Search results for: gene expression profiles

Authors: Harish Rajak, Preeti Patel

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HDAC inhibitors can reactivate gene expression and inhibit the growth and survival of cancer cells. The 3D-QSAR and Pharmacophore modeling studies were performed to identify important pharmacophoric features and correlate 3D-chemical structure with biological activity. The pharmacophore hypotheses were developed using e-pharmacophore script and phase module. Pharmacophore hypothesis represents the 3D arrangement of molecular features necessary for activity. A series of 55 compounds with well-assigned HDAC inhibitory activity was used for 3D-QSAR model development. Best 3D-QSAR model, which is a five PLS factor model with good statistics and predictive ability, acquired Q2 (0.7293), R2 (0.9811) and standard deviation (0.0952). Molecular docking were performed using Histone Deacetylase protein (PDB ID: 1t69) and prepared series of hydroxamic acid based HDAC inhibitors. Docking study of compound 43 show significant binding interactions Ser 276 and oxygen atom of dioxine cap region, Gly 151 and amino group and Asp 267 with carboxyl group of CONHOH, which are essential for anticancer activity. On docking, most of the compounds exhibited better glide score values between -8 to -10.5. We have established structure activity correlation using docking, energetic based pharmacophore modelling, pharmacophore and atom based 3D QSAR model. The results of these studies were further used for the design and testing of new HDAC analogs.

Keywords: Docking, e-pharmacophore, HDACIs, QSAR, Suberoylanilidehydroxamic acid.

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Authors: Suhair Saleh, Ahmad Aljaberi

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Cationic lipid-mediated delivery of nucleic acids represents an exciting approach for developing therapeutically realistic gene medicines. Elucidation of the molecular and formulation requirements for efficient lipofection is a prerequisite to enhance the biological activity of such delivery systems. To this end, the in vitro lipofection activity of the ionizable asymmetric 1,2-dialkoylamidopropane-based derivatives bearing single primary amine group as the cationic head group was evaluated. The electrostatic interactions of these cationic lipids with plasmid DNA in physiologically relevant medium were investigated by means of gel electrophoresis retardation and Eth-Br quenching assays. The effect of the presence of the helper lipid on these interactions was evaluated. The physicochemical properties of these lipids in terms of bilayer fluidity and extent of ionization were investigated using fluorescence anisotropy and surface potential techniques, respectively. The results showed that only the active lipid, 1,2lmp[5], existed in a liquid crystalline state at physiological temperature. Moreover, the extent of ionization of this lipid in assemblies was significantly higher that it's saturated analogues. Inclusion of the helper lipid DOPE improved the encapsulation and association between 1,2lmp[5] and plasmid DNA, which was reflected by the significant boost of lipofection activity of the 1,2lmp[5]/DOPE formulation as compared to the lipid alone. In conclusion, membrane fluidity and sufficient protonation of ionizable cationic lipid are required for efficient association and encapsulation of plasmid DNA and promoting improved in vitro lipofection activity.

Keywords: cationic lipids, gene delivery, lipofection, membrane fluidity, helper lipids, surface potential

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Authors: Soumya S., Manju Nidagodu Jayakumar, Vellore Kannan Gopinath

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Dental pulp inflammation resulting from dental caries often leads to a pathologic condition known as irreversible pulpitis and the currently managed by root canal treatment. Extirpation of the entire pulp tissue is done during this procedure, and the canal space is filled with synthetic materials. Recent studies in the stem cell biology state that some portion of the irreversibly inflamed pulp tissue could be viable with progenitor cells, having the properties similar to that of Mesenchymal stem cells. Hence, we aim to isolate Dental Pulp Stem Cells (DPSCs) from patients diagnosed with severe irreversible pulpitis and characterize the cells for the MSC specific markers. The pulp tissue was collected from the dental clinic and subjected to collagenase/dispase digestion. The isolated cells were expanded in culture, and the phenotypic characterization was done using flow cytometry. MSC specific markers such as CD-90, CD-73, and CD-105 were analysed along with negative markers such as CD-14 and CD-45. The isolated cells expressed positive expression for CD markers with CD90 and CD105 ( > 95%) and CD73 (19%). The cells did not express the negative markers CD-14 and CD-45. The commercially available DPSCs from vital extracted teeth, preferably molar/wisdom teeth with large pulp cavity or incomplete root growth in young patients (aged 15-30 years) showed more than 90% expression for all the CD markers such as CD-90, 73 and 105, whereas negative for CD-14 and CD-45. The DPSCs isolated from inflamed pulp tissue showed a less expression for CD-73 compared to the commercially available DPSCs whereas, as the other two markers were found to show similar percentage of positive expression. This could be attributed to the fact that the pulp population is very heterogeneous and we used the pooled tissue from different patients. Hence the phenotypic characterization and comparison with the commercially available DPSCs proved that the inflamed pulp tissue is a good source of MSC like cells which can be utilized further for regenerative application.

Keywords: collagenase/dispase, dental pulp stem cells, flow cytometry, irreversible pulpitis

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Authors: N. Hoosain, S. Nene, B. Pearce, C. Jacobs, M. Du Plessis, M. Benjeddou

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Organic Cation Transporters play a vital role in the absorption, tissue distribution and elimination of various substrates. Numerous studies have suggested that variations in non-synonymous single nucleotide polymorphisms (SNPs) of SLC22A2 could influence an individual’s response to various treatments, including clinically important drugs. This study is the first to determine the baseline frequency distribution for twenty SNPs of SLC22A2in the Zulu population. DNA was collected from 101 unrelated “healthy” Zulu participants. Genotypes of all samples were determined using a multiplex PCR and SNaPshot assay followed by the generation of the haplotype structure. This is the first time that the baseline frequency distribution of SNPs is reported for the Zulu population. Data from this study could be used in in vitro and in vivo pharmacogenetic and pharmacokinetic studies to evaluate the potential role the studied SNPs play in the therapeutic efficacy of clinically important drugs.

Keywords: SLC22A2 gene, SNaPshot assay, PCR, Zulu population

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Authors: Maddalena Arigoni, Maria Luisa Ratto, Raffaele A. Calogero, Luca Alessandri

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The presence of tumor heterogeneity, where distinct cancer cells exhibit diverse morphological and phenotypic profiles, including gene expression, metabolism, and proliferation, poses challenges for molecular prognostic markers and patient classification for targeted therapies. Understanding the causes and progression of cancer requires research efforts aimed at characterizing heterogeneity, which can be facilitated by evolving single-cell sequencing technologies. However, analyzing single-cell data necessitates computational methods that often lack objective validation. Therefore, the establishment of benchmarking datasets is necessary to provide a controlled environment for validating bioinformatics tools in the field of single-cell oncology. Benchmarking bioinformatics tools for single-cell experiments can be costly due to the high expense involved. Therefore, datasets used for benchmarking are typically sourced from publicly available experiments, which often lack a comprehensive cell annotation. This limitation can affect the accuracy and effectiveness of such experiments as benchmarking tools. To address this issue, we introduce omics benchmark experiments designed to evaluate bioinformatics tools to depict the heterogeneity in single-cell tumor experiments. We conducted single-cell RNA sequencing on six lung cancer tumor cell lines that display resistant clones upon treatment of EGFR mutated tumors and are characterized by driver genes, namely ROS1, ALK, HER2, MET, KRAS, and BRAF. These driver genes are associated with downstream networks controlled by EGFR mutations, such as JAK-STAT, PI3K-AKT-mTOR, and MEK-ERK. The experiment also featured an EGFR-mutated cell line. Using 10XGenomics platform with cellplex technology, we analyzed the seven cell lines together with a pseudo-immunological microenvironment consisting of PBMC cells labeled with the Biolegend TotalSeq™-B Human Universal Cocktail (CITEseq). This technology allowed for independent labeling of each cell line and single-cell analysis of the pooled seven cell lines and the pseudo-microenvironment. The data generated from the aforementioned experiments are available as part of an online tool, which allows users to define cell heterogeneity and generates count tables as an output. The tool provides the cell line derivation for each cell and cell annotations for the pseudo-microenvironment based on CITEseq data by an experienced immunologist. Additionally, we created a range of pseudo-tumor tissues using different ratios of the aforementioned cells embedded in matrigel. These tissues were analyzed using 10XGenomics (FFPE samples) and Curio Bioscience (fresh frozen samples) platforms for spatial transcriptomics, further expanding the scope of our benchmark experiments. The benchmark experiments we conducted provide a unique opportunity to evaluate the performance of bioinformatics tools for detecting and characterizing tumor heterogeneity at the single-cell level. Overall, our experiments provide a controlled and standardized environment for assessing the accuracy and robustness of bioinformatics tools for studying tumor heterogeneity at the single-cell level, which can ultimately lead to more precise and effective cancer diagnosis and treatment.

Keywords: single cell omics, benchmark, spatial transcriptomics, CITEseq

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Authors: Parinaz Arashnia, Farzad Hatami, Saeed Ghaffarpour Jahromi

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Using steel in bridges’ construction because of their desired tensile and compressive strength and light weight especially in large spans was widely popular. Disadvantages of steel such as corrosion, buckling and weaknesses in high temperature and unsuitable weld could be solve with using Fibres Reinforced Polymer (FRP) profiles. The FRP is a remarkable class of composite polymers that can improve structural elements behaviour like corrosion resistance, fir resistance with good proofing and electricity and magnetic non-conductor. Nowadays except FRP reinforced bars and laminates, FRP I-beams are made and studied. The main reason for using FRP profiles is, prevent of corrosion and increase the load carrying capacity and durability, especially in large spans in bridges’ deck. In this paper, behaviour of I-section glass fibres reinforced polymer (GFRP) beam is discussed under point loads with numerical models and results has been compared and verified with experimental tests.

Keywords: glass fibres reinforced polymer, composite, I-section beam, durability, finite element method, numerical model

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Authors: Othman E. Othman, Amira M. Nowier, Medhat El-Denary

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Camels play an important socio-economic role within the pastoral and agricultural system in the dry and semidry zones of Asia and Africa. Camels are economically important animals in Egypt where they are dual purpose animals (meat and milk). The analysis of chemical composition of camel milk showed that the total protein contents ranged from 2.4% to 5.3% and it is divided into casein and whey proteins. The casein fraction constitutes 52% to 89% of total camel milk protein and it divided into 4 fractions namely αs1, αs2, β and κ-caseins which are encoded by four tightly genes. In spite of the important role of casein genes and the effects of their genetic polymorphisms on quantitative traits and technological properties of milk, the studies for the detection of genetic polymorphism of camel milk genes are still limited. Due to this fact, this work focused - using PCR-RFP and sequencing analysis - on the identification of genetic polymorphisms and SNPs of two casein genes in Maghrabi camel breed which is a dual purpose camel breed in Egypt. The amplified fragments at 488-bp of the camel κ-CN gene were digested with AluI endonuclease. The results showed the appearance of three different genotypes in the tested animals; CC with three digested fragments at 203-, 127- and 120-bp, TT with three digested fragments at 203-, 158- and 127-bp and CT with four digested fragments at 203-, 158-, 127- and 120-bp. The frequencies of three detected genotypes were 11.0% for CC, 48.0% for TT and 41.0% for CT genotypes. The sequencing analysis of the two different alleles declared the presence of a single nucleotide polymorphism (C→T) at position 121 in the amplified fragments which is responsible for the destruction of a restriction site (AG/CT) in allele T and resulted in the presence of two different alleles C and T in tested animals. The nucleotide sequences of κ-CN alleles C and T were submitted to GenBank with the accession numbers; KU055605 and KU055606, respectively. The primers used in this study amplified 942-bp fragments spanning from exon 4 to exon 6 of camel αS1-Casein gene. The amplified fragments were digested with two different restriction enzymes; SmlI and AluI. The results of SmlI digestion did not show any restriction site whereas the digestion with AluI endonuclease revealed the presence of two restriction sites AG^CT at positions 68^69 and 631^632 yielding the presence of three digested fragments with sizes 68-, 563- and 293-bp.The nucleotide sequences of this fragment from camel αS1-Casein gene were submitted to GenBank with the accession number KU145820. In conclusion, the genetic characterization of quantitative traits genes which are associated with the production traits like milk yield and composition is considered an important step towards the genetic improvement of livestock species through the selection of superior animals depending on the favorable alleles and genotypes; marker assisted selection (MAS).

Keywords: genetic polymorphism, SNP polymorphism, Maghrabi camels, κ-Casein gene, αS1-Casein gene

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Authors: Yusuf Ali Abdulle, Azhar Uddin Keerio

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Background: Protein elicitors play a key role in signaling or displaying plant defense mechanisms and emerging as vital tools for bio-control of insects. This study was aimed at the characterization of the novel protein elicitor isolated from entomopathogenic fungi Lecanicillium lecanii (V3) strain and its activity against Whitefly, Bemisia tabaci in cotton. The sequence of purified elicitor protein showed 100% similarity with hypothetical protein LEL_00878 [Cordyceps confragosa RCEF 1005], GenBank no (OAA81333.1). This novel protein elicitor has 253 amino acid residues and 762bp with a molecular mass of 29 kDa. The protein recombinant was expressed in Escherichia coli using pET‐28a (+) plasmid. Effects of purified novel protein elicitor on Bemisia tabaci were determined at three concentrations of protein (i.e., 58.32, 41.22, 35.41 μg mL⁻¹) on cotton plants and were exposed to newly molted adult B.tabaci. Bioassay results showed a significant effect of the exogenous application of novel protein elicitor on B. tabaci in cotton. In addition, the gene expression analysis found a significant up-regulation of the major genes associated with salicylic acid (SA) and jasmonic acid (JA) linked plant defense pathways in elicitor protein-treated plants. Our results suggested the potential application of a novel protein elicitor derived from Lecanicillium lecanii as a future bio-intensive controlling approach against the whitefly, Bemisia tabaci.

Keywords: resistance, Lecanicillium lecanii, secondary metabolites, whitefly

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Authors: Daniel Rowe, Christopher R. Vogel, Richard H. J. Willden

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The University of Oxford’s recirculating water flume is a combined wave and current test tank with a 1 m depth, 1.1 m width, and 10 m long working section, and is capable of flow speeds up to 1 ms−1 . This study documents the hydrodynamic characteristics of the facility in preparation for experimental testing of horizontal axis tidal stream turbine models. The turbine to be tested has a rotor diameter of 0.6 m and is a modified version of one of two model-scale turbines tested in previous experimental campaigns. An Acoustic Doppler Velocimeter (ADV) was used to measure the flow at high temporal resolution at various locations throughout the flume, enabling the spatial uniformity and turbulence flow parameters to be investigated. The mean velocity profiles exhibited high levels of spatial uniformity at the design speed of the flume, 0.6 ms−1 , with variations in the three-dimensional velocity components on the order of ±1% at the 95% confidence level, along with a modest streamwise acceleration through the measurement domain, a target 5 m working section of the flume. A high degree of uniformity was also apparent for the turbulence intensity, with values ranging between 1-2% across the intended swept area of the turbine rotor. The integral scales of turbulence exhibited a far higher degree of variation throughout the water column, particularly in the streamwise and vertical scales. This behaviour is believed to be due to the high signal noise content leading to decorrelation in the sampling records. To achieve more realistic levels of vertical velocity shear in the flume, a simple procedure to practically generate target vertical shear profiles in open-channel flows is described. Here, the authors arranged a series of non-uniformly spaced parallel bars placed across the width of the flume and normal to the onset flow. By adjusting the resistance grading across the height of the working section, the downstream profiles could be modified accordingly, characterised by changes in the velocity profile power law exponent, 1/n. Considering the significant temporal variation in a tidal channel, the choice of the exponent denominator, n = 6 and n = 9, effectively provides an achievable range around the much-cited value of n = 7 observed at many tidal sites. The resulting flow profiles, which we intend to use in future turbine tests, have been characterised in detail. The results indicate non-uniform vertical shear across the survey area and reveal substantial corner flows, arising from the differential shear between the target vertical and cross-stream shear profiles throughout the measurement domain. In vertically sheared flow, the rotor-equivalent turbulence intensity ranges between 3.0-3.8% throughout the measurement domain for both bar arrangements, while the streamwise integral length scale grows from a characteristic dimension on the order of the bar width, similar to the flow downstream of a turbulence-generating grid. The experimental tests are well-defined and repeatable and serve as a reference for other researchers who wish to undertake similar investigations.

Keywords: acoustic doppler Velocimeter, experimental hydrodynamics, open-channel flow, shear profiles, tidal stream turbines

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Authors: Zahra Mohajer, Afsaneh Soltani

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Doping is a major issue in competitive sports and is favored by vast groups of athletes. The feeling of being higher-ranking than others and gaining fame has caused many athletes to misuse drugs. The definition of doping is to use prohibited substances and/or methods that help physical or mental performances or both. Doping counts as the illegal use of chemical substances or drugs, excessive amounts of physiological substances to increase the performance at or out of competition or even the use of inappropriate medications to treat an injury to gain the ability to participate in a competition. The International Olympic Committee (IOC) and World Anti-Doping Agency (WADA) have forbidden these substances to ensure fair and equal competition and also the health of the competitors. As of 2004 WADA has published an international list of illegal substances used for doping, which is updated annually. In the process of the Genome Project scientists have gained the ability to treat numerous diseases by gene therapy, which may result in bodily performance increase and therefore a potential opportunity to misuse by some athletes. Gene doping is defined as the non-therapeutic direct and indirect genetic modifications using genetic materials that can improve the performances in sports events. Biosynthetic drugs are a form of indirect genetic engineering. The method can be performed in three ways such as injecting the DNA directly into the muscle, inserting the genetically engineered cells, or transferring the DNA using a virus as a vector. Erythropoietin is a hormone majorly released by the kidney and in small amounts by the liver. Its function is to stimulate the erythropoiesis and therefore the more production of red blood cells (RBC) which causes an increase in Hemoglobin (Hb). During this process, the oxygen delivery to muscles will increase, which will improve athletic performance and postpone exhaustion. There are ways to increase the oxygen transferred to muscles such as blood transfusion, stimulating the production of red blood cells by using Erythropoietin (EPO), and also using allosteric effectors of Hemoglobin. EPO can either be injected as a protein or can be inserted into the cells as the gene which encodes EPO. Adeno-associated viruses have been employed to deliver the EPO gene to the cells. Employing the genes that naturally exist in the human body such as the EPO gene can reduce the risk of detecting gene doping. The first research about blood doping was conducted in 1947. The study has shown that an increase in hematocrit (HCT) up to 55% following homologous transfusion makes it more unchallenging for the body to perform the exercise at the altitude. Thereafter athletes’ attraction to blood infusion escalated. Also, a study has demonstrated that by reinfusing their own blood 4 weeks after being drawn, three men have shown a rise in Hb level which improved the oxygen uptake, and a delay in exhaustion. The list of performance-enhancing drugs is published by WADA annually and includes the following drugs: anabolic agents, hormones, Beta-2 agonists, Beta-blockers, Diuretics, Stimulants, narcotics, cannabinoids, and corticosteroids.

Keywords: doping, PEDs, sports, WADA

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Authors: Indu Gaur, Neha Bhadauria, Shilpi Shilpi, Susmita Goswami, Prem D. Sharma, Prabir K. Paul

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The concept of organic agriculture has been accepted as novelty in Indian society, but there is no data available on the human pathogens colonizing plant parts due to such practices. Also, the pattern and mechanism of their colonization need to be understood in order to devise possible strategies for their prevention. In the present study, human pathogenic bacteria were isolated from organically grown tomato plants and five of them were identified as Klebsiella pneumoniae, Enterobacter ludwigii, Serratia fonticola, Stenotrophomonas maltophilia and Chryseobacterium jejuense. Tomato plants were grown in controlled aseptic conditions with 25±1˚C, 70% humidity and 12 hour L/D photoperiod. Six weeks old plants were divided into 6 groups of 25 plants each and treated as follows: Group 1: K. pneumonia, Group 2: E. ludwigii, Group 3: S. fonticola, Group 4: S. maltophilia, Group 5: C. jejuense, Group 6: Sterile distilled water (control). The inoculums for all treatments were prepared by overnight growth with uniform concentration of 108 cells/ml. Leaf samples from above groups were collected at 0.5, 2, 4, 6 and 24 hours post inoculation for the colony forming unit counts (CFU/cm2 of leaf area) of individual pathogens using leaf impression method. These CFU counts were used for the in vivo colonization assay and adherence assay of individual pathogens. Also, resistance of these pathogens to at least 12 antibiotics was studied. Based on these findings S. fonticola was found to be most prominently colonizing the phylloplane of tomato and was further studied. Tomato plants grown in controlled aseptic conditions same as mentioned above were divided into 2 groups of 25 plants each and treated as follows: Group 1: S. fonticola, Group 2: Sterile distilled water (control). Leaf samples from above groups were collected at 0, 24, 48, 72 and 96 hours post inoculation and homogenized in suitable buffers for surface and cell wall protein isolation. Protein samples thus obtained were subjected to isocratic SDS-gel electrophoresis and analyzed. It was observed that presence of S. fonticola could induce the expression of at least 3 additional cell wall proteins at different time intervals. Surface proteins also showed variation in the expression pattern at different sampling intervals. Further identification of these proteins by MALDI-MS and bioinformatics tools revealed the gene(s) involved in the interaction of S. fonticola with tomato phylloplane.

Keywords: cell wall proteins, human pathogenic bacteria, phylloplane, solanum lycopersicum

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Authors: Alan Ruiz-Padilla, Brando Villalobos-Villalobos, Yeniley Ruiz-Noa, Claudia Mendoza-Macías, Claudia Palafox-Sánchez, Miguel Marín-Rosales, Álvaro Cruz, Rubén Rangel-Salazar

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Introduction: In patients with rheumatoid arthritis, resistance or poor response to disease modifying antirheumatic drugs (DMARD) may be a reflection of the increase in g-P. The expression of g-P may be important in mediating the effluence of DMARD from the cell. In addition, P-glycoprotein is involved in the transport of cytokines, IL-1, IL-2 and IL-4, from normal lymphocytes activated to the surrounding extracellular matrix, thus influencing the activity of RA. The involvement of P-glycoprotein in the transmembrane transport of cytokines can serve as a modulator of the efficacy of DMARD. It was shown that a number of lymphocytes with glycoprotein P activity is increased in patients with RA; therefore, P-glycoprotein expression could be related to the activity of RA and could be a predictor of poor response to therapy. Objective: To evaluate in RA patients, if the G2677T/A MDR1 polymorphisms is associated with differences in the rate of therapeutic response to disease-modifying antirheumatic agents in patients with rheumatoid arthritis. Material and Methods: A prospective cohort study was conducted. Fifty seven patients with RA were included. They had an active disease according to DAS-28 (score >3.2). We excluded patients receiving biological agents. All the patients were followed during 6 months in order to identify the rate of therapeutic response according to the American College of Rheumatology (ACR) criteria. At the baseline peripheral blood samples were taken in order to identify the G2677T/A MDR1 polymorphisms using PCR- Specific allele. The fragment was identified by electrophoresis in polyacrylamide gels stained with ethidium bromide. For statistical analysis, the genotypic and allelic frequencies of MDR1 gene polymorphism between responders and non-responders were determined. Chi-square tests as well as, relative risks with 95% confidence intervals (95%CI) were computed to identify differences in the risk for achieving therapeutic response. Results: RA patients had a mean age of 47.33 ± 12.52 years, 87.7% were women with a mean for DAS-28 score of 6.45 ± 1.12. At the 6 months, the rate of therapeutic response was 68.7 %. The observed genotype frequencies were: for G/G 40%, T/T 32%, A/A 19%, G/T 7% and for A/A genotype 2%. Patients with G allele developed at 6 months of treatment, higher rate for therapeutic response assessed by ACR20 compared to patients with others alleles (p=0.039). Conclusions: Patients with G allele of the - G2677T/A MDR1 polymorphisms had a higher rate of therapeutic response at 6 months with DMARD. These preliminary data support the requirement for a deep evaluation of these and other genotypes as factors that may influence the therapeutic response in RA.

Keywords: pharmacogenetics, MDR1, P-glycoprotein, therapeutic response, rheumatoid arthritis

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Authors: Fengmei Li, Li Xu, Guoliang Xia

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Whey acidic proteins (WAP) domain-containing proteins in crustacean are involved in innate immune response against microbial invasion. In the present study, a novel double WAP domain (DWD)-containing protein gene 3 was identified from Chinese mitten crab Eriocheir sinensis (designated EsDWD3) by expressed sequence tag (EST) analysis and PCR techniques. The full-length cDNA of EsDWD3 was of 1223 bp, consisting of a 5′-terminal untranslated region (UTR) of 74 bp, a 3′ UTR of 727 bp with a polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 423 bp. The ORF encoded a polypeptide of 140 amino acids with a signal peptide of 22 amino acids. The deduced protein sequence EsDWD3 showed 96.4 % amino acid similar to other reported EsDWD1 from E. sinensis, and phylogenetic tree analysis revealed that EsDWD3 had closer relationships with the reported two double WAP domain-containing proteins of E. sinensis species.

Keywords: Chinese mitten crab, Eriocheir sinensis, cloning, double WAP domain-containing protein

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Authors: Ni Luh Putu Harta Wedari

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Background: Clostridium difficile has two main virulence factors, namely TcdA and TcdB. TcdA encoded by the tcdA gene acts as an enterotoxin, pro-inflammatory and fluid accumulation, while TcdB encoded by the tcdB gene is cytotoxic, causes disruption of the actin cytoskeleton, and causes disruption of tight junctions in colon cells. This study aims to explore the relationship between the tcdA and tcdB genes and the duration of diarrhea in elderly patients. Method: This research was an observational analytic with a prospective cross-sectional with samples of elderly diarrhea patients who met the inclusion criteria in Denpasar City health service facilities from 1 December 2022 until 30 June 2023, and then their feces were analyzed using the real-time PCR method. Results: In this study, 40 elderly diarrhea patients met the inclusion criteria and in accordance with the minimum sample size, 28 (70%) men and 12 (30%) women. 5 patients (12.5%) had a history of azithromycin, 4 (10%) levofloxacin, 17 (42.5%) ciprofloxacin, 8 (20%) metronidazole, 1 (2.5%) cefoperazone, 5 (12, 5%) doxycycline. Comorbids, namely 13 (32.5%) type II diabetes mellitus, 4 (10%) chronic kidney disease, 10 (25%) malignancies, 7 (17.5%) urinary tract infections, 3 (7.5%) %) immunocompromised, 2 (5%) cardiac heart failure, and 1 (2.5%) acute on chronic kidney disease. The overall diarrhea duration average was 5 days. 8 samples (20%) were positive for 16s rRNA, and there was no significant difference in diarrhea duration with negative samples (p=0.166). The relationship between the tcdA gene and the duration of diarrhea could not be performed because all samples were negative. Likewise, relationship analysis between the coexistence of tcdA and tcdB could not be performed. There was no significant difference between tcdB positive 3 (7.5%) and negative with diarrhea duration (p=0.739). Conclusion: There is no significant relationship between the presence of the 16s rRNA and tcdB C. difficile genes with the duration of diarrhea in elderly patients.

Keywords: clostridium, difficile, diarrhea, elderly, tcdA, tcdB

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Authors: Hala Ali

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In the evolving landscape of contemporary art, the value of failure and experimentation plays a central role in reshaping artistic research. This paper explores an artistic residency where the focus shifted from traditional practices of ink on canvas to performance art using the human body as a medium of expression. This residency emphasized uncertainty, experimentation, and emotional expression as the core of the process. Through collaboration between a calligrapher and a visual artist, the performance engaged themes of seduction, silence, and the transition between reality and abstraction. In alignment with experimental art practices, the process itself became the artwork, embracing moments of failure and disruption as key components of creative exploration. This research integrates theories from neuroscience, psychology, and artistic failure, drawing on the insights of thinkers like John Cage, Samuel Beckett, and Cornelius Cardew to further contextualize the residency’s impact.

Keywords: embodied art, emotional communication, mindfulness in art, nonverbal communication, performance art

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Authors: Reham K. Sebaih, Afaf I. Shehata , Awatif A. Hindi, Tarek Gheith, Amal A. Hazzani Anas Al-Orjan

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Staphylococci spp are ubiquitous gram-positive bacteria is often associated with infections, especially nosocomial infections, and antibiotic resistanceStudy pathogenic bacteria and its use as a tool in the technology of Nano biology and molecular genetics research of the latest research trends of modern characterization and definition of different multiresistant of bacteria including Staphylococci. The Staphylococci are widespread all over the world and particularly in Saudi Arabia The present work study was conducted to evaluate the effect of five different types of nanoparticles (biosynthesized zinc oxide, Spherical and rod of each silver and gold nanoparticles) and their antibacterial impact on the Staphylococcus species. Ninety-six isolates of Staphylococcus species. Staphylococcus aureus, Staphylococcus epidermidis, MRSA were collected from different sources during the period between March 2011G to June 2011G. All isolates were isolated from inpatients and outpatients departments at Royal Commission Hospital in Yanbu Industrial, Saudi Arabia. High percentage isolation from males(55%) than females (45%). Staphylococcus epidermidis from males was (47%), (28%), and(25%). For Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus (MRSA. Isolates from females were Staphylococcus aureus with higher percent of (47%), (30%), and (23%) for MRSA, Staphylococcus epidermidis. Staphylococcus aureus from wound swab were the highest percent (51.42%) followed by vaginal swab (25.71%). Staphylococcus epidermidis were founded with higher percentage in blood (37.14%) and wound swab (34.21%) respectively related to other. The highest percentage of methicillin-resistant Staphylococcus aureus (MRSA)(80.77%) were isolated from wound swab, while those from nostrils were (19.23%). Staphylococcus species were isolates in highest percentage from hospital Emergency department with Staphylococcus aureus (59.37%), Methicillin-resistant Staphylococcus aureus (MRSA) (28.13%)and Staphylococcus epidermidis (12.5%) respectively. Evaluate the antibacterial property of Zinc oxide, Silver, and Gold nanoparticles as an alternative to conventional antibacterial agents Staphylococci isolates from hospital sources we screened them. Gold and Silver rods Nanoparticles to be sensitive to all isolates of Staphylococcus species. Zinc oxide Nanoparticles gave sensitivity impact range(52%) and (48%). The Gold and Silver spherical nanoparticles did not showed any effect on Staphylococci species. Zinc Oxide Nanoparticles gave bactericidal impact (25%) and bacteriostatic impact (75%) for of Staphylococci species. Detecting the association of nanoparticles with Staphylococci isolates imaging by scanning electron microscope (SEM) of some bacteriostatic isolates for Zinc Oxide nanoparticles on Staphylococcus aureus, Staphylococcus epidermidis and Methicillin resistant Staphylococcus aureus(MRSA), showed some Overlapping Bacterial cells with lower their number and appearing some appendages with deformities in external shape. Molecular analysis was applied by Multiplex polymerase chain reaction (PCR) used for the identification of genes within Staphylococcal pathogens. A multiplex polymerase chain reaction (PCR) method has been developed using six primer pairs to detect different genes using 50bp and 100bp DNA ladder marker. The range of Molecular gene typing ranging between 93 bp to 326 bp for Staphylococcus aureus and Methicillin resistant Staphylococcus aureus by TSST-1,mecA,femA and eta, while the bands border were from 546 bp to 682 bp for Staphylococcus epidermidis using icaAB and atlE. Sixteen isolation of Staphylococcus aureus and Methicillin resistant Staphylococcus aureus were positive for the femA gene at 132bp,this allowed the using of this gene as an internal positive control, fifteen isolates of Staphylococcus aureus and Methicillin resistant Staphylococcus aureus were positive for mecA gene at163bp.This gene was responsible for antibiotic resistant Methicillin, Two isolates of Staphylococcus aureus and Methicillin resistant Staphylococcus aureus were positive for the TSST-1 gene at326bp which is responsible for toxic shock syndrome in some Staphylococcus species, None were positive for eta gene at 102bpto that was responsible for Exfoliative toxins. Six isolates of Staphylococcus epidermidis were positive for atlE gene at 682 bp which is responsible for the initial adherence, three isolates of Staphylococcus epidermidis were positive for icaAB gene at 546bp that are responsible for mediates the formation of the biofilm. In conclusion, this study demonstrates the ability of the detection of the genes to discriminate between infecting Staphylococcus strains and considered biological tests, they may potentiate the clinical criteria used for the diagnosis of septicemia or catheter-related infections.

Keywords: multiplex polymerase chain reaction, toxic shock syndrome, Staphylococcus aureus, nosocomial infections

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Authors: Xinxiu Li, Chuqian Zheng, Yanyan Qian, Hong Fan

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Objectives: In hepatocellular carcinoma (HCC), oncogenes are continuously and robustly transcribed due to aberrant expression of essential components of the trans-acting super-enhancers (SE) complex. Preclinical and clinical trials are now being conducted on small-molecule inhibitors that target core-transcriptional components, including as transcriptional bromodomain protein 4 (BRD4) and cyclin-dependent kinase 7 (CDK7), in a number of malignant tumors. This study aims to explore whether co-overexpression of BRD4 and CDK7 is a potential marker of worse prognosis and a combined therapeutic target in HCC. Methods: The expression pattern of BRD4 and CDK7 and their correlation with prognosis in HCC were analyzed by RNA sequencing data and survival data of HCC patients from TCGA and GEO datasets. The protein levels of BRD4 and CDK7 were determined by immunohistochemistry (IHC), and survival data of patients were analyzed using the Kaplan-Meier method. The mRNA expression levels of genes in HCC cell lines were evaluated by quantitative PCR (q-PCR). CCK-8 and colony formation assays were conducted to assess cell proliferation of HCC upon treatment with BRD4 inhibitor JQ1 or/and CDK7 inhibitor THZ1. Results: It was shown that BRD4 and CDK7 were often overexpressed in HCCs and were associated with poor prognosis of HCC by analyzing the TCGA and GEO datasets. BRD4 or CDK7 overexpression was related to a lower survival rate. It's interesting to note that co-overexpression of CDK7 and BRD4 was a worse prognostic factor in HCC. Treatment with JQ1 or THZ1 alone had an inhibitory effect on cell proliferation; however, when JQ1 and THZ1 were combined, there was a more notable suppression of cell growth. At the same time, the combined use of JQ1 and THZ1 synergistically suppresses the expression of HCC driver genes. Conclusion: Our research revealed that BRD4 and CDK7 coupled can be a useful biomarker in HCC prognosis and the combination of JQ1 and THZ1 can be a promising therapeutic therapy against HCC.

Keywords: BRD4, CDK7, cell proliferation, combined inhibition

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Authors: Chia-Jung Li, Kuan-Hao Tsui

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As women age, the quality of their oocytes deteriorates irreversibly, leading to reduced fertility. To better understand the role of Ferroptosis-related genes in ovarian aging, we employed a multi-omics analysis approach, including spatial transcriptomics, single-cell RNA sequencing, human ovarian pathology, and clinical biopsies. Our study identified excess lipid peroxide accumulation in aging germ cells, metal ion accumulation via oxidative reduction, and the interaction between ferroptosis and cellular energy metabolism. We used multi-histological prediction of ferroptosis key genes to evaluate 75 patients with ovarian aging insufficiency and then analyzed changes in hub genes after supplementing with DHEA, Ubiquinol CoQ10, and Cleo-20 T3 for two months. Our results demonstrated a significant increase in TFRC, GPX4, NCOA4, and SLC3A2, which were consistent with our multi-component prediction. We theorized that these supplements increase the mitochondrial tricarboxylic acid cycle (TCA) or electron transport chain (ETC), thereby increasing antioxidant enzyme GPX4 levels and reducing lipid peroxide accumulation and ferroptosis. Overall, our findings suggest that supplementation intervention significantly improves IVF outcomes in senescent cells by enhancing metal ion and energy metabolism and enhancing oocyte quality in aging women.

Keywords: multi-omics, nutrients, ferroptosis, ovarian aging

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Authors: Valentina V. Zhurikhina, Mihail I. Petrov, Alexandra A. Rtischeva, Mark Dussauze, Thierry Cardinal, Andrey A. Lipovskii

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Polarization of glass, often referred to as thermal poling, is a well-known method to modify the glass physical and chemical properties, that manifest themselves in loosing central symmetry of the medium, glass structure and refractive index modification. The usage of the poling for second optical harmonic generation, fabrication of optical waveguides and electrooptic modulators was also reported. Nevertheless, the detailed description of the poling of glasses, containing multiple charge carriers is still under discussion. In particular, the role of possible migration of electrons in the space charge formation usually remains out of the question. In this work, we performed the numerical simulation of thermal poling of a silicate glass, containing Na, K, Mg, and Ca. We took into consideration the contribution of electrons in the polarization process. The possible explanation of migration of electrons can be the break of non-bridging oxygen bonds. It was found, that the modeled depth of the space charge region is about 10 times higher if the migration of the negative charges is taken under consideration. The simulated profiles of cations, participating in the polarization process, are in a good agreement with the experimental data, obtained by glow discharge spectroscopy.

Keywords: glass poling, charge transport, modeling, concentration profiles

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Authors: Mahmut Ali Gökçe, Oguz Tasbozan, Celal Erbas, Zafer Akpinar, S. Surhan Tabakoglu, Mehmet Celik, Bilge Kaan Tekelioglu

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This study was conducted to investigate proximate compositions and fatty acid profiles of marketable size striped sea bream of obtained from fish cages of aquaculture companies and fishermen. Ten fish samples were used for both groups. The average total weight of farmed and wild samples was 252,75 ± 36,78 g and 193,0 ± 32 g respectively. While the protein level of farmed samples was (23,49±0,15) higher than that of wild fish (21,80±0,18), lipid level was less (1,55±0,08) in farmed group than wild fish samples (2,52±0,07). Amount of Σ SFA was significantly higher in wild group (44,09±0,9) than the farmed (32,79±1,13) group. Total MUFA were 36,38±29,91 in wild and 29,91±1,52 in farmed fish. However, Σ PUFA (27,89±1,53) and EPA+DHA values (15,73±1,63) of farmed samples were significantly higher than the wild (14,06 ±3,67 and 9,7±0,86) counterparts. Σώ3/ώ6 rate was better in farmed group with 2,54±0,84 in comparison with (1,59±0,06) the other group. As a result, it can be speculated that the farmed striped sea bream can be preferred by the consumers. Acknowledgement: This work was supported by the Scientific Research Project Unit of the University of Cukurova, Turkey under grant no FBA-2016-5073.

Keywords: striped sea bream, Litognathus mormyrus, proximate composition, fatty acid profile

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Authors: Zunnu Raen Akhtar

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Cotton Leaf Curl Disease (CLCuD) is from Gemini virus and is transmitted through whiteflies in cotton. Transgenic cotton containing Antisense Coat Protein (ACP) has been found to show better results against CLCuD in cotton. In current research, Antisense Coat Protein was inserted in cotton plants to observe resistance developed in the cotton plants against CLCuD. T1 generation of plants were observed for its expression in plants. Tests were carried out to observe the expression of Antisense Coat Protein using Polymerase Chain Reaction (PCR) technique and by southern blotting. Whiteflies showing positive Cotton Leaf Curl Virus (CLCV) were reared and released in bioassay on ACP expressing cotton plants under laboratory as well as confined semi-field conditions. Results confirmed the expression of AC protein in PCR and southern blotting. Further laboratory results showed that cotton plants expressing AC protein showed rare incidence of CLCuD infection as compared to control. In the confined semi-field, similar results were observed in AC protein expressing cotton as compared to control. These results explicitly show that ACP can help to tackle the CLCuD issue in the future and further studies on biochemical processes involved in these plants and effects of ACP induction on non-target organisms should also be studied for eco-system.

Keywords: cotton, white flies, antisense coat protein, CLCV

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

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Formulation of gliclazide in the form of extended-release tablet in 30 and 60 mg dosage forms was performed using hypromellose (HPMC K4M) as a retarding agent. Drug-release profiles were investigated in comparison with references Diamicron MR 30 and 60 mg tablets. The effect of size of powder particles, the amount of hypromellose in formulation, hardness of tablets, and also the effect of halving the tablets were investigated on drug release profile. A mathematical model which describes hypromellose behavior in initial times of drug release was proposed for the estimation of hypromellose content in modified-release gliclazide 60 mg tablet. This model is based on erosion of hypromellose in dissolution media. The model is applicable to describe release profiles of insoluble drugs. Therefore, by using dissolved amount of drug in initial times of dissolution and the model, the amount of hypromellose in formulation can be predictable. The model was used to predict the HPMC K4M content in modified-release gliclazide 30 mg and extended-release quetiapine 200 mg tablets.

Keywords: Gliclazide, hypromellose, drug release, modified-release tablet, mathematical model

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Authors: G. Blanco-Lizana, C. García-Fernández, J. A. Sánchez

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Growth is a productive trait regulated by a large and complex gene network with very different effect. Some of they (candidate genes) have a higher effect and are excellent resources to search in them polymorphisms correlated with differences in growth rates. This study was focused on the identification of single nucleotide polymorphism (SNP) in MyoD-1 and MyoD-2 genes, members of the family of myogenic regulatory genes with a key role in the differentiation and development of muscular tissue.(MFRs), and its evaluation as potential markers in genetic selection programs for growth in gilthead sea bream (Sparus aurata). Through a sequencing in 30 seabream (classified as unrelated by microsatellite markers) of 1.968bp in MyoD-1 gene [AF478568 .1] and 1.963bp in MyoD-2 gene [AF478569.1], three SNPs were identified in each gene (SaMyoD-1 D2100A (D indicate a deletion) SaMyoD-1 A2143G and SaMyoD-1 A2404G and SaMyoD-2_A785C, SaMyoD-2_C1982T and SaMyoD-2_A2031T). The relationships between SNPs and body weight were evaluated by SNP genotyping of 53 breeders from two broodstocks (A:18♀-9♂; B:16♀-10♂) and 389 offspring divided into two groups (slow- and fast-growth) with significant differences in growth at 18 months of development (A18Slow: N=107, A18Fast: N=103, B18Slow: N=92 and B18Fast: N=87) (Borrell et al., 2011). Haplotype and diplotype were reconstructed from genotype data by Phase 2.1 software. Differences among means of different diplotypes were calculated by one-way ANOVA followed by post-hoc Tukey test. Association analysis indicated that single SNP did not show significant effect on body weight. However, when the analysis is carried out considering haplotype data it was observed that the DGG haplotipe of MyoD-1 gen and CCA haplotipe of MyoD- 2gen were associated to with lower body weight. This haplotype combination always showed the lowest mean body weight (P<0.05) in three (A18Slow, A18Fast & B18Slow) of the four groups tested. Individuals with DGG haplotipe of MyoD-1 gen have a 25,5% and those with CCA haplotipe of MyoD- 2gen showed 14-18% less on mean body weight. Although further studies are need to validate the role of these 3 SNPs as marker for body weight, the polymorphism-trait association established in this work create promising expectations on the use of these variants as genetic tool for future giltead seabream breeding programs.

Keywords: growth, MyoD-1 and MyoD-2 genes, selective breeding, SNP-haplotype

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Authors: Sara A. Al-Jaberi, Salma Ben-Salem, Meriam Messedi, Fatma Ayadi, Lihadh Al-Gazali, Bassam R. Ali

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Background: The chemokine receptor components play crucial roles in the immune system and some of them serve as co-receptors for the HIV virus. Several studies have documented those variants in chemokine receptors are correlated with susceptibility and resistance to infection with HIV virus. For example, mutations in the chemokine receptor 5 gene (CCR5) resulting in loss-of-function (such as the homozygous CCR5Δ32) confer high degree of resistance to HIV infection. Heterozygotes for these variants exhibit slow progression to AIDS. The prevalence of CCR5 polymorphisms varies among ethnic and geographical groups. For example, the CCR5 Δ32 variant is present in 10–15% of north Europeans but is rarely encountered among Africans. This study aims to identify the prevalence of some CCR5 variants in two geographically distant Arab populations (namely Emiratis and Tunisians). Methodology: The prevalence of CCR5 gene variants including CCR5Δ32, FS299, C101X, A29S and C178R has been determined using PCR and direct DNA sequencing. A total of 403 unrelated healthy individuals (253 Emiratis and 150 Tunisians) were genotyped for the CCR5Δ32 variant using PCR amplification and gel electrophoresis. In addition, 200 Emiratis have been screened for other SNPs using Sanger DNA sequencing. Results: Among Emiratis, the allele frequency of the CCR5Δ32 variant has been found to be 0.002. In addition, two variants L55Q and A159 were found at a frequency of 0.002.Moreover, the prevalence of the CCR5Δ32 variant in Tunisians was estimated to be 0.013 which is relatively higher than its frequency in Emiratis but lower than Europeans. Conclusion: We conclude that the allele frequency of the most critical CCR5 polymorphism (Δ32) is extremely low among Emiratis compared to other Arabs and North Europeans. In addition, very low allele frequencies of other CCR5 polymorphisms have been detected among Emiratis.

Keywords: chemokine receptors, CCR5Δ32, CCR5 polymorphisms, Emiratis, Arab populations

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Authors: Aniket Kumar, Jacob Peedicayil

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Major depressive disorder (MDD) is a common and important psychiatric disorder. Several clinical features of MDD suggest an epigenetic basis for its pathogenesis. Since epigenetics (heritable changes in gene expression not involving changes in DNA sequence) may underlie the pathogenesis of MDD, epigenetic drugs such as DNA methyltransferase inhibitors (DNMTi) and histone deactylase inhibitors (HDACi) may be useful for treating MDD. The available literature indexed in Pubmed on preclinical drug trials of epigenetic drugs for the treatment of MDD was investigated. The search terms we used were ‘depression’ or ‘depressive’ and ‘HDACi’ or ‘DNMTi’. Among epigenetic drugs, it was found that there were 3 preclinical trials using HDACi and 3 using DNMTi for the treatment of MDD. All the trials were conducted on rodents (mice or rats). The animal models of depression that were used were: learned helplessness-induced animal model, forced swim test, open field test, and the tail suspension test. One study used a genetic rat model of depression (the Flinders Sensitive Line). The HDACi that were tested were: sodium butyrate, compound 60 (Cpd-60), and valproic acid. The DNMTi that were tested were: 5-azacytidine and decitabine. Among the three preclinical trials using HDACi, all showed an antidepressant effect in animal models of depression. Among the 3 preclinical trials using DNMTi also, all showed an antidepressant effect in animal models of depression. Thus, epigenetic drugs, namely, HDACi and DNMTi, may prove to be useful in the treatment of MDD and merit further investigation for the treatment of this disorder.

Keywords: DNA methylation, drug discovery, epigenetics, major depressive disorder

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Authors: Yasser M. Saad, Amr A. El Hanafy, Saleh A. Alkarim, Hussein A. Almehdar, Elrashdy M. Redwan

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Camels are substantial providers of transport, milk, sport, meat, shelter, security and capital in many countries, particularly in Saudi Arabia. Inter simple sequence repeat technique was used to detect the genetic variations among some camel breeds (Majaheim, Safra, Wadah, and Hamara). Actual number of alleles, effective number of alleles, gene diversity, Shannon’s information index and polymorphic bands were calculated for each evaluated camel breed. Neighbor-joining tree that re-constructed for evaluated these camel breeds showed that, Hamara breed is distantly related from the other evaluated camels. In addition, the polymorphic sites, haplotypes and nucleotide diversity were identified for some camelidae cox1 gene sequences (obtained from NCBI). The distance value between C. bactrianus and C. dromedarius (0.072) was relatively low. Analysis of genetic diversity is an important way for conserving Camelus dromedarius genetic resources.

Keywords: camel, genetics, ISSR, neighbor-joining

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Authors: Xi Chen, King-Yip Cheng

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Hypercholesterolemia, characterized by high low-density lipoprotein cholesterol (LDL-C), raises cardiovascular events in patients with type 2 diabetes (T2D). Although several drugs, such as statin and PCSK9 inhibitors, are available for the treatment of hypercholesterolemia, they exert detrimental effects on glucose metabolism and hence increase the risk of T2D. On the other hand, the drugs used to treat T2D have minimal effect on improving the lipid profile. Therefore, there is an urgent need to develop treatments that can simultaneously improve glucose and lipid homeostasis. Adaptor protein phosphotyrosine interacting with PH domain and leucine zipper 2 (APPL2) causes insulin resistance in the liver and skeletal muscle via inhibiting insulin and adiponectin actions in animal models. Single-nucleotide polymorphisms in the APPL2 gene were associated with LDL-C, non-alcoholic fatty liver disease, and coronary artery disease in humans. The aim of this project is to investigate whether APPL2 antisense oligonucleotide (ASO) can alleviate dietary-induced T2D and hypercholesterolemia. High-fat diet (HFD) was used to induce obesity and insulin resistance in mice. GalNAc-conjugated APPL2 ASO (GalNAc-APPL2-ASO) was used to silence hepatic APPL2 expression in C57/BL6J mice selectively. Glucose, lipid, and energy metabolism were monitored. Immunoblotting and quantitative PCR analysis showed that GalNAc-APPL2-ASO treatment selectively reduced APPL2 expression in the liver instead of other tissues, like adipose tissues, kidneys, muscle, and heart. The glucose tolerance test and insulin sensitivity test revealed that GalNAc-APPL2-ASO improved glucose tolerance and insulin sensitivity progressively. Blood chemistry analysis revealed that the mice treated with GalNAc-APPL2-ASO had significantly lower circulating levels of total cholesterol and LDL cholesterol. However, there was no difference in circulating levels of high-density lipoprotein (HDL) cholesterol, triglyceride, and free fatty acid between the mice treated with GalNac-APPL2-ASO and GalNAc-Control-ASO. No obvious effect on food intake, body weight, and liver injury markers after GalNAc-APPL2-ASO treatment was found, supporting its tolerability and safety. We showed that selectively silencing hepatic APPL2 alleviated insulin resistance and hypercholesterolemia and improved energy metabolism in the dietary-induced obese mouse model, indicating APPL2 as a therapeutic target for metabolic diseases.

Keywords: APPL2, antisense oligonucleotide, hypercholesterolemia, type 2 diabetes

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Authors: Numfon Rakkhumkaew, Takeru Kawasaki, Makoto Fujie, Takashi Yamada

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Chlorella viruses or chloroviruses contain a gene that encodes a function for chitin synthesis, which is expressed early in viral infection to produce chitin polysaccharide, a polymer of β-1, 4-linked GlcNAc, on the outside of Chlorella cell wall. Interestingly, chlorovirus system is an eco-friendly system which converses CO2 and solar energy from the environment into useful materials. However, infected Chlorella cells are lysed at the final stage of viral infection, and this phenomenon is caused the breaking down of polysaccharide. To postpone the lysing period and prolong the synthesis of chitin polysaccharide on cells, the slow growing virus incorporated with aphidicolin treatment, an inhibitor of DNA synthesis, was investigated. In this study, a total of 25 virus isolates from water samples in Japan region were analyzed for CHS (the gene for CH synthase) gene by PCR (polymerase chain reaction). The accumulation and appearance of chitin polysaccharide on infected cells were detected by biotinylated chitin-binding proteins WGA (wheat germ agglutinin)-biotin for chitin in conjunction with avidin-Cy 2 or Cy 3 and investigated by fluorescence microscopy, observed as green or yellow fluorescence over the cell surface. Among all chlorovirus isolates, cells infected with CNF1 revealed the accumulation of chitin over the cell surface within 30 min p.i. and continued to accumulate on cells until 4 h p.i. before cell lyses which was 1.6 times longer accumulation period than cells infected with CVK2 (prototype virus). Furthermore, addition of aphidicolin could extend the chitin accumulation on cells infected with CNF1 until 8 h p.i. before cell lyses. Whereas, CVK2-infected cells treated with aphidicolin could prolong the chitin synthesis only for 6 h p.i. before cell lyses. Therefore, chitin synthesis by Chlorella-virus system could be prolonged by using slow-growing viral isolates and with aphidicolin.

Keywords: chitin, chlorovirus, Chlorella virus, aphidicolin

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Authors: Lena Payati, Maria Kazou, Effie Tsakalidou

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Table olives are one of the most popular fermented vegetables worldwide, which along with olive oil, have a crucial role in the world economy. They are highly appreciated by the consumers for their characteristic taste and pleasant aromas, while several health and nutritional benefits have been reported as well. Until recently, microbial biogeography, i.e., the study of microbial diversity over time and space, has been mainly associated with wine. However, nowadays, the term 'terroir' has been extended to other crops and food products so as to link the geographical origin and environmental conditions to quality aspects of fermented foods. Taking the above into consideration, the present study focuses on the microbial fingerprinting of the most important olive varieties of Greece with the state-of-the-art amplicon-based metagenomics analysis. Towards this, in 2019, 61 samples from 38 different olive varieties were collected at the final stage of ripening from 13 well spread geographical regions in Greece. For the metagenomics analysis, total DNA was extracted from the olive samples, and the 16S rRNA gene and ITS DNA region were sequenced and analyzed using bioinformatics tools for the identification of bacterial and yeasts/fungal diversity, respectively. Furthermore, principal component analysis (PCA) was also performed for data clustering based on the average microbial composition of all samples from each region of origin. According to the composition, results obtained, when samples were analyzed separately, the majority of both bacteria (such as Pantoea, Enterobacter, Roserbergiella, and Pseudomonas) and yeasts/fungi (such as Aureobasidium, Debaromyces, Candida, and Cladosporium) genera identified were found in all 61 samples. Even though interesting differences were observed at the relative abundance level of the identified genera, the bacterial genus Pantoea and the yeast/fungi genus Aureobasidium were the dominant ones in 35 and 40 samples, respectively. Of note, olive samples collected from the same region had similar fingerprint (genera identified and relative abundance level) regardless of the variety, indicating a potential association between the relative abundance of certain taxa and the geographical region. When samples were grouped by region of origin, distinct bacterial profiles per region were observed, which was also evident from the PCA analysis. This was not the case for the yeast/fungi profiles since 10 out of the 13 regions were grouped together mainly due to the dominance of the genus Aureobasidium. A second cluster was formed for the islands Crete and Rhodes, both of which are located in the Southeast Aegean Sea. These two regions clustered together mainly due to the identification of the genus Toxicocladosporium in relatively high abundances. Finally, the Agrinio region was separated from the others as it showed a completely different microbial fingerprinting. However, due to the limited number of olive samples from some regions, a subsequent PCA analysis with more samples from these regions is expected to yield in a more clear clustering. The present study is part of a bigger project, the first of its kind in Greece, with the ultimate goal to analyze a larger set of olive samples of different varieties and from different regions in Greece in order to have a reliable olives’ microbial biogeography.

Keywords: amplicon-based metagenomics analysis, bacteria, microbial biogeography, olive microbiota, yeasts/fungi

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Authors: Tatsiana Ihnatovich, Darya Nizheharodava, Mikalai Halabarodzka, Tatsiana Savitskaya, Marina Zafranskaya

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Liver fibrosis is a complex of histological changes resulting from chronic liver disease accompanied by an excessive production and deposition of extracellular matrix components in the hepatic parenchyma. Liver fibrosis is a serious medical and social problem. Hepatic stellate cells (HSCs) make a significant contribution to the extracellular matrix deposition due to liver injury. Mesenchymal stem cells (MSCs) have a pronounced anti-inflammatory, regenerative and immunomodulatory effect; they are able to differentiate into hepatocytes and induce apoptosis of activated HSCs that opens the prospect of their use for preventing the excessive fibro-formation and the development of liver cirrhosis. The aim of the study is to evaluate the effect of MSCs therapy on the expression of fibrogenesis markers genes in liver tissue and HSCs cultures of rats with experimental liver cirrhosis (ELC). Materials and methods: ELC was induced by the common bile duct ligation (CBDL) in female Wistar rats (n = 19) with an average body weight of 250 (220 ÷ 270) g. Animals from the control group (n = 10) were sham-operated. On the 56th day after the CBDL, the rats of the experimental (n = 12) and the control (n = 5) groups received intraportal MSCs in concentration of 1×106 cells/animal (previously obtained from rat’s bone marrow) or saline, respectively. The animals were taken out of the experiment on the 21st day. HSCs were isolated by sequential liver perfusion in situ with following disaggregation, enzymatic treatment and centrifugation of cell suspension on a two-stage density gradient. The expression of collagen type I (Col1a1) and type III (Col3a1), matrix metalloproteinase type 2 (MMP2) and type 9 (MMP9), tissue inhibitor of matrix metalloproteinases type 1 (TIMP1), transforming growth factor β type 1 (TGFβ1) and type 3 (TGFβ3) was determined by real-time polymerase chain reaction. Statistical analysis was performed using Statistica 10.0. Results: In ELC rats compared to sham-operated animals, a significant increase of all studied markers expression was observed. The administration of MSCs led to a significant decrease of all detectable markers in the experimental group compared to rats without cell therapy. In ELC rats, an increased MMP9/TIMP1 ratio after cell therapy was also detected. The infusion of MSCs in the sham-operated animals did not lead to any changes. In the HSCs from ELC animals, the expression of Col1a1 and Col3a1 exceeded the similar parameters of the control group (p <0.05) and statistically decreased after the MSCs administration. The correlation between Col3a1 (Rs = 0.51, p <0.05), TGFβ1 (Rs = 0.6, p <0.01), and TGFβ3 (Rs = 0.75, p <0.001) expression in HSCs cultures and liver tissue has been found. Conclusion: Intraportal administration of MSCs to rats with ELC leads to a decreased Col1a1 and Col3a1, MMP2 and MMP9, TIMP1, TGFβ1 and TGFβ3 expression. The correlation between the expression of Col3a1, TGFβ1 and TGFβ3 in liver tissue and in HSCs cultures indicates the involvement of activated HSCs in the fibrogenesis that allows considering HSCs to be the main cell therapy target in ELC.

Keywords: cell therapy, experimental liver cirrhosis, hepatic stellate cells, mesenchymal stem cells

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