Search results for: embryos’ chromosome set

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

Abstract:

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

Procedia PDF Downloads 76

Authors: Madhulika Pathak, Polani Seshagiri, Vani Venkatappa

Abstract:

Blastocyst hatching is an indispensable process for successful implantation. One of the major reasons for implantation failure in IVF clinic is poor quality of embryo, which are not development/hatching-competent. Therefore, attempts are required to develop or enhance the culture system with a molecule recapitulating the autocrine/paracrine factors containing the environment of in-vivo endometrial milieu. We have tried to explore the functional molecules involved in the hamster hatching phenomenon. Blastocyst hatching is governed by several molecules that are entwined and regulate this process, among which cytokines are known to be expressed and are still least explored. Two of such cytokines we have used for our study are IL-1β and its natural antagonist IL-1ra to understand the functional dynamics of cytokines involved in the hatching process. Using hamster, an intriguing experimental model for hatching behavior, we have shown the mRNA (qPCR) and protein (ICC) expression of IL-1β, IL-1ra and IL-1 receptor type 1 throughout all the stages of morula, blastocyst and hatched blastocyst. Post-asserting the expression, the functional role is shown by supplementation studies, where IL-1β supplementation showed enhancement in hatching level (IL-1β treated: 84.1 ± 4.2% vs control: 63.7 ± 3.1 %, N=11), further confirmed by the diminishing effect of IL-1ra on hatching rate (IL-1ra treated: 27.5 ± 11.1% vs control: 67.9 ± 3.1%). The exogenous supplementation of IL-1ra decreased the survival rate of embryos and affected the viability in dose-dependent manner, establishing the importance of IL-1β in blastocyst cell survival. Previously, the cathepsin L and B were established as the proteases that were involved in the hamster hatching process. The inducing effect of IL-1β was correlated with enhanced mRNA level, as analyzed by qPCR, for both CAT L (by 1.9 ± 0.5 fold) and CAT B (by 3.5 ± 0.1) fold which was diminished in presence of IL-1ra (Cat L by 88 percent and Cat B by 94 percent. Moreover, using a specific fluorescent substrate-based assay kit, the enzymatic activity of these proteases was found to be increased in presence of IL-1β (Cat L by 2.1 ± 0.1 fold and CAT B by 2.3 ± 0.7 fold) and was curtailed in presence of IL-1ra. These observations provide functional insights with respect to the involvement of cytokines in the hatching process. This has implications in understanding the hatching biology and improving the embryo development quality in IVF clinics.

Keywords: Blastocyst, Cytokines, Hatching, Interleukin

Procedia PDF Downloads 117

Authors: Srishty Gulati, Anju Singh, Shrikant Kukreti

Abstract:

The manifestation of structural polymorphism in DNA depends on the sequence and surrounding environment. Ample of folded DNA structures have been found in the cellular system out of which DNA hairpins are very common, however, are indispensable due to their role in the replication initiation sites, recombination, transcription regulation, and protein recognition. We enumerate this approach in our study, where the two base substitutions and change in temperature embark destabilization of DNA structure and misbalance the equilibrium between two structures of a sequence present at the overlapping region of the human COMT gene and MIR4761 gene. COMT and MIR4761 gene encodes for catechol-O-methyltransferase (COMT) enzyme and microRNAs (miRNAs), respectively. Environmental changes and errors during cell division lead to genetic abnormalities. The COMT gene entailed in dopamine regulation fosters neurological diseases like Parkinson's disease, schizophrenia, velocardiofacial syndrome, etc. A 19-mer deoxyoligonucleotide sequence 5'-AGGACAAGGTGTGCATGCC-3' (COMT19) is located at exon-4 on chromosome 22 and band q11.2 at the intersection of COMT and MIR4761 gene. Bioinformatics studies suggest that this sequence is conserved in humans and few other organisms and is involved in recognition of transcription factors in the vicinity of 3'-end. Non-denaturating gel electrophoresis and CD spectroscopy of COMT sequences indicate the formation of hairpin type DNA structures. Temperature-dependent CD studies revealed an unusual shift in the slipped DNA-Hairpin DNA equilibrium with the change in temperature. Also, UV-thermal melting techniques suggest that the two base substitutions on the complementary strand of COMT19 did not affect the structure but reduces the stability of duplex. This study gives insight about the possibility of existing structurally polymorphic transient states within DNA segments present at the intersection of COMT and MIR4761 gene.

Keywords: base-substitution, catechol-o-methyltransferase (COMT), hairpin-DNA, structural polymorphism

Procedia PDF Downloads 99

Authors: Eda Jazexhiu-Postoli, Gladiola Hoxha, Ada Simeoni, Sonila Biba

Abstract:

Background Neonatal hypotonia represents a commonly encountered issue in the Neonatal Intensive Care Unit and newborn nursery. The differential diagnosis is broad, encompassing chromosome abnormalities, primary muscular dystrophies, neuropathies and inborn errors of metabolism. Aim of study Our study describes some of the main clinical features of hypotonia in newborns and presents clinical cases of neonatal hypotonia we treated in our Neonatal unit in the last 3 years. Case reports Four neonates born in our hospital presented with hypotonia after birth, one preterm newborn 35-36 weeks of gestational age and three other term newborns (38-39 weeks of gestational age). Prenatal data revealed a decrease in fetal movements in both cases. Intrapartum meconium-stained amniotic fluid was found in 75% of our hypotonic newborns. Clinical features included inability to establish effective respiratory movements and need for resuscitation in the delivery room, respiratory distress syndrome, feeding difficulties and need for oro-gastric tube feeding, dysmorphic features, hoarse voice and moderate to severe muscular hypotonia. The genetic workup revealed the diagnosis of Autosomal Recessive Congenital Myasthenic Syndrome 1-B, Sotos Syndrome, Spinal Muscular Atrophy Type 1 and Transient Hypotonia of the Newborn. Two out of four hypotonic neonates were transferred to the Pediatric Intensive Care Unit and died at the age of three to five months old. Conclusion Hypotonia is a concerning finding in neonatal care and it is suggested by decreased intrauterine fetal movements, failure to establish first breaths, respiratory distress and feeding difficulties in the neonate. Prognosis is determined by its etiology and time of diagnosis and intervention.

Keywords: hypotonic neonate, respiratory distress, feeding difficulties, fetal movements

Procedia PDF Downloads 79

Authors: Alejandra Betancur Sánchez, Carmen Elena Zapata Sánchez, Juan Bautista López Ortiz

Abstract:

Adverse effects and increased air pollution has raised concerns about regulatory policies and has fostered the development of new air quality standards; this is due to the complexity of the composition and the poorly understood reactions in the atmospheric environment. Toxic compounds act as environmental agents having various effects, from irritation to death of cells and tissues. A toxic agent is defined an adverse response in a biological system. There is a particular class that produces some kind of alteration in the genetic material or associated components, so they are recognized as genotoxic agents. Within cells, they interact directly or indirectly with DNA, causing mutations or interfere with some enzymatic repair processes or in the genesis or polymerization of proteinaceous material involved in chromosome segregation. An air pollutant may cause or contribute to increased mortality or serious illness and even pose a potential danger to human health. The aim of this study was to evaluate the effect on the viability and the genotoxic potential on the cell lines CHO-K1 and Jurkat and peripheral blood of particulate matter PM T lymphocytes 2.5 obtained from filters collected three monitoring stations network air quality Aburrá Valley. Tests, reduction of MTT, trypan blue, NRU, comet assay, sister chromatid exchange (SCE) and chromosomal aberrations allowed evidence reduction in cell viability in cell lines CHO-K1 and Jurkat and damage to the DNA from cell line CHOK1, however, no significant effects were observed in the number of SCEs and chromosomal aberrations. The results suggest that PM2.5 material has genotoxic potential and can induce cancer development, as has been suggested in other studies.

Keywords: PM2.5, cell line Jurkat, cell line CHO-K1, cytotoxicity, genotoxicity

Procedia PDF Downloads 239

Authors: Vidhi Chandra, Arshpreet Singh Grewal

Abstract:

A five-year-old male child born from non-consanguineous marriage, who presented with complaints of growth retardation and no appreciable increase in the penile size since birth and he was posted for de-gloving of penis with dissection of corpora under anaesthesia. After thorough preoperative evaluation it was revealed that patient had peculiar facial dysmorphism that of Robinow Syndrome, high arched palate, Mallampati grade III, mesomelic limbs, scoliotic spine and short stature. All routine investigation were within normal limit, electrocardiography (ECG) and 2D-Echocardiography (ECHO) were normal. In antero-posterior roentgenogram chest showed butterfly and hemivertebrae at multiple levels. The patient was considered to be ASA II. On the day of surgery after ensuring fasting of 6 hours, patient was taken in operation theatre, all standard ASA monitoring was done with ECG, non-invasive blood pressure, peripheral oxygen saturation (SpO2) and body temperature. The patient was pre-oxygenated with 100% oxygen with anatomical face mask. General anaesthesia was induced with Sevoflurane 1-8%, and airway was secured with an appropriate size supraglottic airway and anaesthesia was maintained with nitrous oxide and oxygen in 1:1 ratio along with sevoflurane 2%. An ultrasound guided caudal block was given owing to the skeletal deformities making it difficult even under USG guidance. Post operatively patient was given supportive care with proper hydration, antibiotics, anti-inflammatory and analgesics. He was discharged the next day and followed up weekly for a month. DISCUSSION Robinow syndrome is genetically inherited as autosomal dominant, autosomal recessive or heterogenous disorder involving tyrosine kinase ROR2 gene located on chromosome 9. It has low incidence with no preponderance for any gender. Though intelligence is normal but developmental delay and mental retardation occurs in 20%cases

Keywords: Robinow Syndrome, dwarfism, paediatric, anaesthesia

Procedia PDF Downloads 85

Authors: Mohammed Y. Elsherbeny, Mohamed Salama, Ahmed Lotfy, Hossam Fareed, Nora Mohammed

Abstract:

Background: Developmental neurotoxicity (DNT) entails the toxic effects imparted by various chemicals on brain during the early childhood when human brains are vulnerable during this period. DNT study in vivo cannot determine the effect of the neurotoxins, as it is not applicable, so using the neurosphere cells of lab animals as an alternative is applicable and time saving. Methods: Cell culture: Rat neural progenitor cells were isolated from rat embryos’ brain. The cortices were aseptically dissected out and the tissues were triturated. The dispersed tissues were allowed to settle. The supernatant was then transferred to a fresh tube and centrifuged. The pellet was placed in Hank’s balanced salt solution and cultured as free-floating neurospheres in proliferation medium. Differentiation was initiated by growth factor withdrawal in differentiation medium and plating onto a poly-d-lysine/ laminin matrix. Chemical Exposure: Neurospheres were treated for 2 weeks with papaverine in proliferation medium. Proliferation analyses: Spheres were cultured. After 0, 4, 5, 11 and 14 days, sphere size was determined by software analyses (CellProfiler, version 2.1; Broad Institute). Diameter of each neurosphere was measured and exported to excel file further to statistical analysis. Viability test: Trypsin-EDTA solution was added to neurospheres to dissociate neurospheres into single cells suspension, then viability evaluated by the Trypan Blue exclusion test. Result: As regards proliferation analysis and percentage of viable cells of papaverin treated groups: There was no significant change in cells proliferation compared to control at 0, 4, 5, 11 and 14 days with concentrations 1, 5 and 10 µM of papaverine, but there is a significant change in cell viability compared to control after 1 week and 2 weeks with the same concentrations of papaverine. Conclusion: Papaverine has toxic effect on viability of neural cell, not on their proliferation, so it may produce focal neural lesions not growth morphological changes.

Keywords: developmental neurotoxicity, neurotoxin, papaverine, neuroshperes

Procedia PDF Downloads 362

Authors: Archana. S, Usha Rani. G, Anand Balakrishnan, Sanjana.R, Solomon F, Vijayalakshmi. J

Abstract:

Background: There is evidence that male factors contribute to the infertility of up to 50% of couples, who are evaluated and treated for infertility using advanced assisted reproductive technologies. Genetic abnormalities, including sperm chromosome aneuploidy as well as structural aberrations, are one of the major causes of male infertility. Recent advances in technology expedite the evaluation of sperm aneuploidy. The purpose of the study was to de-termine the prevalence of sperm aneuploidy in infertile males and the degree of association between DNA fragmentation and sperm aneuploidy. Methods: In this study, 75 infertile men were included, and they were divided into four abnormal groups (Oligospermia, Terato-spermia, Asthenospermia and Oligoasthenoteratospermia (OAT)). Men with children who were normozoospermia served as the control group. The Fluorescence in situ hybridization (FISH) method was used to test for sperm aneuploidy, and the Sperm Chromatin Dispersion Assay (SCDA) was used to measure the fragmentation of sperm DNA. Spearman's correla-tion coefficient was used to evaluate the relationship between sperm aneuploidy and sperm DNA fragmentation along with age. P < 0.05 was regarded as significant. Results: 75 partic-ipants' ages varied from 28 to 48 years old (35.5±5.1). The percentage of spermatozoa bear-ing X and Y was determined to be statistically significant (p-value < 0.05) and was found to be 48.92% and 51.18% of CEP X X 1 – nucish (CEP XX 1) [100] and CEP Y X 1 – nucish (CEP Y X 1) [100]. When compared to the rate of DNA fragmentation, it was discovered that infertile males had a greater frequency of sperm aneuploidy. Asthenospermia and OAT groups in sex chromosomal aneuploidy were significantly correlated (p<0.05). Conclusion: Sperm FISH and SCDA assay results showed increased sperm aneuploidy frequency, and DNA fragmentation index in infertile men compared with fertile men. There is a significant relationship observed between sperm aneuploidy and DNA fragmentation in OAT patients. When evaluating male variables and idiopathic infertility, the sperm FISH screening method can be used as a valuable diagnostic tool.

Keywords: ale infertility, dfi (dna fragmentation assay) (scd-sperm chromatin dispersion).art (artificial reproductive technology), trisomy, aneuploidy, fish (fluorescence in-situ hybridization), oat (oligoasthoteratospermia)

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Authors: Qin Yang, Fan Zhang, Juan Du, Chongkui Sun, Krishna Kota, Yun-Ling Zheng

Abstract:

Telomeres are specialized structures at chromosome ends consisting of tandem repetitive DNA sequences [(TTAGGG)n in humans] and associated proteins, which are necessary for telomere function. Telomere lengths are tightly regulated within a narrow range in normal human somatic cells, the basis of cellular senescence and aging. Previous studies have extensively focused on how short telomeres are extended and have demonstrated that telomerase plays a central role in telomere maintenance through elongating the short telomeres. However, the molecular mechanisms of regulating excessively long telomeres are unknown. Here, we found that telomerase enzymatic component hTERT plays a dual role in the regulation of telomeres length. We analyzed single telomere alterations at each chromosomal end led to the discoveries that hTERT shortens excessively long telomeres and elongates short telomeres simultaneously, thus maintaining the optimal telomere length at each chromosomal end for an efficient protection. The hTERT-mediated telomere shortening removes large segments of telomere DNA rapidly without inducing telomere dysfunction foci or affecting cell proliferation, thus it is mechanistically distinct from rapid telomere deletion. We found that expression of hTERT generates telomeric circular DNA, suggesting that telomere homologous recombination may be involved in this telomere shortening process. Moreover, the hTERT-mediated telomere shortening is required its enzymatic activity, but telomerase RNA component hTR is not involved in it. Furthermore, shelterin protein TPP1 interacts with hTERT and recruits it on telomeres to mediate telomere shortening. In addition, telomere-associated proteins, DKC1 and TCAB1 also play roles in this process. This novel hTERT-mediated telomere shortening mechanism not only exists in cancer cells, but also in primary human cells. Thus, the hTERT-mediated telomere shortening is expected to shift the paradigm on current molecular models of telomere length maintenance, with wide-reaching consequences in cancer and aging fields.

Keywords: aging, hTERT, telomerase, telomeres, human cells

Procedia PDF Downloads 396

Authors: Nemat Sokhandan Bashir

Abstract:

Threats from viruses to agricultural crops could be even larger than the losses caused by the other pathogens because, in many cases, the viral infection is latent but crucial from an epidemic point of view. Wild vegetation can be a source of many viruses that eventually find their destiny in crop plants. Although often asymptomatic in wild plants due to adaptation, they can potentially cause serious losses in crops. Therefore, exploring viruses in wild vegetation is very important. Recently, omics have been quite useful for exploring plant viruses from various plant sources, especially wild vegetation. For instance, we have discovered viruses such as Ambrossia asymptomatic virus I (AAV-1) through the application of metagenomics from Oklahoma Prairie Reserve. Accordingly, extracts from randomly-sampled plants are subjected to high speed and ultracentrifugation to separated virus-like particles (VLP), then nucleic acids in the form of DNA or RNA are extracted from such VLPs by treatment with phenol—chloroform and subsequent precipitation by ethanol. The nucleic acid preparations are separately treated with RNAse or DNAse in order to determine the genome component of VLPs. In the case of RNAs, the complementary cDNAs are synthesized before submitting to DNA sequencing. However, for VLPs with DNA contents, the procedure would be relatively straightforward without making cDNA. Because the length of the nucleic acid content of VPLs can be different, various strategies are employed to achieve sequencing. Techniques similar to so-called "chromosome walking" may be used to achieve sequences of long segments. When the nucleotide sequence data were obtained, they were subjected to BLAST analysis to determine the most related previously reported virus sequences. In one case, we determined that the novel virus was AAV-l because the sequence comparison and analysis revealed that the reads were the closest to the Indian citrus ringspot virus (ICRSV). AAV—l had an RNA genome with 7408 nucleotides in length and contained six open reading frames (ORFs). Based on phylogenies inferred from the replicase and coat protein ORFs of the virus, it was placed in the genus Mandarivirus.

Keywords: wild, plant, novel, metagenomics

Procedia PDF Downloads 47

Authors: Kinnsley Travis, Camerron M. Crowder

Abstract:

Mapk8ip3 (Mitogen-Activated Protein Kinase 8 Interacting Protein 3) is a gene that codes for the JIP3 protein, which is a part of the JIP scaffolding protein family. This protein is involved in axonal vesicle transport, elongation and regeneration. Variants in the Mapk8ip3 gene are associated with a rare-genetic condition that results in a neurodevelopmental disorder that can cause a range of phenotypes including global developmental delay and intellectual disability. Currently, there are 18 known individuals diagnosed to have sequenced confirmed Mapk8ip3 genetic disorders. This project focuses on examining the impact of a subset of missense patient variants on the Jip3 protein function by overexpressing the mRNA of these variants in a zebrafish knockout model for Jip3. Plasmids containing cDNA with individual missense variants were reverse transcribed, purified, and injected into single-cell zebrafish embryos (Wild Type, Jip3 -/+, and Jip3 -/-). At 6-days post mRNA microinjection, morphological, behavioral, and microscopic phenotypes were examined in zebrafish larvae. Morphologically, we compared the size and shape of the zebrafish during their development over a 5-day period. Total locomotive activity was assessed using the Microtracker assay and patterns of movement over time were examined using the DanioVision assay. Lastly, we used confocal microscopy to examine sensory axons for swelling and shortened length, which are phenotypes observed in the loss-of-function knockout Jip3 zebrafish model. Using these assays during embryonic development, we determined the impact of various missense variants on Jip3 protein function, compared to knockout and wild-type zebrafish embryo models. Variants in the gene Mapk8ip3 cause rare-neurodevelopmental disorders due to an essential role in axonal vesicle transport, elongation and regeneration. A subset of missense variants was examined by overexpressing the mRNA of these variants in a Jip3 knock-out zebrafish. Morphological, behavioral, and microscopic phenotypes were examined in zebrafish larvae. Using these assays, the spectrum of disorders can be phenotypically determined and the impact of variant location can be compared to knockout and wild-type zebrafish embryo models.

Keywords: rare disease, neurodevelopmental disorders, mrna overexpression, zebrafish research

Procedia PDF Downloads 92

Authors: L. Musak, J. Valachova, T. Vasicko, O. Osina

Abstract:

Stainless steel is resistant to electrochemical corrosion by passivation. Welders are greatly exposed to welding fumes of toxic metals, which added to this steel. The content of chromium (Cr) is above 11.5%, Ni and Mo from 2 to 6.5%. The aim of the study was the evaluation of occupational exposure to Cr, chromosome analysis and valuation of individual susceptibility polymorphism of gene CCND1 c.870 G>A. The exposed group was consisted from 117 welders of stainless steels. The average age was 38.43 years and average exposure time 7.14 years. Smokers represented 40.17%. The control group consisted of 123 non-exposed workers with an average age of 39.74 years and time employment 16.67 years. Smokers accounted for 22.76%. Analysis of Cr in blood and urine was performed by atomic absorption spectrophotometry (AAS Varian SpectraAA 30P) with electrothermal decomposition of the sample in the graphite furnace. For the evaluation of chromosomal aberrations (CA) cytogenetic analysis of peripheral blood lymphocytes was used. Gene polymorphism was determined by PCR-RFLP reaction using appropriate primers and restriction enzymes. For statistic analysis the Mann-Whitney U-test was used. The mean Cr level in blood of exposed group was 0.095 µmol/l (0.019 min - max 0.504). No value exceeds the average normal value. The mean value Cr in urine was 7.9 µmol/mol creatinine (min 0.026 to max 19.26). The total number of CA was 1.86% in compared to 1.70% controls. (CTA-type 0.90% vs. 0.80% and CSA-type 0.96% vs. 0.90%). In the number of total CA statistical difference was observed between smokers and non-smokers of exposed group (S-1.57% vs. NS-2.04%, P<0.05). In CCND1 gene polymorphisms was observed the increasing of the total CA with wild-type allele (WT) via heterozygous to the VAR genotype (1.44% <1.82% <2.13%). A statistically higher incidence of CTA-type aberrations in variant genotypes between exposed and control groups was observed (1.22% vs. 0.59%, P <0.05). The work place is usually higher source of exposure to harmful factors. Workers need consistent and frequent health control. In assessing the risk of adverse effects of metals it is important to consider their persistence, behavior and bioavailability. Prolonged exposure to carcinogens may not manifest symptoms of poisoning, but delayed effects may occur, which resulted in a higher incidence of malignant tumors.

Keywords: CCND1, genotoxicity, polymorphism, stainless steel, welders

Procedia PDF Downloads 333

Authors: Irena Maus, Katharina Gabriella Cibis, Andreas Bremges, Yvonne Stolze, Geizecler Tomazetto, Daniel Wibberg, Helmut König, Alfred Pühler, Andreas Schlüter

Abstract:

Within the agricultural sector, the production of biogas from organic substrates represents an economically attractive technology to generate bioenergy. Complex consortia of microorganisms are responsible for biomass decomposition and biogas production. Recently, species belonging to the phylum Thermotogae were detected in thermophilic biogas-production plants utilizing renewable primary products for biomethanation. To analyze adaptive genome features of representative Thermotogae strains, Defluviitoga tunisiensis L3 was isolated from a rural thermophilic biogas plant (54°C) and completely sequenced on an Illumina MiSeq system. Sequencing and assembly of the D. tunisiensis L3 genome yielded a circular chromosome with a size of 2,053,097 bp and a mean GC content of 31.38%. Functional annotation of the complete genome sequence revealed that the thermophilic strain L3 encodes several genes predicted to facilitate growth of this microorganism on arabinose, galactose, maltose, mannose, fructose, raffinose, ribose, cellobiose, lactose, xylose, xylan, lactate and mannitol. Acetate, hydrogen (H2) and carbon dioxide (CO2) are supposed to be end products of the fermentation process. The latter gene products are metabolites for methanogenic archaea, the key players in the final step of the anaerobic digestion process. To determine the degree of relatedness of dominant biogas community members within selected digester systems to D. tunisiensis L3, metagenome sequences from corresponding communities were mapped on the L3 genome. These fragment recruitments revealed that metagenome reads originating from a thermophilic biogas plant covered 95% of D. tunisiensis L3 genome sequence. In conclusion, availability of the D. tunisiensis L3 genome sequence and insights into its metabolic capabilities provide the basis for biotechnological exploitation of genome features involved in thermophilic fermentation processes utilizing renewable primary products.

Keywords: genome sequence, thermophilic biogas plant, Thermotogae, Defluviitoga tunisiensis

Procedia PDF Downloads 469

Authors: Bahaa Eldin A. Fouda, Khaled N. Yossef, Mohamed Elhosseny, Ahmed Lotfy, Mohamed Salama, Mohamed Sobh

Abstract:

Developmental neurotoxicity (DNT) entails the toxic effects imparted by various chemicals on the brain during the early childhood period. As human brains are vulnerable during this period, various chemicals would have their maximum effects on brains during early childhood. Some toxicants have been confirmed to induce developmental toxic effects on CNS e.g. lead, however; most of the agents cannot be identified with certainty due the defective nature of predictive toxicology models used. A novel alternative method that can overcome most of the limitations of conventional techniques is the use of 3D neurospheres system. This in-vitro system can recapitulate most of the changes during the period of brain development making it an ideal model for predicting neurotoxic effects. In the present study, we verified the possible DNT of epoxomicin which is a naturally occurring selective proteasome inhibitor with anti-inflammatory activity. Rat neural progenitor cells were isolated from rat embryos (E14) extracted from placental tissue. The cortices were aseptically dissected out from the brains of the fetuses and the tissues were triturated by repeated passage through a fire-polished constricted Pasteur pipette. The dispersed tissues were allowed to settle for 3 min. The supernatant was, then, transferred to a fresh tube and centrifuged at 1,000 g for 5 min. The pellet was placed in Hank’s balanced salt solution cultured as free-floating neurospheres in proliferation medium. Two doses of epoxomicin (1µM and 10µM) were used in cultured neuropsheres for a period of 14 days. For proliferation analysis, spheres were cultured in proliferation medium. After 0, 4, 5, 11, and 14 days, sphere size was determined by software analyses. The diameter of each neurosphere was measured and exported to excel file further to statistical analysis. For viability analysis, trypsin-EDTA solution were added to neurospheres for 3 min to dissociate them into single cells suspension, then viability evaluated by the Trypan Blue exclusion test. Epoxomicin was found to affect proliferation and viability of neuropsheres, these effects were positively correlated to doses and progress of time. This study confirms the DNT effects of epoxomicin on 3D neurospheres model. The effects on proliferation suggest possible gross morphologic changes while the decrease in viability propose possible focal lesion on exposure to epoxomicin during early childhood.

Keywords: neural progentor cells, epoxomicin, neurosphere, medical and health sciences

Procedia PDF Downloads 399

Authors: Yunjia Yang, Peng Li, Gordon Xu, Timothy Mahony, Bing Zhang, Neena Mitter, Karishma Mody

Abstract:

Sheep flystrike is one of the most economically important diseases affecting the Australian sheep and wool industry (>356M/annually). Currently, control of Lucillia cuprina relies almost exclusively on chemicals controls and the parasite has developed resistance to nearly all control chemicals used in the past. It is therefore critical to develop an alternative solution for the sustainable control and management of flystrike. RNA interference (RNAi) technologies have been successfully explored in multiple animal industries for developing parasites controls. This research project aims to develop a RNAi based biological control for sheep blowfly. Double-stranded RNA (dsRNA) has already proven successful against viruses, fungi and insects. However, the environmental instability of dsRNA is a major bottleneck for successful RNAi. Bentonite polymer (BenPol) technology can overcome this problem, as it can be tuned for the controlled release of dsRNA in the gut challenging pH environment of the blowfly larvae, prolonging its exposure time to and uptake by target cells. To investigate the potential of BenPol technology for dsRNA delivery, four different BenPol carriers were tested for their dsRNA loading capabilities, and three of them were found to be capable of affording dsRNA stability under multiple temperatures (4°C, 22°C, 40°C, 55°C) in sheep serum. Based on stability results, dsRNA from potential targeted genes was loaded onto BenPol carriers and tested in larvae feeding assays, three genes resulting in knockdowns. Meanwhile, a primary blowfly embryo cell line (BFEC) derived from L. cuprina embryos was successfully established, aim for an effective insect cell model for testing RNAi efficacy for preliminary assessments and screening. The results of this study establish that the dsRNA is stable when loaded on BenPol particles, unlike naked dsRNA rapidly degraded in sheep serum. The stable nanoparticle delivery system offered by BenPol technology can protect and increase the inherent stability of dsRNA molecules at higher temperatures in a complex biological fluid like serum, providing promise for its future use in enhancing animal protection.

Keywords: flystrike, RNA interference, bentonite polymer technology, Lucillia cuprina

Procedia PDF Downloads 61

Authors: L. Musak, J. Valachova, T. Vasicko, O. Osina

Abstract:

Introduction: Stainless steel is resistant to electrochemical corrosion by passivation. Welders are greatly exposed to welding fumes of toxic metals, which added to this steel. The content of chromium (Cr) in steel was above 11.5%, Ni and Mo from 2 to 6.5%. The aim of the study was the evaluation of occupational exposure to Cr, chromosome analysis and valuation of individual susceptibility polymorphism of gene CCND1 c.870 G>A. Materials and Methods: The exposed group was consisted from 117 welders of stainless steels. The average age was 38.43 years and average exposure time 7.14 years. Smokers represented 40.17%. The control group consisted of 123 non-exposed workers with an average age of 39.74 years and time employment 16.67 years. Smokers accounted for 22.76%. Analysis of Cr in blood and urine was performed by atomic absorption spectrophotometry (AAS Varian SpectraAA 30P) with electrothermal decomposition of the sample in the graphite furnace. For the evaluation of chromosomal aberrations (CA) was used cytogenetic analysis of peripheral blood lymphocytes, gene polymorphism was determined by PCR-RFLP reaction using appropriate primers and restriction enzymes. For statistical analysis was used the Mann-Whitney U-test. Results: The mean Cr level in exposed group was 0.095 mmol/l (0.019 min-max 0.504). No value does exceed the average normal value. The average value Cr in urine was 7.9 mmol/mol creatinine (min 0.026 to max 19.26). The total number of CA was 1.86% in compared to 1.70% controls. (CTA-type 0.90% vs 0.80% and CSA-type 0.96% vs 0.90%). In the number of total CA was observed statistical difference between smokers and non-smokers of exposed group (S-1.57% vs. NS-2.04%, P<0.05). In CCND1 gene polymorphisms was observed the increasing of the total CA with wild-type allele (WT) via heterozygous to the VAR genotype (1.44%<1.82%<2.13%). There was observed a statistically higher incidence of CTA-type aberrations in variant genotypes between exposed and control groups (1.22% vs. 0.59%, P<0.05). Discussion and conclusions: The work place is usually higher source of exposure to harmful factors. Workers need consistently and checked frequently health control. In assessing the risk of adverse effects of metals is important to consider their persistence, behavior and bioavailability. Prolonged exposure to carcinogens may not manifest symptoms of poisoning, but delayed effects may occur, which resulted in a higher incidence of malignant tumors.

Keywords: genotoxicity, chromium, stainless steels, welders

Procedia PDF Downloads 344

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

Abstract:

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

Procedia PDF Downloads 217

Authors: Yunjia Yang, Peng Li, Gordon Xu, Timothy Mahony, Bing Zhang, Neena Mitter, Karishma Mody

Abstract:

Sheep flystrike is one of the most economically important diseases affecting the Australian sheep and wool industry (>356M/annually). Currently, control of Lucillia cuprina relies almost exclusively on chemicals controls, and the parasite has developed resistance to nearly all control chemicals used in the past. It is, therefore, critical to develop an alternative solution for the sustainable control and management of flystrike. RNA interference (RNAi) technologies have been successfully explored in multiple animal industries for developing parasites controls. This research project aims to develop a RNAi based biological control for sheep blowfly. Double-stranded RNA (dsRNA) has already proven successful against viruses, fungi, and insects. However, the environmental instability of dsRNA is a major bottleneck for successful RNAi. Bentonite polymer (BenPol) technology can overcome this problem, as it can be tuned for the controlled release of dsRNA in the gut challenging pH environment of the blowfly larvae, prolonging its exposure time to and uptake by target cells. To investigate the potential of BenPol technology for dsRNA delivery, four different BenPol carriers were tested for their dsRNA loading capabilities, and three of them were found to be capable of affording dsRNA stability under multiple temperatures (4°C, 22°C, 40°C, 55°C) in sheep serum. Based on stability results, dsRNA from potential targeted genes was loaded onto BenPol carriers and tested in larvae feeding assays, three genes resulting in knockdowns. Meanwhile, a primary blowfly embryo cell line (BFEC) derived from L. cuprina embryos was successfully established, aim for an effective insect cell model for testing RNAi efficacy for preliminary assessments and screening. The results of this study establish that the dsRNA is stable when loaded on BenPol particles, unlike naked dsRNA rapidly degraded in sheep serum. The stable nanoparticle delivery system offered by BenPol technology can protect and increase the inherent stability of dsRNA molecules at higher temperatures in a complex biological fluid like serum, providing promise for its future use in enhancing animal protection.

Keywords: lucilia cuprina, primary cell line establishment, RNA interference, insect cell transfection

Procedia PDF Downloads 51

Authors: Sushil Pradhan

Abstract:

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

Procedia PDF Downloads 359

Authors: M. Bhattarai, B. B. Rana, M. Kamimukai, I. Takamure, T. Kawano, M. Murai

Abstract:

The sd1-d allele at the sd1 locus on chromosome 1, originating from Taiwanese variety Dee-geo-woo-gen, has been playing important role for developing short-culm and lodging-resistant indica varieties such as IR36 in rice. The dominant allele SD1 for long culm at the locus is differentiated into SD1-in and SD1-ja which are harbored in indica and japonica subspecies’s, respectively. The sd1-d of an indica variety IR36 was substituted with SD1-in or SD1-ja by recurrent backcrosses of 17 times with IR36, and two isogenic tall lines regarding the respective dominant alleles were developed by using an indica variety IR5867 and a japonica one ‘Koshihikari’ as donors, which were denoted by '5867-36' and 'Koshi-36', respectively. The present study was conducted to examine the effect of sd1-d on yield and related traits as compared with SD1-in and SD1-ja, by using the two isogenic tall lines. Seedlings of IR36 and the two isogenic lines were transplanted on an experimental field of Kochi University, by the planting distance of 30 cm × 15 cm with two seedlings per hill, on May 3, 2017. Chemical fertilizers were supplied by basal application and top-dressing at a rate of 8.00, 6.57 and 7.52 g/m², respectively, for N, P₂O₅ and K₂O in total. Yield, yield components, and other traits were measured. Culm length (cm) was in the order of 5867-36 (101.9) > Koshi-36 (80.1) > IR36 (60.0), where '>' indicates statistically significant difference at the 5% level. Accordingly, sd1-d reduced culm by 41.9 and 20.1 cm, compared with SD1-in and SD1-ja, respectively, and the effect of elongating culm was higher in the former allele than in the latter one. Total brown rice yield (g/m²), including unripened grains, was in the order of IR36 (611) ≧ 5867-36 (586) ≧ Koshi-36 (572), indicating non-significant differences among them. Yield-1.5mm sieve (g/m²) was in the order of IR36 (596) ≧ 5867-36 (575) ≧ Koshi-36 (558). Spikelet number per panicle was in the order of 5867-36 (89.2) ≧ IR36 (84.7) ≧ Koshi-36 (79.8), and 5867-36 > Koshi-36. Panicle number per m² was in the order of IR36 (428) ≧ Koshi-36 (403) ≧ 5867-36 (353), and IR36 > 5867-36, suggesting that sd1-d increased number of panicles compared with SD1-in. Ripened-grain percentage-1.5mm sieve was in the order of Koshi-36 (86.0) ≧ 5867-36 (85.0) ≧ IR36 (82.7), and Koshi-36 > IR36. Thousand brown-rice-grain weight-1.5mm sieve (g) was in the order of 5867-36 (21.5) > Koshi-36 (20.2) ≧ IR36 (19.9). Total dry weight at maturity (g/m²) was in the order of 5867-36 (1404 ) ≧ IR36 (1310) ≧ Kosihi-36 (1290). Harvest index of total brown rice (%) was in the order of IR36 (39.6) > Koshi-36 (37.7) > 5867-36 (35.5). Hence, sd1-d did not exert significant effect on yield in indica genetic background. However, lodging was observed from the late stage of maturity in 5867-36 and Koshi-36, particularly in the former, which was principally due to their long culms. Consequently, sd1-d enables higher yield with higher fertilizer application, by enhancing lodging resistance, particularly in indica subspecies.

Keywords: rice, dwarfing gene, sd1-d, SD1-in, SD1-ja, yield

Procedia PDF Downloads 145

Authors: Satendra Pal Singh, Dalip Kr. Kakru, Jyoti Mishra, Rajesh Thakur, Tarana Sarwat

Abstract:

COVID-19 is still an intrigue as far as morbidity or mortality is concerned. The rate of recovery varies from person to person, & it depends upon the accessibility of the healthcare system and the roles played by the physicians and caregivers. It is envisaged that with the passage of time, people would become immune to this virus, and those who are vulnerable would sustain themselves with the help of vaccines. The proposed study deals with the severeness of COVID-19 is associated with some specific biomarkers linked to correlate age and gender. We will be assessing the overall homeostasis of the persons who were affected by the coronavirus infection and also of those who recovered from it. Some people show more severe effects, while others show very mild symptoms, however, they show low CT values. Thus far, it is unclear why the new strain of Covid has different effects on different people in terms of age, gender, and ABO blood typing. According to data, the fatality rate with heart disease was 10.5 percent, 7.3 percent were diabetic, and 6 percent who are already infected from other comorbidities. However, some COVID-19 cases are worse than others & it is not fully explainable as of date. Overall data show that the ABO blood group is effective or prone to the risk of SARS-COV2 infection, while another study also shows the phenotypic effects of the blood group related to covid. It is an accepted fact that females have more strong immune systems than males, which may be related to the fact that females have two ‘X’ chromosomes, which might contain a more effective immunity booster gene on the X chromosome, and are capable to protect the female. Also specific sex hormones also induce a better immune response in a specific gender. This calls for in-depth analysis to be able to gain insight into this dilemma. COVID-19 is still not fully characterized, and thus we are not very familiar with its biology, mode of infection, susceptibility, and overall viral load in the human body. How many virus particles are needed to infect a person? How, then, comorbidity contribute to coronavirus infection? Since the emergence of this virus in 2020, a large number of papers have been published, and seemingly, vaccines have been prepared. But still, a large number of questions remain unanswered. The proneness of humans for infection by covid-19 needs to be established to be able to develop a better strategy to fight this virus. Our study will be on the Impact of demography on the Severity of covid-19 infection & at the same time, will look into gender-specific sensitivity of Covid-19 and the Operational variation of different biochemical markers in Covid-19 positive patients. Besides, we will be studying the co-relation, if any, of COVID severity & ABO Blood group type and the occurrence of the most common blood group type amongst positive patience.

Keywords: coronavirus, ABO blood group, age, gender

Procedia PDF Downloads 71

Authors: Abiodun Olayinka, Daniel Dzidzienyo, Pangirayi Tongoona, Samuel Offei, Edwige Gaby Nkouaya Mbanjo, Chiedozie Egesi, Ismail Yusuf Rabbi

Abstract:

Cassava (Manihot esculenta Crantz) is a major source of starch for various industrial applications. However, the traditional cultivation and harvesting methods of cassava are labour-intensive and inefficient, limiting the supply of fresh cassava roots for industrial starch production. To achieve improved productivity and quality of fresh cassava roots through mechanized cultivation, cassava cultivars with compact plant architecture and moderate plant height are needed. Plant architecture-related traits, such as plant height, harvest index, stem diameter, branching angle, and lodging tolerance, are critical for crop productivity and suitability for mechanized cultivation. However, the genetics of cassava plant architecture remain poorly understood. This study aimed to identify the genetic bases of the relationships between plant architecture traits and productivity-related traits, particularly starch content. A panel of 453 clones developed at the International Institute of Tropical Agriculture, Nigeria, was genotyped and phenotyped for 18 plant architecture and productivity-related traits at four locations in Nigeria. A genome-wide association study (GWAS) was conducted using the phenotypic data from a panel of 453 clones and 61,238 high-quality Diversity Arrays Technology sequencing (DArTseq) derived Single Nucleotide Polymorphism (SNP) markers that are evenly distributed across the cassava genome. Five significant associations between ten SNPs and three plant architecture component traits were identified through GWAS. We found five SNPs on chromosomes 6 and 16 that were significantly associated with shoot weight, harvest index, and total yield through genome-wide association mapping. We also discovered an essential candidate gene that is co-located with peak SNPs linked to these traits in M. esculenta. A review of the cassava reference genome v7.1 revealed that the SNP on chromosome 6 is in proximity to Manes.06G101600.1, a gene that regulates endodermal differentiation and root development in plants. The findings of this study provide insights into the genetic basis of plant architecture and yield in cassava. Cassava breeders could leverage this knowledge to optimize plant architecture and yield in cassava through marker-assisted selection and targeted manipulation of the candidate gene.

Keywords: Manihot esculenta Crantz, plant architecture, DArtseq, SNP markers, genome-wide association study

Procedia PDF Downloads 43

Authors: Paula I. Buonfiglio, Carlos David Bruque, Lucia Salatino, Vanesa Lotersztein, Sebastián Menazzi, Paola Plazas, Ana Belén Elgoyhen, Viviana Dalamón

Abstract:

Hearing loss (HL) is the most prevalent sensorineural disorder affecting about 10% of the global population, with more than half due to genetic causes. About 1 in 500-1000 newborns present congenital HL. Most of the patients are non-syndromic with an autosomal recessive mode of inheritance. To date, more than 100 genes are related to HL. Therefore, the Whole-exome sequencing (WES) technique has become a cost-effective alternative approach for molecular diagnosis. Nevertheless, new challenges arise from the detection of novel variants, in particular missense changes, which can lead to a spectrum of genotype-to-phenotype correlations, which is not always straightforward. In this work, we aimed to identify the genetic causes of HL in isolated and familial cases by designing a multistep approach to analyze target genes related to hearing impairment. Moreover, we performed in silico and in vivo analyses in order to further study the effect of some of the novel variants identified in the hair cell function using the zebrafish model. A total of 650 patients were studied by Sanger Sequencing and Gap-PCR in GJB2 and GJB6 genes, respectively, diagnosing 15.5% of sporadic cases and 36% of familial ones. Overall, 50 different sequence variants were detected. Fifty of the undiagnosed patients with moderate HL were tested for deletions in STRC gene by Multiplex ligation-dependent probe amplification technique (MLPA), leading to 6% of diagnosis. After this initial screening, 50 families were selected to be analyzed by WES, achieving diagnosis in 44% of them. Half of the identified variants were novel. A missense variant in MYO6 gene detected in a family with postlingual HL was selected to be further analyzed. A protein modeling with AlphaFold2 software was performed, proving its pathogenic effect. In order to functionally validate this novel variant, a knockdown phenotype rescue assay in zebrafish was carried out. Injection of wild-type MYO6 mRNA in embryos rescued the phenotype, whereas using the mutant MYO6 mRNA (carrying c.2782C>A variant) had no effect. These results strongly suggest the deleterious effect of this variant on the mobility of stereocilia in zebrafish neuromasts, and hence on the auditory system. In the present work, we demonstrated that our algorithm is suitable for the sequential multigenic approach to HL in our cohort. These results highlight the importance of a combined strategy in order to identify candidate variants as well as the in silico and in vivo studies to analyze and prove their pathogenicity and accomplish a better understanding of the mechanisms underlying the physiopathology of the hearing impairment.

Keywords: diagnosis, genetics, hearing loss, in silico analysis, in vivo analysis, WES, zebrafish

Procedia PDF Downloads 61

Authors: Habtamu Abera Goshu, Ping Yan

Abstract:

Copy number variation (CNV) is a significant marker of the genetic and phenotypic diversity among individuals that accounts for complex quantitative traits of phenotype and diseases via modulating gene dosage, position effects, alteration of downstream pathways, modification of chromosome structure, and position within the nucleus and disrupting coding regions in the genome. Associating copy number variations (CNVs) with growth and gene expression are a powerful approach for identifying genomic characteristics that contribute to phenotypic and genotypic variation. A previous study using next-generation sequencing illustrated that the choline kinase beta (CHKB), Krüpple-like factor 6 (KLF6), glypican 1(GPC1), and cholinergic receptor muscarinic 3 (CHRM3) genes reside within copy number variable regions (CNVRs) of yak populations that overlap with quantitative trait loci (QTLs) of meat quality and growth. As a result, this research aimed to determine the association of CNVs of the KLF6, CHKB, GPC1, and CHRM3 genes with growth traits in the Datong yak breed. The association between the CNV types of the KLF6, CHKB, GPC1, and CHRM3 genes and the growth traits in the Datong yak breed was determined by one-way analysis of variance (ANOVA) using SPSS software. The CNV types were classified as a loss (a copy number of 0 or 1), gain (a copy number >2), and normal (a copy number of 2) relative to the reference gene, BTF3 in the 387 individuals of Datong yak. These results indicated that the normal CNV types of the CHKB and GPC1 genes were significantly (P<0.05) associated with high body length, height and weight, and chest girth in six-month-old and five-year-old Datong yaks. On the other hand, the loss CNV types of the KLF6 gene is significantly (P<0.05) associated with body weight and length and chest girth at six-month-old and five-year-old Datong yaks. In the contrary, the gain CNV type of the CHRM3 gene is highly (P<0.05) associated with body weight, length, height, and chest girth in six-month-old and five-year-old. This work provides the first observation of the biological role of CNVs of the CHKB, KLF6, GPC1, and CHRM3 genes in the Datong yak breed and might, therefore, provide a novel opportunity to utilize data on CNVs in designing molecular markers for the selection of animal breeding programs for larger populations of various yak breeds. Therefore, we hypothesized that this study provided inclusive information on the application of CNVs of the CHKB, KLF6, GPC1, and CHRM3 genes in growth traits in Datong yaks and its possible function in bovine species.

Keywords: Copy number variation, growth traits, yak, genes

Procedia PDF Downloads 140

Authors: Abiodun Olayinka, Daniel Dzidzienyo, Pangirayi Tongoona, Samuel Offei, Edwige Gaby Nkouaya Mbanjo, Chiedozie Egesi, Ismail Yusuf Rabbi

Abstract:

Cassava (Manihot esculenta Crantz) is a major source of starch for various industrial applications. However, the traditional cultivation and harvesting methods of cassava are labour-intensive and inefficient, limiting the supply of fresh cassava roots for industrial starch production. To achieve improved productivity and quality of fresh cassava roots through mechanized cultivation, cassava cultivars with compact plant architecture and moderate plant height are needed. Plant architecture-related traits, such as plant height, harvest index, stem diameter, branching angle, and lodging tolerance, are critical for crop productivity and suitability for mechanized cultivation. However, the genetics of cassava plant architecture remain poorly understood. This study aimed to identify the genetic bases of the relationships between plant architecture traits and productivity-related traits, particularly starch content. A panel of 453 clones developed at the International Institute of Tropical Agriculture, Nigeria, was genotyped and phenotyped for 18 plant architecture and productivity-related traits at four locations in Nigeria. A genome-wide association study (GWAS) was conducted using the phenotypic data from a panel of 453 clones and 61,238 high-quality Diversity Arrays Technology sequencing (DArTseq) derived Single Nucleotide Polymorphism (SNP) markers that are evenly distributed across the cassava genome. Five significant associations between ten SNPs and three plant architecture component traits were identified through GWAS. We found five SNPs on chromosomes 6 and 16 that were significantly associated with shoot weight, harvest index, and total yield through genome-wide association mapping. We also discovered an essential candidate gene that is co-located with peak SNPs linked to these traits in M. esculenta. A review of the cassava reference genome v7.1 revealed that the SNP on chromosome 6 is in proximity to Manes.06G101600.1, a gene that regulates endodermal differentiation and root development in plants. The findings of this study provide insights into the genetic basis of plant architecture and yield in cassava. Cassava breeders could leverage this knowledge to optimize plant architecture and yield in cassava through marker-assisted selection and targeted manipulation of the candidate gene.

Keywords: manihot esculenta crantz, plant architecture, dartseq, snp markers, genome-wide association study

Procedia PDF Downloads 54

Authors: Leo Nnamdi Ozurumba-Dwight

Abstract:

Single-cell genomic analytical systems have proved to be a platform to isolate bulk cells into selected single cells for genomic, proteomic, and related metabolomic studies. This is enabling systematic investigations of the level of heterogeneity in a diverse and wide pool of cell populations. Single cell technologies, embracing techniques such as high parameter flow cytometry, single-cell sequencing, and high-resolution images are playing vital roles in these investigations on messenger ribonucleic acid (mRNA) molecules and related gene expressions in tracking the nature and course of disease conditions. This entails targeted molecular investigations on unit cells that help us understand cell behavoiur and expressions, which can be examined for their health implications on the health state of patients. One of the vital good sides of single-cell RNA sequencing (scRNA seq) is its probing capacity to detect deranged or abnormal cell populations present within homogenously perceived pooled cells, which would have evaded cursory screening on the pooled cell populations of biological samples obtained as part of diagnostic procedures. Despite conduction of just single-cell transcriptome analysis, scRNAseq now permits comparison of the transcriptome of the individual cells, which can be evaluated for gene expressional patterns that depict areas of heterogeneity with pharmaceutical drug discovery and clinical treatment applications. It is vital to strictly work through the tools of investigations from wet lab to bioinformatics and computational tooled analyses. In the precise steps for scRNAseq, it is critical to do thorough and effective isolation of viable single cells from the tissues of interest using dependable techniques (such as FACS) before proceeding to lysis, as this enhances the appropriate picking of quality mRNA molecules for subsequent sequencing (such as by the use of Polymerase Chain Reaction machine). Interestingly, scRNAseq can be deployed to analyze various types of biological samples such as embryos, nervous systems, tumour cells, stem cells, lymphocytes, and haematopoietic cells. In haematopoietic cells, it can be used to stratify acute myeloid leukemia patterns in patients, sorting them out into cohorts that enable re-modeling of treatment regimens based on stratified presentations. In immunotherapy, it can furnish specialist clinician-immunologist with tools to re-model treatment for each patient, an attribute of precision medicine. Finally, the good predictive attribute of scRNAseq can help reduce the cost of treatment for patients, thus attracting more patients who would have otherwise been discouraged from seeking quality clinical consultation help due to perceived high cost. This is a positive paradigm shift for patients’ attitudes primed towards seeking treatment.

Keywords: immunotherapy, transcriptome, re-modeling, mRNA, scRNA-seq

Procedia PDF Downloads 145

Authors: Z. M. Biyasheva, M. Zh. Tleubergenova, Y. A. Zaripova, A. L. Shakirov, V. V. Dyachkov

Abstract:

In recent years, interest in ecogenetic and biomedical problems related to the effects on the population of radon and its daughter decay products has increased significantly. Of particular interest is the assessment of the consequence of irradiation at hazardous radon areas, which includes the Almaty region due to the large number of tectonic faults that enhance radon emanation. In connection with the foregoing, the purpose of this work was to study the genetic effects of exposure to supernormal radon doses on the alpha-radiation model. Irradiation does not affect the growth of the cell, but rather its ability to differentiate. In addition, irradiation can lead to somatic mutations, morphoses and modifications. These damages most likely occur from changes in the composition of the substances of the cell. Such changes are epigenetic since they affect the regulatory processes of ontogenesis. Variability in the expression of regulatory genes refers to conditional mutations that modify the formation of signs of intraspecific similarity. Characteristic features of these conditional mutations are the dominant type of their manifestation, phenotypic asymmetry and their instability in the generations. Currently, the terms “morphosis” and “modification” are used to describe epigenetic variability, which are maintained in Drosophila melanogaster cultures using linkaged X- chromosomes, and the mutant X-chromosome is transmitted along the paternal line. In this paper, we investigated the epigenetic effects of alpha particles, whose source in nature is mainly radon and its daughter decay products. In the experiment, an isotope of plutonium-238 (Pu²³⁸), generating radiation with an energy of about 5500 eV, was used as a source of alpha particles. In an experiment in the first generation (F₁), deformities or morphoses were found, which can be called "radiation syndromes" or mutations, the manifestation of which is similar to the pleiotropic action of genes. The proportion of morphoses in the experiment was 1.8%, and in control 0.4%. In this experiment, the morphoses in the flies of the first and second generation looked like black spots, or melanomas on different parts of the imago body; "generalized" melanomas; curled, curved wings; shortened wing; bubble on one wing; absence of one wing, deformation of thorax, interruption and violation of tergite patterns, disruption of distribution of ocular facets and bristles; absence of pigmentation of the second and third legs. Statistical analysis by the Chi-square method showed the reliability of the difference in experiment and control at P ≤ 0.01. On the basis of this, it can be considered that alpha particles, which in the environment are mainly generated by radon and its isotopes, have a mutagenic effect that manifests itself, mainly in the formation of morphoses or deformities.

Keywords: alpha-radiation, genotoxicity, morphoses, radioecology, radon

Procedia PDF Downloads 122

Authors: Vladimir Berezin, Andrey Bogoyavlenskiy, Pavel Alexyuk, Madina Alexyuk, Aizhan Turmagambetova, Irina Zaitseva, Nadezhda Sokolova, Elmira Omirtaeva

Abstract:

Introduction: Triterpene saponins of plant origin are one of the most promising candidates for elaboration of novel adjuvants. Due to the combination of immunostimulating activity and the capacity interact with amphipathic molecules with formation of highly immunogenic nanocomplexes, triterpene saponins could serve as a good adjuvant/delivery system for vaccine use. In the research presented adjuvants on the base of nanocomplexes contained triterpene saponins isolated from Glycyrrhiza glabra and Saponaria officinalis plants indigenous to Kazakhstan were elaborated for influenza vaccine use. Methods: Purified triterpene saponins 'Glabilox' and 'SO1' with low toxicity and high immunostimulatory activity were isolated from plants Glycyrrhiza glabra L. and Saponaria officinalis L. by high-performance liquid chromatography (HPLC) and identified using electrospray ionization mass spectrometry (ESI-MS). Influenza virus A/St-Petersburg/5/09 (H1N1) propagated in 9-days old chicken embryos was concentrated and purified by centrifugation in sucrose gradient. Nanocomplexes contained lipids, and triterpene saponins Glabilox or SO1 were prepared by dialysis technique. Immunostimulating activity of experimental vaccine preparations was studied in vaccination/challenge experiments in mice. Results: Humoral and cellular immune responses and protection against influenza virus infection were examined after single subcutaneous and intranasal immunization. Mice were immunized subunit influenza vaccine (HA+NA) or whole virus inactivated influenza vaccine in doses 3.0/5.0/10.0 µg antigen/animal mixed with adjuvant in dose 15.0 µg/animal. Sera were taken 14-21 days following single immunization and mice challenged by A/St-Petersburg/5/09 influenza virus in dose 100 EID₅₀. Study of experimental influenza vaccine preparations in animal immunization experiments has shown that subcutaneous and intranasal immunization with subunit influenza vaccine mixed with nanocomplexes contained Glabilox or SO1 saponins stimulated high levels of humoral immune response (IgM, IgA, IgG1, IgG2a, and IgG2b antibody) and cellular immune response (IL-2, IL-4, IL-10, and IFN-γ cytokines) and resulted 80-90% protection against lethal influenza infection. Also, single intranasal and single subcutaneous immunization with whole virus inactivated influenza vaccine mixed with nanoparticulated adjuvants stimulated high levels of humoral and cellular immune responses and provided 100% protection against lethal influenza infection. Conclusion: The results of study have shown that nanocomplexes contained purified triterpene saponins Glabilox and SO1 isolated from plants indigenous to Kazakhstan can stimulate a broad spectrum of humoral and cellular immune responses and induce protection against lethal influenza infection. Both elaborated adjuvants are promising for incorporation to influenza vaccine intended for subcutaneous and intranasal routes of immunization.

Keywords: influenza vaccine, adjuvants, triterpene saponins, immunostimulating activity

Procedia PDF Downloads 104

Authors: Sandra Smieszek, Jonathan Haines

Abstract:

Introduction: Numerous research efforts have focused on searching for ‘longevity genes’. However, attempting to decipher the genetic component of the longevous phenotype have resulted in limited success and the mechanisms governing longevity remain to be explained. We conducted a genome-wide homozygosity analysis (GWHA) of the founder population of the Amish community in central Ohio. While genome-wide association studies using unrelated individuals have revealed many interesting longevity associated variants, these variants are typically of small effect and cannot explain the observed patterns of heritability for this complex trait. The Amish provide a large cohort of extended kinships allowing for in depth analysis via family-based approach excellent population due to its. Heritability of longevity increases with age with significant genetic contribution being seen in individuals living beyond 60 years of age. In our present analysis we show that the heritability of longevity is estimated to be increasing with age particularly on the paternal side. Methods: The present analysis integrated both phenotypic and genotypic data and led to the discovery of a series of variants, distinct for stratified populations across ages and distinct for paternal and maternal cohorts. Specifically 5437 subjects were analyzed and a subset of 893 successfully genotyped individuals was used to assess CHIP heritability. We have conducted the homozygosity analysis to examine if homozygosity is associated with increased risk of living beyond 90. We analyzed AMISH cohort genotyped for 614,957 SNPs. Results: We delineated 10 significant regions of homozygosity (ROH) specific for the age group of interest (>90). Of particular interest was ROH on chromosome 13, P < 0.0001. The lead SNPs rs7318486 and rs9645914 point to COL4A2 and our lead SNP. COL25A1 encodes one of the six subunits of type IV collagen, the C-terminal portion of the protein, known as canstatin, is an inhibitor of angiogenesis and tumor growth. COL4A2 mutations have been reported with a broader spectrum of cerebrovascular, renal, ophthalmological, cardiac, and muscular abnormalities. The second region of interest points to IRS2. Furthermore we built a classifier using the obtained SNPs from the significant ROH region with 0.945 AUC giving ability to discriminate between those living beyond to 90 years of age and beyond. Conclusion: In conclusion our results suggest that a history of longevity does indeed contribute to increasing the odds of individual longevity. Preliminary results are consistent with conjecture that heritability of longevity is substantial when we start looking at oldest fifth and smaller percentiles of survival specifically in males. We will validate all the candidate variants in independent cohorts of centenarians, to test whether they are robustly associated with human longevity. The identified regions of interest via ROH analysis could be of profound importance for the understanding of genetic underpinnings of longevity.

Keywords: regions of homozygosity, longevity, SNP, Amish

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Authors: S. Najafi, E. Bertini, M. Pezzotti, G.B. Tornielli, S. Zenoni

Abstract:

Grapevine is an important agricultural fruit crop plant consumed worldwide and with a key role in the global economy. Grapevine is strongly affected by both biotic and abiotic stresses, which impact grape growth at different stages, such as during plant and berry development and pre- and post-harvest, consequently causing significant economic losses. Recently global warming has propelled the anticipation of the onset of berry ripening, determining the reduction of a grape color and increased volatilization of aroma compounds. Climate change could negatively alter the physiological characteristics of the grape and affect the berry and wine quality. Modern plant breeding can provide tools such as genome editing for improving grape resilience traits while maintaining intact the viticultural and oenological quality characteristics of the genotype. This study aims at developing a platform for genome editing application in grapevine plants with the final goal to improve berry quality, biotic, and abiotic resilience traits. We chose to directly deliver ribonucleoproteins (RNP, preassembled Cas protein and guide RNA) into plant protoplasts, and, from these cell structures, regenerate grapevine plants edited in specific selected genes controlling traits of interest. Edited plants regenerated by somatic embryogenesis from protoplasts will then be sequenced and molecularly characterized. Embryogenic calli of Sultana and Shiraz cultivars were initiated from unopened leaves of in-vitro shoot tip cultures and from stamens, respectively. Leaves were placed on NB2 medium while stamens on callus initiation medium (PIV) medium and incubated in the dark at 28 °C for three months. Viable protoplasts, tested by FDA staining, isolated from embryogenic calli were cultured by disc method at 1*105 protoplasts/ml. Mature well-shaped somatic embryos developed directly in the protoplast culture medium two months later and were transferred in the light into to shooting medium for further growth. Regenerated plants were then transferred to the greenhouse; no phenotypic alterations were observed when compared to non in-vitro cultured plants. The performed experiments allowed to established an efficient protocol of embryogenic calli production, protoplast isolation, and regeneration of the whole plant through somatic embryogenesis in both Sultana and Shiraz. Regenerated plants, through direct somatic embryogenesis deriving from a single cell, avoid the risk of chimerism during the regeneration process, therefore improving the genome editing process. As pre-requisite of genome editing, an efficient method for transfection of protoplast by yellow fluorescent protein (YFP) marker genes was also established and experiments of direct delivery of CRISPR–Cas9 ribonucleoproteins (RNPs) in protoplasts to achieve efficient DNA-free targeted mutations are in progress.

Keywords: CRISPR-cas9, plant regeneration, protoplast isolation, Vitis vinifera

Procedia PDF Downloads 121