Search results for: gene sequencing

Authors: Seraj Makkawi, Abdulaziz A. Alqarni, Himyan Alghaythee, Suzan Y. Alharbi, Anmar Fatani, Reem Adas, Ahmad R. Abuzinadah

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Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is a neurodegenerative disease that involves both the upper and lower motor neurons. Familial ALS, including superoxide dismutase 1 (SOD1) mutation, accounts for 5-10% of all cases of ALS. Typically, the symptoms of ALS are purely motor, though coexistent sensory symptoms have been reported in rare cases. In this report, we describe the case of a 47- year-old man who presented with progressive bilateral lower limb weakness and numbness for the last four years. A nerve conduction study (NCS) showed evidence of coexistent axonal sensorimotor polyneuropathy in addition to the typical findings of ALS in needle electromyography. Genetic testing confirmed the diagnosis of familial ALS secondary to the SOD1 genetic mutation. This report highlights that the presence of sensory symptoms should not exclude the possibility of ALS in an appropriate clinical setting.

Keywords: Saudi Arabia, polyneuropathy, SOD1 gene mutation, familial amyotrophic lateral sclerosis, amyotrophic lateral sclerosis

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Authors: Titus A. Beu

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Many modern gene-delivery protocols rely on condensed complexes of DNA with polycations to introduce the genetic payload into cells by endocytosis. In particular, polyethyleneimine (PEI) stands out by a high buffering capacity (enabling the efficient condensation of DNA) and relatively simple fabrication. Realistic computational studies can offer essential insights into the formation process of DNA-PEI polyplexes, providing hints on efficient designs and engineering routes. We present comprehensive computational investigations of solvated PEI and DNA-PEI polyplexes involving calculations at three levels: ab initio, all-atom (AA), and coarse-grained (CG) molecular mechanics. In the first stage, we developed a rigorous AA CHARMM (Chemistry at Harvard Macromolecular Mechanics) force field (FF) for PEI on the basis of accurate ab initio calculations on protonated model pentamers. We validated this atomistic FF by matching the results of extensive molecular dynamics (MD) simulations of structural and dynamical properties of PEI with experimental data. In a second stage, we developed a CG MARTINI FF for PEI by Boltzmann inversion techniques from bead-based probability distributions obtained from AA simulations and ensuring an optimal match between the AA and CG structural and dynamical properties. In a third stage, we combined the developed CG FF for PEI with the standard MARTINI FF for DNA and performed comprehensive CG simulations of DNA-PEI complex formation and condensation. Various technical aspects which are crucial for the realistic modeling of DNA-PEI polyplexes, such as options of treating electrostatics and the relevance of polarizable water models, are discussed in detail. Massive CG simulations (with up to 500 000 beads) shed light on the mechanism and provide time scales for DNA polyplex formation independence of PEI chain size and protonation pattern. The DNA-PEI condensation mechanism is shown to primarily rely on the formation of DNA bundles, rather than by changes of the DNA-strand curvature. The gained insights are expected to be of significant help for designing effective gene-delivery applications.

Keywords: DNA condensation, gene-delivery, polyethylene-imine, molecular dynamics.

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Authors: Soultana Konstantinidou, Tiziana Schmidt, Elena Landi, Alessandro De Carli, Giovanni Maltinti, Darius Witt, Alicja Dziadosz, Agnieszka Lindstaedt, Michele Lai, Mauro Pistello, Valentina Cappello, Luciana Dente, Chiara Gabellini, Piotr Barski, Vittoria Raffa

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CRISPR/Cas9 technology has gained the interest of researchers in the field of biotechnology for genome editing. Since its discovery as a microbial adaptive immune defense, this system has been widely adopted and is acknowledged for having a variety of applications. However, critical barriers related to safety and delivery are persisting. Here, we propose a new concept of genome engineering, which is based on a nano-formulation of Cas9. The Cas9 enzyme was conjugated to a gold nanoparticle (AuNP-Cas9). The AuNP-Cas9 maintained its cleavage efficiency in vitro, to the same extent as the ribonucleoprotein, including non-conjugated Cas9 enzyme, and showed high gene editing efficiency in vivo in zebrafish embryos. Since CRISPR/Cas9 technology is extensively used in cancer research, melanoma was selected as a validation target. Cell studies were performed in A375 human melanoma cells. Particles per se had no impact on cell metabolism and proliferation. Intriguingly, the AuNP-Cas9 internalized spontaneously in cells and localized as a single particle in the cytoplasm and organelles. More importantly, the AuNP-Cas9 showed a high nuclear localization signal. The AuNP-Cas9, overcoming the delivery difficulties of Cas9, could be used in cellular biology and localization studies. Taking advantage of the plasmonic properties of gold nanoparticles, this technology could potentially be a bio-tool for combining gene editing and photothermal therapy in cancer cells. Further work will be focused on intracellular interactions of the nano-formulation and characterization of the optical properties.

Keywords: CRISPR/Cas9, gene editing, gold nanoparticles, nanotechnology

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Authors: Kamila Weissova, Jitka Skrabalova, Katerina Skalova, Jana Koprivova, Zdenka Bendova

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Any Arctic visitor has to deal with extreme conditions, including constant light during the summer season or constant darkness during winter time. Light/dark cycle is the most powerful synchronizing signal for biological clock and the absence of daily dark period during the polar day can significantly alter the functional state of the internal clock. However, the inner clock can be synchronized by other zeitgebers such as physical activity, food intake or social interactions. Here, we investigated the effect of polar day on circadian clock of 10 researchers attending the polar base station in the Svalbard region during July. The data obtained on Svalbard were compared with the data obtained before the researchers left for the expedition (in the Czech Republic). To determine the state of circadian clock we used wrist actigraphy followed by sleep diaries, saliva, and buccal mucosa samples, both collected every 4 hours during 24h-interval to detect melatonin by radioimmunoassay and clock gene (PER1, BMAL1, NR1D1, DBP) mRNA levels by RT-qPCR. The clock gene expression was analyzed using cosinor analysis. From our results, it is apparent that the constant sunlight delayed melatonin onset and postponed the physical activity in the same order. Nevertheless, the clock gene expression displayed higher amplitude on Svalbard compared to the amplitude detected in the Czech Republic. These results have suggested that the common daily schedule at the Svalbard expedition can strengthen circadian rhythm in the environment that is lacking light/dark cycle. In conclusion, the constant sunlight delays melatonin onset, but it still maintains its rhythmic secretion. The effect of constant sunlight on circadian clock can be minimalized by common daily scheduled activity.

Keywords: actighraph, clock genes, human, melatonin, polar day

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Authors: Anna D. Kotrova, Alexandr N. Shishkin, Elena I. Ermolenko

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The aim. To study the quantitative and qualitative colon bacteria ratio from patients with metabolic syndrome. Materials and methods. Fecal samples from patients of 2 groups were identified and analyzed: the first group was formed by patients with metabolic syndrome, the second one - by healthy individuals. The metagenomics method was used with the analysis of 16S rRNA gene sequences. The libraries of the variable sites (V3 and V4) gene 16S RNA were analyzed using the MiSeq device (Illumina). To prepare the libraries was used the standard recommended by Illumina, a method based on two rounds of PCR. Results. At the phylum level in the microbiota of patients with metabolic syndrome compared to healthy individuals, the proportion of Tenericutes was reduced, the proportion of Actinobacteria was increased. At the genus level, in the group with metabolic syndrome, relative to the second group was increased the proportion of Lachnospira. Conclusion. Changes in the colon bacteria ratio in the gut microbiota of patients with metabolic syndrome were found both at the type and the genus level. In the metabolic syndrome group, there is a decrease in the proportion of bacteria that do not have a cell wall. To confirm the revealed microbiota features in patients with metabolic syndrome, further study with a larger number of samples is required.

Keywords: gut microbiota, metabolic syndrome, metagenomics, tenericutes

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Authors: Haruka Ito, Toru Maruyama, Michihiro Ito, Chuya Shinzato, Hiroyuki Fujimura, Yoshikatsu Nakano, Shoichiro Suda, Sachiyo Aburatani, Haruko Takeyama

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Clarifying symbiotic relationships is one of the most important theme for understanding the marine eco-system. Coral reef has been regarded as an important environmental resource. Coral holobiont composed by coral, symbiotic microalgae zooxanthellae, and bacteria have complexed relationship. Zooxanthellae mainly supply organic matter to the host corals through their photosynthetic activity. The symbiotic relationship is indispensable for corals but may easily collapses due to the rise of seawater temperature. However, the molecular mechanism how seawater temperature influences their relationships still remain unclear. In this study, the transcriptomic analysis has applied to elucidate the coral-zooxanthellae relationships under high seawater temperature stress. To observe reactions of corals and zooxanthellae against the rise of seawater temperature, meta-gene expression in coral have been analyzed. The branches from six different colonies of a stony coral, Acropora tenuis, were sampled at nine times by 2016 at two locations, Ishikawabaru and South of Sesoko Island, Okinawa, Japan. The mRNAs extracted from the branches including zooxanthellae were sequenced by illumina HiSeq. Gene Set Enrichment Analysis (GSEA) based on hyper geometric distribution was performed. The seawater temperature at 2016 summer was unusually high, which was caused by El Niño event, and the number of zooxanthellae in coral was decreased in August. GSEA derived the several specific genes expressed in A. tenuis under heat stress conditions. The upregulated genes under heat stress highly related with infection immunity. The downregulated genes significantly contained cell cycle related genes. Thu, it is considered that heat stress cause disorder in cell metabolism of A. tenuis, resulting in serious influence to coral holobiont.

Keywords: coral, symbiosis, thermal stress response, transcriptome analysis

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Authors: Liaqat Ali, Hubert E. Blum, Türkân Sakinc

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Enterococcus faecium is an emerging multidrug-resistant nosocomial pathogen increased dramatically worldwide and causing bacteremia, endocarditis, urinary tract and surgical site infections in immunocomprised patients. The capsular polysaccharides that contribute to pathogenesis through evasion of the host innate immune system are also involved in hindering leukocyte killing of enterococci. The gene cluster (enterococcal polysaccharide antigen) of E. faecalis encoding homologues of many genes involved in polysaccharide biosynthesis. We identified two putative loci with 22 kb and 19 kb which contained 11 genes encoding for glycosyltransferases (GTFs); this was confirmed by using genome comparison of already sequenced strains that has no homology to known capsule genes and the epa-locus. The polysaccharide-conjugate vaccines have rapidly emerged as a suitable strategy to combat different pathogenic bacteria, therefore, we investigated a polysaccharide biosynthesis CapD protein in E. faecium contains 336 amino acids and had putative function for N-linked glycosylation. The deletion/knock-out capD mutant was constructed and complemented by homologues recombination method and confirmed by using PCR and sequencing. For further characterization and functional analysis, in-vitro cell culture and in-vivo a mouse infection models were used. Our ΔcapD mutant shows a strong hydrophobicity and all strains exhibited biofilm production. Subsequently, the opsonic activity was tested in an opsonophagocytic assay which shows increased in mutant compared complemented and wild type strains but more than two fold decreased in colonization and adherence was seen on surface of uroepithelial cells. However, a significant higher bacterial colonialization was observed in capD mutant during animal bacteremia infection. Unlike other polysaccharides biosynthesis proteins, CapD does not seems to be a major virulence factor in enterococci but further experiments and attention is needed to clarify its function, exact mechanism and involvement in pathogenesis of enteroccocal nosocomial infections eventually to develop a vaccine/ or targeted therapy.

Keywords: E. faecium, pathogenesis, polysaccharides, biofilm formation

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Authors: Karol Rodriguez-Peña, Martha L. Macias-Rubalcava, Leticia Rocha-Zavaleta, Sergio Sanchez

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A bioassay-guided study for anti-cancer compounds from endophytes of the Mexican medicinal plant Amphipteryygium adstringens resulted in the isolation of a streptomycete capable of producing a group of compounds with high cytotoxic activity. Microorganisms from surface sterilized samples of various sections of the plant were isolated and all the actinomycetes found were evaluated for their potential to produce compounds with cytotoxic activity against cancer cell lines MCF7 (breast cancer) and HeLa (cervical cancer) as well as the non-tumoural cell line HaCaT (keratinocyte). The most active microorganism was picked for further evaluation. The identification of the microorganism was carried out by 16S rDNA gene sequencing, finding the closest proximity to Streptomyces scabrisporus, but with the additional characteristic that the strain isolated in this study was capable of producing colorful compounds never described for this species. Crude extracts of dichloromethane and ethyl acetate showed IC50 values of 0.29 and 0.96 μg/mL for MCF7, 0.51 and 1.98 μg/mL for HeLa and 0.96 and 2.7 μg/mL for HaCaT. Scaling the fermentation to 10 L in a bioreactor generated 1 g of total crude extract, which was fractionated by silica gel open column to yield 14 fractions. Nine of the fractions showed cytotoxic activity. Fraction 4 was chosen for subsequent purification because of its high activity against cancerous cell lines, lower activity against keratinocytes. HPLC-UV-MS/ESI was used for the evaluation of this fraction, finding at least 10 different compounds with high values of m/z (≈588). Purification of the compounds was carried out by preparative thin-layer chromatography. The prevalent compound was Steffimycin B, a molecule known for its antibiotic and cytotoxic activities and also for its low solubility in aqueous solutions. Along with steffimycin B, another five compounds belonging to the steffimycin family were isolated and at this moment their structures are being elucidated, some of which display better solubility in water: an attractive property for the pharmaceutical industry. As a conclusion to this study, the isolation of endophytes resulted in the discovery of a strain capable of producing compounds with high cytotoxic activity that need to be studied for their possible utilization.

Keywords: amphipterygium adstringens, cytotoxicity, streptomyces scabrisporus, steffimycin

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Authors: Rahaba Marima, Clement Penny

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The Health-related quality of life (HRQoL) for HIV positive patients has improved since the introduction of the highly active antiretroviral treatment (HAART). However, in the present HAART era, HIV co-morbidities such as lung cancer, a non-AIDS (NAIDS) defining cancer have been documented to be on the rise. Under normal physiological conditions, cells grow, repair and proliferate through the cell-cycle as cellular homeostasis is important in the maintenance and proper regulation of tissues and organs. Contrarily, the deregulation of the cell-cycle is a hallmark of cancer, including lung cancer. The association between lung cancer and the use of HAART components such as Efavirenz (EFV) is poorly understood. This study aimed at elucidating the effects of EFV on the cell-cycle genes’ expression in lung cancer. For this purpose, the human cell-cycle gene array composed of 84 genes was evaluated on both normal lung fibroblasts (MRC-5) cells and adenocarcinoma (A549) lung cells, in response to 13µM EFV or 0.01% vehicle. The ±2 up or down fold change was used as a basis of target selection, with p < 0.05. Additionally, RT-qPCR was done to validate the gene array results. Next, In-silico bio-informatics tools, Search Tool for the Retrieval of Interacting Genes/Proteins (STRING), Reactome, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Ingenuity Pathway Analysis (IPA) were used for gene/gene interaction studies as well as to map the molecular and biological pathways influenced by the identified targets. Interestingly, the DNA damage response (DDR) pathway genes such as p53, Ataxia telangiectasia mutated and Rad3 related (ATR), Growth arrest and DNA damage inducible alpha (GADD45A), HUS1 checkpoint homolog (HUS1) and Role of radiation (RAD) genes were shown to be upregulated following EFV treatment, as revealed by STRING analysis. Additionally, functional enrichment analysis by the KEGG pathway revealed that most of the differentially expressed gene targets function at the cell-cycle checkpoint such as p21, Aurora kinase B (AURKB) and Mitotic Arrest Deficient-Like 2 (MAD2L2). Core analysis by IPA revealed that p53 downstream targets such as survivin, Bcl2, and cyclin/cyclin dependent kinases (CDKs) complexes are down-regulated, following exposure to EFV. Furthermore, Reactome analysis showed a significant increase in cellular response to stress genes, DNA repair genes, and apoptosis genes, as observed in both normal and cancerous cells. These findings implicate the genotoxic effects of EFV on lung cells, provoking the DDR pathway. Notably, the constitutive expression of this pathway (DDR) often leads to uncontrolled cell proliferation and eventually tumourigenesis, which could be the attribute of HAART components’ (such as EFV) effect on human cancers. Targeting the cell-cycle and its regulation holds a promising therapeutic intervention to the potential HAART associated carcinogenesis, particularly lung cancer.

Keywords: cell-cycle, DNA damage response, Efavirenz, lung cancer

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Authors: Micheale Yifter Weldemichael, Hailay Mehari Gebremedhn, Teklehaimanot Hailesslasie Teklu

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Recent developments in targeted genome editing accelerated genetic research and opened new potentials to improve crops for better yields and quality. Given the significance of cereal crops as a primary source of food for the global population, the utilization of contemporary genome editing techniques like CRISPR/Cas9 is timely and crucial. CRISPR/Cas technology has enabled targeted genomic modifications, revolutionizing genetic research and exploration. Application of gene editing through CRISPR/Cas9 in enhancing sorghum is particularly vital given the current ecological, environmental, and agricultural challenges exacerbated by climate change. As sorghum is one of the main staple foods of our region and is known to be a resilient crop with a high potential to overcome the above challenges, the application of genome editing technology will enhance the investigation of gene functionality. CRISPR/Cas9 enables the improvement of desirable sorghum traits, including nutritional value, yield, resistance to pests and diseases, and tolerance to various abiotic stresses. Furthermore, CRISPR/Cas9 has the potential to perform intricate editing and reshape the existing elite sorghum varieties, and introduce new genetic variations. However, current research primarily focuses on improving the efficacy of the CRISPR/Cas9 system in successfully editing endogenous sorghum genes, making it a feasible and successful undertaking in sorghum improvement. Recent advancements and developments in CRISPR/Cas9 techniques have further empowered researchers to modify additional genes in sorghum with greater efficiency. Successful application and advancement of CRISPR techniques in sorghum will aid not only in gene discovery and the creation of novel traits that regulate gene expression and functional genomics but also in facilitating site-specific integration events. The purpose of this review is, therefore, to elucidate the current advances in sorghum genome editing and highlight its potential in addressing food security issues. It also assesses the efficiency of CRISPR-mediated improvement and its long-term effects on crop improvement and host resistance against parasites, including tissue-specific activity and the ability to induce resistance. This review ends by emphasizing the challenges and opportunities of CRISPR technology in combating parasitic plants and proposing directions for future research to safeguard global agricultural productivity.

Keywords: CRISPR/Cas9, genome editing, quality, sorghum, stress, yield

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Authors: Muhammad Saad Shoaib Khan, Rasbin Basnet, Qingyao Shu

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Phospholipids are one of the major classes of lipid comprising 10% of total grain lipid in rice. Phospholipids are the main phosphorus containing lipid in the rice endosperm, contributing to rice palatability and seed storage property. However, in the rice grain, the majority of phosphorus occur in the form of phytic acid and are highly abundant in the bran. Phytic acid, also known as hexaphosphorylated inositol (IP6), are strong chelating agents which reduces the bioavailability of essential dietary nutrients and are therefore less desirable by rice breeders. We used the CRISPR/Cas9 system to generate mutants of a phospholipase D gene (PLDα1), which is responsible for the degradation of phospholipids into phosphatidic acid (PA). In the mutants, we found a significant reduction in the concentration of phytic acid in the grain as compared to the wild-type. The biochemical analysis of the PLDα1 mutants showed that the decrease in production of phosphatidic acid is due to reduced accumulation of CDP-diacylglycerolderived phosphatidylinositol (PI), ultimately leading to lower accumulation of phytic acid in mutants. These results showed that loss of function of PLD in rice leads to lower production of phytic acid, suggesting the potential application of Ospldα1 in breeding rice with less phytic acid.

Keywords: CRISPR/Cas9, phospholipase D, phytic acid, rice

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Authors: Ji-Young Choi, Jungjin Kim, Sang-Sun Yun, Sangmee Ahn Jo

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Intracellular adhesion molecule1 (ICAM1) is a mediator of inflammation and involved in adhesion and transmigration of leukocytes to endothelial cells, resulting in enhancement of brain inflammation. We hypothesized that increase of ICAM1 expression in endothelial cells is an early step in the pathogenesis of brain diseases such as Alzheimer’s disease. Here, we report that ICAM1 expression is regulated by pro-inflammatory cytokine TNFa in human microvascular endothelial cell (HBMVEC). TNFa significantly increased ICAM1 mRNA and protein levels at the concentrations showing no cell toxicity. This increase was also shown in micro vessels of mouse brain 24 hours after treatment with TNFa (8 mg/kg, i.v). We then investigated the epigenetic mechanism involved in the induction of ICAM1 expression. Chromatin immunoprecipitation assay revealed that TNFa reduced methylation of histone3K9 (H3K9-2me) and histone3K27 (H3K27-3me), well-known modification as gene suppression, with in the ICAM1 promoter region. However, acetylation of H3K9 and H3K14, well-known modification as gene activation, was not changed by TNFa. Treatment of BIX01294, a specific inhibitor of histone methyltransferase G9a responsible for H3K9-2me, dramatically increased in ICAM1 mRNA and protein levels and overexpression of G9a gene suppressed TNFa-induced ICAM1 expression. In contrast, GSK126, an inhibitor of histone methyltransferase EZH2 responsible for H3K27-3me and valproic acid, an inhibitor of histone deacetylase (HDAC) did not affect ICAM1 expression. These results suggested that histone3 methylation is involved in ICAM1 repression. Moreover, TNFa or BIX01294-induced ICAM induction resulted in both enhancements in adhesion and transmigration of leukocyte on endothelial cell. This study demonstrates that TNFa upregulates ICAM1 expression through H3K9-2me and H3K27-3me within the ICAM1 promoter region, in which G9a is likely to play a pivotal role in ICAM1 transcription. Our study provides a novel mechanism for ICAM1 transcription regulation in HBMVEC.

Keywords: ICAM1, TNFa, HBMVEC, H3K9-2me

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Authors: Muhammad Fiaz, Muhammad Saqlain, Bernard M. Y. Cheung, S. M. Saqlan Naqvi, Ghazala Kaukab Raja

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Background: Association of C allele of rs662799 SNP of APOA5 gene with metabolic syndrome (MetS) has been reported in different populations around the world. A case control study was conducted to explore the relationship of rs662799 variants (T/C) with the MetS and the associated risk phenotypes in a population of Pakistani origin. Methods: MetS was defined according to the IDF criteria. Blood samples were collected from the Pakistan Institute of Medical Sciences, Islamabad, Pakistan for biochemical profiling and DNA extraction. Genotyping of rs662799 was performed using mass ARRAY, iPEX Gold technology. A total of 712 unrelated case and control subjects were genotyped. Data were analyzed using Plink software and SPSS 16.0. Results: The risk allele C of rs662799 showed highly significant association with MetS (OR=1.5, Ρ=0.002). Among risk phenotypes, dyslipidemia, and obesity showed strong association with SNP (OR=1.49, p=0.03; OR =1.46, p=0.01) respectively in models adjusted for age and gender. Conclusion: The rs662799C allele is a significant risk marker for MetS in the local Pakistani population studied. The effect of the SNP is more on dyslipidemia than the other components of the MetS.

Keywords: metabolic syndrome, APOA5, rs662799, dyslipidemia, obesity

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Authors: Ali Bayram, Burak Uz, Ilhan Dolasik, Remzi Yiğiter

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The GABARAP (GABAA-receptor-associated protein) family consists of GABARAP, GABARAPL1 (GABARAP-like 1) and GABARAPL2 (GABARAP-like 2). GABARAPL1, like GABARAP, was described to interact with both GABAA receptor and tubulin, and to be involved in intracellular GABAA receptor trafficking and promoting tubulin polymerization. In addition, GABARAPL1 is thought to be involved in various physiological (autophagosome closure, regulation of circadian rhythms) and/or pathological mechanisms (cancer, neurodegeneration). Alzheimer’s disease (AD) is a progressive neuro degenerative disorder characterized with impaired cognitive functions. Disruption of the GABAergic neuro transmission as well as cholinergic and glutamatergic interactions, may also be involved in the pathogenesis of AD. GABARAPL1 presents a regulated tissue expression and is the most expressed gene among the GABARAP family members in the central nervous system. We, herein, conducted a study to investigate the GABARAPL1 mRNA expression levels in patients with AD. 50 patients with AD and 49 control patients were enrolled to the present study. Messenger RNA expression levels of GABARAPL1 were detected by real-time polymerase chain reaction. GABARAPL1 mRNA expression in AD / control patients was 0,495 (95% confidence interval: 0,404-0,607), p= 0,00000002646. Reduced activity of GABARAPL1 gene might play a role, at least partly, in the pathophysiology of AD.

Keywords: Alzheimer’s disease, GABARAPL1, mRNA expression, RT-PCR

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Authors: Srijoni Banerjee

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Sequences of some of the candidate genes (e.g., CPE, CDKAL1, GCKR, HSD11B1, IGF2BP2, IRS1, LPIN1, PKLR, TNF, PPARG) implicated in some of the complex disease, e.g. Type II diabetes in man has been compared with other species to investigate phylogenetic affinity. Based on mRNA sequence of these genes of 7 to 8 species, using bioinformatics tools Mega 5, Bioedit, Clustal W, distance matrix was obtained. Phylogenetic trees were obtained by NJ and UPGMA clustering methods. The results of the phylogenetic analyses show that of the species compared: Xenopus l., Danio r., Macaca m., Homo sapiens s., Rattus n., Mus m. and Gallus g., Bos taurus, both NJ and UPGMA clustering show close affinity between clustering of Homo sapiens s. (Man) with Rattus n. (Rat), Mus m. species for the candidate genes, except in case of Lipin1 gene. The results support the functional similarity of these genes in physiological and biochemical process involving man and mouse/rat. Therefore, in understanding the complex etiology and treatment of the complex disease mouse/rate model is the best laboratory choice for experimentation.

Keywords: phylogeny, candidate gene of type-2 diabetes, CPE, CDKAL1, GCKR, HSD11B1, IGF2BP2, IRS1, LPIN1, PKLR, TNF, PPARG

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Authors: Alime Cengiz, Talip Kahyaoglu

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Roasting process has the major importance to obtain desired aromatic taste of nuts. In this study, two kinds of roasting process were applied to hulled sesame seeds - vacuum oven and hot air roasting. Efficiency of Gene Expression Programming (GEP), a new soft computing technique of evolutionary algorithm that describes the cause and effect relationships in the data modelling system, and response surface methodology (RSM) were examined in the modelling of roasting processes over a range of temperature (120-180°C) for various times (30-60 min). Color attributes (L*, a*, b*, Browning Index (BI)), textural properties (hardness and fracturability) and moisture content were evaluated and modelled by RSM and GEP. The GEP-based formulations and RSM approach were compared with experimental results and evaluated according to correlation coefficients. The results showed that both GEP and RSM were found to be able to adequately learn the relation between roasting conditions and physical and textural parameters of roasted seeds. However, GEP had better prediction performance than the RSM with the high correlation coefficients (R2 >0.92) for the all quality parameters. This result indicates that the soft computing techniques have better capability for describing the physical changes occuring in sesame seeds during roasting process.

Keywords: genetic expression programming, response surface methodology, roasting, sesame seed

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Authors: Zi-Han Li, Zi-Jun Sun, Dong-Yuan Chang, Li Zhu, Min Chen, Ming-Hui Zhao

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Aims: A genome-wide association study (GWAS) reported that patients with the rs55703767 minor allele in collagen type IV α3 chain encoding gene COL4A3 showed protection against diabetic kidney disease (DKD) in type 1 diabetes mellitus (T1DM). However, the role of rs55703767 in type 2 DKD has not been elucidated. The aim of the current study was to investigate the association between COL4A3 variant rs55703767 and DKD risk in Chinese patients with type 2 diabetes mellitus (T2DM). Methods: This nested case-control study was performed on 1311 patients who had T2DM for at least 10 years, including 580 with DKD and 731 without DKD. We detected the genotypes of all patients by TaqMan SNP Genotyping Assay and analyzed the association between COL4A3 variant rs55703767 and DKD risk. Results: Genetic analysis revealed that there was no significant difference between T2DM patients with DKD and those without DKD regarding allele or genotype frequencies of rs55703767, and the effect of this variant was not hyperglycemia specific. Conclusion: Our findings suggested that there was no detectable association between the COL4A3 variant rs55703767 and the susceptibility to DKD in the Chinese T2DM population.

Keywords: collagen type IV α3 chain, gene polymorphism, type 2 diabetes, diabetic kidney disease

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Authors: Akbar Nawaz Khan, Abdul Ghaffar, Muhammad Naeem Riaz

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The study was conducted to investigate the phenotypic features, morphometry and production potentialities of indigenous naked neck chicken (NN) of Pakistan under intensive management condition. A total of 35 NN chicks were randomly selected, and the experiment was performed at Poultry and wildlife research section NARC Islamabad for a period of 22 weeks. The predominant plumage color was black and golden while skin color was observed white. The average shank length, leg length, thigh length, keel length, chest breadth, head width, wing space, wing length, body length, body girth, body height and pubic bone width in adult males and females were 69.19 ± 3.34mm, 117.93 ± 4.42mm, 117.93 ± 4.42mm, 90.87 ± 6.53mm, 95.03 ± 4.56mm, 49.77 ± 2.53mm, 30.63 ± 1.50cm, 27.24 ± 2.71cm, 18.88 ± 0.65cm, 17.77 ± 1.01cm, 25.96 ± 0.56cm, 47.81 ± 1.41cm and 35.69 ± 4.09mm respectively. The average age and live body weight of NN chicken at sexual maturity were recorded as 165.85 days and 1269.38 g. While hen-day egg production of NN was recorded as 45%. The present study was aimed to investigate the existence of polymorphism at IGF-I gene in indigenous naked neck chicken through PCR based Restriction Fragment Length Polymorphism. Based on restriction analysis using Hinf I restriction enzyme, three genotypes were detected designated as AA, AC, and CC. Restriction analysis of PCR amplified product showed the presence of DNA fragments of 622, 378, 244 and 191, (genotypes). The PCR-RFLP analysis is easy, cost effective method which permits the easy characterization of IGF-I gene. This showed the investigated IGF-I genes can serve as good molecular markers for marker assisted selection (MAS) concerning growth related traits in chicken.

Keywords: Desi chicken, naked neck, morphology, morphometry, production potential, egg traits, egg geometry, IGF-I, growth, PCR- RFLP, chicken

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Authors: Ali Bagherpour

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The aim of this study was to determinate the variety of Anaplasma species among cattle of Khuzestan province, Iran. From April 2013 to June 2013, a total of 200 blood samples were collected via the jugular vein from healthy cattle (100), randomly. The extracted DNA from blood cells were amplified by Anaplasma-all primers, which amplify an approximately 1468bp DNA fragment from region of 16S rRNA gene from various members of the genus Anaplasma. For raising the test sensivity, the PCR products were amplified with the primers, which were designed from the region flanked by the first primers. The amplified nested PCR product had an expected PCR product with 345 nucleotides in length. 44 out of 100 cattle blood samples were Anaplasma spp. positive by first PCR and nested PCR. All cattle positive samples were further analyzed for the presence of A. centrale, A. bovis and A. phagocytophilum by specific nested PCR. A.phagocytophilum was identified by specific nested PCR in 3% of cattle blood samples. The extracted DNA from positive Anaplasma spp. samples were amplified by Anaplasma marginale/ovis specific primers, which amplify an approximately 866bp DNA fragment from region of msp4 gene. 41 out of 100 cattle blood samples (41%) were positive for Anaplasma marginale and Anaplasma ovis, respectively.

Keywords: Iran, Khuzestan, Anaplasma species, Cattle, A. marginale, A. ovis, A. phagocytophilum, PCR

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Authors: Sajewicz-Krukowska Joanna, Domańska-Blicharz Katarzyna, Tarasiuk Karolina, Marzec-Kotarska Barbara

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Astroviral infections pose a significant problem in the poultry industry, leading to multiple adverse effects such as decreased egg production, breeding disorders, poor weight gain, and even increased mortality. Commonly observed chicken astrovirus (CAstV) was recently reported to be responsible for "white chicks syndrome" associated with increased embryo/chick mortality. The CAstV-mediated pathogenesis in chicken occurs due to complex interactions between the infectious pathogen and the immune system. Many aspects of CAstV-chicken interactions remain unclear, and there is no information available regarding gene expression changes in the chicken's spleen in response to CAstV infection. We aimed to investigate the molecular background triggered by CAstV infection. Ten 21-day-old SPF White Leghorn chickens were divided into two groups of 5 birds each. One group was inoculated with CAstV, and the other was used as the negative control. On 4th dpi, spleen samples were collected and immediately frozen at -70°C for RNA isolation. We analysed transcriptional profiles of the chickens' spleens at the 4th day following infection using RNA-seq to establish differentially expressed genes (DEGs). The RNA-seq findings were verified by quantitative real-time PCR (qRT-PCR). A total of 31959 transcripts were identified in response to CAstV infection. Eventually 45 DEGs (p-value<0.05; Log2Foldchange>1)were recognized in the spleen after CAstV infection (26 upregulated DEGs and 19 downregulated DEGs). qRT-PCR performed on 4 genes (IFIT5, OASL, RASD1, DDX60) confirmed RNAseq results. Top differentially expressed genes belonged to novel putative IFN-induced CAstV restriction factors. Most of the DEGs were associated with RIG-I–like signalling pathway or, more generally, with an innate antiviral response(upregulated: BLEC3, CMPK2, IFIT5, OASL, DDX60, IFI6, and downregulated: SPIK5, SELENOP, HSPA2, TMEM158, RASD1, YWHAB). The study provided a global analysis of host transcriptional changes that occur during CAstV infection in vivo and proved the cell cycle in the spleen and immune signalling in chickens were predominantly affected upon CAstV infection.

Keywords: chicken astrovirus, CastV, RNA-seq, transcriptome, spleen

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Authors: Hae Jeong Park, Joo-Ho Chung

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In this study, the effect of essential oil from Chamaecyparis obtuse (EOCO) on early life stress using maternal separation (MS) rats was investigated. Anxiety-related behaviors were examined in MS rats using the elevated plus-maze (EPM) test. The changes of gene expressions by EOCO in the hippocampus of MS rats were analyzed using a microarray method. Rats in the MS groups were separated from their respective mothers from postnatal day (pnd) 14 to 28. Rats in the EOCO-treated groups were exposed to EOCO for 1 h or 2 h by inhalation from pnd 21 to 28. The EOCO-treated MS rats showed decreased anxiety-related behaviors compared to the MS rats in the EPM test. In the microarray analysis, EOCO downregulated the expressions of cytokine genes such as Ccl2, Il6, Cxcl10, Ccl19, and Il1rl in the hippocampus of MS rats, and it was also confirmed through RT-PCR. In particular, the expressions of Ccl2 and Il6 were predominantly decreased by EOCO in the hippocampus of MS rats. Interestingly, their protein expressions were also reduced by EOCO in MS rats. These results indicate that EOCO decreases MS-induced anxiety-related behaviors, and modulate cytokines, particularly Ccl2 and Il6, in the hippocampus of MS rats.

Keywords: anxiety-related behavior, Chamaecyparis obtuse, cytokine gene, early-life stress, maternal separation

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Authors: Yahia I. Mohamed, Ahmed I. Marzouk, Mohamed A. Yacout

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Aim of this work was to study the genetic basis for oil accumulation in olive fruit via tracking DGAT2 (Diacylglycerol acyltransferase type-2) gene in three Egyptian Origen Olive cultivars namely Toffahi, Hamed and Maraki using molecular marker techniques and bioinformatics tools. Results illustrate that, firstly: specific genomic band of Maraki cultivars was identified as DGAT2 (Diacylglycerol acyltransferase type-2) and identical for this gene in Olea europaea with 100 % of similarity. Secondly, differential genomic band of Maraki cultivars which produced from RAPD fingerprinting technique reflected predicted distinguished sequence which identified as DGAT2 (Diacylglycerol acyltransferase type-2) in Fragaria vesca subsp. Vesca with 76% of sequential similarity. Third and finally, specific genomic specific band of Hamed cultivars was indentified as two fragments, 1-Olea europaea cultivar Koroneiki diacylglycerol acyltransferase type 2 mRNA, complete cds with two matches regions with 99% or 2-PREDICTED: Fragaria vesca subsp. vesca diacylglycerol O-acyltransferase 2-like (LOC101313050), mRNA with 86% of similarity.

Keywords: Olea europaea, fingerprinting, diacylglycerol acyltransferase type-2 (DGAT2), Egypt

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Authors: Tayaba Dastgeer, Farhan Rasheed, Muhammad Saeed, Maqsood Ahmad, Zia Ashraf, Abdul Waheed, Muhammad Kamran, Mohsin Khurshid

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Objectives: The study aimed to determine the efficacy of linezolid against clinical isolates of methicillin-resistant staphylococcus aureus (MRSA). Methodology: This cross-sectional study was conducted in the microbiology department of Allama Iqbal Medical College Lahore from August 2017 to September 2019. Isolates were confirmed as MRSA via the presence of the mec-A gene. Confirmed MRSA isolates were processed for susceptibility testing against different antimicrobials, especially linezolid, via the disc diffusion method. Zone sizes were interpreted according to CLSI guidelines. Results: Various types of clinical samples were included in the study; however, the highest frequency of MRSA isolates was found in pus samples, followed by other clinical samples. Among hospitalized patients, most MRSA isolates were obtained from patients in the surgical ward. Of 243 mec-A gene detected isolates, Vancomycin and linezolid showed 100% susceptibility, chloramphenicol showed declining resistance 78 (32.09%), and emerging sensitivity 165 (67.90%) against MRSA. Conclusion: Linezolid is a very efficient drug against MRSA, but the use of this novel drug must be conserved for vancomycin-resistant Staphylococcus aureus or when more resistant pathogens are suspected.

Keywords: MRSA, chloramphenicol, linezolid, nosocomial infections

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Authors: Clotilde Guyon, Nada Jmari, Yen-Chin Li, Jean Denoyel, Noriyuki Fujikado, Christophe Blanchet, David Root, Matthieu Giraud

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Aire induces ectopic expression of a large repertoire of tissue-specific antigen (TSA) genes in thymic medullary epithelial cells (MECs), driving immunological self-tolerance in maturing T cells. Although important mechanisms of Aire-induced transcription have recently been disclosed through the identification and the study of Aire’s partners, the fine transcriptional functions underlied by a number of them and conferred to Aire are still unknown. Alternative cleavage and polyadenylation (APA) is an essential mRNA processing step regulated by the termination complex consisting of 85 proteins, 10 of them have been related to Aire. We evaluated APA in MECs in vivo by microarray analysis with mRNA-spanning probes and RNA deep sequencing. We uncovered the preference of Aire-dependent transcripts for short-3’UTR isoforms and for proximal poly(A) site selection marked by the increased binding of the cleavage factor Cstf-64. RNA interference of the 10 Aire-related proteins revealed that Clp1, a member of the core termination complex, exerts a profound effect on short 3’UTR isoform preference. Clp1 is also significantly upregulated in the MECs compared to 25 mouse tissues in which we found that TSA expression is associated with longer 3’UTR isoforms. Aire-dependent transcripts escape a global 3’UTR lengthening associated with MEC differentiation, thereby potentiating the repressive effect of microRNAs that are globally upregulated in mature MECs. Consistent with these findings, RNA deep sequencing of actinomycinD-treated MECs revealed the increased stability of short 3’UTR Aire-induced transcripts, resulting in TSA transcripts accumulation and contributing for their enrichment in the MECs.

Keywords: Aire, central tolerance, miRNAs, transcription termination

Procedia PDF Downloads 377

Authors: Mahsa Jalili, Essa Ehsan Khan, Signe Dille Lovmo, Augustine Akruwe, Egil Lien, Rolf Erik Olsen, Trygve Sigholt, Atle Magnus Bones

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Data from the Norwegian Directorate of Fisheries reported that >1.2 million tons of Atlantic salmon were produced in Norway aquaculture industry in 2016. Peroxisome proliferator-activated receptors (PPARs) are one of the key transcription factor families that respond to nutritional ligands. Recent studies have shown the connection between PPARs with lipid and carbohydrate metabolism in aquaculture. To our knowledge, there is no published data about the effects of krill meal, soybean meal, Bactocell ® and butyrate feedings compared to control group on PPARs gene and protein expressions in Atlantic salmon. Fish, 1year +postsmolt, average weight 250 gram were cultured for 12 weeks after acclimatization by control commercial feeding in 2 weeks after hatchery. Water oxygen rate, salinity, and temperature were monitored every second day. At the end of the trial, fish were taken from tanks randomly, and four replicates per group were collected and stored in -80 freezers until analysis. Total RNA extracted from posterior part of dorsal fin muscle tissues and Nanodrop and Bioanalyzer was used to check the quality of RNA. Gene expression of PPAR α, β and γ were determined by RT-PCR. The expression of genes of interest was measured relative to control group after normalization to three reference genes. Total protein concentration was calculated by Bradford method, and protein expression was determined with primary PPARγ antibody by western blot. All data were analyzed by ANOVA followed by Benjamini-Hochberg and Bonferroni tests. Probability values <0.05 considered significant. Bactocell® and butyrate groups showed significantly lower PPARα expression. PPARβ and γ were not significantly different among groups. PPARγ mRNA expression was approximately consistent with protein expression pattern, except than butyrate group showed lower mRNA level. The order of PPARγ expression was Bactocell® > soy meal > butyrate > krill meal > control respectively. PPARβ gene expression decreased more in soy meal > butyrate > krill meal > Bactocell® > control groups respectively. In conclusion, the increased expression of PPARγ and α is proposed to represent a reduction tendency of lipid storage in fish fed by Bactocell®, butyrate, soy and krill meal.

Keywords: aquaculture, blotting western, gene expression, krill protein extract, prebiotics, probiotics, Salmo salar

Procedia PDF Downloads 219

Authors: Pattanapon Kayansamruaj, Ha Thanh Dong, Nopadon Pirarat, Channarong Rodkhum

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Streptococcus iniae (SI) is a devastating pathogenic bacteria causing heavy mortality in farmed fish. The application of commercialized bacterin vaccine has been reported failures as the outbreaks of the new serotype of SI were emerged in farms after vaccination and subsequently caused severe losses. In the present study, we attempted to develop effective DNA vaccines against SI infection using Nile tilapia (Oreochromis niloticus) as an animal model. Two monovalent DNA vaccines were constructed by the insertion of coding sequences of cell wall-associated virulence factors-encoding genes, comprised of eno (α-enolase) and mtsB (hydrophobic membrane protein), into cytomegalovirus expression vector (pCI-neo). In the animal trial, 30-g Nile tilapia were injected intramuscularly with 15 µg of each vaccine (mock vaccine group was injected by naked pCI-neo) and maintained for 35 days prior challenging with pathogenic SI at the dosage of 107 CFU/fish. At 13 days post-challenge, the relative percent survival of pEno, pMtsB and mock vaccine were 57%, 45% and 27%, respectively. The expression levels of immune responses-associated genes, namely, IL1β, TNF-α, TGF-β, COX2, IL-6, IL-12 and IL-13, were investigated from the spleen of experimental animal at 7 days post-vaccination (PV) and 7 days post-challenge (PC) using quantitative RT-PCR technique. Generally, at 7 days PV, the pEno vaccinated group exhibited highest level of up-regulation (1.7 to 2.9 folds) of every gene, but TGF-β, comparing to pMtsB and mock vaccine groups. However, at 7 days PC, pEno group showed significant up-regulation (1.4 to 8.5 folds) of immune-related genes as similar as mock vaccine group, while pMtsB group had lowest level of up-regulation (0.7 to 3.3 folds). Summarily, this study indicated that the pEno and pMtsB vaccines could elicit the immune responses of the fish and the magnitude of gene expression at 7 days PV was also consistent with the protection level conferred by the vaccine.

Keywords: gene expression, DNA vaccine, Nile tilapia, Streptococcus iniae

Procedia PDF Downloads 324

Authors: Srijata Mitra, Mobina Parveen, Pranab Roy, Narayan Chandra Chattopadhyay

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Chlorinated hydrocarbon can be a major pollution problem in groundwater as well as soil. Many people interact with these chemicals on daily accidentally or by professionally in the laboratory. One of the most common sources for Chlorinated hydrocarbon contamination of soil and groundwater are industrial effluents. The wide use and discharge of Trichloroethylene (TCE), a volatile chlorohydrocarbon from chemical industry, led to major water pollution in rural areas. TCE is an mainly used as an industrial metal degreaser in industries. Biotransformation of TCE to the potent carcinogen vinyl chloride (VC) by consortia of anaerobic bacteria might have role for the above purpose. For these reasons, the aim of current study was to isolate and characterized the genes involved in TCE metabolism and also to investigate the in silico study of those genes. To our knowledge, only one aromatic dioxygenase system, the toluene dioxygenase in Pseudomonas putida F1 has been shown to be involved in TCE degradation. This is first instance where Bacillus cereus group being used in biodegradation of trichloroethylene. A novel bacterial strain 2479 was isolated from oil depot site at Rajbandh, Durgapur (West Bengal, India) by enrichment culture technique. It was identified based on polyphasic approach and ribotyping. The bacterium was gram positive, rod shaped, endospore forming and capable of degrading trichloroethylene as the sole carbon source. On the basis of phylogenetic data and Fatty Acid Methyl Ester Analysis, strain 2479 should be placed within the genus Bacillus and species cereus. However, the present isolate (strain 2479) is unique and sharply different from the usual Bacillus strains in its biodegrading nature. Fujiwara test was done to estimate that the strain 2479 could degrade TCE efficiently. The gene for TCE biodegradation was PCR amplified from genomic DNA of Bacillus cereus 2479 by using todC1 gene specific primers. The 600bp amplicon was cloned into expression vector pUC I8 in the E. coli host XL1-Blue and expressed under the control of lac promoter and nucleotide sequence was determined. The gene sequence was deposited at NCBI under the Accession no. GU183105. In Silico approach involved predicting the physico-chemical properties of deduced Tce1 protein by using ProtParam tool. The tce1 gene contained 342 bp long ORF encoding 114 amino acids with a predicted molecular weight 12.6 kDa and the theoretical pI value of the polypeptide was 5.17, molecular formula: C559H886N152O165S8, total number of atoms: 1770, aliphatic index: 101.93, instability index: 28.60, Grand Average of Hydropathicity (GRAVY): 0.152. Three differentially expressed proteins (97.1, 40 and 30 kDa) were directly involved in TCE biodegradation, found to react immunologically to the antibodies raised against TCE inducible proteins in Western blot analysis. The present study suggested that cloned gene product (TCE1) was capable of degrading TCE as verified chemically.

Keywords: cloning, Bacillus cereus, in silico analysis, TCE

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Authors: Ruwani N. Nugara, Masashi Inafuku, Kensaku Takara, Hironori Iwasaki, Hirosuke Oku

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Pteryxin from the partially purified hexane phase (HP) of Peucedanum japonicum Thunb (PJT) was identified as the active compound related to anti-obesity. Thus, in this study we investigated the mechanisms related to anti-obesity activity in-vitro. The HP was fractionated, and effect on the triglyceride (TG) content was evaluated in 3T3-L1 and HepG2 cells. Comprehensive spectroscopic analyses were used to identify the structure of the active compound. The dose dependent effect of active constituent on the TG content, and the gene expressions related to adipogenesis, fatty acid catabolism, energy expenditure, lipolysis and lipogenesis (20 μg/mL) were examined in-vitro. Furthermore, higher dosage of pteryxin (50μg/mL) was tested against 20μg/mL in 3T3-L1 adipocytes. The mRNA were subjected to SOLiD next generation sequencer and the obtained data were analyzed by Ingenuity Pathway Analysis (IPA). The active constituent was identified as pteryxin, a known compound in PJT. However, its biological activities against obesity have not been reported previously. Pteryxin dose dependently suppressed TG content in both 3T3-L1 adipocytes and HepG2 hepatocytes (P < 0.05). Sterol regulatory element-binding protein-1 (SREBP1 c), Fatty acid synthase (FASN), and acetyl-CoA carboxylase-1 (ACC1) were downregulated in pteryxin-treated adipocytes (by 18.0, 36.1 and 38.2%; P < 0.05, respectively) and hepatocytes (by 72.3, 62.9 and 38.8%, respectively; P < 0.05) indicating its suppressive effects on fatty acid synthesis. The hormone-sensitive lipase (HSL), a lipid catabolising gene was upregulated (by 15.1%; P < 0.05) in pteryxin-treated adipocytes suggesting improved lipolysis. Concordantly, the adipocyte size marker gene, paternally expressed gene1/mesoderm specific transcript (MEST) was downregulated (by 42.8%; P < 0.05), further accelerating the lipolytic activity. The upregulated trend of uncoupling protein 2 (UCP2; by 77.5%; P < 0.05) reflected the improved energy expenditure due to pteryxin. The 50μg/mL dosage of pteryxin completely suppressed PPARγ, MEST, SREBP 1C, HSL, Adiponectin, Fatty Acid Binding Protein (FABP) 4, and UCP’s in 3T3-L1 adipocytes. The IPA suggested that pteryxin at 20μg/mL and 50μg/mL suppress obesity in two different pathways, whereas the WNT signaling pathway play a key role in the higher dose of pteryxin in preadipocyte stage. Pteryxin in PJT play the key role in regulating lipid metabolism related gene network and improving energy production in vitro. Thus, the results suggests pteryxin as a new natural compound to be used as an anti-obesity drug in pharmaceutical industry.

Keywords: obesity, peucedanum japonicum thunb, pteryxin, food science

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Authors: Vincent Murray

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The glycopeptide bleomycin is used in the treatment of testicular cancer, Hodgkin's lymphoma, and squamous cell carcinoma. Bleomycin damages and cleaves DNA in human cells, and this is considered to be the main mode of action for bleomycin's anti-tumor activity. In particular, double-strand breaks are thought to be the main mechanism for the cellular toxicity of bleomycin. Using Illumina next-generation DNA sequencing techniques, the genome-wide sequence specificity of bleomycin-induced double-strand breaks was determined in human cells. The degree of bleomycin cleavage was also assessed at the transcription start sites (TSSs) of actively transcribed genes and compared with non-transcribed genes. It was observed that bleomycin preferentially cleaved at the TSSs of actively transcribed human genes. There was a correlation between the degree of this enhanced cleavage at TSSs and the level of transcriptional activity. Bleomycin cleavage is also affected by chromatin structure and at TSSs, the peaks of bleomycin cleavage were approximately 200 bp apart. This indicated that bleomycin was able to detect phased nucleosomes at the TSSs of actively transcribed human genes. The genome-wide cleavage pattern of the bleomycin analogues 6′-deoxy-BLM Z and zorbamycin was also investigated in human cells. As found for bleomycin, these bleomycin analogues also preferentially cleaved at the TSSs of actively transcribed human genes. The cytotoxicity (IC₅₀ values) of these bleomycin analogues was determined. It was found that the degree of enhanced cleavage at TSSs was inversely correlated with the IC₅₀ values of the bleomycin analogues. This suggested that the level of cleavage at the TSSs of actively transcribed human genes was important for the cytotoxicity of bleomycin and analogues. Hence this study provided a deeper understanding of the cellular processes involved in the cancer chemotherapeutic activity of bleomycin.

Keywords: anti-tumour activity, bleomycin analogues, chromatin structure, genome-wide study, Illumina DNA sequencing

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Authors: L. K. Davis

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The effects of the evolution force are observable in nature at all structural levels ranging from small molecular systems to conversely enormous biospheric systems. However, the evolution force and work associated with formation of biological structures has yet to be described mathematically or theoretically. In addressing the conundrum, we consider evolution from a unique perspective and in doing so we introduce the “Fundamental Theory of the Evolution Force: FTEF”. We utilized synthetic evolution artificial intelligence (SYN-AI) to identify genomic building blocks and to engineer 14-3-3 ζ docking proteins by transforming gene sequences into time-based DNA codes derived from protein hierarchical structural levels. The aforementioned served as templates for random DNA hybridizations and genetic assembly. The application of hierarchical DNA codes allowed us to fast forward evolution, while dampening the effect of point mutations. Natural selection was performed at each hierarchical structural level and mutations screened using Blosum 80 mutation frequency-based algorithms. Notably, SYN-AI engineered a set of three architecturally conserved docking proteins that retained motion and vibrational dynamics of native Bos taurus 14-3-3 ζ.

Keywords: 14-3-3 docking genes, synthetic protein design, time-based DNA codes, writing DNA code from scratch

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