, El M. El Fahim, S. Mrani

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Three Moroccan children fully vaccinated with Rotarix™ have been hospitalized for Rotavirus Gastroenteritis (RVGE) in the pediatric division of the Farabi Hospital, Oujda. Rotavirus G/P genotypes could not be typed because of their delayed crossing threshold (Ct) resolute with a group A rotavirus (RVA) real time RT-PCR. These strains were adapted to cell culture. All viruses replicated and caused extensive cytopathic effects after four or five passages in MA104 cell lines. Significant improvements have been obtained in the amount of viral particles. Each virus multiplied to a high titer (7.5 TCID50/ml). VP7 and VP4 partial gene sequencing revealed distinct genotypes compared to the Rotarix(®) vaccine strain. Two strains were of G2P[4] genotype whereas the third was G9P[8] genotype. Virus isolation while labor intensive, is recommended as a second test, especially when higher sensitivity for conventional RVA genotyping RT-PCR is needed. VP7 antigenic similarities between these strains and Rotarix were determined.

Keywords: esacpe-vaccine, Morocco, Rotarix, G2P[4], G9P[8]

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Authors: Md. Samsuddin Ansari, Ashish Arora

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Every year 10 million people fall ill with one of the oldest diseases known as tuberculosis, caused by Mycobacterium tuberculosis. The success of M. tuberculosis as a pathogen is because of its ability to persist in host tissues. Multidrug resistance (MDR) mycobacteria cases increase every day, which is associated with efflux pumps controlled at the level of transcription. The transcription regulators of MDR transporters in bacteria belong to one of the following four regulatory protein families: AraC, MarR, MerR, and TetR. Phenolic acid decarboxylase repressor (PadR), like a family of transcription regulators, is closely related to the MarR family. Phenolic acid decarboxylase repressor (PadR) was first identified as a transcription factor involved in the regulation of phenolic acid stress response in various microorganisms (including Mycobacterium tuberculosis H37Rv). Recently research has shown that the PadR family transcription factors are global, multifunction transcription regulators. Rv0047c is a PadR subfamily-1 protein. We are exploring the biophysical and structural characterization of Rv0047c. The Rv0047 gene was amplified by PCR using the primers containing EcoRI and HindIII restriction enzyme sites cloned in pET-NH6 vector and overexpressed in DH5α and BL21 (λDE3) cells of E. coli following purification with Ni2+-NTA column and size exclusion chromatography. We did DSC to know the thermal stability; the Tm (transition temperature) of protein is 55.29ºC, and ΔH (enthalpy change) of 6.92 kcal/mol. Circular dichroism to know the secondary structure and conformation and fluorescence spectroscopy for tertiary structure study of protein. To understand the effect of pH on the structure, function, and stability of Rv0047c we employed spectroscopy techniques such as circular dichroism, fluorescence, and absorbance measurements in a wide range of pH (from pH-2.0 to pH-12). At low and high pH, it shows drastic changes in the secondary and tertiary structure of the protein. EMSA studies showed the specific binding of Rv0047c with its own 30-bp promoter region. To determine the effect of complex formation on the secondary structure of Rv0047c, we examined the CD spectra of the complex of Rv0047c with promoter DNA of rv0047. The functional role of Rv0047c was characterized by over-expressing the Rv0047c gene under the control of hsp60 promoter in Mycobacterium tuberculosis H37Rv. We have predicted the three-dimensional structure of Rv0047c using the Swiss Model and Modeller, with validity checked by the Ramachandra plot. We did molecular docking of Rv0047c with dnaA, through PatchDock following refinement through FireDock. Through this, it is possible to easily identify the binding hot-stop of the receptor molecule with that of the ligand, the nature of the interface itself, and the conformational change undergone by the protein pattern. We are using X-crystallography to unravel the structure of Rv0047c. Overall the studies show that Rv0047c may have transcription regulation along with providing an insight into the activity of Rv0047c in the pH range of subcellular environment and helps to understand the protein-protein interaction, a novel target to kill dormant bacteria and potential strategy for tuberculosis control.

Keywords: mycobacterium tuberculosis, phenolic acid decarboxylase repressor, Rv0047c, Circular dichroism, fluorescence spectroscopy, docking, protein-protein interaction

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Authors: Michelle C. S. Azevedo, Priscila M. Colavite, Carolina F. Francisconi, Ana P. Trombone, Gustavo P. Garlet

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VIP (vasoactive intestinal peptide) know as a potential protective factor in the view of its marked immunosuppressive properties. In this work, we investigated a possible association of VIP with the clinical status of experimental periapical granulomas and the association with expression markers in the lesions potentially associated with periapical lesions pathogenesis. C57BL/6WT mice were treated or not with recombinant VIP. Animals with active/progressive (N=40), inactive/stable (N=70) periapical granulomas and controls (N=50) were anesthetized and the right mandibular first molar was surgically opened, allowing exposure of dental pulp. Endodontic pathogenic bacterial strains were inoculated: Porphyromonas gingivalis, Prevotella nigrescens, Actinomyces viscosus, and Fusobacterium nucleatum subsp. polymorphum. The cavity was not sealed after bacterial inoculation. During lesion development, animals were treated or not with recombinant VIP 3 days post infection. Animals were killed after 3, 7, 14, and 21 days of infection and the jaws were dissected. The extraction of total RNA from periodontal tissues was performed and the integrity of samples was checked. qPCR reaction using TaqMan chemistry with inventoried primers were performed in ViiA7 equipment. The results, depicted as the relative levels of gene expression, were calculated in reference to GAPDH and β-actin expression. Periodontal tissues from upper molars were vested and incubated supplemented RPMI, followed by processing with 0.05% DNase. Cell viability and couting were determined by Neubauer chamber analysis. For flow cytometry analysis, after cell counting the cells were stained with the optimal dilution of each antibody; (PE)-conjugated and (FITC)-conjugated antibodies against CD4, CD25, FOXP3, IL-4, IL-17 and IFN-γ antibodies, as well their respective isotype controls. Cells were analyzed by FACScan and CellQuest software. Results are presented as the number of cells in the periodontal tissues or the number of positive cells for each marker in the CD4+FOXp3+, CD4+IL-4+, CD4+IFNg+ and CD4+IL-17+ subpopulations. The levels mRNA were measured by qPCR. The VIP expression was predominated in inactive lesions, as well part of the clusters of cytokine/Th markers identified as protective factors and a negative correlation between VIP expression and lesion evolution was observed. A quantitative analysis of IL1β, IL17, TNF, IFN, MMP2, RANKL, OPG, IL10, TGFβ, CTLA4, COL5A1, CTGF, CXCL11, FGF7, ITGA4, ITGA5, SERP1 and VTN expression was measured in experimental periapical lesions treated with VIP 7 and 14 days after lesion induction and healthy animals. After 7 days, all targets presented a significate increase in comparison to untreated animals. About migration kinetics, profile of chemokine receptors expression of TCD4+ subsets and phenotypic analysis of Tregs, Th1, Th2 and Th17 cells during the course of experimental periodontal disease evaluated by flow cytometry and depicted as the number of positive cells for each marker. CD4+IFNg+ and CD4+FOXp3+ cells migration were significate increased 7 days post VIP treatment. CD4+IL17+ cells migration were significate increased 7 and 14 days post VIP treatment, CD4+IL4+ cells migration were significate increased 14 and 21 days post VIP treatment compared to the control group. In conclusion, our experimental data support VIP involvement in determining the inactivity of periapical lesions. Financial support: FAPESP #2015/25618-2.

Keywords: chronic inflammation, cytokines, osteolytic lesions, VIP (Vasoactive Intestinal Peptide)

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Authors: Thoudam Jomita Devi

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This paper is an attempt to analyze the problems of translating Manipuri folktales into English and the strategies deployed. In Manipuri, folktales are known as Fungawari/Phungawari, which is similar to a western bed time story. The work is with the special reference to folktales of Meetei community. Meetei are the majority ethnic group of Manipur, India. For this paper’s purpose, two folktales Shandrembi Cheisra and Pebet will be chosen for analysis and discussion. The translation of folktales can contribute to intercultural communication and bridge the gap between the generations. Translating Manipuri Folktales is problematic on both cultural and linguistic levels. Therefore, the aim of this analysis is to understand, how the idea of André Lefevere (1992) translation could be implicated in translating Manipuri folktales.

Keywords: cultural, folktales, intercultural, interlingual, translation

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Authors: Getenet Aseged Zeleke

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The aim of this project was to study the synthesis of activated carbon from low-cost animal beef and the characterization of the product obtained. The bone was carbonized in an inert atmosphere at three different temperatures (500°C, 700oC and 900°C) in an electric furnace, followed by activation with hydrochloric acid. The activated animal bone charcoals obtained were characterized by using scanning electron microscopy (SEM)to observe the effect of activation compared to the unactivated bone charcoal. The following parameters were also determined: ash content, moisture content, volatile content, fixed carbon, pH, pore volume and bulk (apparent) density. The characterization result showed that the activated bone charcoal has good properties and is compared favorably with other reference activated carbons.

Keywords: bones, carbonization, activation, characterization, activated carbon

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Authors: Sandy Elsayed, Aya Mohamed, Noha Nassar

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Introduction: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by social deficits and repetitive behavior. Multiple studies suggest that oxidative stress and neuroinflammation are key factors in the etiology of ASD and often associated with worsening of ASD-related behaviors. Nuclear factor erythroid 2-related factor 2 (NRF-2) is a transcription factor that promotes expression of antioxidant response element genes in oxidative stress. In ASD subjects, decreased expression of NRF-2 in frontal cortex shifted the redox homeostasis towards oxidative stress, and resulted in inflammation evidenced by elevation of nuclear factor kappa B (NF-κB) transcriptional activity. Dimethyl fumarate (DMF) is a NRF-2 activator that is used in the treatment of psoriasis and multiple sclerosis. It participates in the transcriptional control of inflammatory factors via inhibition of NF-κB and its downstream targets. This study aimed to investigate the role of DMF in alleviating the cognitive impairments and behavior deficits associated with ASD through mitigation of oxidative stress and inflammation in prenatal valproic acid (VPA) rat model of autism. Methods: Pregnant female Wistar rats received a single intraperitoneal injection of VPA (600 mg/kg) to induce autistic-like-behavioral and neurobiological alterations in their offspring. Chronic oral gavage of DMF (150mg/kg/day) started from postnatal day (PND) 24 till PND62 (39 days). Prenatal VPA exposure elicited autistic behaviors including decreased social interaction and stereotyped behavior. Social interaction was evaluated using three-chamber sociability test and calculation of sociability index (SI), while stereotyped repetitive behavior and anxiety associated with ASD were assessed using marble burying test (MBT). Biochemical analyses were done on prefrontal cortex homogenates including NRF-2, and NF-κB expression. Moreover, inducible nitric oxide synthase (iNOS) gene expression and tumor necrosis factor (TNF-) protein expression were evaluated as markers of inflammation. Results: Prenatal VPA elicited decreased social interaction shown by decreased SI compared to control group (p < 0.001) and DMF enhanced SI (p < 0.05). In MBT, prenatal injection of VPA manifested stereotyped behavior and enhanced number of buried marbles compared to control (p < 0.05) and DMF reduced the anxiety-related behavior in rats exhibiting ASD-like behaviors (p < 0.05). In prefrontal cortex, NRF-2 expression was downregulated in prenatal VPA model (p < 0.0001) and DMF reversed this effect (p < 0.0001). The inflammatory transcription factor NF-κB was elevated in prenatal VPA model (p < 0.0001) and reduced (p < 0.0001) upon NRF-2 activation by DMF. Prenatal VPA expressed higher levels of proinflammatory cytokine TNF- compared to control group (p < 0.0001) and DMF reduced it (p < 0.0001). Finally, the gene expression of iNOS was downregulated upon NRF-2 activation by DMF (p < 0.01). Conclusion: This study proposes that DMF is a potential agent that can be used to ameliorate autistic-like-changes through NRF-2 activation along with NF-κB downregulation and therefore, it is a promising novel therapy for ASD.

Keywords: autism spectrum disorders, dimethyl fumarate, neuroinflammation, NRF-2

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Authors: Jing Zhang

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To reduce the carbon footprint of mankind and become more sustainable is one of the major challenges in our era. Internet of Things (IoT) mainly resolves three problems: Things to Things (T2T), Human to Things, H2T), and Human to Human (H2H). Borrowing the classification of IoT, we can find carbon prints of industries also can be divided in these three ways. Therefore, monitoring the routes of generation and circulation of products may help calculate product carbon print. This paper does not consider any technique used by IoT itself, but the ideas of it look at the connection of products. Carbon prints are like a gene or mark of a product from raw materials to the final products, which never leave the products. The contribution of this paper is to combine the characteristics of IoT and the methodology of network science to find a way to calculate the product's carbon footprint. Life cycle assessment, LCA is a traditional and main tool to calculate the carbon print of products. LCA is a traditional but main tool, which includes three kinds.

Keywords: product carbon footprint, Internet of Things, network science, life cycle assessment

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Authors: Honggang Qu, Rongmei Liu, Bin Wang, Yong Xu, Zhenji Gao

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There is a big gap in the ability and level of the informatization technology innovation of cores and samples compared with developed countries. Under the current background of promoting the technology innovation, how to strengthen the informatization technology innovation of cores and samples for National Cores and Samples Archives, which is a national innovation research center, is an important research topic. The paper summarizes the development status of cores and samples informatization technology, and finds the gaps and deficiencies, and proposes the innovation research directions and content, including data extraction, recognition, processing, integration, application and so on, so as to provide some reference and guidance for the future innovation research of the archives and support better the geological technology innovation in China.

Keywords: cores and samples;, informatization technology;, innovation;, suggestion

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Authors: B. Atika, S. Lehmann, E. Wimmer

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The defense strategy is very common in the insect world. Defensive substances play a wide variety of functions for beetles, such as repellents, toxicants, insecticides, and antimicrobics. Beetles react to predators, invaders, and parasitic microbes with the release of toxic and repellent substances. Defensive substances are directed against a large array of potential target organisms or may function for boiling bombardment or as surfactants. Usually, Coleoptera biosynthesize and store their defensive compounds in a complex secretory organ, known as odoriferous defensive stink glands. The red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae), uses these glands to produce antimicrobial p-benzoquinones and 1-alkenes. In the past, the morphology of stink gland has been studied in detail in tenebrionid beetles; however, very little is known about the genes that are involved in the production of gland secretion. In this study, we studied a subset of genes that are essential for the benzoquinone production in red flour beetle. In the first phase, we selected 74 potential candidate genes from a genome-wide RNA interference (RNAi) knockdown screen named 'iBeetle.' All these 74 candidate genes were functionally characterized by RNAi-mediated gene knockdown. Therefore, they were selected for a subsequent gas chromatography-mass spectrometry (GC-MS) analysis of secretion volatiles in respective RNAi knockdown glands. 33 of them were observed to alter the phenotype of stink gland. In the GC-MS analysis, 7 candidate genes were noted to display a strongly altered gland, in terms of secretion color and chemical composition, upon knockdown, showing their key role in the biosynthesis of gland secretion. Morphologically altered stink glands were found for odorant receptor and protein kinase superfamily. Subsequent GC-MS analysis of secretion volatiles revealed reduced benzoquinone levels in LIM domain, PDZ domain, PBP/GOBP family knockdowns and a complete lack of benzoquinones in the knockdown of sulfatase-modifying factor enzyme 1, sulfate transporter family. Based on stink gland transcriptome data, we analyzed the function of sulfatase-modifying factor enzyme 1 and sulfate transporter family via RNAi-mediated gene knockdowns, GC-MS, in situ hybridization, and enzymatic activity assays. Morphologically altered stink glands were noted in knockdown of both these genes. Furthermore, GC-MS analysis of secretion volatiles showed a complete lack of benzoquinones in the knockdown of these two genes. In situ hybridization showed that these two genes are expressed around the vesicle of certain subgroup of secretory stink gland cells. Enzymatic activity assays on stink gland tissue showed that these genes are involved in p-benzoquinone biosynthesis. These results suggest that sulfatase-modifying factor enzyme 1 and sulfate transporter family play a role specifically in benzoquinone biosynthesis in red flour beetles.

Keywords: Red Flour Beetle, defensive stink gland, benzoquinones, sulfate transporter, sulfatase-modifying factor enzyme 1

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Authors: Z. Giagkazoglou, D. Loukovitis, C. Gubili, A. Imsiridou

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Along with the increase in human population, demand for seafood has increased. Despite the strict labeling regulations that exist for most marketed species in the European Union, seafood substitution remains a persistent global issue. Food fraud occurs when food products are traded in a false or misleading way. Mislabeling occurs when one species is substituted and traded under the name of another, and it can be intentional or unintentional. Crustaceans are one of the most regularly consumed seafood in Greece. Shrimps, prawns, lobsters, crayfish, and crabs are considered a delicacy and can be encountered in a variety of market presentations (fresh, frozen, pre-cooked, peeled, etc.). With most of the external traits removed, products as such are susceptible to species substitution. DNA barcoding has proven to be the most accurate method for the detection of fraudulent seafood products. To our best knowledge, the DNA barcoding methodology is used for the first time in Greece, in order to investigate the labeling practices for crustacean products available in the market. A total of 100 tissue samples were collected from various retailers and markets across four Greek cities. In an effort to cover the highest range of products possible, different market presentations were targeted (fresh, frozen and cooked). Genomic DNA was extracted using the DNeasy Blood & Tissue Kit, according to the manufacturer's instructions. The mitochondrial gene selected as the target region of the analysis was the cytochrome c oxidase subunit I (COI). PCR products were purified and sequenced using an ABI 3500 Genetic Analyzer. Sequences were manually checked and edited using BioEdit software and compared against the ones available in GenBank and BOLD databases. Statistical analyses were conducted in R and PAST software. For most samples, COI amplification was successful, and species-level identification was possible. The preliminary results estimate moderate mislabeling rates (25%) in the identified samples. Mislabeling was most commonly detected in fresh products, with 50% of the samples in this category labeled incorrectly. Overall, the mislabeling rates detected by our study probably relate to some degree of unintentional misidentification, and lack of knowledge surrounding the legal designations by both retailers and consumers. For some species of crustaceans (i.e. Squila mantis) the mislabeling appears to be also affected by the local labeling practices. Across Greece, S. mantis is sold in the market under two common names, but only one is recognized by the country's legislation, and therefore any mislabeling is probably not profit-motivated. However, the substitution of the speckled shrimp (Metapenaus monoceros) for the distinct, giant river prawn (Macrobranchium rosenbergii), is a clear example of deliberate fraudulent substitution, aiming for profit. To our best knowledge, no scientific study investigating substitution and mislabeling rates in crustaceans has been conducted in Greece. For a better understanding of Greece's seafood market, similar DNA barcoding studies in other regions with increased touristic importance (e.g., the Greek islands) should be conducted. Regardless, the expansion of the list of species-specific designations for crustaceans in the country is advised.

Keywords: COI gene, food fraud, labelling control, molecular identification

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Authors: Alaa Abdellatif, Gabrièle Breda

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Background. Advanced therapy medicinal products (ATMPs) are innovative therapies that mainly target orphan diseases and high unmet medical needs. ATMP includes gene therapy medicinal products (GTMP), somatic cell therapy medicinal products (CTMP), and tissue-engineered therapies (TEP). Since legislation opened the way in 2007, 25 ATMPs have been approved in the EU, which is about the same amount as the U.S. Food and Drug Administration. However, not all of the ATMPs that have been approved have successfully reached the market and retained their approval. Objectives. We aim to understand all the factors limiting the market access to very promising therapies in a systemic approach, to be able to overcome these problems, in the future, with scientific, regulatory and commercial innovations. Further to recent reviews that focus either on specific countries, products, or dimensions, we will address all the challenges faced by ATMP development today. Methodology. We used mixed methods and a multi-level approach for data collection. First, we performed an updated academic literature review on ATMP development and their scientific and market access challenges (papers published between 2018 and April 2023). Second, we analyzed industry feedback from cell and gene therapy webinars and white papers published by providers and pharmaceutical industries. Finally, we established a comparative analysis of the regulatory guidelines published by EMA and the FDA for ATMP approval. Results: The main challenges in bringing these therapies to market are the high development costs. Developing ATMPs is expensive due to the need for specialized manufacturing processes. Furthermore, the regulatory pathways for ATMPs are often complex and can vary between countries, making it challenging to obtain approval and ensure compliance with different regulations. As a result of the high costs associated with ATMPs, challenges in obtaining reimbursement from healthcare payers lead to limited patient access to these treatments. ATMPs are often developed for orphan diseases, which means that the patient population is limited for clinical trials which can make it challenging to demonstrate their safety and efficacy. In addition, the complex manufacturing processes required for ATMPs can make it challenging to scale up production to meet demand, which can limit their availability and increase costs. Finally, ATMPs face safety and efficacy challenges: dangerous adverse events of these therapies like toxicity related to the use of viral vectors or cell therapy, starting material and donor-related aspects. Conclusion. As a result of our mixed method analysis, we found that ATMPs face a number of challenges in their development, regulatory approval, and commercialization and that addressing these challenges requires collaboration between industry, regulators, healthcare providers, and patient groups. This first analysis will help us to address, for each challenge, proper and innovative solution(s) in order to increase the number of ATMPs approved and reach the patients

Keywords: advanced therapy medicinal products (ATMPs), product development, market access, innovation

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Authors: Serpil Kilic, Ihsan Burak Cam, Murat Kilic, Timur Tongur

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Interest in the lead (Pb) has considerably increased due to knowledge about the potential toxic effects of this element, recently. Exposure to heavy metals above the acceptable limit affects human health. Indeed, Pb is accumulated through food chains up to toxic concentrations; therefore, it can pose an adverse potential threat to human health. A sensitive and reliable method for determination of Pb element in red pepper were improved in the present study. Samples (33 red pepper products having different brands) were purchased from different markets in Turkey. The selected method validation criteria (linearity, Limit of Detection, Limit of Quantification, recovery, and trueness) demonstrated. Recovery values close to 100% showed adequate precision and accuracy for analysis. According to the results of red pepper analysis, all of the tested lead element in the samples was determined at various concentrations. A Perkin- Elmer ELAN DRC-e model ICP-MS system was used for detection of Pb. Organic red pepper was used to obtain a matrix for all method validation studies. The certified reference material, Fapas chili powder, was digested and analyzed, together with the different sample batches. Three replicates from each sample were digested and analyzed. The results of the exposure levels of the elements were discussed considering the scientific opinions of the European Food Safety Authority (EFSA), which is the European Union’s (EU) risk assessment source associated with food safety. The Target Hazard Quotient (THQ) was described by the United States Environmental Protection Agency (USEPA) for the calculation of potential health risks associated with long-term exposure to chemical pollutants. THQ value contains intake of elements, exposure frequency and duration, body weight and the oral reference dose (RfD). If the THQ value is lower than one, it means that the exposed population is assumed to be safe and 1 < THQ < 5 means that the exposed population is in a level of concern interval. In this study, the THQ of Pb was obtained as < 1. The results of THQ calculations showed that the values were below one for all the tested, meaning the samples did not pose a health risk to the local population. This work was supported by The Scientific Research Projects Coordination Unit of Akdeniz University. Project Number: FBA-2017-2494.

Keywords: lead analyses, red pepper, risk assessment, daily exposure

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Authors: Berengere Vire, Nicolas Salvetat, Yoann Lannay, Guillaume Marcellin, Siem Van Der Laan, Franck Molina, Dinah Weissmann

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Predicting suicidal behaviors is one of the most complex challenges of daily psychiatric practices. Today, suicide risk prediction using biological tools is not validated and is only based on subjective clinical reports of the at-risk individual. Therefore, there is a great need to identify biomarkers that would allow early identification of individuals at risk of suicide. Alterations of adenosine-to-inosine (A-to-I) RNA editing of neurotransmitter receptors and other proteins have been shown to be involved in etiology of different psychiatric disorders and linked to suicidal behavior. RNA editing is a co- or post-transcriptional process leading to a site-specific alteration in RNA sequences. It plays an important role in the epi transcriptomic regulation of RNA metabolism. On postmortem human brain tissue (prefrontal cortex) of depressed suicide victims, Alcediag found specific alterations of RNA editing activity on the mRNA coding for the serotonin 2C receptor (5-HT2cR). Additionally, an increase in expression levels of ADARs, the RNA editing enzymes, and modifications of RNA editing profiles of prime targets, such as phosphodiesterase 8A (PDE8A) mRNA, have also been observed. Interestingly, the PDE8A gene is located on chromosome 15q25.3, a genomic region that has recurrently been associated with the early-onset major depressive disorder (MDD). In the current study, we examined whether modifications in RNA editing profile of prime targets allow identifying disease-relevant blood biomarkers and evaluating suicide risk in patients. To address this question, we performed a clinical study to identify an RNA editing signature in blood of depressed patients with and without the history of suicide attempts. Patient’s samples were drawn in PAXgene tubes and analyzed on Alcediag’s proprietary RNA editing platform using next generation sequencing technology. In addition, gene expression analysis by quantitative PCR was performed. We generated a multivariate algorithm comprising various selected biomarkers to detect patients with a high risk to attempt suicide. We evaluated the diagnostic performance using the relative proportion of PDE8A mRNA editing at different sites and/or isoforms as well as the expression of PDE8A and the ADARs. The significance of these biomarkers for suicidality was evaluated using the area under the receiver-operating characteristic curve (AUC). The generated algorithm comprising the biomarkers was found to have strong diagnostic performances with high specificity and sensitivity. In conclusion, we developed tools to measure disease-specific biomarkers in blood samples of patients for identifying individuals at the greatest risk for future suicide attempts. This technology not only fosters patient management but is also suitable to predict the risk of drug-induced psychiatric side effects such as iatrogenic increase of suicidal ideas/behaviors.

Keywords: blood biomarker, next-generation-sequencing, RNA editing, suicide

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Authors: William Castillo, Hugo Viza, Arturo Vargas

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The Pacific Alliance is a trade bloc that is composed of four emerging economies: Chile, Colombia, Peru, and Mexico. These economies have gained macroeconomic stability in the past decade and as a consequence present future economic progress. Under this positive scenario, international business firms have flourished. However, the literature in this region has been widely unexamined. Therefore, it is critical to fill this theoretical gap, especially considering that Latin America is starting to become a global player and it possesses a different institutional context than developed markets. This paper analyzes the effect of international entrepreneurial orientation and institutionalism on international performance, for the Pacific Alliance small-to-medium enterprises (SMEs). The literature considers international entrepreneurial orientation to be a powerful managerial capability – along the resource based view- that firms can leverage to obtain a satisfactory international performance. Thereby, obtaining a competitive advantage through the correct allocation of key resources to exploit the capabilities here involved. Entrepreneurial Orientation is defined around five factors: innovation, proactiveness, risk-taking, competitive aggressiveness, and autonomy. Nevertheless, the institutional environment – both local and foreign, adversely affects International Performance; this is especially the case for emerging markets with uncertain scenarios. In this way, the study analyzes an Entrepreneurial Orientation, key endogenous variable of international performance, and Institutionalism, an exogenous variable. The survey data consists of Pacific Alliance SMEs that have foreign operations in at least another country in the trade bloc. Findings are still in an ongoing research process. Later, the study will undertake a structural equation modeling (SEM) using the variance-based partial least square estimation procedure. The software that is going to be used is the SmartPLS. This research contributes to the theoretical discussion of a largely postponed topic: SMEs in Latin America, that has had limited academic research. Also, it has practical implication for decision-makers and policy-makers, providing insights into what is behind international performance.

Keywords: institutional theory, international entrepreneurial orientation, international performance, SMEs, Pacific Alliance

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Authors: Tengku Sepora Tengku Mahadi

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Where the speed of book writing lags behind the high need for such material for tertiary studies, translation offers a way to enhance the equilibrium in this demand-supply equation. Nevertheless, translation is confronted by obstacles that threaten its effectiveness. The primary challenge to the production of efficient translations may well be related to the text-type and in terms of its complexity. A text that is intricately written with unique rhetorical devices, subject-matter foundation and cultural references will undoubtedly challenge the translator. Longer time and greater effort would be the consequence. To understand these text-related challenges, the present paper set out to analyze a lawbook entitled Learning the Law by David Melinkoff. The book is chosen because it has often been used as a textbook or for reference in many law courses in the United Kingdom and has seen over thirteen editions; therefore, it can be said to be a worthy book for studies in law. Another reason is the existence of a ready translation in Malay. Reference to this translation enables confirmation to some extent of the potential problems that might occur in its translation. Understanding the organization and the language of the book will help translators to prepare themselves better for the task. They can anticipate the research and time that may be needed to produce an effective translation. Another premise here is that this text-type implies certain ways of writing and organization. Accordingly, it seems practicable to adopt the functional theory of language as suggested by Michael Halliday as its theoretical framework. Concepts of the context of culture, the context of situation and measures of the field, tenor and mode form the instruments for analysis. Additional examples from similar materials can also be used to validate the findings. Some interesting findings include the presence of several other text-types or sub-text-types in the book and the dependence on literary discourse and devices to capture the meanings better or add color to the dry field of law. In addition, many elements of culture can be seen, for example, the use of familiar alternatives, allusions, and even terminology and references that date back to various periods of time and languages. Also found are parts which discuss origins of words and terms that may be relevant to readers within the United Kingdom but make little sense to readers of the book in other languages. In conclusion, the textual analysis in terms of its functions and the linguistic and textual devices used to achieve them can then be applied as a guide to determine the effectiveness of the translation that is produced.

Keywords: functional theory of language, lawbook text-type, rhetorical devices, culture

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Authors: Morello Sara, Pederiva Sabina, Bianchi Manila, Martucci Francesca, Marchis Daniela, Decastelli Lucia

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Vegetables represent an integral part of a healthy diet due to their valuable nutritional properties and the growth in consumer demand in recent years is particularly remarkable for a diet rich in vitamins and micronutrients. However, plant-based products are involved in several food outbreaks connected to various sources of contamination and quite often, bacteria responsible for side effects showed high resistance to antibiotics. The abuse of antibiotics can be one of the main mechanisms responsible for increasing antibiotic resistance (AR). Plants grown for food use can be contaminated directly by spraying antibiotics on crops or indirectly by treatments with antibiotics due to the use of manure, which may contain both antibiotics and genes of antibiotic resistance (ARG). Antibiotic residues could represent a potential way of human health risk due to exposure through the consumption of plant-based foods. The presence of antibiotic-resistant bacteria might pose a particular risk to consumers. The present work aims to investigate through a multidisciplinary approach the occurrence of ARG by means of a biomolecular approach (PCR) and the prevalence of antibiotic residues using a multi residues LC-MS/MS method, both in different plant-based products. During the period from July 2020 to October 2021, a total of 74 plant samples (33 lettuces and 41 tomatoes) were collected from 57 farms located throughout the Piedmont area, and18 out of 74 samples (11 lettuces and 7 tomatoes) were selected to LC-MS/MS analyses. DNA extracted (ExtractME, Blirt, Poland) from plants used on crops and isolated bacteria were analyzed with 6 sets of end-point multiplex PCR (Qiagen, Germany) to detect the presence of resistance genes of the main antibiotic families, such as tet genes (tetracyclines), bla (β-lactams) and mcr (colistin). Simultaneous detection of 43 molecules of antibiotics belonging to 10 different classes (tetracyclines, sulphonamides, quinolones, penicillins, amphenicols, macrolides, pleuromotilines, lincosamides, diaminopyrimidines) was performed using Exion LC system AB SCIEX coupled to a triple quadrupole mass spectrometer QTRAP 5500 from AB SCIEX. The PCR assays showed the presence of ARG in 57% (n=42): tetB (4.8%; n=2), tetA (9.5%; n=4), tetE (2.4%; n=1), tetL (12%; n=5), tetM (26%; n=11), blaSHV (21.5%; n=9), blaTEM (4.8%; n =2) and blaCTX-M (19%; n=8). In none of the analyzed samples was the mcr gene responsible for colistin resistance detected. Results obtained from LC-MS/MS analyses showed that none of the tested antibiotics appear to exceed the LOQ (100 ppb). Data obtained confirmed the presence of bacterial populations containing antibiotic resistance determinants such as tet gene (tetracycline) and bla genes (beta-lactams), widely used in human medicine, which can join the food chain and represent a risk for consumers, especially with raw products. The presence of traces of antibiotic residues in vegetables, in concentration below the LOQ of the LC-MS/MS method applied, cannot be excluded. In conclusion, traces of antibiotic residues could be a health risk to the consumer due to potential involvement in the spread of AR. PCR represents a useful and effective approach to characterize and monitor AR carried by bacteria from the entire food chain.

Keywords: plant-based products, ARG, PCR, antibiotic residues

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Authors: Farzad Khajavi, Farzaneh Jalilfar, Faranak Jafari, Leila Shokrani

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Formulation of Ranolazine in the form of extended-release tablet in 500 mg dosage form was performed using Eudragit L100-55 as a retarding agent. Drug-release profiles were investigated in comparison with the reference Ranexa extended-release 500 mg tablet. F₂ and f₁ were calculated as 64.16 and 8.53, respectively. According to Peppas equation, the release of drug is controlled by diffusion (n=0.5). The tablets were put into accelerated stability conditions (40 °C, 75% humidity) for 3 and 6 months. The dissolution release profiles and other physical and chemical characteristics of the tablets confirmed the robustness and stability of formulation in this condition.

Keywords: drug release, extended-release tablet, ranolazine, stability

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Authors: Rohita Gupta, G. S. Brah, R. Verma, C. S. Mukhopadhayay

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Although chicken strains show differences in susceptibility to a number of diseases, the underlying immunological basis is yet to be elucidated. In the present study, heterophils were subjected to LPS stimulation and total RNA extraction, further differential gene expression was studied in broiler, layer and indigenous Aseel strain by Real Time RT-PCR at different time periods before and after induction. The expression of the 14 AvBDs and chTLR 1, 2, 3, 4, 5, 7, 15 and 21 was detectable in heterophils. The expression level of most of the AvBDs significantly increased (P<0.05) 3 hours post in vitro lipopolysaccharide challenge. Higher expression level and stronger activation of most AvBDs, NFkB-1 and IRF-3 in heterophils was observed, with the stimulation of LPS in layer compared to broiler, and in Aseel compared to both layer and broiler. This investigation will allow more refined interpretation of immuno-genetic basis of the variable disease resistance/susceptibility in divergent stock of chicken including indigenous breed. Moreover this study will be helpful in formulation of strategy for isolation of antimicrobial peptides from heterophils.

Keywords: differential expression, heterophils, cytokines, defensin, TLR

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Authors: R. Daïra, V. Chalvidan

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We present the speckle interferometry method to determine the deformation of a piece. This method of holographic imaging using a CCD camera for simultaneous digital recording of two states object and reference. The reconstruction is obtained numerically. This latest method has the advantage of being simpler than the methods currently available, and it does not suffer the holographic configuration faults online. Furthermore, it is entirely digital and avoids heavy analysis after recording the hologram. This work was carried out in the laboratory HOLO 3 (optical metrology laboratory in Saint Louis, France) and it consists in controlling qualitatively and quantitatively the deformation of object by using a camera CCD connected to a computer equipped with software of Fringe Analysis.

Keywords: speckle, nondestructive testing, interferometry, image processing

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Authors: Gabriella Kengyel

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This study aims to describe the differences between the attitudes of the right-wing extremists with internal or external control towards migrants. They both have a significantly higher score on Rotter's Locus of Control Scale, and they are quite xenophobic (54%) according to Bogardus Social Distance Scale. Present research suggests their motives are different. Principle components analysis shows that extremists with internal control reject migrants because of welfare chauvinism and they think that there is some kind of political conspirationism behind the European Refugee Crisis. Contrarily extremist with external control believe in a common enemy and they are significantly more ethnocentric and less skeptical in politics. Results suggest that extremist with internal control shows hostility toward minorities and migrants mainly because of their own reference group.

Keywords: control, extremist, migrant, right-wing

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Authors: Emmanuel Pedler, Elena Raevskikh, Maxime Jaffré

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Can the cultural geography of European cities be durably managed by European policies? To answer this question, two hypotheses can be proposed. (1) Either European cultural policies are able to erase cultural inequalities between the territories through the creation of new areas of cultural attractiveness in each beneficiary neighborhood, city or country. Or, (2) each European region historically rooted in a number of endogenous socio-historical, political or demographic factors is not receptive to exogenous political influences. Thus, the cultural attractiveness of a territory is difficult to measure and to impact by top-down policies in the long term. How do these two logics - European and local - interact and contribute to the emergence of a valued, popular sense of a common European cultural identity? Does this constant interaction between historical backgrounds and new political concepts encourage a positive identification with the European project? The European cultural policy programs, such as ECC (European Capital of Culture), seek to develop new forms of civic cohesion through inclusive and participative cultural events. The cultural assets of a city elected ‘ECC’ are mobilized to attract a wide range of new audiences, including populations poorly integrated into local cultural life – and consequently distant from pre-existing cultural offers. In the current context of increasingly heterogeneous individual perceptions of Europe, the ECC program aims to promote cultural forms and institutions that should accelerate both territorial and cross-border European cohesion. The new cultural consumption pattern is conceived to stimulate integration and mobility, but also to create a legitimate and transnational ideal European citizen type. Our comparative research confronts contrasting cases of ‘European Capitals of Culture’ from the south and from the north of Europe, cities recently concerned by the ECC political mechanism and cities that were elected ECC in the past, multi-centered cultural models vs. highly centralized cultural models. We aim to explore the impacts of European policies on the urban cultural geography, but also to understand the current obstacles for its efficient implementation.

Keywords: urbanism, cultural policies, cultural institutions, european cultural capitals, heritage industries, exclusion effects

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Authors: Maria Giovanna Lupo, Marina Camera, Marcello Rattazzi, Nicola Ferri

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A complex interplay among chronic kidney disease, lipid metabolism and aortic calcification has been recognized starting from results of many clinical and experimental studies. Here we investigated the influence of kidney function on PCSK9 levels, both in uremic rats and in clinical observation study, and its potential direct action on cultured smooth muscle cells (SMCs) calcification. In a cohort of 594 subjects enrolled in a single centre, observational, cross-sectional and longitudinal study, a negative association between GFR and plasma PCSK9 was found. Atherosclerotic cardiovascular disease (ASCVD), as co-morbidity, further increased PCSK9 plasma levels. Diet-induced uremic condition in rats, induced aortic calcification and increased total cholesterol and PCSK9 levels in plasma, livers and kidneys. Immunohistochemical analysis confirmed PCSK9 expression in aortic SMCs. SMCs overexpressing PCSK9 (SMCsPCSK9), cultured for 7-days in a pro-calcification environment (2.0mM or 2.4mM inorganic phosphate, Pi) showed a significantly higher extracellular calcium (Ca2+) deposition compared to mocked SMCs. Under the same experimental conditions, the addition of exogenous recombinant PCSK9 did not increase the extracellular calcification of SMCs. By flow cytometry analysis we showed that SMCsPCSK9, in response to 2.4mM Pi, released higher number of extracellular vesicles (EVs) positive for three tetraspanin molecules, such as CD63, CD9, and CD81. EVs derived from SMCsPCSK9 tended to be more enriched in calcium and alkaline phosphatase (ALPL), compared to EVs from mocks SMCs. In conclusion, our study reveals a direct role of PCSK9 on vascular calcification induced by higher inorganic phosphate levels associated to CKD condition. This effect appears to be mediated by a positive effect of endogenous PCSK9 on the release of EVs containing Ca2+ and ALP, which facilitate the deposition inorganic calcium phosphate crystals.

Keywords: PCSK9, calcification, extracellular vesicles, chronic kidney disease

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Authors: Inas Raafat, Azza El Bassiouny, Waldemar L. Olszewsky, Nagui E. Mikhail, Mona Nossier, Nora E. I. El-Bassiouni, Mona Zoheiry, Houda Abou Taleb, Noha Abd El-Aal, Ali Baioumy, Shimaa Attia

Abstract:

Background: Orthotopic liver transplantation is an established treatment for patients with severe acute and end-stage chronic liver disease. The shortage of donor organs continues to be the rate-limiting factor for liver transplantation throughout the world. Hepatocyte transplantation is a promising treatment for several liver diseases and can, also, be used as a "bridge" to liver transplantation in cases of liver failure. Aim of the work: This study was designed to develop a highly efficient protocol for isolation and transplantation of hepatocytes in experimental Lewis rat model to provide satisfactory guidelines for future application on humans.Materials and Methods: Hepatocytes were isolated from the liver by double perfusion technique and bone marrow cells were isolated by centrifugation of shafts of tibia and femur of donor Lewis rats. Recipient rats were subjected to sub-lethal dose of irradiation 2 days before transplantation. In a laparotomy operation the spleen was injected by freshly isolated hepatocytes and bone marrow cells were injected intravenously. The animals were sacrificed 45 day latter and splenic sections were prepared and stained with H & E, PAS AFP and Prox1. Results: The data obtained from this study showed that the double perfusion technique is successful in separation of hepatocytes regarding cell number and viability. Also the method used for bone marrow cells separation gave excellent results regarding cell number and viability. Intrasplenic engraftment of hepatocytes and live tissue formation within the splenic tissue were found in 70% of cases. Hematoxylin and eosin stained splenic sections from 7 rats showed sheets and clusters of cells among the splenic tissues. Periodic Acid Schiff stained splenic sections from 7 rats showed clusters of hepatocytes with intensely stained pink cytoplasmic granules denoting the presence of glycogen. Splenic sections from 7 rats stained with anti-α-fetoprotein antibody showed brownish cytoplasmic staining of the hepatocytes denoting positive expression of AFP. Splenic sections from 7 rats stained with anti-Prox1 showed brownish nuclear staining of the hepatocytes denoting positive expression of Prox1 gene on these cells. Also, positive expression of Prox1 gene was detected on lymphocytes aggregations in the spleens. Conclusions: Isolation of liver cells by double perfusion technique using collagenase buffer is a reliable method that has a very satisfactory yield regarding cell number and viability. The intrasplenic route of transplantation of the freshly isolated liver cells in an immunocompromised model was found to give good results regarding cell engraftment and tissue formation. Further studies are needed to assess function of engrafted hepatocytes by measuring prothrombin time, serum albumin and bilirubin levels.

Keywords: Lewis rats, hepatocytes, BMCs, transplantation, AFP, Prox1

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Authors: Foad Alzoughool

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Conflicting studies on the association between the rs5219 polymorphism and type 2 diabetes, some studies have confirmed a strong relationship between this variant and type2 diabetes, on the other hand, many studies denied the existence of this association. This study aimed to provide evidence about whether the rs5219 polymorphism has or hasn't a role as a risk factor for diabetes and meta-analysis to investigate the role of the control age group in the association. Genotyping of the rs5219 polymorphism was performed in a cohort of 266 healthy elderly subjects with a mean age (60.2 ± 5.1) with no history of diabetes (HbA1c < 6%) using standard Sanger sequencing methods. Lys/Lys alleles were detected in 20 persons (7.5%), Lys/Glu alleles in 96 persons (36.1%), and Glu/Glu in 150 persons (56.4%). The genotype distribution was consistent with Hardy–Weinberg equilibrium (P =0.7). Meta-analysis notably indicates no association between rs5219 polymorphism and type 2 diabetes in all studies used the younger age of the control group compared to the patient's age. In conclusion, our study sheds light on the importance of age factor among the control group recruited in case-control studies.

Keywords: Type 2 diabetes, rs5219 polymorphism, E23K, KCNJ11 gene

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Authors: Mohaya Farzin, Parvin Babaei, Mohammad Rostampour

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Ghrelin plays a considerable role in important neurological effects related to food intake and energy homeostasis. As was found, regular physical activity may make available significant improvements to cognitive functions in various behavioral situations. Anxiety is one of the main concerns of the modern world, affecting millions of individuals’ health. There are contradictory results regarding ghrelin's effects on anxiety-like behavior, and the plasma level of this peptide is increased during physical activity. Here we aimed to evaluate the coincident effects of exogenous ghrelin and aerobic exercise on anxiety-like behavior and passive avoidance memory in Wistar rats. Forty-five male Wistar rats (250 ± 20 g) were divided into 9 groups (n=5) and received intra-hippocampal injections of 3.0 nmol ghrelin and performed aerobic exercise training for 8 weeks. Control groups received the same volume of saline and diazepam as negative and positive control groups, respectively. Learning and memory were estimated using a shuttle box apparatus, and anxiety-like behavior was recorded by an elevated plus-maze test (EPM). Data were analyzed by ANOVA test, and p<0.05 was considered significant. Our findings showed that the combined effect of ghrelin and aerobic exercise improves the acquisition, consolidation, and retrieval of passive avoidance memory in Wistar rats. Furthermore, it is supposed that the ghrelin receiving group spent less time in open arms and fewer open arms entries compared with the control group (p<0.05). However, exercising Wistar rats spent more time in the open arm zone in comparison with the control group (p<0.05). The exercise + Ghrelin administration established reduced anxiety (p<0.05). The results of this study demonstrate that aerobic exercise contributes to an increase in the endogenous production of ghrelin, and physical activity alleviates anxiety-related behaviors induced by intra-hippocampal injection of ghrelin. In general, exercise and ghrelin can reduce anxiety and improve memory.

Keywords: anxiety, ghrelin, aerobic exercise, learning, passive avoidance memory

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Authors: Talah Aissa

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This paper reports an experimental study of the influence of marble powder used as a partial substitute for Portland cement (PC) on the mechanical properties and durability of high-performance concretes. The analysis of the experimental results on concrete at 15% content of marble powder with a fineness modulus of 11500 cm2/g, in a chloride environment, showed that it contributes positively to the perfection of its mechanical characteristics, its durability with respect to migration of chloride ions and oxygen permeability. On the basis of the experiments performed, it can be concluded that the marble powder is suitable for formulation of high performance concretes (HPC) and their properties are significantly better compared to the reference concrete (RC).

Keywords: marble powder, durability, concrete, cement

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Authors: M. N. Ervina Efzan, H. J. Kong, C. K. Kok

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The present work deals with a study on the influences of hybrid modifier on LM 6 added through ladle metallurgy. In this study, LM 6 served as the reference alloy while Na2CO3 and Ti powders were used as the hybrid modifier. The effects of hybrid modifier on the micro structural enhancement of LM 6 were investigated using optical microscope (OM) and Scanning Electron Microscope (SEM). The results showed fragmented Si-rich needles and strength enhanced petal/ globular-like structures without obvious formation of soft primary α-Al and β-Fe-rich inter metallic compound (IMC) after the hybrid modification. Hardness test was conducted to examine the mechanical improvement of hybrid modified LM 6. 10% of hardness improvement was recorded in the hybrid modified LM 6 through ladle metallurgy.

Keywords: Al-Si, hybrid modifier, ladle metallurgy, hardness

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Authors: Baljeet Singh Saharan

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Bioremediation is the demand of the day. Tannery and textile effluents/waste waters have lots of pollution due to presence of hexavalent Chromium. Methodologies used in the present investigations include isolation, cultivation and purification of bacterial strain. Further characterization techniques and 16S rRNA sequencing were performed. Efficient bacterial strain capable of reducing hexavalent chromium was obtained. The strain can be used for bioremediation of industrial effluents containing hexavalent Cr. A gram negative, rod shaped and yellowish pigment producing bacterial strain from tannery effluent was isolated using nutrient agar. The 16S rRNA gene sequence similarity indicated that isolate SA13A is associated with genus Luteimonas (99%). This isolate has been found to reduce 100% of hexavalent chromium Cr (VI) (100 mg L-1) 100% in 16 h. Growth conditions were optimized for Cr (VI) reduction. Maximum reduction was observed at a temperature of 37 °C and pH 8.0. Additionally, Luteimonas aestuarii SA13A showed resistance against various heavy metals like Cr+6, Cr+3, Cu+2, Zn+2, Co+2, Ni+2 and Cd+2 . Hence, Luteimonas aestuarii SA13A could be used as potent Cr (VI) reducing strain as well as significant bioremediator in heavy metal contaminated sites.

Keywords: bioremediation, chromium, eco-friendly, heavy metals

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Authors: H. Sakamoto, Y. Nakagawara, S. Oguri

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Drought stress is a critical environmental factor that adversely affects crop productivity and quality. Because of their immobile nature, plants have evolved mechanisms to sense and respond to drought stress. We identified a novel locus of Arabidopsis, designated DRA1 (drought responsive ankyrin 1), whose disruption leads to increased drought stress tolerance. DRA1 encodes a transmembrane protein with an ankyrin repeat motif that has been implicated in diverse cellular processes such as signal transduction. RT-PCR analysis revealed that there were at least two splicing variants of DRA1 transcripts in wild type plants. In response to drought stress, the levels of DRA1 transcripts retaining second and third introns were increased, whereas these introns were removed under unstressed conditions. These results suggest that DRA1 protein may negatively regulate plant drought tolerance and that the expression of DRA1 is regulated in response to drought stress by alternative splicing.

Keywords: alternative splicing, ankyrin repeat, Arabidopsis, drought tolerance

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Authors: Chung-Young Lee, Se-Hee Ahn, Ilhwan Kim, Du-Min Go, Dae-Yong Kim, Jun-Gu Choi, Youn-Jeong Lee, Jae-Hong Kim, Hyuk-Joon Kwon

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The polymerase of avian influenza A virus (AIV) is a heterotrimer composed of PB2, PB1 and PA. PB2 plays a role in overcoming the host barrier; however, the genetic prerequisites for avian PB2 to acquire mammalian pathogenic mutations have not been well elucidated. Here, we demonstrated that key amino acid mutations (I66M, I109V and I133V, collectively referred to as MVV) of prototypic avian PB2 increase the replication efficiency of recombinant PR8 virus carrying the mutated PB2 in both avian and mammalian hosts. The MVV mutations caused no weight loss in mice, but they did allow replication in infected lungs, and the viruses acquired fatal mammalian pathogenic mutations such as Q591R/K, E627K, or D701N in the infected lungs. The MVV mutations are located at the interfaces of the trimer and are predicted to increase the strength of this structure. Thus, gaining MVV mutations might be the first step for AIV to acquire mammalian pathogenicity. These results provide new insights into the evolution of AIV in birds and mammals.

Keywords: avian influenza A virus, prototypic PB2, polymerase activity, mammalian pathogenicity, first-step mutations

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