Search results for: de novo transcriptome sequencing

Authors: Yew Mun Yip, Dawei Zhang

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Proteins are the biological machinery that executes specific vital functions in every cell of the human body by folding into their 3D structures. When a protein misfolds from its native structure, the machinery will malfunction and lead to misfolding diseases. Although in vitro experiments are able to conclude that the mutations of the amino acid sequence lead to incorrectly folded protein structures, these experiments are unable to decipher the folding process. Therefore, molecular dynamic (MD) simulations are employed to simulate the folding process so that our improved understanding of the folding process will enable us to contemplate better treatments for misfolding diseases. MD simulations make use of force fields to simulate the folding process of peptides. Secondary structures are formed via the hydrogen bonds formed between the backbone atoms (C, O, N, H). It is important that the hydrogen bond energy computed during the MD simulation is accurate in order to direct the folding process to the native structure. Since the atoms involved in a hydrogen bond possess very dissimilar electronegativities, the more electronegative atom will attract greater electron density from the less electronegative atom towards itself. This is known as the polarization effect. Since the polarization effect changes the electron density of the two atoms in close proximity, the atomic charges of the two atoms should also vary based on the strength of the polarization effect. However, the fixed atomic charge scheme in force fields does not account for the polarization effect. In this study, we introduce the polarized structure-specific backbone charge (PSBC) model. The PSBC model accounts for the polarization effect in MD simulation by updating the atomic charges of the backbone hydrogen bond atoms according to equations derived between the amount of charge transferred to the atom and the length of the hydrogen bond, which are calculated from quantum-mechanical calculations. Compared to other polarizable models, the PSBC model does not require quantum-mechanical calculations of the peptide simulated at every time-step of the simulation and maintains the dynamic update of atomic charges, thereby reducing the computational cost and time while accounting for the polarization effect dynamically at the same time. The PSBC model is applied to two different β-peptides, namely the Beta3s/GS peptide, a de novo designed three-stranded β-sheet whose structure is folded in vitro and studied by NMR, and the trpzip peptides, a double-stranded β-sheet where a correlation is found between the type of amino acids that constitute the β-turn and the β-propensity.

Keywords: hydrogen bond, polarization effect, protein folding, PSBC

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Authors: Ghazanfar Ali, Fahad N Almajhdi

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This study provides data on the viral diagnosis and molecular epidemiology of influenza A(H3N2) virus isolated in Riyadh, Saudi Arabia. Nasopharyngeal aspirates from 80 clinically infected patients in the peak of the 2010-2011 winter seasons were processed for viral diagnosis by RT-PCR. Sequencing of entire HA and NA genes of representative isolates and molecular epidemiological analysis were performed. A total of 06 patients were positive for influenza A, B and respiratory syncytial viruses by RT-PCR assays; out of these only one sample was positive for influenza A(H3N2) by RT-PCR. Phylogenetic analysis of the HA and NA gene sequences showed identities higher than 99-98.8 % in both genes. They were also similar to reference isolates in HA sequences (99 % identity) and in NA sequences (99 % identity). Amino acid sequences predicted for the HA gene were highly identical to reference strains. The NA amino acid substitutions identified did not include the oseltamivir-resistant H275Y substitution. Conclusion: Viral isolation and RT-PCR together were useful for diagnosis of the influenza A (H3N2) virus. Variations in HA and NA sequences are similar to those identified in worldwide reference isolates and no drug resistance was found.

Keywords: influenza A (H3N2), genetic characterization, viral isolation, RT-PCR, Saudi Arabia

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Authors: Narin Salehiyan

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The later sequencing of the complete genomes of Mycoplasma genitalium and M. pneumoniae has pulled in significant consideration to the atomic science of mycoplasmas, the littlest self-replicating living beings. It shows up that we are presently much closer to the objective of defining, in atomic terms, the complete apparatus of a self-replicating cell. Comparative genomics based on comparison of the genomic cosmetics of mycoplasmal genomes with those of other microbes, has opened better approaches of looking at the developmental history of the mycoplasmas. There's presently strong hereditary bolster for the speculation that mycoplasmas have advanced as a department of gram-positive microbes by a handle of reductive advancement. Amid this prepare, the mycoplasmas misplaced significant parcels of their ancestors’ chromosomes but held the qualities basic for life. In this way, the mycoplasmal genomes carry a tall rate of preserved qualities, incredibly encouraging quality comment. The critical genome compaction that happened in mycoplasmas was made conceivable by receiving a parasitic mode of life. The supply of supplements from their has clearly empowered mycoplasmas to lose, amid advancement, the qualities for numerous assimilative forms. Amid their advancement and adjustment to a parasitic mode of life, the mycoplasmas have created different hereditary frameworks giving a profoundly plastic set of variable surface proteins to avoid the have safe framework.

Keywords: mycoplasma, plasma, pathogen, genome

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Authors: Mohit Kumar, Beklashwar Salona, Shiv Murti, Mukesh Singh

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We report here a case of five-months old male baby, born as second child of non-consanguineous parents with no considerable history of genetic abnormality which was referred to our cytogenetic laboratory for chromosomal analysis. Physical dysmorphic facial features including mongoloid face, cleft palate, simian crease, and developmental delay were observed. We present this case with unique balanced autosomal translocation of t(3;10)(p21;p13). The risk of phenotypic abnormalities based on de novo balanced translocation was estimated to be 7%. The association of balanced chromosomal rearrangement with Down syndrome features such as multiple congenital anomalies, facial dysmorphism and congenital heart anomalies are very rare in a 5-months old male child. Trisomy-21 is not uncommon in chromosomal abnormality with the birth defect and balanced translocations are frequently observed in patients with secondary infertility or recurrent spontaneous abortion (RSA). Two ml heparinized peripheral blood cells cultured in RPMI-1640 for 72 hours supplemented with 20% fetal bovine serum, phytohemagglutinin (PHA), and antibiotics were used for chromosomal analysis. A total 30 metaphases images were captured using Olympus-BX51 microscope and analyzed using Bio-view karyotyping software through GTG-banding (G bands by trypsin and Giemsa) according to International System for Human Cytogenetic Nomenclature 2016. The results showed balanced translocation between short arm of chromosome # 3 and short arm of chromosome # 10. The karyotype of the child was found to be 46,XY,t(3;10)(p21; p13). Chromosomal abnormalities are one of the major causes of birth defect in new born babies. Also, balanced translocations are frequently observed in patients with secondary infertility or recurrent spontaneous abortion. The index case presented with dysmorphic facial features and had a balanced translocation 46,XY,t(3;10)(p21;p13). This translocation with break points at (p21; p13) has not been reported in the literature in a child with facial dysmorphism. To the best of our knowledge, this is the first report of novel balanced translocation t(3;10) with break points in a child with dysmorphic features. We found balanced chromosomal translocation instead of any trisomy or unbalanced aberrations along with some phenotypic abnormalities. Therefore, we suggest that such novel balanced translocation with abnormal phenotype should be reported in order to enable the pathologist, pediatrician, and gynecologist to have a better insight into the intricacies of chromosomal abnormalities and their associated phenotypic features. We hypothesized that dysmorphic features as seen in this case may be the result of change in the pattern of genes located at the breakpoint area in balanced translocations or may be due to deletion or mutation of genes located on the p-arm of chromosome # 3 and p-arm of chromosome # 10.

Keywords: balanced translocation, karyotyping, phenotypic abnormalities, facial dimorphisms

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Authors: Vladimir Ryabov, Mikhail Baryshevs, Mikhail Voskresenskey, Boris Popov

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The human prostate gland (PG) samples were obtained from patients who had undergone radical prostatectomy for prostate cancer (PC) and used to extract total RNA and prepare the prostate stromal cell cultures (PSCC) and patients-derived organoids (PDO). Growth of the cell cultures was accessed under microscopic evaluation in transmitted light and the marker expression by reverse polymerase chain reaction (RT-PCR), immunofluorescence, and immunoblotting. Some PCR products from prostate tissue, PSCC, and PDO were cloned and sequenced. We found that the cells of early and late passages of PSCC and corresponding PDO expressed luminal (androgen receptor, AR; cytokeratin 18, CK18) and basal (CK5, p63) epithelial markers, the production of which decreased or disappeared in late PSCC and PDO. The PSCC and PDO of early passages from cancer tissue additionally produced cancer markers AMACR, TMPRSS2-ERG, and Ezh2. The expression of TMPRSS2-ERG fusion transcripts was verified by cloning and sequencing the PCR products. The results obtained suggest that early passages of PSCC might be used as a pre-clinical model for the evaluation of early markers of prostate cancer.

Keywords: localized prostate cancer, prostate epithelial markers, prostate cancer markers, AMACR, TMPRSS2-ERG, prostate stromal cell cultures, PDO

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Authors: Huong M. T. Nguyen, Hoa A. P. Nguyen, Chi V. Phan, Mai P. T. Nguyen, Ngoc D. Ngo, Van T. Ta, Hai T. Le

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Wilson’s disease is an autosomal recessive disorder of copper metabolism, which is caused by mutation in copper- transporting P-type ATPase (ATP7B). The mechanism of this disease is a failure of hepatic excretion of copper to the bile, and it leads to copper deposits in the liver and other organs. Most clinical symptoms of Wilson’s disease can present as liver disease and/or neurologic disease. Objective: The goal of the study is prenatal diagnosis for pregnant women at high risk of Wilson’s disease in Northern Vietnam. Material and method: Three probands with clinically diagnosed liver disease were detected in the mutations of 21 exons and exon-intron boundaries of the ATP7B gene by direct Sanger-sequencing. Prenatal diagnoses were performed by amniotic fluid sampling from pregnant women in the 16th-18th weeks of pregnancy after the genotypes of parents with the probands were identified. Result: A total of three different mutations of the probands, including of S105*, P1052L, P1273G, were detected. Among three fetuses which underwent prenatal genetic testing, one fetus was homozygote; two fetuses were carriers. Conclusion: Genetic testing provided a useful method for prenatal diagnosis, and is a basis for genetic counseling.

Keywords: ATP7B gene, genetic testing, prenatal diagnosis, pedigree, Wilson disease

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Authors: Sami Ali Metwally, Bedour Helmy Abou-Leila, Hussien I.Abdel-Shafy

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The work was carried out at the greenhouse of National Research Centre, Pot experiment was carried out during of 2020 and 2021 seasons aimed to study the effect of two types of water (two recycling gray water treatments((SMR (Sequencing Batch Reactor) and MBR(Membrane Biology Reactor) and three watering intervals 15, 20 and 25 days on Syangonium plants growth. Examination of data cleared that, (MBR) recorded increase in vegetative growth parameters, osmotic pressure, transpiration rate chlorophyll a,b,carotenoids and carbohydrate)in compared with SBR.As for water, intervalsthe highest values of most growth parameters were obtained from plants irrigated with after (20 days) compared with other treatments.15 days irrigation intervals recorded significantly increased in osmotic pressure, transpiration rate and photosynthetic pigments, while carbohydrate values recorded decreased. Interaction between water type and water intervals(SBR) recorded the highest values of most growth parameters by irrigation after 20 days. While the treatment (MBR)and irrigated after 25 days showed the highest values on leaf area and leaves fresh weight compared with other treatments.

Keywords: grey water, water intervals, Syngonium plant, recycling water, vegetative growth

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Authors: T. Warinpramote, J. Sanguanjeen, P. Pholphakwaen, S. Kittisorayut, J. Kerdtubtim, S. Palachote, M. Taweechotipatr

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Cholesterol is a type of lipid molecule which is the significant risk factor for coronary heart disease. Currently, there are many cholesterol-lowering alternative treatments especially bile salt hydrolase positive lactobacilli. BSH can cleave the peptide linkage of bile salt, which results in removal of the amino acid group from the steroid core and increases production of the new bile acid by using more cholesterol. The purpose of this study was to isolate, and screen probiotic characteristics of lactobacilli from fermented Thai foods and further investigated for their comparative BSH activity. The result showed that 2 of 81 Lactobacillus strains, JPK2-2 and JPK3-2, isolated from Brassica juncea (L.) had significantly exhibited high BSH activity. In addition, these Lactobacillus strains showed their results that the ability to tolerate acid and bile salt. Furthermore, the using of 16S rDNA sequencing for definitive microbial identifications showed that these 2 strains belong to Lactobacillus plantarum. In the future, the L. plantarum with BSH activity strains JPK2-2 and JPK3-2 may be the candidate probiotics for application in functional foods and dairy products to improve in the patient with cardiovascular diseases.

Keywords: Lactobacillus plantarum, probiotics, bile salt hydrolase, cholesterol-lowering, fermented Thai food

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Authors: Kaustuvmani Patowary, Suresh Deka

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Oil pollution accidents, nowadays, have become a common phenomenon and have caused ecological and social disasters. Microorganisms with high oil-degrading performance are essential for bioremediation of petroleum hydrocarbon. In this investigation, an effective biosurfactant producer and hydrocarbon degrading bacterial strain, Pseudomonas sp.PG1 (identified by 16s rDNA sequencing) was isolated from hydrocarbon contaminated garage soil of Pathsala, Assam, India, using crude oil enrichment technique. The growth parameters such as pH and temperature were optimized for the strain and upto 81.8% degradation of total petroleum hydrocarbon (TPH) has been achieved after 5 weeks when grown in mineral salt media (MSM) containing 2% (w/v) crude oil as the carbon source. The biosurfactant production during the course of hydrocarbon degradation was monitored by surface tension measurement and emulsification activity. The produced biosurfactant had the ability to decrease the surface tension of MSM from 72 mN/m to 29.6 mN/m, with the critical micelle concentration (CMC)of 56 mg/L. The biosurfactant exhibited 100% emulsification activity on crude oil. FTIR spectroscopy and LCMS-MS analysis of the purified biosurfactant revealed that the biosurfactant is Rhamnolipidic in nature with several rhamnolipid congeners. Gas Chromatography-Mass spectroscopy (GC-MS) analysis clearly demonstrated that the strain PG1 efficiently degrade different hydrocarbon fractions of the crude oil. The study suggeststhat application of the biosurfactant producing strain PG1 as an appropriate candidate for bioremediation of crude oil contaminants.

Keywords: petroleum hydrocarbon, hydrocarbon contamination, bioremediation, biosurfactant, rhamnolipid

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Authors: Christian Liebchen

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Both from an environmental perspective and with respect to road traffic flow quality, planning so-called green waves along major road axes is a well-established target for traffic engineers. For one-way road axes (e.g. the Avenues in Manhattan), this is a trivial downstream task. For bidirectional arterials, the well-known necessary condition for establishing a green wave in both directions is that the driving times between two subsequent crossings must be an integer multiple of half of the cycle time of the signal programs at the nodes. In this paper, we propose an integer linear optimization model to establish fixed-time green waves in both directions that are as long and as wide as possible, even in the situation where the driving time condition is not fulfilled. In particular, we are considering an arterial along whose nodes separate left-turn signal groups are realized. In our computational results, we show that scheduling left-turn phases before or after the straight phases can reduce waiting times along the arterial. Moreover, we show that there is always a solution with green waves in both directions that are as long and as wide as possible, where absolute priority is put on just one direction. Compared to optimizing both directions together, establishing an ideal green wave into one direction can only provide suboptimal quality when considering prioritized parts of a green band (e.g., first few seconds).

Keywords: traffic light coordination, synchronization, phase sequencing, green waves, integer programming

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Authors: Domingos Viegas, Miguel Almeida, Carmen Rocha, Ilda Novo, Yolanda Luna

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Forest fires along the more than 1200km of the Spanish-Portuguese border are more and more frequent, currently achieving around 2000 fire events per year. Some of these events develop to large international wildfire requiring concerted operations based on shared information between the two countries. The fire event of Valencia de Alcantara (2003) causing several fatalities and more than 13000ha burnt, is a reference example of these international events. Currently, Portugal and Spain have a specific cross-border cooperation protocol on wildfires response for a strip of about 30km (15 km for each side). It is recognized by public authorities the successfulness of this collaboration however it is also assumed that this cooperation should include more functionalities such as the development of a common risk information system for transboundary fire events. Since Portuguese and Spanish authorities use different approaches to determine the fire risk indexes inputs and different methodologies to assess the fire risk, sometimes the conjoint firefighting operations are jeopardized since the information is not harmonized and the understanding of the situation by the civil protection agents from both countries is not unique. Thus, a methodology aiming the harmonization of the fire risk calculation and perception by Portuguese and Spanish Civil protection authorities is hereby presented. The final results are presented as well. The fire risk index used in this work is the Canadian Fire Weather Index (FWI), which is based on meteorological data. The FWI is limited on its application as it does not take into account other important factors with great effect on the fire appearance and development. The combination of these factors is very complex since, besides the meteorology, it addresses several parameters of different topics, namely: sociology, topography, vegetation and soil cover. Therefore, the meaning of FWI values is different from region to region, according the specific characteristics of each region. In this work, a methodology for FWI calibration based on the number of fire occurrences and on the burnt area in the transboundary regions of Portugal and Spain, in order to assess the fire risk based on calibrated FWI values, is proposed. As previously mentioned, the cooperative firefighting operations require a common perception of the information shared. Therefore, a common classification of the fire risk for the fire events occurred in the transboundary strip is proposed with the objective of harmonizing this type of information. This work is integrated in the ECHO project SpitFire - Spanish-Portuguese Meteorological Information System for Transboundary Operations in Forest Fires, which aims the development of a web platform for the sharing of information and supporting decision tools to be used in international fire events involving Portugal and Spain.

Keywords: data harmonization, FWI, international collaboration, transboundary wildfires

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Authors: A. Alyami, S. Christmas, K. Neeltje, G. Pollakis, B. Paxton, Z. Al-Bayati

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The Human leukocyte antigen-G (HLA-G) molecule plays an important role in immunomodulation. To date, 16 untranslated regions (UTR) HLA-G haplotypes have been previously defined by sequenced SNPs in the coding region. From these, UTR-1, UTR-2, UTR-3, UTR-4, UTR-5, UTR-6 and UTR-7 are the most frequent 3’UTR haplotypes at the global level. UTR-1 is associated with higher levels of soluble HLA-G and HLA-G expression, whereas UTR-5 and UTR-7 are linked with low levels of soluble HLA-G and HLA-G expression. Human immunodeficiency virus type 1 (HIV-1) infection results in the progressive loss of immune function in infected individuals. The virus escape mechanism typically includes T lymphocytes and NK cell recognition and lyses by classical HLA-A and B down-regulation, which has been associated with non-classical HLA-G molecule up-regulation, respectively. We evaluated the haplotypes of the HLA-G 3′ untranslated region frequencies observed in three HIV-1 groups from the Netherlands and their susceptibility to develop infection. The three groups are made up of mainly men who have sex with men (MSM), injection drug users (IDU) and a high-risk-seronegative (HRSN) group. DNA samples were amplified with published primers prior sequencing. According to our results, the low expresser frequencies show higher in HRSN compared to other groups. This is indicating that 3’UTR polymorphisms may be identified as potential prognostic biomarkers to determine susceptibility to HIV.

Keywords: Human leukocyte antigen-G (HLA-G) , men who have sex with men (MSM), injection drug users (IDU), high-risk-seronegative (HRSN) group, high-untranslated region (UTR)

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Authors: Katarina Pogacnik, Ursa Zakrajsek, Nejc Sirk, Ziga Lampret

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Due to an increase in living standards along with global population growth and a trend of urbanization, municipalities and regions are faced with an ever rising energy demand. A challenge has arisen for cities around the world to modify the energy supply chain in order to reduce its consumption and CO₂ emissions. The aim of our work is the development of a computational-analytical platform for dynamic support in decision-making and the determination of economic and technical indicators of energy efficiency in a smart city, named Energy Atlas. Similar products in this field focuse on a narrower approach, whereas in order to achieve its aim, this platform encompasses a wider spectrum of beneficial and important information for energy planning on a local or regional scale. GIS based interactive maps provide an extensive database on the potential, use and supply of energy and renewable energy sources along with climate, transport and spatial data of the selected municipality. Beneficiaries of Energy atlas are local communities, companies, investors, contractors as well as residents. The Energy Atlas platform consists of three modules named E-Planning, E-Indicators and E-Cooperation. The E-Planning module is a comprehensive data service, which represents a support towards optimal decision-making and offers a sum of solutions and feasibility of measures and their effects in the area of efficient use of energy and renewable energy sources. The E-Indicators module identifies, collects and develops optimal data and key performance indicators and develops an analytical application service for dynamic support in managing a smart city in regards to energy use and sustainable environment. In order to support cooperation and direct involvement of citizens of the smart city, the E-cooperation is developed with the purpose of integrating the interdisciplinary and sociological aspects of energy end-users. Interaction of all the above-described modules contributes to regional development because it enables for a precise assessment of the current situation, strategic planning, detection of potential future difficulties and also the possibility of public involvement in decision-making. From the implementation of the technology in Slovenian municipalities of Ljubljana, Piran, and Novo mesto, there is evidence to suggest that the set goals are to be achieved to a great extent. Such thorough urban energy planning tool is viewed as an important piece of the puzzle towards achieving a low-carbon society, circular economy and therefore, sustainable society.

Keywords: circular economy, energy atlas, energy management, energy planning, low-carbon society

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Authors: Gao Qin, Hu Liwei, Dong Xiangzhou, Zhu Qifa, Cheng Tingming, Zhao Limei, Yang Mengmeng, Zhai Zhen, Dai Huaxin, Liang Taibo, Zhang Shixiang, Xue Chaoqun

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The functional strain T976 for starch degradation was isolated from Nicotiana tabacum L. Yunyan 97 tobacco leaves, the ratio of starch hydrolysis transparent circle diameter to colony diameter of the strain was 4.14, 16S rDNA sequencing identified these strains as Enterobacter ludwigii. Then Enterobacter ludwigii T976 was fermented and spaying Yunyan 97 plant in vigorous growing stage. The results of once spraying fermentation broth of Enterobacter ludwigii T976 showed that starch content of upper leaves decreased slightly, from 3.77% to 3.1%, the reducing sugar content increased from 4.39% to 5.53%, and the total sugar content increased from 5.82% to 7.39%. The chemical content was also checked after three time spraying. The starch content of middle leaves decreased from 5.63% to 3.74%, while the content of total sugar and reducing sugar decreased slightly. And the starch content of upper leaves decreased from 7.62% to 4.78%, the total sugar and reducing sugar decreased slightly, and starch content of middle leaf decreased from 6.27% to 3.62%, the total sugar and reducing sugar did not change much, and other chemical components were in a suitable range.

Keywords: nicotiana tabacum, yunyan 97, leaf, starch, degradation, enterobacter ludwigii

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Authors: Abeer A. Q. Ahmed, Tracey McKay

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The increasing demand for renewable fuels and chemicals is pressuring manufacturing industry toward finding more sustainable cost-effective resources. Lignocellulose, such as agro-food wastes, is a suitable equivalent to petroleum for fine chemicals and fuels production. The complex structure of lignocellulose, however, requires a variety of enzymes in order to degrade its components into their respective building blocks that can be used further for the production of various value added products. This study aimed to isolate bacterial strain with the ability to produce a variety of lignocellulosic enzymes. One bacterial isolate was identified by 16S rRNA gene sequencing and phylogenetic analysis as Bacillus safensis LCX found to have CMCase, xylanase, manganese peroxidase, lignin peroxidase, and laccase activities. The enzymes production was induced by growing Bacillus safensis LCX in solid state fermentation using wheat straw, wheat bran, and corn stover. The activities of enzymes were determined by specific colorimetric assays. This study presents Bacillus safensis LCX as a promising source for lignocellulosic enzymes. These findings can extend the knowledge on agro-food wastes valorization strategies toward a sustainable production of fuels and chemicals.

Keywords: Bacillus safensis LCX, high valued chemicals, lignocellulosic enzymes, solid state fermentation

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Authors: H.-H. Doh, J.-M. Yu, Y.-J. Kwon, J.-H. Shin, H.-W. Kim, S.-H. Nam, D.-H. Lee

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This paper suggests a decision tree based approach for flexible job shop scheduling with multiple process plans, i. e. each job can be processed through alternative operations, each of which can be processed on alternative machines. The main decision variables are: (a) selecting operation/machine pair; and (b) sequencing the jobs assigned to each machine. As an extension of the priority scheduling approach that selects the best priority rule combination after many simulation runs, this study suggests a decision tree based approach in which a decision tree is used to select a priority rule combination adequate for a specific system state and hence the burdens required for developing simulation models and carrying out simulation runs can be eliminated. The decision tree based scheduling approach consists of construction and scheduling modules. In the construction module, a decision tree is constructed using a four-stage algorithm, and in the scheduling module, a priority rule combination is selected using the decision tree. To show the performance of the decision tree based approach suggested in this study, a case study was done on a flexible job shop with reconfigurable manufacturing cells and a conventional job shop, and the results are reported by comparing it with individual priority rule combinations for the objectives of minimizing total flow time and total tardiness.

Keywords: flexible job shop scheduling, decision tree, priority rules, case study

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Authors: A. I. Khalafalla, K. A. Al-Busada, I. M. El-Sabagh

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Pox and pox-like diseases of camels are a group of exanthematous skin conditions that have become increasingly important economically. They may be caused by three distinct viruses: camelpox virus (CMPV), camel contagious ecthyma virus (CCEV) and camel papillomavirus (CAPV). These diseases are difficult to differentiate based on clinical presentation in disease outbreaks. Molecular methods such as PCR targeting species-specific genes have been developed and used to identify CMPV and CCEV, but not simultaneously in a single tube. Recently, multiplex PCR has gained reputation as a convenient diagnostic method with cost- and time–saving benefits. In the present communication, we describe the development, optimization and validation a multiplex PCR assays able to detect simultaneously the genome of the three viruses in one single test allowing for rapid and efficient molecular diagnosis. The assay was developed based on the evaluation and combination of published and new primer sets, and was applied to the detection of 110 tissue samples. The method showed high sensitivity, and the specificity was confirmed by PCR-product sequencing. In conclusion, this rapid, sensitive and specific assay is considered a useful method for identifying three important viruses in specimens from camels and as part of a molecular diagnostic regime.

Keywords: multiplex PCR, diagnosis, pox and pox-like diseases, camels

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Authors: Abhishikta Ghosh Roy

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Breast cancer is the most common malignancy among women globally, including India. Human breast cancer results from the genetic and environmental interaction. The present study attempts to understand the molecular heterogeneity of BRCA1 and BRCA2 genes, as well as to understand the association of various lifestyle and reproductive variables for the Breast Cancer risk. The study was conducted amongst 110 patients and 128 controls with total DNA sequencing of flanking and coding regions of BRCA1 BRCA2 genes that revealed ten Single Nucleotide Polymorphisms (SNPs) (6 novels). The controls selected for the study were age, sex and ethnic group matched. After written and informed consent biological samples were collected from the subjects. After detailed molecular analysis, significant (p < 0.005) molecular heterogeneity is revealed in terms of SNPs in BRCA1 (4 Exonic & 1 Intronic) and BRCA2 (2exonic and 3 Intronic) genes. The augmentation study investigated significant (p < 0.05) association with positive family history, early age at menarche, irregular menstrual periods, menopause, prolong contraceptive use, nulliparity, history of abortions, consumption of alcohol and smoking for breast cancer risk. To the best of authors knowledge, this study is the first of its kind, envisaged that the identification of the SNPs and modification of the lifestyle factors might aid to minimize the risk among the Bengalee Hindu females.

Keywords: breast cancer, BRCA, lifestyle, India

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Authors: Syed M. Shahid, Rozeena Shaikh, Syeda N. Nawab, Abid Azhar

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Diabetes mellitus (DM) is a prevalent non-communicable disease worldwide. DM may lead to many vascular complications like hypertension, nephropathy, retinopathy, neuropathy and foot infections. Pathogenesis of diabetic nephropathy (DN) is implicated by the polymorphisms in genes encoding the specific components of renin angiotensin aldosterone system (RAAS) which include angiotensinogen (AGT), angiotensin-II receptor and angiotensin converting enzyme (ACE) genes. This study was designed to explore the possible association of AG (M268T) polymorphism in the patients of diabetes and nephropathy in Pakistan. Study subjects included 100 controls, 260 diabetic patients without renal insufficiency and 190 diabetic nephropathy patients with persistent albuminuria. Fasting blood samples were collected from all the subjects after getting institutional ethical approval and informed consent. The biochemical estimations, PCR amplification and direct sequencing for the specific region of AGT gene was carried out. A significantly high frequency of TT genotype and T allele of AGT (M268T) was observed in the patients of diabetes with nephropathy as compared to controls and diabetic patients without any known renal impairment. The TT genotype and T allele of AGT (M268T) polymorphism may be considered as a genetic risk factor for the development and progression of nephropathy in diabetes. Further cross sectional population studies would be of help to establish and confirm the observed possible association of AGT gene variations with development of nephropathy in diabetes.

Keywords: RAAS, AGT (M268T), diabetes, nephropathy

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Authors: Nan Deng, Zhenqiu Liu

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Thousands of patients with metastatic tumors were diagnosed with cancers of unknown primary sites each year. The inability to identify the primary cancer site may lead to inappropriate treatment and unexpected prognosis. Nowadays, a large amount of genomics and transcriptomics cancer data has been generated by next-generation sequencing (NGS) technologies, and The Cancer Genome Atlas (TCGA) database has accrued thousands of human cancer tumors and healthy controls, which provides an abundance of resource to differentiate cancer types. Meanwhile, deep convolutional neural networks (CNNs) have shown high accuracy on classification among a large number of image object categories. Here, we utilize 25 cancer primary tumors and 3 normal tissues from TCGA and convert their RNA-Seq gene expression profiling to color images; train, validate and test a CNN classifier directly from these images. The performance result shows that our CNN classifier can archive >80% test accuracy on most of the tumors and normal tissues. Since the gene expression pattern of distant metastases is similar to their primary tumors, the CNN classifier may provide a potential computational strategy on identifying the unknown primary origin of metastatic cancer in order to plan appropriate treatment for patients.

Keywords: bioinformatics, cancer, convolutional neural network, deep leaning, gene expression pattern

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Authors: Dalia. G. Aseel

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Begomoviruses are economically important plant viruses that infect dicotyledonous plants and exclusively transmitted by the whitefly Bemisia tabaci. Here, replicative form was isolated from Okra, Cotton, Tomato plants and whitefly infected with Begomoviruses. Using coat protein specific primers (AV1), the viral infection was verified with amplicon at 450 bp. The sequence of OLCuV-AV1 gene was recorded and received an accession number (FJ441605) from Genebank. The phylogenetic tree of OLCuV was closely related to Okra leaf curl virus previously isolated from Cameroon and USA with nucleotide sequence identity of 92%. The protein purification was carried out using His-Tag methodology by using Affinity Chromatography. The purified protein was separated on SDS-PAGE analysis and an enriched expected size of band at 30 kDa was observed. Furthermore, RAPD and SDS-PAGE were used to detect genetic variability between different hosts of okra leaf curl virus (OLCuV), cotton leaf curl virus (CLCuV), tomato yellow leaf curl virus (TYLCuV) and the whitefly vector. Finally, the present study would help to understand the relationship between the whitefly and different economical crops in Egypt.

Keywords: okra leaf curl virus, AV1 gene, sequencing, phylogenetic, cloning, purified protein, genetic diversity and viral proteins

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Authors: S. Hallal, Z. Chami, A. Hadji-Lehtihet, S. Sokhal-Boudella, A. Berhoune, L. Yargui

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Gaucher disease is the most common lysosomal storage in our population, it is due to a deficiency of β –glucosidase acid. The enzyme deficiency causes a pathological accumulation of undegraded substrate in lysosomes. This metabolic overload is responsible for a multisystemic disease with hepatosplenomegaly, anemia, thrombocytopenia, and bone involvement. Neurological involvement is rare. The laboratory diagnosis of Gaucher disease consists of phenotypic diagnosis by determining the enzymatic activity of β - glucosidase by fluorimetric method, a study by genotypic diagnosis in the GBA gene, limiting the search recurrent mutations (N370S, L444P, 84 GG); PCR followed by an enzymatic digestion. Abnormal profiles were verified by sequencing. Monitoring of treated patients is provided by the determination of chitotriosidase. Our experience spaning a period of 6 years (2007-2014) has enabled us to diagnose 78 patients out of a total of 328 requests from the various departments of pediatrics, internal medicine, neurology. Genotypic diagnosis focused on the entire family of 9 children treated at pediatric CHU Mustapha, which help define the clinical form; or 5 of them had type III disease, carrying the L444P mutation in the homozygous state. Three others were composite (N370/L444P) (N370S/other unintended mutation in our study), and only in one family no recurrent mutation has been found. This molecular study permits screening of heterozygous essential for genetic counseling.

Keywords: Gaucher disease, mutations, N370S, L444P

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Authors: Isaac Quiros-Fernandez, Angel Cid-Arregui

Abstract:

Tumor-reactive T cell receptors (TCRs) are being subject of intense investigation since they offer great potential in adoptive cell therapies against cancer. However, the identification of tumor-specific TCRs has proven challenging, for instance, due to the limited expansion capacity of tumor-infiltrating T cells (TILs) and the extremely low frequencies of tumor-reactive T cells in the repertoire of patients and healthy donors. We have developed an approach for rapid identification and characterization of neoepitope-reactive TCRs from the T cell repertoire of healthy donors. CD8 T cells isolated from multiple donors are subjected to a first sorting step after staining with HLA multimers carrying the peptide of interest. The isolated cells are expanded for two weeks, after which a second sorting is performed using the same peptide-HLA multimers. The cells isolated in this way are then processed for single-cell sequencing of their TCR alpha and beta chains. Newly identified TCRs are cloned in appropriate expression vectors for functional analysis on Jurkat, NK92, and primary CD8 T cells and tumor cells expressing the appropriate antigen. We have identified TCRs specifically binding HLA-A2 presenting epitopes of tumor antigens, which are capable of inducing TCR-mediated cell activation and cytotoxicity in target cancer cell lines. This method allows the identification of tumor-reactive TCRs in about two to three weeks, starting from peripheral blood samples of readily available healthy donors.

Keywords: cancer, TCR, tumor antigens, immunotherapy

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Authors: Muna A. Eissawi, Safaa Abed Elfataah, Haytham Hago, Fatima E Abukunna, Ibtisam Amin Goreish, Nahid Gornas

Abstract:

The study examined and analyzed the genetic diversity of DRB1locus of exon 2 of major histocompatibility complex of Sudanese desert sheep using PCR-RFLP and DNA sequencing. Five hundred samples belonging to five ecotypes of Desert Sudanese sheep (Abrag (Ab), Ashgar (Ash), Hamari (H), Kabashi (K) and Watish (W) were included. Amplification of exon 2 of the DRB1 gene yielded (300bp) amplified product in different ecotypes. Nine different digestion patterns corresponding to Five distinct alleles were observed with Rsa1 digestion. Genotype (ag) was the most common among all ecotypes, with a percentage comprised (40.4 %). The Hardy-Weinberg equilibrium (HWE) test showed that the studied ecotypes have significantly deviated from the theoretical proportions of Rsa1 patterns; probability values of the Chi-square test for HWE for MHC-DRB1 gene in SDS were 0.00 in all ecotypes. The constructed phylogenetic tree revealed the relation of 22 Sudanese isolates with each other and showed the shared sequences with 47 published foreign sequences randomly selected from different geographic regions. The results of this study highlight the effect of heterozygosity of MHC genes of the Desert sheep of Sudan which may clarify some of genetic back ground of their disease resistance and adaptation to environment.

Keywords: desert sheep, MHC, Ovar-DRB1, polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP)

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Authors: Parmjit S. Panesar, Rupinder Kaur, Ram S. Singh

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Enzymes are the biocatalysts which catalyze the biochemical processes and thus have a wide variety of applications in the industrial sector. β-Galactosidase (E.C. 3.2.1.23) also known as lactase, is one of the prime enzymes, which has significant potential in the dairy and food processing industries. It has the capability to catalyze both the hydrolytic reaction for the production of lactose hydrolyzed milk and transgalactosylation reaction for the synthesis of prebiotics such as lactulose and galactooligosaccharides. These prebiotics have various nutritional and technological benefits. Although, the enzyme is naturally present in almonds, peaches, apricots and other variety of fruits and animals, the extraction of enzyme from these sources increases the cost of enzyme. Therefore, focus has been shifted towards the production of low cost enzyme from the microorganisms such as bacteria, yeast and fungi. As compared to yeast and bacteria, fungal β-galactosidase is generally preferred as being extracellular and thermostable in nature. Keeping the above in view, the present study was carried out for the isolation of the β-galactosidase producing fungal strain from the food as well as the agricultural wastes. A total of more than 100 fungal cultures were examined for their potential in enzyme production. All the fungal strains were screened using X-gal and IPTG as inducers in the modified Czapek Dox Agar medium. Among the various isolated fungal strains, the strain exhibiting the highest enzyme activity was chosen for further phenotypic and genotypic characterization. The strain was identified as Rhizomucor pusillus on the basis of 5.8s RNA gene sequencing data.

Keywords: beta-galactosidase, enzyme, fungal, isolation

Procedia PDF Downloads 223

Authors: M. Błoch, P. Karpiński, P. Gasperowicz, R. Płoski, A. Lebioda, P. Skiba, A. Rozensztrauch, D. Patkowski, R. Śmigiel

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Introduction: The cause of the isolated form of esophageal atresia has been yet unknown. Objectives: The primary objective of this study was to indicate epigenetic factors which may play an important role in the etiopathogenesis of esophageal atresia. Methods: We recruited a group of 6 pairs of twins, among whom one of the twins developed EA. The selection of such a group for testing allows for excluding external factors (e.g., infections, drugs, toxins) as the cause of the birth defect. The analyzes were performed with the use of genetic material isolated from the whole blood and esophagus tissue of a patient with EA. The reduced representation bisulphite sequencing (RRBS) technique was used to study the change in the genomic imprinting -a change in the expression of genes, which may be the epigenetic cause of EA. Results: In the course of the analyzes, significant hypomethylation and hypermethylation regions were identified. 65 genes with probably increased expression and 65 with decreased expression were selected. These genes have not been marked in literature as possibly pathogenic in esophageal atresia. However, their participation in the pathogenesis of esophageal atresia cannot be clearly excluded. Conclusion: We suggest a role of hypomethylation or hypermethylation of selected genes as one of the possible epigenetic factors in EA pathogenesis. The use of the RRBS technique in the search for the cause of EA is pioneer research; therefore, it seems necessary to extend the research group to new patients with EA. Acknowledgment: The work was supported by the National Science Centre, Poland, under research project 2016/21/N/NZ5/01927.

Keywords: esophageal atresia, epigenetics, embryonic development, surgery, genes expression, twins

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Authors: Mia Bahar, Özlem Bozkurt

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Obsessive-compulsive disorder (OCD) is a typical, persistent, and long-lasting mental health condition in which a person experiences uncontrollable, recurrent thoughts (or "obsessions") and/or activities (or "compulsions") that they feel compelled to engage in repeatedly. Obsessive-compulsive disorder is both underdiagnosed and undertreated. It frequently manifests in a variety of medical settings and is persistent, expensive, and burdensome. Obsessive-compulsive neurosis was long believed to be a condition that offered valuable insight into the inner workings of the unconscious mind. Obsessive-compulsive disorder is now recognized as a prime example of a neuropsychiatric condition susceptible to particular pharmacotherapeutic and psychotherapy therapies and mediated by pathology in particular neural circuits. An obsessive-compulsive disorder which is called OCD, usually has two components, one cognitive and the other behavioral, although either can occur alone. Obsessions are often repetitive and intrusive thoughts that invade consciousness. These obsessions are incredibly hard to control or dismiss. People who have OCD often engage in rituals to reduce anxiety associated with intrusive thoughts. Once the ritual is formed, the person may feel extreme relief and be free from anxiety until the thoughts of contamination intrude once again. These thoughts are strengthened through a manifestation of negative reinforcement because they allow the person to avoid anxiety and obscurity. These thoughts are described as autogenous, meaning they most likely come from nowhere. These unwelcome thoughts are related to actions which we can describe as Thought Action Fusion. The thought becomes equated with an action, such as if they refuse to perform the ritual, something bad might happen, and so people perform the ritual to escape the intrusive thought. In almost all cases of OCD, the person's life gets extremely disturbed by compulsions and obsessions. Studies show OCD is an estimated 1.1% prevalence, making it a challenging issue with high co-morbidities with other issues like depressive episodes, panic disorders, and specific phobias. The first to reveal brain anomalies in OCD were numerous CT investigations, although the results were inconsistent. A few studies have focused on the orbitofrontal cortex (OFC), anterior cingulate gyrus (AC), and thalamus, structures also implicated in the pathophysiology of OCD by functional neuroimaging studies, but few have found consistent results. However, some studies have found abnormalities in the basal ganglion. There have also been some discussions that OCD might be genetic. OCD has been linked to families in studies of family aggregation, and findings from twin studies show that this relationship is somewhat influenced by genetic variables. Some Research has shown that OCD is a heritable, polygenic condition that can result from de novo harmful mutations as well as common and unusual variants. Numerous studies have also presented solid evidence in favor of a significant additive genetic component to OCD risk, with distinct OCD symptom dimensions showing both common and individual genetic risks.

Keywords: compulsions, obsessions, neuropsychiatric, genetic

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Authors: James Sinclair, Katy Soapi, Brad Carte

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The marine environment has continuously demonstrated to be a rich source of secondary metabolites and bioactive compounds that can address the many pharmaceutical problems facing mankind. The emergence of multidrug resistant pathogens has caused scientists to explore contemporary ways of combating these super bugs. A red-pigmented bacterial strain isolated from a marine alga collected in Fiji was identified to be Pseudoalteromonas rubra from 16s rRNA sequencing. This bacterial strain was cultured using a yeast-peptone media and incubated for five days. The ethyl acetate extract of this bacterium was subjected to chromatographic separation techniques such as vacuum liquid chromatography, flash chromatography, size exclusion chromatography and high-pressure liquid chromatography to yield the pure compound and a number of semi-pure fractions. The crude extract and subsequent purified fractions were analyzed by ultraviolet/visible spectroscopy and mass spectroscopy and was found to contain the compounds ivermectin, stenothricin, cyclo-L-pro-L-val, prodigiosin, mycophenolic acid, phenazine-1-carboxylic acid, eplerenone, staurosporine and pseudoalteromone A. The structure of the pure compound, pseudoalteromone A, was elucidated using NMR 1H, 13C, 1H-1H COSY, HSQC and HMBC spectroscopic data.

Keywords: Pseudoalteromonas rubra, Pseudoalteromone A, secondary metabolites, structure elucidation

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Authors: M. Zafar, S. Rasheed, Imran Hashmi

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Very little is concerned about the bacterial contamination in drinking water biofilm which provide a potential source for bacteria to grow and increase rapidly. So as to understand the microbial density in DWDs, a three-month study was carried out. The aim of this study was to examine biofilm in three different pipe materials including PVC, PPR and GI. A set of all these pipe materials was installed in DWDs at nine different locations and assessed on monthly basis. Drinking water quality was evaluated by different parameters and characterization of biofilm. Among various parameters are Temperature, pH, turbidity, TDS, electrical conductivity, BOD, COD, total phosphates, total nitrates, total organic carbon (TOC) free chlorine and total chlorine, coliforms and spread plate counts (SPC) according to standard methods. Predominant species were Bacillus thuringiensis, Pseudomonas fluorescens , Staphylococcus haemolyticus, Bacillus safensis and significant increase in bacterial population was observed in PVC pipes while least in cement pipes. The quantity of DWDs bacteria was directly depended on biofilm bacteria and its increase was correlated with growth and detachment of bacteria from biofilms. Pipe material also affected the microbial community in drinking water distribution network biofilm while Similarity in bacterial species was observed between systems due to same disinfectant dose, time period and plumbing pipes.

Keywords: biofilm, DWDs, pipe material, bacterial population

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Authors: Sarisha Devnath, Oluwatosin A. Ijabadeniyi

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In recent years milk contamination has become a major problem in households; due to the likely occurrence of bacteria, even after the milk has been processed. One such genus of bacteria causing unwanted growth is Bacillus. This research project looks at the presence of spore formers in processed milk from household refrigerators and the effect of pasteurization and high temperature on Bacillus spores activation. 24 samples each of UHT milk and pasteurised milk from 24 households were sampled for the presence of spore formers. While anaerobic spore formers were not found in any of the samples, the average aerobic spore formers in UHT milk and pasteurized milk however were 5.77 cfu/ml and 5.88 cfu/ml respectively. After sequencing, it was detected that the mixed culture contained Bacillus cereus, for both pasteurised and UHT milk samples. For the activation study, raw milk samples were collected and subjected to four different temperatures; 65˚C, 72˚C, 80˚C, 100˚C respectively. Samples were stored for 7 days at 5˚C and 10˚C and analysed daily. The average aerobic spore formers in raw milk for samples stored at 5˚C range between 4.67-6.00 cfu/ml while it ranges between 4.84-6.00 cfu/ml at 10˚C, signifying that the high temperatures could have resulted in germination of dominant spores. Statistical analysis conducted on these results indicated a significant difference between the numbers of colonies present at the different treatment temperatures the bacterium was exposed to. This work showed that household milk may constitute public health risk furthermore; pasteurization and higher temperatures may not be effective to remove aerobic spore formers because of Bacillus spores activation.

Keywords: sporeformers, bacillus, spores, activation, milk

Procedia PDF Downloads 413