Search results for: non-coding genome

Authors: Fei Sun, Meilan Xu, Jianghui Zhu, Maria Stefanie Dwiyanti, Cheolwoo Park, Fanjiang Kong, Baohui Liu, Tetsuya Yamada, Jun Abe

Abstract:

Reduced or lack of sensitivity to long daylengths is a key character for soybean, a short-day crop, to adapt to higher latitudinal environments. However, the photoperiod-insensitivity often results in a reduction of the duration of vegetative growth and final yield. To overcome this limitation, a photoperiod insensitive line (RIL16) was developed in this study that delayed flowering from the recombinant inbred population derived from a cross between a photoperiod-insensitive cultivar AGS292 and a late-flowering Thai cultivar K3. Expression analyses under SD and LD conditions revealed that the expression levels of FLOWERING LOCUS T (FT) orthologues, FT2a and FT5a, were lowered in RIL16 relative to AGS292, although the expression of E1, a soybean-specific suppressor for FTs, was inhibited in both conditions. A soybean orthologue of TARGET OF EAT1 (TOE1), another suppressor of FT, showed an upregulated expression in RIL16, which appeared to reflect a lower expression of miR172a. Our data suggest that the delayed flowering of RIL16 most likely is controlled by genes involved in an age-dependent pathway in flowering. The QTL analysis based on 1,125 SNPs obtained from Restriction Site Associated DNA Sequencing revealed two major QTLs for flowering dates in Chromosome 16 and two minor QTLs in Chromosome 4, all of which accounted for 55% and 48% of the whole variations observed in natural day length and artificially-induced long day length conditions, respectively. The intervals of the major QTLs harbored FT2a and FT5a, respectively, on the basis of annotated genes in the Williams 82 reference genome. Sequencing analysis further revealed a nonsynonymous mutation in FT2a and an SNP in the 3′ UTR region of FT5a. A further study may elucidate a detailed mechanism underlying the QTL for late flowering. The alleles from K3 at the two QTLs can be used singly or in combination to retain an appropriate duration of vegetative growth to maximize the final yield of photoperiod-insensitive soybeans.

Keywords: FT genes, miR72a, photoperiod-insensitive, soybean flowering

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Authors: Mohammad Nazri Abdul Bahari, Nurshafika Mohd Sakeh, Siti Nor Akmar Abdullah

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Basal stem rot (BSR) is the most devastating disease in oil palm. Among the oil palm pathogenic fungi, the most prevalent and virulent species associated with BSR is Ganoderma boninense. Early detection of G. boninense attack in oil palm wherein physical symptoms has not yet appeared can offer opportunities to prevent the spread of the necrotrophic fungus. However, poor understanding of molecular defense responses and roles of antifungal metabolites in oil palm against G. boninense has complicated the resolving measures. Hence, characterization of defense-related molecular responses and production of antifungal compounds during early interaction with G. boninense is of utmost important. Four month-old oil palm (Elaeis guineensis) seedlings were artificially infected with G. boninense-inoculated rubber wood block via sitting technique. RNA of samples were extracted from roots and leaves tissues at 0, 3, 7 and 11 days post inoculation (d.p.i) followed with sequencing using RNA-Seq method. Differentially-expressed genes (DEGs) of oil palm-G. boninense interaction were identified, while changes in metabolite profile will be scrutinized related to the DEGs. The RNA-Seq data generated a total of 113,829,376 and 313,293,229 paired-end clean reads from untreated (0 d.p.i) and treated (3, 7, 11 d.p.i) samples respectively, each with two biological replicates. The paired-end reads were mapped to Elaeis guineensis reference genome to screen out non-oil palm genes and subsequently generated 74,794 coding sequences. DEG analysis of phytohormone biosynthetic genes in oil palm roots revealed that at p-value ≤ 0.01, ethylene and jasmonic acid may act in antagonistic manner with salicylic acid to coordinate defense response at early interaction with G. boninense. Findings on metabolite profiling of G. boninense-infected oil palm roots and leaves are hoped to explain the defense-related compounds elicited by Elaeis guineensis in response to G. boninense colonization. The study aims to shed light on molecular defense response of oil palm at early interaction with G. boninense and promote prevention measures against Ganoderma infection.

Keywords: Ganoderma boninense, metabolites, phytohormones, RNA-Seq

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Authors: Daniel Aberdam

Abstract:

Haploinsufficiency of PAX6 in humans is the main cause of congenital aniridia, a rare eye disease characterized by reduced visual acuity. Patients have also progressive disorders including cataract, glaucoma and corneal abnormalities making their condition very challenging to manage. Aniridia-related keratopathy (ARK), caused by a combination of factors including limbal stem-cell deficiency, impaired healing response, abnormal differentiation, and infiltration of conjunctival cells onto the corneal surface, affects up to 95% of patients. It usually begins in the first decade of life resulting in recurrent corneal erosions, sub-epithelial fibrosis with corneal decompensation and opacification. Unfortunately, current treatment options for aniridia patients are currently limited. Although animal models partially recapitulate this disease, there is no in vitro cellular model of AKT needed for drug/therapeutic tools screening and validation. We used genome editing (CRISPR/Cas9 technology) to introduce a nonsense mutation found in patients into one allele of the PAX6 gene into limbal stem cells. Resulting mutated clones, expressing half of the amount of PAX6 protein and thus representative of haploinsufficiency were further characterized. Sequencing analysis showed that no off-target mutations were induced. The mutated cells displayed reduced cell proliferation and cell migration but enhanced cell adhesion. Known PAX6 targets expression was also reduced. Remarkably, addition of soluble recombinant PAX6 protein into the culture medium was sufficient to activate endogenous PAX6 gene and, as a consequence, rescue the phenotype. It strongly suggests that our in vitro model recapitulates well the epithelial defect and becomes a powerful tool to identify drugs that could rescue the corneal defect in patients. Furthermore, we demonstrate that the homeotic transcription factor Pax6 is able to be uptake naturally by recipient cells to function into the nucleus.

Keywords: Pax6, crispr/cas9, limbal stem cells, aniridia, gene therapy

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Authors: Safee Chaudhary, Mahnoor Naseer Gondal, Hira Anees Awan, Abdul Rehman, Ammar Arif, Risham Hussain, Huma Khawar, Zainab Arshad, Muhammad Faizyab Ali Chaudhary, Waleed Ahmed, Muhammad Umer Sultan, Bibi Amina, Salaar Khan, Muhammad Moaz Ahmad, Osama Shiraz Shah, Hadia Hameed, Muhammad Farooq Ahmad Butt, Muhammad Ahmad, Sameer Ahmed, Fayyaz Ahmed, Omer Ishaq, Waqar Nabi, Wim Vanderbauwhede, Bilal Wajid, Huma Shehwana, Muhammad Tariq, Amir Faisal

Abstract:

Cancer is a manifestation of multifactorial deregulations in biomolecular pathways. These deregulations arise from the complex multi-scale interplay between cellular and extracellular factors. Such multifactorial aberrations at gene, protein, and extracellular scales need to be investigated systematically towards decoding the underlying mechanisms and orchestrating therapeutic interventions for patient treatment. In this work, we propose ‘TISON’, a next-generation web-based multiscale modeling platform for clinical systems oncology. TISON’s unique modeling abstraction allows a seamless coupling of information from biomolecular networks, cell decision circuits, extra-cellular environments, and tissue geometries. The platform can undertake multiscale sensitivity analysis towards in silico biomarker identification and drug evaluation on cellular phenotypes in user-defined tissue geometries. Furthermore, integration of cancer expression databases such as The Cancer Genome Atlas (TCGA) and Human Proteome Atlas (HPA) facilitates in the development of personalized therapeutics. TISON is the next-evolution of multiscale cancer modeling and simulation platforms and provides a ‘zero-code’ model development, simulation, and analysis environment for application in clinical settings.

Keywords: systems oncology, cancer systems biology, cancer therapeutics, personalized therapeutics, cancer modelling

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Authors: Neslihan Taskale Karatug, Mustafa Akcelik

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Some literature data show that misL protein play a role on host immune response formed against Salmonella Typhimurium. The aim of the present study is to learn the role of the protein in S. Typhimurium pathogenicity. To describe certain functions of the protein, primarily recombinant misL protein was produced and purified. PCR was performed using a primer set targeted to passenger domain of the misL gene on S. Typhimurium LT2 genome. Amplicon and pet28a vector were enzymatically cleaved with EcoRI and NheI. The digested DNA materials were purified with High Pure PCR Product Purification Kit. The ligation reaction was achieved with the pure products. After preparation of competent Escherichia coli Dh5α, ligation mix was transformed into the cell by electroporation. To confirm the existence of insert gene, recombinant plasmid DNA of Dh5α was isolated with high pure plasmid DNA kit. Proved the correctness of recombinant plasmid was electroporated to BL21. The cell was induced by IPTG. After induction, the presence of recombinant protein was checked by SDS-PAGE. The recombinant misL protein was purified using HisPur Ni-NTA spin colon. The pure protein was shown by SDS-PAGE and western blot immünoassay. The concentration of the protein was measured BCA Protein Assay kit. In the wake of ligation with digested products (2 kb misL and 5.4 kb pet28a) visualised on gel size of the band was about 7.4 kb and was named as pNT01. The pNT01 recombinant plasmid was transformed into Dh5α and colonies were chosen in selective medium. Plasmid DNA isolation from them was carried out. PCR was achieved on the pNT01 to check misL and 2 kb band was observed on the agarose gel. After electroporation of the plasmid and induction of the cell, 68 kDa misL protein was seen. Subsequent to the purification of the protein, only a band was observed on SDS-PAGE. Association of the pure protein with anti-his antibody was verified by the western blot assay. The concentration of the pure misL protein was determined as 345 μg/mL. Production of polyclonal antibody will be achieved by using the obtained pure recombinant misL protein as next step. The role of the protein will come out on the immune system together some assays.

Keywords: cloning, Escherichia coli, recombinant protein purification, Salmonella Typhimurium

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Authors: Sohan Sengupta, Arnab Pramanik, Abhrajyoti Ghosh, Maitree Bhattacharyya

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Newly emerged phyto-pathogens and multi drug resistance have been threating the world for last few decades. Actinomycetes, the most endowed group of microorganisms isolated from unexplored regions of the world may be the ultimate solution to these problems. Thus the aim of this study was to isolate several bioactive actinomycetes strains capable of producing antimicrobial secondary metabolite from Sundarbans, the only mangrove tiger land of the world. Fifty four actinomycetes were isolated and analyzed for antimicrobial activity against fifteen test organisms including three phytopathogens. Nine morphologically distinct and biologically active isolates were subjected to polyphasic identification study. 16s rDNA sequencing indicated eight isolates to reveal maximum similarity to the genus streptomyces, whereas one isolate presented only 93.57% similarity with Streptomyces albogriseolus NRRL B-1305T. Seventy-one carbon sources and twenty-three chemical sources utilization assay revealed their metabolic relatedness. Among these nine isolates three specific strains were found to have notably higher degree of antimicrobial potential effective in a broader range including phyto-pathogenic fungus. PCR base whole genome screen for PKS and NRPS genes, confirmed the occurrence of bio-synthetic gene cluster in some of the isolates for novel antibiotic production. Finally the strain SMS_SU21, which showed antimicrobial activity with MIC value of 0.05 mg ml-1and antioxidant activity with IC50 value of 0.242±0.33 mg ml-1 was detected to be the most potential one. True prospective of this strain was evaluated utilizing GC-MS and the bioactive compound responsible for antimicrobial activity was purified and characterized. Rare bioactive actinomycetes were isolated from unexplored heritage site. Diversity of the biosynthetic gene cluster for antimicrobial compound production has also been evaluated. Antimicrobial compound SU21-C has been identified and purified which is active against a broad range of pathogens.

Keywords: actinomycetes, sundarbans, antimicrobial, pks nrps, phyto-pathogens, GC-MS

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Authors: E. Syngelaki, C. C. F. Schinkel, S. Klatt, E. Hörandl

Abstract:

Environmental influence can alter the conditions for plant development and can trigger changes in epigenetic variation. Thus, the exposure to abiotic environmental stress can lead to different DNA methylation profiles and may have evolutionary consequences for adaptation. Epigenetic control mechanisms may further influence mode of reproduction. The alpine species R. kuepferi has diploid and tetraploid cytotypes, that are mostly sexual and facultative apomicts, respectively. Hence, it is a suitable model system for studying the correlations of mode of reproduction, ploidy, and environmental stress. Diploid and tetraploid individuals were placed in two climate chambers and treated with low (+7°C day/+2°C night, -1°C cold shocks for three nights per week) and warm (control) temperatures (+15°C day/+10°C night). Subsequently, methylation sensitive-Amplified Fragment-Length Polymorphism (AFPL) markers were used to screen genome-wide methylation alterations triggered by stress treatments. The dataset was analyzed for four groups regarding treatment (cold/warm) and ploidy level (diploid/tetraploid), and also separately for full methylated, hemi-methylated and unmethylated sites. Patterns of epigenetic variation suggested that diploids differed significantly in their profiles from tetraploids independent from treatment, while treatments did not differ significantly within cytotypes. Furthermore, diploids are more differentiated than the tetraploids in overall methylation profiles of both treatments. This observation is in accordance with the increased frequency of apomictic seed formation in diploids and maintenance of facultative apomixis in tetraploids during the experiment. Global analysis of molecular variance showed higher epigenetic variation within groups than among them, while locus-by-locus analysis of molecular variance showed a high number (54.7%) of significantly differentiated un-methylated loci. To summarise, epigenetic variation seems to depend on ploidy level, and in diploids may be correlated to changes in mode of reproduction. However, further studies are needed to elucidate the mechanism and possible functional significance of these correlations.

Keywords: apomixis, cold stress, DNA methylation, Ranunculus kuepferi

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Authors: Reema K. AlEssa, Sahar Alshomer, Abdullah Alfaleh, Sultan ALkhenaizan, Mohammed Albalwi

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Ichthyosis is a disorder of abnormal keratinization, characterized by excessive scaling, and consists of more than twenty subtypes varied in severity, mode of inheritance, and the genes involved. There is insufficient data in the literature about the epidemiology and characteristics of ichthyosis locally. Our aim is to identify the histopathological features and genetic profile of ichthyosis. Method: It is an observational retrospective case series study conducted in March 2020, included all patients who were diagnosed with Ichthyosis and confirmed by histological and molecular findings over the last 20 years in King Abdulaziz Medical City (KAMC), Riyadh, Saudi Arabia. Molecular analysis was performed by testing genomic DNA and checking genetic variations using the AmpliSeq panel. All disease-causing variants were checked against HGMD, ClinVar, Genome Aggregation Database (gnomAD), and Exome Aggregation Consortium (ExAC) databases. Result: A total of 60 cases of Ichthyosis were identified with a mean age of 13 ± 9.2. There is an almost equal distribution between female patients 29 (48%) and males 31 (52%). The majority of them were Saudis, 94%. More than half of patients presented with general scaling 33 (55%), followed by dryness and coarse skin 19 (31.6%) and hyperlinearity 5 (8.33%). Family history and history of consanguinity were seen in 26 (43.3% ), 13 (22%), respectively. History of colloidal babies was found in 6 (10%) cases of ichthyosis. The most frequent genes were ALOX12B, ALOXE3, CERS3, CYP4F22, DOLK, FLG2, GJB2, PNPLA1, SLC27A4, SPINK5, STS, SUMF1, TGM1, TGM5, VPS33B. Most frequent variations were detected in CYP4F22 in 16 cases (26.6%) followed by ALOXE3 6 (10%) and STS 6 (10%) then TGM1 5 (8.3) and ALOX12B 5 (8.3). The analysis of molecular genetic identified 23 different genetic variations in the genes of ichthyosis, of which 13 were novel mutations. Homozygous mutations were detected in the majority of ichthyosis cases, 54 (90%), and only 1 case was heterozygous. Few cases, 4 (6.6%) had an unknown type of ichthyosis with a negative genetic result. Conclusion: 13 novel mutations were discovered. Also, about half of ichthyosis patients had a positive history of consanguinity.

Keywords: ichthyosis, genetic profile, molecular characterization, congenital ichthyosis

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Authors: Janneth Gonzalez, Andres Pinzon Velasco, Maria Angarita, Nicolas Mendoza

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Astrocytes play an important role in various processes in the brain, including pathological conditions such as neurodegenerative diseases. Recent studies have shown that the increase in saturated fatty acids such as palmitic acid (PA) triggers pro-inflammatory pathways in the brain. The use of synthetic neurosteroids such as tibolone has demonstrated neuro-protective mechanisms. However, there are few studies on the neuro-protective mechanisms of tibolone, especially at the systemic (omic) level. In this study, we performed the integration of multi-omic data (transcriptome and proteome) into a human astrocyte genomic scale metabolic model to study the astrocytic response during palmitate treatment. We evaluated metabolic fluxes in three scenarios (healthy, induced inflammation by PA, and tibolone treatment under PA inflammation). We also use control theory to identify those reactions that control the astrocytic system. Our results suggest that PA generates a modulation of central and secondary metabolism, showing a change in energy source use through inhibition of folate cycle and fatty acid β-oxidation and upregulation of ketone bodies formation.We found 25 metabolic switches under PA-mediated cellular regulation, 9 of which were critical only in the inflammatory scenario but not in the protective tibolone one. Within these reactions, inhibitory, total, and directional coupling profiles were key findings, playing a fundamental role in the (de)regulation in metabolic pathways that increase neurotoxicity and represent potential treatment targets. Finally, this study framework facilitates the understanding of metabolic regulation strategies, andit can be used for in silico exploring the mechanisms of astrocytic cell regulation, directing a more complex future experimental work in neurodegenerative diseases.

Keywords: astrocytes, data integration, palmitic acid, computational model, multi-omics, control theory

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

Abstract:

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: M. Shemis, O. Maher, G. Casterou, F. Gauffre

Abstract:

Hepatitis C virus (HCV) is a major cause of chronic liver disease with a global 170 million chronic carriers at risk of developing liver cirrhosis and/or liver cancer. Egypt reports the highest prevalence of HCV worldwide. Currently, two classes of assays are used in the diagnosis and management of HCV infection. Despite the high sensitivity and specificity of the available diagnostic assays, they are time-consuming, labor-intensive, expensive, and require specialized equipment and highly qualified personal. It is therefore important for clinical and economic terms to develop a low-tech assay for the direct detection of HCV RNA with acceptable sensitivity and specificity, short turnaround time, and cost-effectiveness. Such an assay would be critical to control HCV in developing countries with limited resources and high infection rates, such as Egypt. The unique optical and physical properties of gold nanoparticles (AuNPs) have allowed the use of these nanoparticles in developing simple and rapid colorimetric assays for clinical diagnosis offering higher sensitivity and specificity than current detection techniques. The current research aims to develop a detection assay for HCV RNA using gold nanoparticles (AuNPs). Methods: 200 anti-HCV positive samples and 50 anti-HCV negative plasma samples were collected from Egyptian patients. HCV viral load was quantified using m2000rt (Abbott Molecular Inc., Des Plaines, IL). HCV genotypes were determined using multiplex nested RT- PCR. The assay is based on the aggregation of AuNPs in presence of the target RNA. Aggregation of AuNPs causes a color shift from red to blue. AuNPs were synthesized using citrate reduction method. Different sets of probes within the 5’ UTR conserved region of the HCV genome were designed, grafted on AuNPs and optimized for the efficient detection of HCV RNA. Results: The nano-gold assay could colorimetrically detect HCV RNA down to 125 IU/ml with sensitivity and specificity of 91.1% and 93.8% respectively. The turnaround time of the assay is < 30 min. Conclusions: The assay allows sensitive and rapid detection of HCV RNA and represents an inexpensive and simple point-of-care assay for resource-limited settings.

Keywords: HCV, gold nanoparticles, point of care, viral load

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Authors: Ananya Gupta, Sangeeta Bhaskar

Abstract:

Mycobacterium indicus pranii (MIP) is a saprophytic mycobacterium. It is a predecessor of M. avium complex (MAC). Whole genome analysis and growth kinetics studies have placed MIP in between pathogenic and non-pathogenic species. It shares significant antigenic repertoire with M. tuberculosis and have unique immunomodulatory properties. MIP provides better protection than BCG against pulmonary tuberculosis in animal models. Immunization with MIP by aerosol route provides significantly higher protection as compared to immunization by subcutaneous (s.c.) route. However, mechanism behind differential protection has not been studied. In this study, using mice model we have evaluated and compared the M.tb specific immune response in lung compartments (airway lumen / lung interstitium) as well as spleen following MIP immunization via nasal (i.n.) and s.c. route. MIP i.n. vaccination resulted in increased seeding of memory T cells (CD4+ and CD8+ T-cells) in the airway lumen. Frequency of CD4+ T cells expressing Th1 migratory marker (CXCR3) and activation marker (CD69) were also high in airway lumen of MIP i.n. group. Significantly high ex vivo secretion of cytokines- IFN-, IL-12, IL-17 and TNF- from cells of airway luminal spaces provides evidence of antigen-specific lung immune response, besides generating systemic immunity comparable to MIP s.c. group. Analysis of T cell response on per cell basis revealed that antigen specific T-cells of MIP i.n. group were functionally superior as higher percentage of these cells simultaneously secreted IFN-gamma, IL-2 and TNF-alpha cytokines as compared to MIP s.c. group. T-cells secreting more than one of the cytokines simultaneously are believed to have robust effector response and crucial for protection, compared with single cytokine secreting T-cells. Adoptive transfer of airway luminal T-cells from MIP i.n. group into trachea of naive B6 mice revealed that MIP induced CD8 T-cells play crucial role in providing long term protection. Thus the study demonstrates that MIP intranasal vaccination induces M.tb specific memory T-cells in the airway lumen that results in an early and robust recall response against M.tb infection.

Keywords: airway lumen, Mycobacterium indicus pranii, Th1 migratory markers, vaccination

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Authors: Muhammad Tanveer Altaf, Mehmet Bedir, Waqas Liaqat, Gönül Cömertpay, Volkan Çatalkaya, Celaluddin Barutçular, Nergiz Çoban, Ibrahim Cerit, Muhammad Azhar Nadeem, Tolga Karaköy, Faheem Shehzad Baloch

Abstract:

Food production needs to be almost double by 2050 in order to feed around 9 billion people around the Globe. Plant production mostly relies on fertilizers, which also have one of the main roles in environmental pollution. In addition to this, climatic conditions are unpredictable, and the earth is expected to face severe drought conditions in the future. Therefore, water and fertilizers, especially nitrogen are considered as main constraints for future food security. To face these challenges, developing integrative approaches for germplasm characterization and selecting the resilient genotypes performing under limiting conditions is very crucial for effective breeding to meet the food requirement under climatic change scenarios. This study is part of the European Research Area Network (ERANET) project for the characterization of the diversity panel of 172 sorghum accessions and six hybrids as control cultivars under limiting (+N/-H2O, -N/+H2O) and non-limiting conditions (+N+H2O). This study was planned to characterize the sorghum diversity in relation to resource Use Efficiency (RUE), with special attention on harnessing the interaction between genotype and environment (GxE) from a physiological and agronomic perspective. Experiments were conducted at Adana, a Mediterranean climate, with augmented design, and data on various agronomic and physiological parameters were recorded. Plentiful diversity was observed in the sorghum diversity panel and significant variations were seen among the limiting water and nitrogen conditions in comparison with the control experiment. Potential genotypes with the best performance are identified under limiting conditions. Whole genome resequencing was performed for whole germplasm under investigation for diversity analysis. GWAS analysis will be performed using genotypic and phenotypic data and linked markers will be identified. The results of this study will show the adaptation and improvement of sorghum under climate change conditions for future food security.

Keywords: germplasm, sorghum, drought, nitrogen, resources use efficiency, sequencing

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Authors: Janneth Gonzalez, Andrés Pinzon Velasco, Maria Angarita

Abstract:

Astrocytes play an important role in various processes in the brain, including pathological conditions such as neurodegenerative diseases. Recent studies have shown that the increase in saturated fatty acids such as palmitic acid (PA) triggers pro-inflammatorypathways in the brain. The use of synthetic neurosteroids such as tibolone has demonstrated neuro-protective mechanisms. However, broad studies with a systemic point of view on the neurodegenerative role of PA and the neuro-protective mechanisms of tibolone are lacking. In this study, we performed the integration of multi-omic data (transcriptome and proteome) into a human astrocyte genomic scale metabolic model to study the astrocytic response during palmitate treatment. We evaluated metabolic fluxes in three scenarios (healthy, induced inflammation by PA, and tibolone treatment under PA inflammation). We also applied a control theory approach to identify those reactions that exert more control in the astrocytic system. Our results suggest that PA generates a modulation of central and secondary metabolism, showing a switch in energy source use through inhibition of folate cycle and fatty acid β‐oxidation and upregulation of ketone bodies formation. We found 25 metabolic switches under PA‐mediated cellular regulation, 9 of which were critical only in the inflammatory scenario but not in the protective tibolone one. Within these reactions, inhibitory, total, and directional coupling profiles were key findings, playing a fundamental role in the (de)regulation of metabolic pathways that may increase neurotoxicity and represent potential treatment targets. Finally, the overall framework of our approach facilitates the understanding of complex metabolic regulation, and it can be used for in silico exploration of the mechanisms of astrocytic cell regulation, directing a more complex future experimental work in neurodegenerative diseases.

Keywords: astrocytes, data integration, palmitic acid, computational model, multi-omics

Procedia PDF Downloads 97

Authors: Engin Berber, Nurettin Canakoglu, Ibrahim Sozdutmaz, Merve Caliskan, Shaikh Terkis Islam Pavel, Hazel Yetiskin, Aykut Ozdarendeli

Abstract:

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus in the family Bunyaviridae, genus Nairovirus. The CCHFV genome consists of three molecules of negative-sense single-stranded RNA, each encapsulated separately. The virion particle contains viral RNA polymerase (L segment), surface glycoproteins Gn and Gc (Msegment), and a nucleocapsid protein NP (S segment). CCHF is characterized by high case mortality, occurring in Asia, Africa, the Middle East and Eastern Europe. Clinical CCHF was first recognized in Turkey in 2002. The numbers of CCHF cases have gradually increased in Turkey making the virus a public health concern. Between 2002 and 2014, more than 8000 the CCHF cases have been reported in Turkey and mortality rate is around 5%. So, Turkey is one of the countries where the epidemy has become spread to the wider geography and the biggest outbreaks of CCHF have occurred in the world. We have recently developed an inactivated cell-culture based vaccine against CCHF. We have showed that the Balb/c mice immunized with the CCHF vaccine induced the high level of neutralizing antibodies. In this study, we aimed to determine the immunodominant regions of nucleoprotein (NP) CCHFV Kelkit06 strain which stimulate T cells. For this purpose, pools of overlapping NP were used for an IFN- γ ELISPOT assay. Balb/c mice were divided into two groups for the experiment. Two groups (n = 10 each) were immunized via the intraperitoneal route with 5, or 10μg of the cell culture-based vaccine. The control group (n = 6) was mock immunized with PBS. Booster injections with the same formulation were given on days 21 and 42 after the first immunization. The higher reactivity against the CCHFV NP pools 31-40 and 80-90 was determined in the two dose groups. In order to analyze the vaccine-induced T cell responses in Balb/c mice immunized with varying doses of the vaccine, we have been also currently working on CD4+, CD8+ and CD3 + T cells by flow cytometry.

Keywords: Crimean-Congo hemorrhagic fever virus, immunodominant regions of NP, T cell response, vaccine

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Authors: Atlal El-Assaad

Abstract:

Viruses change with time through mutations and result in new variants that may persist or disappear. A Mutation refers to an actual change in the virus genetic sequence, and a variant is a viral genome that may contain one or more mutations. Critical mutations may cause the virus to be more transmissible, with high disease severity, and more vulnerable to diagnostics, therapeutics, and vaccines. Thus, variants carrying such mutations may increase the risk to human health and are considered variants of concern (VOC). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) - the contagious in humans, positive-sense single-stranded RNA virus that caused coronavirus disease 2019 (COVID-19) - has been studied thoroughly, and several variants were revealed across the world with their corresponding mutations. SARS-CoV-2 has four structural proteins, known as the S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins, but prior study and vaccines development focused on genetic mutations in the S protein due to its vital role in allowing the virus to attach and fuse with the membrane of a host cell. Specifically, subunit S1 catalyzes attachment, whereas subunit S2 mediates fusion. In this perspective, we studied all charged amino acid mutations of the SARS-CoV-2 viral spike protein S1 when bound to Antibody CC12.1 in a crystal structure and assessed the effect of different mutations. We generated all missense mutants of SARS-CoV-2 protein amino acids (AAs) within the SARS-CoV-2:CC12.1 complex model. To generate the family of mutants in each complex, we mutated every charged amino acid with all other charged amino acids (Lysine (K), Arginine (R), Glutamic Acid (E), and Aspartic Acid (D)) and studied the new binding of the complex after each mutation. We applied Poisson-Boltzmann electrostatic calculations feeding into free energy calculations to determine the effect of each mutation on binding. After analyzing our data, we identified charged amino acids keys for binding. Furthermore, we validated those findings against published experimental genetic data. Our results are the first to propose in silico potential life-threatening mutations of SARS-CoV-2 beyond the present mutations found in the five common variants found worldwide.

Keywords: SARS-CoV-2, variant, ionic amino acid, protein-protein interactions, missense mutation, AESOP

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Authors: Ridwaan N. Milase, Leonardo J. Van Zyl, Marla Trindade

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American foulbrood (AFB) is the world’s most devastating honeybee disease that has drastically reduced the population of Apis mellifera capensis since 2009. The outbreak has jeopardized the South African bee keeping industry as well as the agricultural sector dependent on honeybees for honey production and pollination, leading to significant economic losses. AFB is caused by Paenibacillus larvae, a spore-forming, Gram positive facultative anaerobic and flagellated bacterium. The use of antibiotics within beehives has selected for resistant strains of P. larvae, while the current practice of burning spore contaminated beehives and equipment contributes to the economic losses in the honeybee-keeping industry. Therefore, phage therapy is proposed as a promising alternative to combat P. larvae strains affecting A. mellifera capensis. The genomes of two P. larvae strains isolated from infected combs in the Western Cape have been sequenced and annotated using bioinformatics tools. Genome analyses has revealed that these P. larvae strains are lysogens to more than 6 different prophages and possess different type of clustered regularly interspaced short palindromic repeat (CRISPRs) regions per strain. Active prophages from one of the two P. larvae strains were detected and identified using PCR. Electron microscopy was used to determine the family of the identified active prophages. Lytic bacteriophages that specifically target the two P. larvae strains were purified from sewage wastewater, beehive materials, and soil samples to investigate their potential development as anti-P. larvae agents. Another alternative treatment being investigated is the development of a prophage endolysin cocktail. Endolysin genes of the prophages have been targeted, cloned and expressed in Escherichia coli. The heterologously expressed endolysins have been purified and are currently being assessed for their lytic activity against P. larvae strains and other commensal microorganisms that compose the honeybee larvae microbiota. The study has shown that phage therapy and endolysins have a great potential as alternative control methods for AFB disease affecting A. mellifera capensis.

Keywords: American foulbrood, bacteriophage, honeybee, Paenibacillus larvae

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Authors: Allan Kiptanui Kimisto, Geoffrey Odhiambo Ongondo, Anastasia Wairimu Muia, Cyrus Ndungu Kimani

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The major challenge to proper sewage sludge treatment processes is the poor understanding of sludge microbiome diversities. This study applied the whole-genome. shotgun metagenomics technique to profile the microbial composition of sewage sludge in two active digestion lagoons at the Nyeri-Kangemi Wastewater Treatment Plant in Nyeri County, Kenya. Total microbial community DNA was extracted from samples using the available ZymoBIOMICS™ DNA Miniprep Kit and sequenced using Shotgun metagenomics. Samples were analyzed using MG-RAST software (Project ID: mgp100988), which allowed for comparing taxonomic diversity before β-diversities studies for Bacteria, Archaea and Eukaryotes. The study identified 57 phyla, 145 classes, 301 orders, 506 families, 963 genera, and 1980 species. Bacteria dominated the microbes and comprised 28 species, 51 classes, 110 orders, 243 families, 597 genera, and 1518 species. The Bacteroides(6.77%) were dominant, followed by Acinetobacter(1.44%) belonging to the Gammaproteobacteria and Acidororax (1.36%), Bacillus (1.24%) and Clostridium (1.02%) belonging to Betaproteobacteria. Archaea recorded 5 phyla, 13 classes, 19 orders, 29 families, 60 genera,and87 species, with the dominant genera being Methanospirillum (16.01%), methanosarcina (15.70%), and Methanoregula(14.80%) and Methanosaeta (8.74%), Methanosphaerula(5.48%) and Methanobrevibacter(5.03%) being the subdominant group. The eukaryotes were the least in abundance and comprised 24 phyla, 81 classes, 301 orders, 506 families, 963 genera, and 980 species. Arabidopsis (4.91%) and Caenorhabditis (4.81%) dominated the eukaryotes, while Dityostelium (3.63%) and Drosophila(2.08%) were the subdominant genera. All these microbes play distinct roles in the anaerobic treatment process of sewage sludge. The local sludge microbial composition and abundance variations may be due to age difference differences between the two digestion lagoons in operation at the plant and the different degradation rales played by the taxa. The information presented in this study can help in the genetic manipulation or formulation of optimal microbial ratios to improve their effectiveness in sewage sludge treatment. This study recommends further research on how the different taxa respond to environmental changes over time and space.

Keywords: shotgun metagenomics, sludge, bacteria, archaea, eukaryotes

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Authors: Nurulhikma Md Isa, Intan Elya Suka, Nur Farhana Roslan, Chew Bee Lynn

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The evolutionarily conserved N-end rule pathway marks proteins for degradation by the Ubiquitin Proteosome System (UPS) based on the nature of their N-terminal residue. Proteins with a destabilizing N-terminal residue undergo a series of condition-dependent N-terminal modifications, resulting in their ubiquitination and degradation. Intensive research has been carried out in Arabidopsis previously. The group VII Ethylene Response Factor (ERFs) transcription factors are the first N-end rule pathway substrates found in Arabidopsis and their role in regulating oxygen sensing. ERFs also function as central hubs for the perception of gaseous signals in plants and control different plant developmental including germination, stomatal aperture, hypocotyl elongation and stress responses. However, nothing is known about the role of this pathway during fruit development and ripening aspect. The plant model system Arabidopsis cannot represent fleshy fruit model system therefore tomato is the best model plant to study. PROTEOLYSIS6 (PRT6) is an E3 ubiquitin ligase of the N-end rule pathway. Two homologs of PRT6 sequences have been identified in tomato genome database using the PRT6 protein sequence from model plant Arabidopsis thaliana. Homology search against Ensemble Plant database (tomato) showed Solyc09g010830.2 is the best hit with highest score of 1143, e-value of 0.0 and 61.3% identity compare to the second hit Solyc10g084760.1. Further homology search was done using NCBI Blast database to validate the data. The result showed best gene hit was XP_010325853.1 of uncharacterized protein LOC101255129 (Solanum lycopersicum) with highest score of 1601, e-value 0.0 and 48% identity. Both Solyc09g010830.2 and uncharacterized protein LOC101255129 were genes located at chromosome 9. Further validation was carried out using BLASTP program between these two sequences (Solyc09g010830.2 and uncharacterized protein LOC101255129) to investigate whether they were the same proteins represent PRT6 in tomato. Results showed that both proteins have 100 % identity, indicates that they were the same gene represents PRT6 in tomato. In addition, we used two different RNAi constructs that were driven under 35S and Polygalacturonase (PG) promoters to study the function of PRT6 during tomato developmental stages and ripening processes.

Keywords: ERFs, PRT6, tomato, ubiquitin

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Authors: Vandana Deopanée Tomar, Gurpreet Kaur Sidhu, Panchsheela Nogia, Rajesh Mehrotra, Sandhya Mehrotra

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The cyanobacterial CO₂ concentrating mechanism (CCM) operates to raise the levels of CO₂ in the vicinity of the main carboxylation enzyme Rubisco which is encapsulated in protein micro compartments called carboxysomes. Thus, due to the presence of CCM, cyanobacterial cells are able to work with high photosynthetic efficiency even at low Ci conditions and can accumulate 1000 folds high internal concentrations of Ci than external environment. Engineering of some useful CCM components into higher plants is one of the plausible approaches to improve their photosynthetic performance. The first step and the simplest approach for attaining this objective would be the transfer of cyanobacterial bicarbonate transporter such as SbtA to inner chloroplast envelope of C₃ plants. For this, SbtA transporter gene from Synechococcus elongatus PCC 7942 was fused to a transit peptide element to generate chimeric constructs in order to direct it to chloroplast inner envelope. Two transit peptides namely, TnaXTP (transit peptide from AT3G56160) and TMDTP (transit peptide from AT2G02590) were shortlisted from Arabidopsis thaliana genome and cloned in plant expression vector pCAMBIA1302 having mgfp5 as a reporter gene. Plant transformation was done by agro infiltration and Agrobacterium mediated co-culture. DNA, RNA, and protein were isolated from the leaves four days post infiltration, and the presence of transgene was confirmed by gene specific PCR (Polymerase Chain Reaction) analysis and by RT-PCR (Reverse Transcription Polymerase Chain Reaction). The expression was confirmed at the protein level by western blotting using anti-GFP primary antibody and horseradish peroxidase (HRP) conjugated secondary antibody. The localization of the protein was detected by confocal microscopy of isolated protoplasts. We observed chloroplastic expression for both the fusion constructs which suggest that the transit peptide sequences are capable of taking the cargo protein to the chloroplasts. These constructs are now being used to generate stable transgenic plants by Agrobacterium mediated transformation. The stability of transgene expression will be analyzed from T₀ to T₂ generation.

Keywords: agro infiltration, bicarbonate transporter, carbon concentrating mechanisms, cyanobacteria, SbtA

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Authors: Seyedahmad Hosseini, Mohammadjavad Sotoudeheian

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Introduction: Allergic asthma is a heterogeneous immuno-inflammatory disease based on Th-2-mediated inflammation. Histopathologic abnormalities of the airways characteristic of asthma include epithelial damage and subepithelial collagen deposition. Objectives: Human bronchial epithelial cell genome expression of TNF‑α, IL‑6, ICAM‑1, VCAM‑1, nuclear factor (NF)‑κB signaling pathways up-regulate during inflammatory cascades. Moreover, immunofluorescence assays confirmed the nuclear translocation of NF‑κB p65 during inflammatory responses. An absolute LDH leakage assays suggestedLPS-inducedcells injury, and the associated mechanisms are co-incident events. LPS-induced phosphorylation of ERKand JNK causes inflammation in epithelial cells through inhibition of ERK and JNK activation and NF-κB signaling pathway. Furthermore, the inhibition of NF-κB mRNA expression and the nuclear translocation of NF-κB lead to anti-inflammatory events. Likewise, activation of SUMF2 which inhibits IL-13 and reduces Th2-cytokines, NF-κB, and IgE levels to ameliorate asthma. On the other hand, TNFα-induced mucus production reduced NF-κB activation through inhibition of the activation status of Rac1 and IκBα phosphorylation. In addition, bradykinin B2 receptor (B2R), which mediates airway remodeling, regulates through NF-κB. Bronchial B2R expression is constitutively elevated in allergic asthma. In addition, certain NF-κB -dependent chemokines function to recruit eosinophils in the airway. Besides, bromodomain containing 4 (BRD4) plays a significant role in mediating innate immune response in human small airway epithelial cells as well as transglutaminase 2 (TG2), which is detectable in saliva. So, the guanine nucleotide-binding regulatory protein α-subunit, Gα16, expresses a κB-driven luciferase reporter. This response was accompanied by phosphorylation of IκBα. Furthermore, expression of Gα16 in saliva markedly enhanced TNF-α-induced κB reporter activity. Methods: The applied method to form NF-κB activation is the electromobility shift assay (EMSA). Also, B2R-BRD4-TG2 complex detection by immunoassay method within saliva with EMSA of NF-κB activation may be a novel biomarker for asthma diagnosis and follow up. Conclusion: This concept introduces NF-κB signaling pathway as potential asthma biomarkers and promising targets for the development of new therapeutic strategies against asthma.

Keywords: NF-κB, asthma, saliva, T-helper

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Authors: O. Ekrami, P. Claes, S. Van Dongen

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Fluctuating asymmetry (FA) has been studied for many years as an indicator of developmental stability or ‘genetic quality’ based on the assumption that perfect symmetry is ideally the expected outcome for a bilateral organism. Further studies have also investigated the possible link between FA and attractiveness or levels of masculinity or femininity. These hypotheses have been mostly examined using 2D images, and the structure of interest is usually presented using a limited number of landmarks. Such methods have the downside of simplifying and reducing the dimensionality of the structure, which will in return increase the error of the analysis. In an attempt to reach more conclusive and accurate results, in this study we have used high-resolution 3D scans of human faces and have developed an algorithm to measure and localize FA, taking a spatially-dense approach. A symmetric spatially dense anthropometric mask with paired vertices is non-rigidly mapped on target faces using an Iterative Closest Point (ICP) registration algorithm. A set of 19 manually indicated landmarks were used to examine the precision of our mapping step. The protocol’s accuracy in measurement and localizing FA is assessed using simulated faces with known amounts of asymmetry added to them. The results of validation of our approach show that the algorithm is perfectly capable of locating and measuring FA in 3D simulated faces. With the use of such algorithm, the additional captured information on asymmetry can be used to improve the studies of FA as an indicator of fitness or attractiveness. This algorithm can especially be of great benefit in studies of high number of subjects due to its automated and time-efficient nature. Additionally, taking a spatially dense approach provides us with information about the locality of FA, which is impossible to obtain using conventional methods. It also enables us to analyze the asymmetry of a morphological structures in a multivariate manner; This can be achieved by using methods such as Principal Components Analysis (PCA) or Factor Analysis, which can be a step towards understanding the underlying processes of asymmetry. This method can also be used in combination with genome wide association studies to help unravel the genetic bases of FA. To conclude, we introduced an algorithm to study and analyze asymmetry in human faces, with the possibility of extending the application to other morphological structures, in an automated, accurate and multi-variate framework.

Keywords: developmental stability, fluctuating asymmetry, morphometrics, 3D image processing

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Authors: Javed Mohammed

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Right now, bio-medical infrastructure lags well behind the curve. Our healthcare system is dispersed and disjointed; medical records are a bit of a mess; and we do not yet have the capacity to store and process the crazy amounts of data coming our way from widespread whole-genome sequencing. And then there are privacy issues. Despite these infrastructure challenges, some researchers are plunging into bio medical Big Data now, in hopes of extracting new and actionable knowledge. They are doing delving into molecular-level data to discover bio markers that help classify patients based on their response to existing treatments; and pushing their results out to physicians in novel and creative ways. Computer scientists and bio medical researchers are able to transform data into models and simulations that will enable scientists for the first time to gain a profound under-standing of the deepest biological functions. Solving biological problems may require High-Performance Computing HPC due either to the massive parallel computation required to solve a particular problem or to algorithmic complexity that may range from difficult to intractable. Many problems involve seemingly well-behaved polynomial time algorithms (such as all-to-all comparisons) but have massive computational requirements due to the large data sets that must be analyzed. High-throughput techniques for DNA sequencing and analysis of gene expression have led to exponential growth in the amount of publicly available genomic data. With the increased availability of genomic data traditional database approaches are no longer sufficient for rapidly performing life science queries involving the fusion of data types. Computing systems are now so powerful it is possible for researchers to consider modeling the folding of a protein or even the simulation of an entire human body. This research paper emphasizes the computational biology's growing need for high-performance computing and Big Data. It illustrates this article’s indispensability in meeting the scientific and engineering challenges of the twenty-first century, and how Protein Folding (the structure and function of proteins) and Phylogeny Reconstruction (evolutionary history of a group of genes) can use HPC that provides sufficient capability for evaluating or solving more limited but meaningful instances. This article also indicates solutions to optimization problems, and benefits Big Data and Computational Biology. The article illustrates the Current State-of-the-Art and Future-Generation Biology of HPC Computing with Big Data.

Keywords: high performance, big data, parallel computation, molecular data, computational biology

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Authors: Rosa Tsegaye Aga, Xuan Jiang, Pavel Vazquez Faci, Siqing Liu, Simon Rayner, Endalkachew Alemu, Markos Abebe

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Tuberculosis (TB) is a major cause of disease globally. In most cases, TB is treatable and curable, but only with the proper treatment. There is a time when drug-resistant TB occurs when bacteria become resistant to the drugs that are used to treat TB. Current strategies to identify drug-resistant TB bacteria are laboratory-based, and it takes a longer time to identify the drug-resistant bacteria and treat the patient accordingly. But machine learning (ML) and data science approaches can offer new approaches to the problem. In this study, we propose to develop an ML-based model to predict the antibiotic resistance phenotypes of TB isolates in minutes and give the right treatment to the patient immediately. The study has been using the whole genome sequence (WGS) of TB isolates as training data that have been extracted from the NCBI repository and contain different countries’ samples to build the ML models. The reason that different countries’ samples have been included is to generalize the large group of TB isolates from different regions in the world. This supports the model to train different behaviors of the TB bacteria and makes the model robust. The model training has been considering three pieces of information that have been extracted from the WGS data to train the model. These are all variants that have been found within the candidate genes (F1), predetermined resistance-associated variants (F2), and only resistance-associated gene information for the particular drug. Two major datasets have been constructed using these three information. F1 and F2 information have been considered as two independent datasets, and the third information is used as a class to label the two datasets. Five machine learning algorithms have been considered to train the model. These are Support Vector Machine (SVM), Random forest (RF), Logistic regression (LR), Gradient Boosting, and Ada boost algorithms. The models have been trained on the datasets F1, F2, and F1F2 that is the F1 and the F2 dataset merged. Additionally, an ensemble approach has been used to train the model. The ensemble approach has been considered to run F1 and F2 datasets on gradient boosting algorithm and use the output as one dataset that is called F1F2 ensemble dataset and train a model using this dataset on the five algorithms. As the experiment shows, the ensemble approach model that has been trained on the Gradient Boosting algorithm outperformed the rest of the models. In conclusion, this study suggests the ensemble approach, that is, the RF + Gradient boosting model, to predict the antibiotic resistance phenotypes of TB isolates by outperforming the rest of the models.

Keywords: machine learning, MTB, WGS, drug resistant TB

Procedia PDF Downloads 52

Authors: Gabriela C. Caldas, Fernanda C. Jacome, Arthur da C. Rasinhas, Ortrud M. Barth, Flavia B. dos Santos, Priscila C. G. Nunes, Yuli R. M. de Souza, Pedro Paulo de A. Manso, Marcelo P. Machado, Debora F. Barreto-Vieira

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The establishment of animal models for studies of DENV infections has been challenging, since circulating epidemic viruses do not naturally infect nonhuman species. Such studies are of great relevance to the various areas of dengue research, including immunopathogenesis, drug development and vaccines. In this scenario, the main objective of this study is to verify possible morphological changes, as well as the presence of antigens and viral RNA in lung samples from BALB/c mice experimentally infected with an epidemic and non-neuroadapted DENV-3 strain. Male BALB/c mice, 2 months old, were inoculated with DENV-3 by intravenous route. After 72 hours of infection, the animals were euthanized and the lungs were collected. Part of the samples was processed by standard technique for analysis by light and transmission electronic microscopies and another part was processed for real-time PCR analysis. Morphological analyzes of lungs from uninfected mice showed preserved tissue areas. In mice infected with DENV-3, the analyzes revealed interalveolar septum thickening with presence of inflammatory infiltrate, foci of alveolar atelectasis and hyperventilation, bleeding foci in the interalveolar septum and bronchioles, peripheral capillary congestion, accumulation of fluid in the blood capillary, signs of interstitial cell necrosis presence of platelets and mononuclear inflammatory cells circulating in the capillaries and/or adhered to the endothelium. In addition, activation of endothelial cells, platelets, mononuclear inflammatory cell and neutrophil-type polymorphonuclear inflammatory cell evidenced by the emission of cytoplasmic membrane prolongation was observed. DEN-like particles were seen in the cytoplasm of endothelial cells. The viral genome was recovered from 3 in 12 lung samples. These results demonstrate that the BALB / c mouse represents a suitable model for the study of the histopathological changes induced by DENV infection in the lung, with tissue alterations similar to those observed in human cases of DEN.

Keywords: BALB/c mice, dengue, histopathology, lung, ultrastructure

Procedia PDF Downloads 253

Authors: Selisha A. Sooklal, Phelelani Mpangase, Shaun Aron, Karl Rumbold

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Organically bound fluorine (C-F bond) is extremely rare in nature. Despite this, the first fluorinated secondary metabolite, fluoroacetate, was isolated from the plant Dichapetalum cymosum (commonly known as Gifblaar). However, the enzyme responsible for fluorination (fluorinase) in Gifblaar was never isolated and very little progress has been achieved in understanding this process in higher plants. Fluorinated compounds have vast applications in the pharmaceutical, agrochemical and fine chemicals industries. Consequently, an enzyme capable of catalysing a C-F bond has great potential as a biocatalyst in the industry considering that the field of fluorination is virtually synthetic. As with any biocatalyst, a range of these enzymes are required. Therefore, it is imperative to expand the exploration for novel fluorinases. This study aimed to gain molecular insights into secondary metabolite biosynthesis in Gifblaar using a high-throughput sequencing-based approach. Mechanical wounding studies were performed using Gifblaar leaf tissue in order to induce expression of the fluorinase. The transcriptome of the wounded and unwounded plant was then sequenced on the Illumina HiSeq platform. A total of 26.4 million short sequence reads were assembled into 77 845 transcripts using Trinity. Overall, 68.6 % of transcripts were annotated with gene identities using public databases (SwissProt, TrEMBL, GO, COG, Pfam, EC) with an E-value threshold of 1E-05. Sequences exhibited the greatest homology to the model plant, Arabidopsis thaliana (27 %). A total of 244 annotated transcripts were found to be differentially expressed between the wounded and unwounded plant. In addition, secondary metabolic pathways present in Gifblaar were successfully reconstructed using Pathway tools. Due to lack of genetic information for plant fluorinases, a transcript failed to be annotated as a fluorinating enzyme. Thus, a local database containing the 5 existing bacterial fluorinases was created. Fifteen transcripts having homology to partial regions of existing fluorinases were found. In efforts to obtain the full coding sequence of the Gifblaar fluorinase, primers were designed targeting the regions of homology and genome walking will be performed to amplify the unknown regions. This is the first genetic data available for Gifblaar. It has provided novel insights into the mechanisms of metabolite biosynthesis and will allow for the discovery of the first eukaryotic fluorinase.

Keywords: biocatalyst, fluorinase, gifblaar, transcriptome

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Authors: Aidin Foroutan, David S. Wishart, Leluo L. Guan, Carolyn Fitzsimmons

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Maximizing efficiency and growth potential of beef cattle requires not only genetic selection (i.e. residual feed intake (RFI)) but also adequate nutrition throughout all stages of growth and development. Nutrient restriction during gestation has been shown to negatively affect post-natal growth and development as well as fertility of the offspring. This, when combined with RFI may affect progeny traits. This study aims to investigate the impact of selection for divergent genetic potential for RFI and maternal nutrition during early- to mid-gestation, on bull calf traits such as fertility and muscle development using multiple ‘omics’ approaches. Comparisons were made between High-diet vs. Low-diet and between High-RFI vs. Low-RFI animals. An epigenetics experiment on semen samples identified 891 biomarkers associated with growth and development. A gene expression study on Longissimus thoracis muscle, semimembranosus muscle, liver, and testis identified 4 genes associated with muscle development and immunity of which Myocyte enhancer factor 2A [MEF2A; induces myogenesis and control muscle differentiation] was the only differentially expressed gene identified in all four tissues. An initial metabolomics experiment on serum samples using nuclear magnetic resonance (NMR) identified 4 metabolite biomarkers related to energy and protein metabolism. Once all the biomarkers are identified, bioinformatics approaches will be used to create a database covering all the ‘omics’ data collected from this project. This database will be broadened by adding other information obtained from relevant literature reviews. Association analyses with these data sets will be performed to reveal key biological pathways affected by RFI and maternal nutrition. Through these association studies between the genome and metabolome, it is expected that candidate biomarker genes and metabolites for feed efficiency, fertility, and/or muscle development are identified. If these gene/metabolite biomarkers are validated in a larger animal population, they could potentially be used in breeding programs to select superior animals. It is also expected that this work will lead to the development of an online tool that could be used to predict future traits of interest in an animal given its measurable ‘omics’ traits.

Keywords: biomarker, maternal nutrition, omics, residual feed intake

Procedia PDF Downloads 191

Authors: Eriko Ochiai, Fumiko Satoh, Keiko Miyashita, Yu Kakimoto, Motoki Osawa

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Sudden and unexpected deaths from unknown causes occur in infants and youths. Recently, molecular links between a part of these deaths and several genetic diseases are examined in the postmortem. For instance, hereditary long QT syndrome and Burgada syndrome are occasionally fatal through critical ventricular tachyarrhythmia. There are a large number of target genes responsible for such diseases, the conventional analysis using the Sanger’s method has been laborious. In this report, we attempted to analyze sudden deaths comprehensively using the next generation sequencing (NGS) technique. Multiplex PCR to subject’s DNA was performed using Ion AmpliSeq Library Kits 2.0 and Ion AmpliSeq Inherited Disease Panel (Life Technologies). After the library was constructed by emulsion PCR, the amplicons were sequenced 500 flows on Ion Personal Genome Machine System (Life Technologies) according to the manufacture instruction. SNPs and indels were analyzed to the sequence reads that were mapped on hg19 of reference sequences. This project has been approved by the ethical committee of Tokai University School of Medicine. As a representative case, the molecular analysis to a 40 years old male who received a diagnosis of Brugada syndrome demonstrated a total of 584 SNPs or indels. Non-synonymous and frameshift nucleotide substitutions were selected in the coding region of heart disease related genes of ANK2, AKAP9, CACNA1C, DSC2, KCNQ1, MYLK, SCN1B, and STARD3. In particular, c.629T-C transition in exon 3 of the SCN1B gene, resulting in a leu210-to-pro (L210P) substitution is predicted “damaging” by the SIFT program. Because the mutation has not been reported, it was unclear if the substitution was pathogenic. Sudden death that failed in determining the cause of death constitutes one of the most important unsolved subjects in forensic pathology. The Ion AmpliSeq Inherited Disease Panel can amplify the exons of 328 genes at one time. We realized the difficulty in selection of the true source from a number of candidates, but postmortem genetic testing using NGS analysis deserves of a diagnostic to date. We now extend this analysis to SIDS suspected subjects and young sudden death victims.

Keywords: postmortem genetic testing, sudden death, SIDS, next generation sequencing

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Authors: Sushil Pradhan

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Biotechnology contributes to sustainable development in several ways such as biofertilizer production, biopesticide production and management of environmental pollution, tissue culture and biodiversity conservation in vitro, in vivo and in situ, Insectivorous medicinal plant Drosera burmannii Vahl belongs to the Family-Droseraceae under Order-Caryophyllales, Dicotyledoneae, Angiospermeae which has 31 (thirty one) living genera and 194 species besides 7 (seven) extinct (fossil) genera. Locally it is known as “Patkanduri” in Odia. Its Hindi name is “Mukhajali” and its English name is “Sundew”. The earliest species of Drosera was first reported in 1753 by Carolous Linnaeus called Drosera indica L (Indian Sundew). The latest species of Drosera reported by Fleisch A, Robinson, AS, McPherson S, Heinrich V, Gironella E and Madulida D.A. (2011) is Drosera ultramafica from Malaysia. More than 50 % species of Drosera have been reported from Australia and next to Australia is South Africa. India harbours only 3 species such as D. indica L, Drosera burmannii Vahl and D. peltata L. From our Odisha only D. burmannii Vahl is being reported for the first time from the district of Subarnapur near Sonepur (Arjunpur Reserve Forest Area). Drosera plant is autotrophic but to supplement its Nitrogen (N2) requirement it adopts heterotrophic mode of nutrition (insectivorous/carnivorous) as well. The colour of plant in mostly red and about 20-30cm in height with beautiful pink or white pentamerous flowers. Plants grow luxuriantly during November to February in shady and moist places near small water bodies of running water stream. Medicinally it is a popular herb in the locality for the treatment of cold and cough in children in rainy season by the local Doctors (Kabiraj and Baidya). In the present field investigation an attempt has been made to understand the unique reproductive phase and life cycle of the plant thereby planning for its conservation and propagation through various techniques of tissue culture and biotechnology. More importantly besides morphological and anatomical studies, cytological investigation is being carried out to find out the number of chromosomes in the cell and its genomics as there is no such report as yet for Drosera burmannii Vahl. The ecological significance and biodiversity conservation of Drosera with special reference to energy, environmental and chemical engineering has been discussed in the research paper presentation.

Keywords: insectivorous, medicinal, drosera, biotechnology, chromosome, genome

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Authors: Priscila A. Bernardes, Marcos E. Buzanskas, Luciana C. A. Regitano, Ricardo V. Ventura, Danisio P. Munari

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Genotype imputation has been used to reduce genomic selections costs. In order to increase haplotype detection accuracy in methods that considers the linkage disequilibrium, another approach could be used, such as combined genotype data from different panels. Therefore, this study aimed to evaluate the linkage disequilibrium and haplotype blocks in two high-density panels before and after the imputation to a combined panel in Nelore beef cattle. A total of 814 animals were genotyped with the Illumina BovineHD BeadChip (IHD), wherein 93 animals (23 bulls and 70 progenies) were also genotyped with the Affymetrix Axion Genome-Wide BOS 1 Array Plate (AHD). After the quality control, 809 IHD animals (509,107 SNPs) and 93 AHD (427,875 SNPs) remained for analyses. The combined genotype panel (CP) was constructed by merging both panels after quality control, resulting in 880,336 SNPs. Imputation analysis was conducted using software FImpute v.2.2b. The reference (CP) and target (IHD) populations consisted of 23 bulls and 786 animals, respectively. The linkage disequilibrium and haplotype blocks studies were carried out for IHD, AHD, and imputed CP. Two linkage disequilibrium measures were considered; the correlation coefficient between alleles from two loci (r²) and the |D’|. Both measures were calculated using the software PLINK. The haplotypes' blocks were estimated using the software Haploview. The r² measurement presented different decay when compared to |D’|, wherein AHD and IHD had almost the same decay. For r², even with possible overestimation by the sample size for AHD (93 animals), the IHD presented higher values when compared to AHD for shorter distances, but with the increase of distance, both panels presented similar values. The r² measurement is influenced by the minor allele frequency of the pair of SNPs, which can cause the observed difference comparing the r² decay and |D’| decay. As a sum of the combinations between Illumina and Affymetrix panels, the CP presented a decay equivalent to a mean of these combinations. The estimated haplotype blocks detected for IHD, AHD, and CP were 84,529, 63,967, and 140,336, respectively. The IHD were composed by haplotype blocks with mean of 137.70 ± 219.05kb, the AHD with mean of 102.10kb ± 155.47, and the CP with mean of 107.10kb ± 169.14. The majority of the haplotype blocks of these three panels were composed by less than 10 SNPs, with only 3,882 (IHD), 193 (AHD) and 8,462 (CP) haplotype blocks composed by 10 SNPs or more. There was an increase in the number of chromosomes covered with long haplotypes when CP was used as well as an increase in haplotype coverage for short chromosomes (23-29), which can contribute for studies that explore haplotype blocks. In general, using CP could be an alternative to increase density and number of haplotype blocks, increasing the probability to obtain a marker close to a quantitative trait loci of interest.

Keywords: Bos taurus indicus, decay, genotype imputation, single nucleotide polymorphism

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