Search results for: tumor suppressor gene

Authors: Axel Mancebo, Ana M. Bada, Angel Casacó, Bárbara González, Avelina León, María E. Arteaga, Consuelo González, Belinda Sánchez, Adriana Carr, Nuris Ledón, Arianna Iglesias

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Despite the many preventive and therapeutic modalities aimed at curing cancer, it remains as a serious world health problem. Promising recent developments suggest that cancer immunotherapy may be the next great hope for cancer treatment. EGFRs are receptor tyrosine kinases and it is considered an important therapeutic target related with tumor progression, and several types of molecular therapies, including monoclonal antibodies, small molecules, and vaccines, have been developed to target the HER family of receptors. On the other hand, gangliosides are membrane glycosphingolipids that contain two variants of sialic acid, the N-acetylated (NeuAc) and the N-glycolylated (NeuGc) variant. The high expression of this antigen-specific molecule has been associated with malignant tumor progression and immunosuppressive mechanisms, so ganglioside could be considered as the target for cancer immunotherapy. We have been working for several years in the safety evaluation of cancer vaccines targeting these two systems, the EGF receptor and ganglioside. We presented in this work results of repeated dose toxicity studies performed in Sprague Dawley rats and Cynomolgus monkeys, including clinical observations, body weight and rectal temperature measuring, clinical pathology analysis, gross necropsy and histological examination in rodent studies, and immunological evaluation. Immunizations were capable of inducing mainly inflammatory effects at the injection site, with findings largely attributable to the adjuvants used and probably enhanced by the immunological properties of the antigens. In general, these vaccines were shown to be well tolerated, and these studies in relevant species allow treating cancer patients with tumors during long periods with relative weight safety margin.

Keywords: cancer vaccines, safety, toxicology, rats, non human primates

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Authors: S. M. M. Syairah, M. H. Rajikin, A. R. Sharaniza

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Several embryonic cellular mechanism including cell cycle, growth and apoptosis are regulated by phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway. The goal of present study is to determine the effects of annatto (Bixa orellana)-derived δ-tocotrienol (δ-TCT) on the regulations of PI3K/Akt genes in murine morula. Twenty four 6-8 week old (23-25g) female balb/c mice were randomly divided into four groups (G1-G4; n=6). Those groups were subjected to the following treatments for 7 consecutive days: G1 (control) received tocopherol stripped corn oil, G2 was given 60 mg/kg/day of δ-TCT mixture (contains 90% delta & 10% gamma isomers), G3 was given 60 mg/kg/day of pure δ-TCT (>98% purity) and G4 received 60 mg/kg/day α-TOC. On Day 8, females were superovulated with 5 IU Pregnant Mare’s Serum Gonadotropin (PMSG) for 48 hours followed with 5 IU human Chorionic Gonadotropin (hCG) before mated with males at the ratio of 1:1. Females were sacrificed by cervical dislocation for embryo collection 48 hours post-coitum. About fifty morula from each group were used in the gene expression analyses using Affymetrix QuantiGene Plex 2.0 Assay. Present data showed a significant increase (p<0.05) in the average number (mean + SEM) of morula produced in G2 (26.0 + 0.45), G3 (23.0 + 0.63) and G4 (25.0 + 0.73) compared to control group (G1 – 16.0 + 0.63). This is parallel with the high expression of PDK1 gene with increase of 2.75-fold (G2), 3.07-fold (G3) and 3.59-fold (G4) compared to G1 (1.78-fold). From the present data, it can be concluded that supplementation with δ-TCT(s) and α-TOC induced high expression of PDK1 in G2-G4 which enhanced the PI3K/Akt signaling activity, resulting in the increased number of morula.

Keywords: delta-tocotrienol, embryonic development, nicotine, vitamin E

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Authors: Naoki Yamamoto, Hidenobu Ozaki, Taiichiro Ookawa, Youming Liu, Kazunori Okada, Aiping Zheng

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Rice sheath blight is one of the destructive fungal diseases in rice. We have thought that rice sheath blight resistance is a polygenic trait. Host-pathogen interactions and secondary metabolites such as lignin and phytoalexins are likely to be involved in defense against R. solani. However, to our knowledge, it is still unknown how sheath blight resistance can be enhanced in rice breeding. To seek for an alternative genetic factor that contribute to sheath blight resistance, we mined relevant allelic variations from rice core collections created in Japan. Based on disease lesion length on detached leaf sheath, we selected 30 varieties of the top tail-end and the bottom tail-end, respectively, from the core collections to perform genome-wide association mapping. Re-sequencing reads for these varieties were used for calling single nucleotide polymorphisms among the 60 varieties to create a SNP panel, which contained 1,137,131 homozygous variant sites after filitering. Association mapping highlighted a locus on the long arm of chromosome 11, which is co-localized with three sheath blight QTLs, qShB11-2-TX, qShB11, and qSBR-11-2. Based on the localization of the trait-associated alleles, we identified an ankyryn repeat-containing protein gene (ANK-M) as an uncharacterized candidate factor for rice sheath blight resistance. Allelic distributions for ANK-M in the whole rice population supported the reliability of trait-allele associations. Gene expression characteristics were checked to evaluiate the functionality of ANK-M. Since an ANK-M homolog (OsPIANK1) in rice seems a basal defense regulator against rice blast and bacterial leaf blight, ANK-M may also play a role in the rice immune system.

Keywords: allele mining, GWAS, QTL, rice sheath blight

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Authors: Camilo Cerda Sarabia, Fernanda Bravo Cornejo, Diego Santibanez Oyarce, Hugo Osses Prado, Esteban Gómez Terán, Belén Diaz Diaz, Raúl Caulier-Cisterna, Jorge Vergara-Quezada, Ana Moya-Beltrán

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Antimicrobial resistance (AMR) represents a significant and rapidly escalating global health threat. Projections estimate that by 2050, AMR infections could claim up to 10 million lives annually. Respiratory infections, in particular, pose a severe risk not only to individual patients but also to the broader public health system. Despite the alarming rise in resistant respiratory infections, AMR within the lung microbiome (microbial community) remains underexplored and poorly characterized. The lungs, as a complex and dynamic microbial environment, host diverse communities of microorganisms whose interactions and resistance mechanisms are not fully understood. Unlike studies that focus on individual genomes, analyzing the entire microbiome provides a comprehensive perspective on microbial interactions, resistance gene transfer, and community dynamics, which are crucial for understanding AMR. However, this holistic approach introduces significant computational challenges and exposes the limitations of traditional analytical methods such as the difficulty of identifying the AMR. Machine learning has emerged as a powerful tool to overcome these challenges, offering the ability to analyze complex genomic data and uncover novel insights into AMR that might be overlooked by conventional approaches. This study investigates microbial resistance within the lung microbiome using unsupervised machine learning approaches to uncover resistance patterns and potential clinical associations. it downloaded and selected lung microbiome data from HumanMetagenomeDB based on metadata characteristics such as relevant clinical information, patient demographics, environmental factors, and sample collection methods. The metadata was further complemented by details on antibiotic usage, disease status, and other relevant descriptions. The sequencing data underwent stringent quality control, followed by a functional profiling focus on identifying resistance genes through specialized databases like Antibiotic Resistance Database (CARD) which contains sequences of AMR gene sequence and resistance profiles. Subsequent analyses employed unsupervised machine learning techniques to unravel the structure and diversity of resistomes in the microbial community. Some of the methods employed were clustering methods such as K-Means and Hierarchical Clustering enabled the identification of sample groups based on their resistance gene profiles. The work was implemented in python, leveraging a range of libraries such as biopython for biological sequence manipulation, NumPy for numerical operations, Scikit-learn for machine learning, Matplotlib for data visualization and Pandas for data manipulation. The findings from this study provide insights into the distribution and dynamics of antimicrobial resistance within the lung microbiome. By leveraging unsupervised machine learning, we identified novel resistance patterns and potential drivers within the microbial community.

Keywords: antibiotic resistance, microbial community, unsupervised machine learning., sequences of AMR gene

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Authors: Sammy Kibor, Filip Huyghe, Marc Kochzius, James Kairo

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Goldstripe Sardinella, Sardinella gibbosa, (Bleeker, 1849) is a commercially and ecologically important small pelagic fish common in the Western Indian Ocean region. The present study aimed to assess genetic diversity and population structure of the species in the Kenya-Tanzania transboundary area using mtDNA and msDNA markers. Some 630 bp sequence in the mitochondrial DNA (mtDNA) Cytochrome C Oxidase I (COI) and five polymorphic microsatellite DNA loci were analyzed. Fin clips of 309 individuals from eight locations within the transboundary area were collected between July and December 2018. The S. gibbosa individuals from the different locations were distinguishable from one another based on the mtDNA variation, as demonstrated with a neighbor-joining tree and minimum spanning network analysis. None of the identified 22 haplotypes were shared between Kenya and Tanzania. Gene diversity per locus was relatively high (0.271-0.751), highest Fis was 0.391. The structure analysis, discriminant analysis of Principal component (DAPC) and the pair-wise (FST = 0.136 P < 0.001) values after Bonferroni correction using five microsatellite loci provided clear inference on genetic differentiation and thus evidence of population structure of S. gibbosa along the Kenya-Tanzania coast. This study shows a high level of genetic diversity and the presence of population structure (Φst =0.078 P < 0.001) resulting to the existence of four populations giving a clear indication of minimum gene flow among the population. This information has application in the designing of marine protected areas, an important tool for marine conservation.

Keywords: marine connectivity, microsatellites, population genetics, transboundary

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Authors: Varsha Shinde, Belle Damodara Shenoy

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Tarballs are crude oil remnants found in oceans after long term weathering process and are a global concern since several decades as potential marine pollutant. Being complicated in structure microbial remediation of tarballs in natural environment is a slow process. They are rich in high molecular weight alkanes and poly aromatic hydrocarbons which are resistant to microbial attack and other environmental factors, therefore remain in environment for long time. However, it has been found that many bacteria and fungi inhabit on tarballs for nutrients and shelter. Many of them are supposed to be oil degraders, while others are supposed to be getting benefited by byproducts formed during hydrocarbon metabolism. Thus tarballs are forming special interesting ecological niche of microbes. This work aimed to study diversity of bacteria and fungi from tarballs and to see their potential application in crude oil degradation. The samples of tarballs were collected from Betul beach of south Goa (India). Different methods were used to isolate culturable fraction of bacteria and fungi from it. Those were sequenced for 16S rRNA gene and ITS for molecular level identification. The 16S rRNA gene sequence analysis revealed the presence of 13 bacterial genera/clades (Alcanivorax, Brevibacterium, Bacillus, Cellulomonas, Enterobacter, Klebsiella, Marinobacter, Nitratireductor, Pantoea, Pseudomonas, Pseudoxanthomonas, Tistrella and Vibrio), while the ITS sequence analysis placed the fungi in 8 diverse genera/ clades (Aspergillus, Byssochlamys, Monascus, Paecilomyces, Penicillium, Scytalidium/ Xylogone, Talaromyces and Trichoderma). All bacterial isolates were screened for oil degradation capacity. Potential strains were subjected to crude oil degradation experiment for quantification. Results were analyzed by GC-MS-MS.

Keywords: bacteria, biodegradation, crude oil, diversity, fungi, tarballs

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Authors: Hamzeh Alipour, Masoumeh Bagheri, Tahereh Karamzadeh, Abbasali Raz, Kourosh Azizi

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Netrin-A, a protein identified for conducting commissural axons, has a similar role in angiogenesis. In addition, studies have shown that one of the netrin-A receptors is expressed in the growing cells of small capillaries. It will be interesting to study this new group of molecules because their role in wound healing will become clearer in the future due to angiogenesis. The greenbottle blowfly Luciliasericata (L. sericata) larvae are increasingly used in maggot therapy of chronic wounds. This aim of this was the identification of moleculareatures of Netrin-A in L. sericata larvae. Larvae were reared under standard maggotarium conditions. The nucleic acid sequence of L. sericataNetrin-A (LSN-A) was then identified using Rapid Amplification of cDNA Ends (RACE) and Rapid Amplification of Genomic Ends (RAGE). Parts of the Netrin-A gene, including the middle, 3′-, and 5′-ends were identified, TA cloned in pTG19 plasmid, and transferred into DH5ɑ Escherichia coli. Each part was sequenced and assembled using SeqMan software. This gene structure was further subjected to in silico analysis. The DNA of LSN-A was identified to be 2407 bp, while its mRNA sequence was recognized as 2115 bp by Oligo0.7 software. It translated the Netrin-A protein with 704 amino acid residues. Its molecular weight is estimated to be 78.6 kDa. The 3-D structure ofNetrin-A drawn by SWISS-MODEL revealed its similarity to the Netrin-1 of humans with 66.8% identity. The LSN-A protein conduces to repair the myelin membrane in neuronal cells. Ultimately, it can be an effective candidate in neural regeneration and wound healing. Furthermore, our next attempt is to deplore recombinant proteins for use in medical sciences.

Keywords: maggot therapy, netrin-A, RACE, RAGE, lucilia sericata

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Authors: Ahmed M. Debela, Mayreli Ortiz , Ciara K. O´Sullivan

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The completion of the human genome Project (HGP) has paved the way for mapping the diversity in the overall genome sequence which helps to understand the genetic causes of inherited diseases and susceptibility to drugs or environmental toxins. Arrayed primer extension (APEX) is a microarray based minisequencing strategy for screening disease causing mutations. It is derived from Sanger DNA sequencing and uses fluorescently dideoxynucleotides (ddNTPs) for termination of a growing DNA strand from a primer with its 3´- end designed immediately upstream of a site where single nucleotide polymorphism (SNP) occurs. The use of DNA polymerase offers a very high accuracy and specificity to APEX which in turn happens to be a method of choice for multiplex SNP detection. Coupling the high specificity of this method with the high sensitivity, low cost and compatibility for miniaturization of electrochemical techniques would offer an excellent platform for detection of mutation as well as sequencing of DNA templates. We are developing an electrochemical APEX for the analysis of SNPs found in the MYH7 gene for group of cardiomyopathy patients. ddNTPs were labeled with four different redox active compounds with four distinct potentials. Thiolated oligonucleotide probes were immobilised on gold and glassy carbon substrates which are followed by hybridisation with complementary target DNA just adjacent to the base to be extended by polymerase. Electrochemical interrogation was performed after the incorporation of the redox labelled dedioxynucleotide. The work involved the synthesis and characterisation of the redox labelled ddNTPs, optimisation and characterisation of surface functionalisation strategies and the nucleotide incorporation assays.

Keywords: array based primer extension, labelled ddNTPs, electrochemical, mutations

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Authors: Mosaab A. Omara, Layla O. Elmajdoubb, Mohammad Saleh Al-Aboodyc, Ahmed ElSifyd, Ahmed O. Elkhtamd

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The aim of this study is to present the molecular characterization of cysticercus tenuicolis of Taenia hydatigena from livestock isolates in Egypt, using the amplification of sequencing of the mt-CO1 gene. We introduce a detailed image of the Cysticercus tenuicolis infection in ruminant animals in Upper Egypt. Cysticercus tenuicolis inhabits such organs in ruminants as the omentum, viscera, and liver. In the present study, the infection rate of Cysticercus tenuicolis was found to be 16% and 19% in sheep and goat sample respectively. Firstly we report one larval stage of Taenia hydatigena detected in the camel liver in Egypt. Cysticercus tenuicolis infection manifested a higher prevalence in females than in males. Those above 2 years of age manifested a higher infection rate than younger animals. The preferred site for the infection was the omentum: a 70% preference in sheep and a 68% preference in goat samples. The molecular characterization using the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene of isolates from sheep, goats and camels corresponded to T. hydatigena. For this study, molecular characterizations of T. hydatigena were done for the first time in Egypt. Molecular tools are of great assistance in characterizing the Cysticercus tenuicolis parasite especially when the morphological character cannot be detected because the metacestodes are frequently confused with infection by the Hydatid cyst, especially when these occur in the visceral organs. In the present study, Cysticercus tenuicolis manifested high identity in the goat and sheep samples, while differences were found more frequently in the camel samples (10 pairbase). Clearly molecular diagnosis for Cysticercus tenuicolis infection significantly helps to differentiate it from such other metacestodes.

Keywords: cysticercus tenuicolis, its2, genetic, qena, molecular and taenia hydatigena

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Authors: Vijay Kumar Singh, Pooja Kushwaha, Prabhat Ranjan, Krishna Kumar Ojha, Jitendra Kumar

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Present day high-throughput genomic technologies, NGS/microarrays, are producing large volume of data that require improved analysis methods to make sense of the data. The correlation between genes and samples has been regularly used to gain insight into many biological phenomena including, but not limited to, co-expression/co-regulation, gene regulatory networks, clustering and pattern identification. However, presence of outliers and violation of assumptions underlying Pearson correlation is frequent and may distort the actual correlation between the genes and lead to spurious conclusions. Here, we report a method to measure the strength of association between genes. The method assumes that the expression values of a gene are Bernoulli random variables whose outcome depends on the sample being probed. The method considers the two genes as uncorrelated if the number of sample with same outcome for both the genes (Ns) is equal to certainly expected number (Es). The extent of correlation depends on how far Ns can deviate from the Es. The method does not assume normality for the parent population, fairly unaffected by the presence of outliers, can be applied to qualitative data and it uses the binomial distribution to assess the significance of association. At this stage, we would not claim about the superiority of the method over other existing correlation methods, but our method could be another way of calculating correlation in addition to existing methods. The method uses binomial distribution, which has not been used until yet, to assess the significance of association between two variables. We are evaluating the performance of our method on NGS/microarray data, which is noisy and pierce by the outliers, to see if our method can differentiate between spurious and actual correlation. While working with the method, it has not escaped our notice that the method could also be generalized to measure the association of more than two variables which has been proven difficult with the existing methods.

Keywords: binomial distribution, correlation, microarray, outliers, transcriptome

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Authors: Lubna Azmi, Ila Shukla, Shyam Sundar Gupta, Padam Kant, C. V. Rao

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To investigate the chemoprotective potential of NBRIQU16 chemotype isolated from Argyreia speciosa (Family: Convolvulaceae) on N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and NaCl-induced gastric carcinomas in Wistar rats. Forty-six male 6-week-old Wistar rats were divided into two groups. Thirty rats in group A were fed with a diet supplemented with 8 % NaCl for 20 weeks and simultaneously given N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) in drinking water at a concentration of 100 ug/ml for the first 17 weeks. After administration of the carcinogen, 200 and 400 mg/kg of NBRIQU16 were administered orally once a day throughout the study. From week 18, these rats were given normal water. From week 21, these rats were fed with a normal diet for 15 weeks. Group B containing 16 rats was fed standard diet for thirty-five days. It served as control. Ten rats from group A were sacrificed after 20 weeks. Scarification of remaining animals was conducted after 35 weeks. Entire stomach and some part of the duodenum were incised parallel to the greater curvature, and the samples were collected. After opening the stomach location and size of tumors were recorded. The number of tumors with their locations and sizes were recorded. Expression of survivin was examined by recording the Immunohistochemistry of the specimens. The treatment with NBRIQU16 significantly reduced the nodule incidence and nodule multiplicity in the rats after MNNG administration. Surviving expression in glandular stomachs of normal rats, of rats in middle induction period, in adenocarcinomas and NBRIQU16 treated tissues adjacent to tumor were 0, 42.0 %, 79.3%, and 36.4 %, respectively. Expression of survivin was significantly different as compared to the normal rats. Histological observations of stomach tissues too correlated with the biochemical observations.These finding powerfully supports that NBRIQU16 chemopreventive effect by suppressing the tumor burden and restoring the activities of gastric cancer marker enzymes on MNNG and NaCl-induced gastric carcinomas in Wistar rats.

Keywords: Argyreia speciosa, gastric carcinoma, immunochemistry, NBRIQU16

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Authors: Lay Poh Tan, Chor Yong Tay, Haiyang Yu

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Micro-contact printing is a form of soft lithography that uses the relief patterns on a master polydimethylsiloxane (PDMS) stamp to form patterns of self-assembled monolayers (SAMs) of ink on the surface of a substrate through conformal contact technique. Here, we adopt this method to print proteins of different dimensions on our biodegradable polymer substrates. We started off with printing 20-500 μm scale lanes of fibronectin to engineer the shape of bone marrow derived human mesenchymal stem cell (hMSCs). After 8 hours of culture, the hMSCs adopted elongated shapes, and upon analysis of the gene expressions, genes commonly associated with myogenesis (GATA-4, MyoD1, cTnT and β-MHC) and neurogenesis (NeuroD, Nestin, GFAP, and MAP2) were up-regulated but gene expression associated to osteogenesis (ALPL, RUNX2, and SPARC) were either down modulated or remained at the nominal level. This is the first evidence that cellular morphology control via micropatterning could be used to modulate stem cell fate without external biochemical stimuli. We further our studies to modulate the focal adhesion (FA) instead of the macro shape of cells. Micro-contact printed islands of different smaller dimensions were investigated. We successfully regulated the FAs into dense FAs and elongated FAs by micropatterning. Additionally, the combined effects of hard (40.4 kPa), and intermediate (10.6 kPa) PA gel and FAs patterning on hMSCs differentiation were studied. Results showed that FA and matrix compliance plays an important role in hMSCs differentiation, and there is a cross-talk between different physical stimulants and the significance of these stimuli can only be realized if they are combined at the optimum level.

Keywords: micro-contact printing, polymer substrate, cell-material interaction, stem cell differentiation

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Authors: P. M. Sreekanth

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Teak (Tectona grandis L. f.) is a tree species indigenous to India and other Southeastern countries. It produces high-value timber and is easily established in plantations. Reforestation requires a constant supply of high quality seeds. Seed Production Areas (SPA) of teak are improved stands used for collection of open-pollinated quality seeds in large quantities. Information on the genetic diversity of major teak SPAs in India is scanty. The genetic structure of four important seed production areas of Kerala State in Southern India was analyzed employing amplified fragment length polymorphism markers using ten selective primer combinations on 80 samples (4 populations X 20 trees). The study revealed that the gene diversity of the SPAs varied from 0.169 (Konni SPA) to 0.203 (Wayanad SPA). The percentage of polymorphic loci ranged from 74.42 (Parambikulam SPA) to 84.06 (Konni SPA). The mean total gene diversity index (HT) of all the four SPAs was 0.2296 ±0.02. A high proportion of genetic diversity was observed within the populations (83%) while diversity between populations was lower (17%) (GST = 0.17). Principal coordinate analysis and STRUCTURE analysis of the genotypes indicated that the pattern of clustering was in accordance with the origin and geographic location of SPAs, indicating specific identity of each population. A UPGMA dendrogram was prepared and showed that all the twenty samples from each of Konni and Parambikulam SPAs clustered into two separate groups, respectively. However, five Nilambur genotypes and one Wayanad genotype intruded into the Konni cluster. The higher gene flow estimated (Nm = 2.4) reflected the inclusion of Konni origin planting stock in the Nilambur and Wayanad plantations. Evidence for population structure investigated using 3D Principal Coordinate Analysis of FAMD software 1.30 indicated that the pattern of clustering was in accordance with the origin of SPAs. The present study showed that assessment of genetic diversity in seed production plantations can be achieved using AFLP markers. The AFLP fingerprinting was also capable of identifying the geographical origin of planting stock and there by revealing the occurrence of the errors in genotype labeling. Molecular marker-based selective culling of genetically similar trees from a stand so as to increase the genetic base of seed production areas could be a new proposition to improve quality of seeds required for raising commercial plantations of teak. The technique can also be used to assess the genetic diversity status of plus trees within provenances during their selection for raising clonal seed orchards for assuring the quality of seeds available for raising future plantations.

Keywords: AFLP, genetic structure, spa, teak

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Authors: Rachana Tank, Donald. M. Lyall, Kristin Flegal, Joey Ward, Jonathan Cavanagh

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Alzheimer’s research UK estimates by 2050, 2 million individuals will be living with Late Onset Alzheimer’s disease (LOAD). However, individuals experience considerable cognitive deficits and brain pathology over decades before reaching clinically diagnosable LOAD and studies have utilised gene candidate studies such as genome wide association studies (GWAS) and polygenic risk (PGR) scores to identify high risk individuals and potential pathways. This investigation aims to determine whether high genetic risk of LOAD is associated with worse brain MRI and cognitive performance in healthy older adults within the UK Biobank cohort. Previous studies investigating associations of PGR for LOAD and measures of MRI or cognitive functioning have focused on specific aspects of hippocampal structure, in relatively small sample sizes and with poor ‘controlling’ for confounders such as smoking. Both the sample size of this study and the discovery GWAS sample are bigger than previous studies to our knowledge. Genetic interaction between loci showing largest effects in GWAS have not been extensively studied and it is known that APOE e4 poses the largest genetic risk of LOAD with potential gene-gene and gene-environment interactions of e4, for this reason we  also analyse genetic interactions of PGR with the APOE e4 genotype. High genetic loading based on a polygenic risk score of 21 SNPs for LOAD is associated with worse brain MRI and cognitive outcomes in healthy individuals within the UK Biobank cohort. Summary statistics from Kunkle et al., GWAS meta-analyses (case: n=30,344, control: n=52,427) will be used to create polygenic risk scores based on 21 SNPs and analyses will be carried out in N=37,000 participants in the UK Biobank. This will be the largest study to date investigating PGR of LOAD in relation to MRI. MRI outcome measures include WM tracts, structural volumes. Cognitive function measures include reaction time, pairs matching, trail making, digit symbol substitution and prospective memory. Interaction of the APOE e4 alleles and PGR will be analysed by including APOE status as an interaction term coded as either 0, 1 or 2 e4 alleles. Models will be adjusted partially for adjusted for age, BMI, sex, genotyping chip, smoking, depression and social deprivation. Preliminary results suggest PGR score for LOAD is associated with decreased hippocampal volumes including hippocampal body (standardised beta = -0.04, P = 0.022) and tail (standardised beta = -0.037, P = 0.030), but not with hippocampal head. There were also associations of genetic risk with decreased cognitive performance including fluid intelligence (standardised beta = -0.08, P<0.01) and reaction time (standardised beta = 2.04, P<0.01). No genetic interactions were found between APOE e4 dose and PGR score for MRI or cognitive measures. The generalisability of these results is limited by selection bias within the UK Biobank as participants are less likely to be obese, smoke, be socioeconomically deprived and have fewer self-reported health conditions when compared to the general population. Lack of a unified approach or standardised method for calculating genetic risk scores may also be a limitation of these analyses. Further discussion and results are pending.

Keywords: Alzheimer's dementia, cognition, polygenic risk, MRI

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Authors: Eric Gismondi, Aurelie Bigot-Clivot

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Endocrine disruptors (EDCs) are well known to disrupt the development and the reproduction of exposed organisms. Although this point has been studied in vertebrate models, the limited knowledge of the endocrine system of invertebrates makes the evaluation of EDCs effects difficult. However, invertebrates represent the major part of aquatic ecosystems, such as amphipods Gammaridea, which are crucial for their functioning (e.g., litter degradation, food resource). Moreover, gammarids are hosts of parasites such as vertically-transmitted microsporidia (microsporidia VT), which could be confounding factors in assessment of EDC effects. Indeed, some microsporidia VT could have endocrine effects by their own present in the host since it was observed for example, a feminization of juvenile males, which become phenotypic females. This work evaluated the impact of ethinylestradiol (EE₂, estrogenic), cyproterone acetate (CPA, anti-androgenic), 4-hydroxytamoxifen (4HT, anti-estrogenic) and 17α-methyltestosterone (17MT - androgenic), on the 20-hydroxyecdysone concentration (i.e. 20HE - molt process) and the vitellogenin gene expression (i.e. reproduction) in the freshwater amphipod Gammarus pulex, after a 96h laboratory exposure. In addition, the presence of microsporidia VT was verified in order to analyze the effect of this confounding factor. Results of this study shown that, although endocrine systems of invertebrates and vertebrates are different, EDCs proved in vertebrates could also affect biological functions hormonally controlled in invertebrates. Indeed, the molt process of crustaceans was disrupted in the first stage (i.e. 20-HE concentration) and therefore, could affect, at the long term, the population dynamic. In addition, it was observed that G. pulex was differently impacted according to the gender and parasitism, which underline the importance to take into account these confounding factors to better evaluate the EDCs impact on invertebrate populations.

Keywords: endocrine disruption, gammarus sp., molt, parasitism

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Authors: Manjula Weerasekera, Chris Sissons, Lisa Wong, Sally Anderson, Ann Holmes, Richard Cannon

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The aim of this study was to characterize bacterial population and yeasts in saliva by Polymerase chain reaction followed by denaturing gradient gel electrophoresis (PCR-DGGE) and measure yeast levels by culture. PCR-DGGE was performed to identify oral bacteria and yeasts in 24 saliva samples. DNA was extracted and used to generate DNA amplicons of the V2–V3 hypervariable region of the bacterial 16S rDNA gene using PCR. Further universal primers targeting the large subunit rDNA gene (25S-28S) of fungi were used to amplify yeasts present in human saliva. Resulting PCR products were subjected to denaturing gradient gel electrophoresis using Universal mutation detection system. DGGE bands were extracted and sequenced using Sanger method. A potential relationship was evaluated between groups of bacteria identified by cluster analysis of DGGE fingerprints with the yeast levels and with their diversity. Significant interpersonal variation of salivary microbiome was observed. Cluster and principal component analysis of the bacterial DGGE patterns yielded three significant major clusters, and outliers. Seventeen of the 24 (71%) saliva samples were yeast positive going up to 10³ cfu/mL. Predominately, C. albicans, and six other species of yeast were detected. The presence, amount and species of yeast showed no clear relationship to the bacterial clusters. Microbial community in saliva showed a significant variation between individuals. A lack of association between yeasts and the bacterial fingerprints in saliva suggests the significant ecological person-specific independence in highly complex oral biofilm systems under normal oral conditions.

Keywords: bacteria, denaturing gradient gel electrophoresis, oral biofilm, yeasts

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Authors: Mahima Dubey, Girish Chandel

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Minor millets are highly nutritious and climate resilient cereal crops. These features make them ideal candidates to excavate the physiology of the underlying mechanism. In an attempt to understand the basis of mineral nutrition in minor millets, a set of five Barnyard millet genotypes were analyzed for grain Fe and Zn content under contrasting Fe-Zn supply to identify genotypes proficient in tolerating mineral deficiency. This resulted in the identification of Melghat-1 genotype to be nutritionally superior with better ability to withstand deficiency. Expression analysis of several Nicotianamine synthase (NAS) genes showed that HvNAS1 and OsNAS2 genes were prominent in positively mediating mineral deficiency response in Barnyard millet. Further, strategic efforts were employed for fast-track identification of more effective orthologous NAS genes from Barnyard millet. This resulted in the identification of two genes namely EfNAS1 (orthologous to HvNAS1 of barley) and EfNAS2 (orthologous to OsNAS2 gene of rice). Sequencing and thorough characterization of these sequences revealed the presence of intact NAS domain and signature tyrosine and di-leucine motifs in their predicted proteins and thus established their candidature as functional NAS genes in Barnyard millet. Moreover, EfNAS1 showed structural superiority over previously known NAS genes and is anticipated to have role in more efficient metal transport. Findings of the study provide insight into Fe-Zn deficiency response and mineral nutrition in millets. This provides millets with a physiological edge over micronutrient deficient staple cereals such as rice in withstanding Fe-Zn deficiency and subsequently accumulating higher levels of Fe and Zn in millet grains.

Keywords: gene expression, micronutrient, millet, ortholog

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Authors: Prasamsha Panta, Da Yeon Kim, Ja Yong Jang, Min Jae Kim, Jae Ho Kim, Moon Suk Kim

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Introduction: Among the many anticancer drugs used clinically, doxorubicin (Dox), was one of widely used drugs to treat many types of solid tumors such as liver, colon, breast, or lung. Intratumoral injection of chemotherapeutic agents is a potentially more effective alternative to systemic administration because direct delivery of the anticancer drug to the target may improve both the stability and efficacy of anticancer drugs. Injectable in situ-forming gels have attracted considerable attention because they can achieve site specific drug delivery, long term action periods, and improved patient compliance. Objective: Objective of present study is to confirm clinical benefit of intratumoral chemotherapy using injectable in situ-forming poly(ethylene glycol)-b-polycaprolactone diblock copolymer (MP) and Dox with increase in efficacy and reducing the toxicity in patients with cancer diseases. Methods and methodology: We prepared biodegradable MP hydrogel and measured viscosity for the evaluation of thermo-sensitive property. In vivo antitumor activity was performed with normal saline, MP only, single free Dox, repeat free Dox, and Dox-loaded MP gel. The remaining amount of Dox drug was measured using HPLC after the mouse was sacrified. For cytotoxicity studies WST-1 assay was performed. Histological analysis was done with H&E and TUNEL processes respectively. Results: The works in this experiment showed that Dox-loaded MP have biodegradable drug depot property. Dox-loaded MP gels showed remarkable in vitro cytotoxicity activities against cancer cells. Finally, this work indicates that injection of Dox-loaded MP allowed Dox to act effectively in the tumor and induced long-lasting supression of tumor growth. Conclusion: This work has examined the potential clinical utility of intratumorally injected Dox-loaded MP gel, which shows significant effect of higher local Dox retention compared with systemically administered Dox.

Keywords: injectable in-situ forming hydrogel, anticancer, doxorubicin, intratumoral injection

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Authors: Nicole C. Valdez, Vincent L. Borromeo, Conrad C. Chong, Ahmad F. Mazahery

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Breast cancer is the most prevalent cancer worldwide, where the majority of cases are estrogen-receptor positive and involve 2 receptor proteins. The binding of estrogen to estrogen receptor alpha (ERα) promotes breast cancer growth, while it's binding to estrogen-receptor beta (ERβ) inhibits tumor growth. While natural products have been a promising source of chemotherapeutic agents, the challenge remains in finding a bioactive compound that specifically targets cancer cells, minimizing side effects on normal cells. Flavonoids are natural products that act as phytoestrogens and induce the same response as estrogen. They are able to compete with estrogen for binding to ERα; however, it has a higher binding affinity for ERβ. Their abundance in nature and low toxicity make them a potential candidate for breast cancer treatment. This study aimed to determine which particular flavonoids can specifically recognize ERβ and potentially be used for breast cancer treatment through molecular docking. A total of 206 flavonoids comprised of 97 isoflavones and 109 flavanones were collected from ZINC15, while the 3D structures of ERβ and ERα were obtained from Protein Data Bank. These flavonoid subclasses were chosen as they bind more strongly to ERs due to their chemical structure. The structures of the flavonoid ligands were converted using Open Babel, while the estrogen receptor protein structures were prepared using Autodock MGL Tools. The optimal binding site was found using BIOVIA Discovery Studio Visualizer before docking all flavonoids on both ERβ and ERα through Autodock Vina. Genistein is a flavonoid that exhibits anticancer effects by binding to ERβ, so its binding affinity was used as a baseline. Eriodictyol and 4”,6”-Di-O-Galloylprunin both exceeded genistein’s binding affinity for ERβ and was lower than its binding affinity for ERα. Of the two, eriodictyol was pursued due to its antitumor properties on a lung cancer cell line and on glioma cells. It is able to arrest the cell cycle at the G2/M phase by inhibiting the mTOR/PI3k/Akt cascade and is able to induce apoptosis via the PI3K/Akt/NF-kB pathway. Protein pathway and gene analysis were also conducted using ChEMBL and PANTHER and it was shown that eriodictyol might induce anticancer effects through the ROS1, CA7, KMO, and KDM1A genes which are involved in cell proliferation in breast cancer, non-small cell lung cancer, and other diseases. The high binding affinity of eriodictyol to ERβ, as well as its potential affected genes and antitumor effects, therefore, make it a candidate for the development of new breast cancer treatment. Verification through in vitro experiments such as checking the upregulation and downregulation of genes through qPCR and checking cell cycle arrest using a flow cytometry assay is recommended.

Keywords: breast cancer, estrogen receptor, flavonoid, molecular docking

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Authors: Jianhua Zhang, Weiping Chen, Guanjie Chen, Jason Flannick, Emma Fikse, Glenda Smerin, Yanqin Yang, Yulong Li, John A. Hanover, William F. Simonds

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Ethnic disparities in many diseases are well recognized and reflect the consequences of genetic, behavior, and environmental factors. However, direct scientific evidence connecting the ethnic genetic variations and the disease disparities has been elusive, which may have led to the ethnic inequalities in large scale genetic studies. Through the genome-wide analysis of data representing 185,934 subjects, including 14,955 from our own studies of the African America Diabetes Mellitus, we discovered sets of genetic variants either unique to or conserved in all ethnicities. We further developed a quantitative gene function-based high-risk variant index (hrVI) of 20,428 genes to establish profiles that strongly correlate with the subjects' self-identified ethnicities. With respect to the ability to detect human essential and pathogenic genes, the hrVI analysis method is both comparable with and complementary to the well-known genetic analysis methods, pLI and VIRlof. Application of the ethnicity-specific hrVI analysis to the type 2 diabetes mellitus (T2DM) national repository, containing 20,791 cases and 24,440 controls, identified 114 candidate T2DM-associated genes, 8.8-fold greater than that of ethnicity-blind analysis. All the genes identified are defined as either pathogenic or likely-pathogenic in ClinVar database, with 33.3% diabetes-associated and 54.4% obesity-associated genes. These results demonstrate the utility of hrVI analysis and provide the first genetic evidence by clustering patterns of how genetic variations among ethnicities may impede the discovery of diabetes and foreseeably other disease-associated genes.

Keywords: diabetes-associated genes, ethnic health disparities, high-risk variant index, hrVI, T2DM

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Authors: Gustavo Axel Elizalde-Velázquez

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Metformin is the first line of oral therapy to treat type II diabetes and is also employed as a treatment for other indications, such as polycystic ovary syndrome, cancer, and COVID-19. Recent data suggest it is the aspirin of the 21st century due to its antioxidant and anti-aging effects. However, increasingly current articles indicate its long-term consumption generates mitochondrial impairment. Up to date, it is known metformin increases the biogenesis of Alzheimer's amyloid peptides via up-regulating BACE1 transcription, but further information related to brain damage after its consumption is missing. Bearing in mind the above, this work aimed to establish whether or not chronic exposure to metformin may alter swimming behavior and induce neurotoxicity in Danio rerio adults. For this purpose, 250 Danio rerio grown-ups were assigned to six tanks of 50 L of capacity. Four of the six systems contained 50 fish, while the remaining two had 25 fish (≈1 male:1 female ratio). Every system with 50 fish was allocated one of the three metformin treatment concentrations (1, 20, and 40 μg/L), with one system as the control treatment. Systems with 25 fish, on the other hand, were used as positive controls for acetylcholinesterase (10 μg/L of Atrazine) and oxidative stress (3 μg/L of Atrazine). After four months of exposure, a mean of 32 fish (S.D. ± 2) per group of MET treatment survived, which were used for the evaluation of behavior with the Novel Tank test. Moreover, after the behavioral assessment, we aimed to collect the blood and brains of all fish from all treatment groups. For blood collection, fish were anesthetized with an MS-222 solution (150 mg/L), while for brain gathering, fish were euthanized using the hypothermic shock method (2–4 °C). Blood was employed to determine CASP3 activity and the percentage of apoptotic cells with the TUNEL assay, and brains were used to evaluate acetylcholinesterase activity, oxidative damage, and gene expression. After chronic exposure, MET-exposed fish exhibited less swimming activity when compared to control fish. Moreover, compared with the control group, MET significantly inhibited the activity of AChE and induced oxidative damage in the brain of fish. Concerning gene expression, MET significantly upregulated the expression of Nrf1, Nrf2, BAX, p53, BACE1, APP, PSEN1, and downregulated CASP3 and CASP9. Although MET did not overexpress the CASP3 gene, we saw a meaningful rise in the activity of this enzyme in the blood of fish exposed to MET compared to the control group, which we then confirmed by a high number of apoptotic cells in the TUNEL assay. To the best of our understanding, this is the first study that delivers evidence of oxidative impairment, apoptosis, AChE alteration, and overexpression of B- amyloid-related genes in the brain of fish exposed to metformin.

Keywords: AChE inhibition, CASP3 activity, NovelTank test, oxidative damage, TUNEL assay

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Authors: Agustyas Tjiptaningrum, Intanri Kurniati, Fadilah Fadilah, Linda Erlina, Tiwuk Susantiningsih

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Coronavirus disease-19 (COVID-19) is still one of the health problems. It can be a severe condition that is caused by a cytokine storm. In a cytokine storm, several proinflammatory cytokines are released massively. It destroys epithelial cells, and subsequently, it can cause death. The anti-inflammatory agent can be used to decrease the number of severe Covid-19 conditions. Melaleuca cajuputi is a plant that has antiviral, antibiotic, antioxidant, and anti-inflammatory activities. This study was carried out to analyze the prediction mechanism of the M. cajuputi extract from Lampung, Indonesia, as an anti-inflammatory agent for COVID-19. This study constructed a database of protein host target that was involved in the inflammation process of COVID-19 using data retrieval from GeneCards with the keyword “SARS-CoV2”, “inflammation,” “cytokine storm,” and “acute respiratory distress syndrome.” Subsequent protein-protein interaction was generated by using Cytoscape version 3.9.1. It can predict the significant target protein. Then the analysis of the Gene Ontology (GO) and KEGG pathways was conducted to generate the genes and components that play a role in COVID-19. The result of this study was 30 nodes representing significant proteins, namely NF-κβ, IL-6, IL-6R, IL-2RA, IL-2, IFN2, C3, TRAF6, IFNAR1, and DOX58. From the KEGG pathway, we obtained the result that NF-κβ has a role in the production of proinflammatory cytokines, which play a role in the COVID-19 cytokine storm. It is an important factor for macrophage transcription; therefore, it will induce inflammatory gene expression that encodes proinflammatory cytokines such as IL-6, TNF-α, and IL-1β. In conclusion, the blocking of NF-κβ is the prediction mechanism of the M. cajuputi extract as an anti-inflammation agent for COVID-19.

Keywords: antiinflammation, COVID-19, cytokine storm, NF-κβ, M. cajuputi

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Authors: B. J. Mahen, N. E. Lloyd-Gale, S. Johnson, W. P. Rakowicz, M. J. Harris, A. D. Miller

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Background: Migraine is a leading cause of disability in the world. Calcitonin gene-related peptide (CGRP) receptor antagonists offer an approach to migraine prophylaxis by inhibiting the inflammatory and vasodilatory effects of CGRP. In recent years, NICE licensed the use of three CGRP-receptor antagonists: Fremanezumab, Galcanezumab, and Erenumab. Here, we present the outcomes of CGRP-antagonist treatment in a cohort of patients who suffer from episodic or chronic migraine and have failed at least three oral prophylactic therapies. Methods: We offered CGRP antagonists to 86 patients who met the NICE criteria to start therapy. We recorded the number of headache days per month (HDPM) at 0 weeks, 3 months, and 12 months. Of those, 26 patients were switched to an alternative treatment due to poor response or side effects. Of the 112 total cases, 9 cases did not sufficiently maintain their headache diary, and 5 cases were not followed up at 3 months. We have therefore included 98 sets of data in our analysis. Results: Fremanezumab achieved a reduction in HDPM by 51.7% at 3 months (p<0.0001), with 63.7% of patients meeting NICE criteria to continue therapy. Patients trialed on Galcanezumab attained a reduction in HDPM by 47.0% (p=0.0019), with 51.6% of patients meeting NICE criteria to continue therapy. Erenumab, however, only achieved a reduction in HDPM by 17.0% (p=0.29), and this was not statistically significant. Furthermore, 34.4%, 9.7%, and 4.9% of patients taking Fremanezumab, Galcanezumab, and Erenumab, respectively, continued therapy beyond 12 months. Of those who attempted drug holidays following 12 months of treatment, migraine symptoms relapsed in 100% of cases. Conclusion: We observed a significant improvement in HDPM amongst episodic and chronic migraine patients following treatment with Fremanezumab or Galcanezumab.

Keywords: migraine, CGRP, fremanezumab, galcanezumab, erenumab

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Authors: Karolis Leonavičius, Dalius Kučiauskas, Dangiras Lukošius, Arnoldas Jasiūnas, Kostas Zdanys, Rokas Stanislovas, Emilis Gegevičius, Žana Kapustina, Juozas Nainys

Abstract:

Screening throughput is a common bottleneck in many research areas, including functional genomics, drug discovery, and directed evolution. High-throughput screening techniques can be classified into two main categories: (i) affinity-based screening and (ii) functional screening. The first one relies on binding assays that provide information about the affinity of a test molecule for a target binding site. Binding assays are relatively easy to establish; however, they reveal no functional activity. In contrast, functional assays show an effect triggered by the interaction of a ligand at a target binding site. Functional assays might be based on a broad range of readouts, such as cell proliferation, reporter gene expression, downstream signaling, and other effects that are a consequence of ligand binding. Screening of large cell or gene libraries based on direct activity rather than binding affinity is now a preferred strategy in many areas of research as functional assays more closely resemble the context where entities of interest are anticipated to act. Droplet sorting is the basis of high-throughput functional biological screening, yet its applicability is limited due to the technical complexity of integrating high-performance droplet analysis and manipulation systems. As a solution, the Droplet Genomics Styx platform enables custom droplet sorting workflows, which are necessary for the development of early-stage or complex biological therapeutics or industrially important biocatalysts. The poster will focus on the technical design considerations of Styx in the context of its application spectra.

Keywords: functional screening, droplet microfluidics, droplet sorting, dielectrophoresis

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Authors: Esther Friedlander, Philip Friedlander

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The use of immunotherapy that inhibits programmed death-1 (PD-1) or its ligand PD-L1 confers survival benefits in patients with non-small cell lung cancer (NSCLC). However, approximately 45% of patients experience primary treatment resistance, necessitating the development of strategies to improve efficacy. While the renin-angiotensin system (RAS) has systemic hemodynamic effects, tissue-specific regulation exists along with modulation of immune activity in part through regulation of myeloid cell activity, leading to the hypothesis that RAS inhibition may improve anti-PD-1/L-1 efficacy. A retrospective analysis was conducted that included 173 advanced solid tumor cancer patients treated at Valley Hospital, a community Hospital in New Jersey, USA, who were treated with a PD-1/L-1 inhibitor in a defined time period showing a statistically significant relationship between RAS pathway inhibition (RASi through concomitant treatment with an ACE inhibitor or angiotensin receptor blocker) and positive efficacy to the immunotherapy that was independent of age, gender and cancer type. Subset analysis revealed strong numerical benefit for efficacy in both patients with squamous and nonsquamous NSCLC as determined by documented clinician assessment of efficacy and by duration of therapy. A PUBMED literature search was now conducted to identify studies assessing the effect of RAS pathway inhibition on anti-PD-1/L1 efficacy in advanced solid tumor patients and compare these findings to those seen in the Valley Hospital retrospective study with a focus on NSCLC specifically. A total of 11 articles were identified assessing the effects of RAS pathway inhibition on the efficacy of checkpoint inhibitor immunotherapy in advanced cancer patients. Of the 11 studies, 10 assessed the effect on survival of RASi in the context of treatment with anti-PD-1/PD-L1, while one assessed the effect on CTLA-4 inhibition. Eight of the studies included patients with NSCLC, while the remaining 2 were specific to genitourinary malignancies. Of the 8 studies, two were specific to NSCLC patients, with the remaining 6 studies including a range of cancer types, of which NSCLC was one. Of these 6 studies, only 2 reported specific survival data for the NSCLC subpopulation. Patient characteristics, multivariate analysis data and efficacy data seen in the 2 NSLCLC specific studies and in the 2 basket studies, which provided data on the NSCLC subpopulation, were compared to that seen in the Valley Hospital retrospective study supporting a broader effect of RASi on anti-PD-1/L1 efficacy in advanced NSLCLC with the majority of studies showing statistically significant benefit or strong statistical trends but with one study demonstrating worsened outcomes. This comparison of studies extends published findings to the community hospital setting and supports prospective assessment through randomized clinical trials of efficacy in NSCLC patients with pharmacodynamic components to determine the effect on immune cell activity in tumors and on the composition of the tumor microenvironment.

Keywords: immunotherapy, cancer, angiotensin, efficacy, PD-1, lung cancer, NSCLC

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Authors: Anjan Hazra, Nirjhar Dasgupta, Chandan Sengupta, Sauren Das

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Tea (Camellia sinensis) has received substantial attention from the scientific world time to time, not only for its commercial importance, but also for its demand to the health-conscious people across the world for its extensive use as potential sources of antioxidant supplement. These health-benefit traits primarily rely on some regulatory networks of different metabolic pathways. Development of microsatellite markers from the conserved genomic regions is being worthwhile for studying the genetic diversity of closely related species or self-pollinated species. Although several SSR markers have been reported, in tea the trait-specific Simple Sequence Repeats (SSRs) are yet to be identified, which can be used for marker assisted breeding technique. MicroRNAs are endogenous, noncoding, short RNAs directly involved in regulating gene expressions at the post-transcriptional level. It has been found that diversity in miRNA gene interferes the formation of its characteristic hair pin structure and the subsequent function. In the present study, the precursors of small regulatory RNAs (microRNAs) has been fished out from tea Expressed Sequence Tag (EST) database. Furthermore, the simple sequence repeat motifs within the putative miRNA precursor genes are also identified in order to experimentally validate their existence and function. It is already known that genic-SSR markers are very adept and breeder-friendly source for genetic diversity analysis. So, the potential outcome of this in-silico study would provide some novel clues in understanding the miRNA-triggered polymorphic genic expression controlling specific metabolic pathways, accountable for tea quality.

Keywords: micro RNA, simple sequence repeats, tea quality, trait specific marker

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Authors: Yeon Ho Je

Abstract:

A novel recombinant baculovirus, NeuroBactrus, was constructed to develop an improved baculovirus insecticide with additional beneficial properties, such as a higher insecticidal activity and improved recovery, compared to wild-type baculovirus. For the construction of NeuroBactrus, the Bacillus thuringiensis crystal protein gene (here termed cry1-5) was introduced into the Autographa californica nucleopolyhedrovirus (AcMNPV) genome by fusion of the polyhedrin–cry1-5–polyhedrin genes under the control of the polyhedrin promoter. In the opposite direction, an insect-specific neurotoxin gene, AaIT, from Androctonus australis was introduced under the control of an early promoter from Cotesia plutellae bracovirus by fusion of a partial fragment of orf603. The polyhedrin–Cry1-5–polyhedrin fusion protein expressed by the NeuroBactrus was not only occluded into the polyhedra, but it was also activated by treatment with trypsin, resulting in an_65-kDa active toxin. In addition, quantitative PCR revealed that the neurotoxin was expressed from the early phase of infection. NeuroBactrus showed a high level of insecticidal activity against Plutella xylostella larvae and a significant reduction in the median lethal time against Spodoptera exigua larvae compared to those of wild-type AcMNPV. Rerecombinant mutants derived from NeuroBactrus in which AaIT and/or cry1-5 were deleted were generated by serial passages in vitro. Expression of the foreign proteins (B. thuringiensis toxin and AaIT) was continuously reduced during the serial passage of the NeuroBactrus. Moreover, polyhedra collected from S. exigua larvae infected with the serially passaged NeuroBactrus showed insecticidal activity similar to that of wild-type AcMNPV. These results suggested that NeuroBactrus could be recovered to wild-type AcMNPV through serial passaging.

Keywords: baculovirus, insecticide, neurotoxin, neurobactrus

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Authors: Zakaria Baka, Halima Alem

Abstract:

Cancer is a major worldwide health problem that has caused around ten million deaths in 2020. In addition, efforts to develop new anti-cancer drugs still face a high failure rate. This is partly due to the lack of preclinical models that recapitulate in-vivo drug responses. Indeed conventional cell culture approach (known as 2D cell culture) is far from reproducing the complex, dynamic and three-dimensional environment of tumors. To set up more in-vivo-like cancer models, 3D bioprinting seems to be a promising technology due to its ability to achieve 3D scaffolds containing different cell types with controlled distribution and precise architecture. Moreover, the introduction of microfluidic technology makes it possible to simulate in-vivo dynamic conditions through the so-called “cancer-on-chip” platforms. Whereas several cancer types have been modeled through the cancer-on-chip approach, such as lung cancer and breast cancer, only a few works describing ovarian cancer models have been described. The aim of this work is to combine 3D bioprinting and microfluidic technics with setting up a 3D dynamic model of ovarian cancer. In the first phase, alginate-gelatin hydrogel containing SKOV3 cells was used to achieve tumor-like structures through an extrusion-based bioprinter. The desired form of the tumor-like mass was first designed on 3D CAD software. The hydrogel composition was then optimized for ensuring good and reproducible printability. Cell viability in the bioprinted structures was assessed using Live/Dead assay and WST1 assay. In the second phase, these bioprinted structures will be included in a microfluidic device that allows simultaneous testing of different drug concentrations. This microfluidic dispositive was first designed through computational fluid dynamics (CFD) simulations for fixing its precise dimensions. It was then be manufactured through a molding method based on a 3D printed template. To confirm the results of CFD simulations, doxorubicin (DOX) solutions were perfused through the dispositive and DOX concentration in each culture chamber was determined. Once completely characterized, this model will be used to assess the efficacy of anti-cancer nanoparticles developed in the Jean Lamour institute.

Keywords: 3D bioprinting, ovarian cancer, cancer-on-chip models, microfluidic techniques

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Authors: Patricia M. B. Saint-Vincent, Anna Furches, Stephanie Galanie, Erica Teixeira Prates, Piet Jones, Nancy Engle, David Kainer, Wellington Muchero, Daniel Jacobson, Timothy J. Tschaplinski

Abstract:

Uridine diphosphate-glycosyltransferases (UGTs) are enzymes that catalyze sugar transfer to a variety of plant metabolites. UGT substrates, which include plant secondary metabolites involved in lignification, demonstrate new activities and incorporation when glycosylated. Knowledge of UGT function, substrate specificity, and enzyme products is important for plant engineering efforts, especially related to increasing plant biomass through lignification. UGTs in Populus trichocarpa, a biofuel feedstock, and model woody plant, were selected from a pool of gene candidates using rapid prioritization strategies. A functional genomics workflow, consisting of a metabolite genome-wide association study (mGWAS), expression of synthetic codon-optimized genes, and high-throughput biochemical assays with mass spectrometry-based analysis, was developed for determining the substrates and products of previously-uncharacterized enzymes. A total of 40 UGTs from P. trichocarpa were profiled, and the biochemical assay results were compared to predicted mGWAS connections. Assay results confirmed seven of 11 leaf mGWAS associations and demonstrated varying levels of substrate specificity among candidate UGTs. P. trichocarpa UGT substrate processing confirms the role of these newly-characterized enzymes in lignan, flavonoid, and phytohormone metabolism, with potential implications for cell wall biosynthesis, nitrogen uptake, and biotic and abiotic stress responses.

Keywords: Populus, metabolite-gene associations, GWAS, bio feedstocks, glycosyltransferase

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Authors: Heba Esaily, Rawhia Eledl, Daila Aboelela, Rasha Noreldin

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Objective: Assess serum level of Transforming growth factor beta 2 (TGF-β2) and Matrilin-3 (MATN3) SNP6 polymorphism in osteoarthritic patients Background: Osteoarthritis (OA) is a musculoskeletal disease characterized by pain and joint stiffness. TGF-β 2 is involved in chondrogenesis and osteogenesis, It has found that MATN3 gene and protein expression was correlated with the extent of tissue damage in OA. Findings suggest that regulation of MATN3 expression is essential for maintenance of the cartilage extracellular matrix microenvironment Subjects and Methods: 72 cases of primary OA (56 with knee OA and 16 with generalized OA were compared with that of 18 healthy controls. Radiographs were scored with the Kellgren-Lawrence scale. Serum TGF-β2 was measured by using (ELISA), levels of marker were correlated to radiographic grading of disease and MATN3 SNP6 polymorphism was determined by (PCR-RFLP). Results: MATN3 SNP6 polymorphism and serum level of TGF-β2 were higher in OA compared with controls. Genotype, NN and N allele frequency were higher in patients with OA compared with controls. NN genotype and N allele frequency were higher in knee osteoarthritis than generalized OA. Significant positive correlation between level of TGFβ2 and radiographic grading in group with knee OA, but no correlation between serum level of TGFβ2 and radiographic grading in generalized OA. Conclusion: MATN3 SNP6 polymorphism and TGF-β2 implicated in the pathogenesis of osteoarthritis. Association of N/N genotype with primary osteoarthritis emphasizes on the need for prospective study include larger sample size to confirm the results of the present study.

Keywords: Matrilin-3, transforming growth factor beta 2, primary osteoarthritis, knee osteoarthritis

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