Search results for: gene amplification

Authors: Etienne K. Ngoy

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This analysis suggests that the comprehensive degradation caused by any environmental factor on fiber reinforced plastics under mechanical stress can be measured as a change in viscoelastic properties of the material. The change in viscoelastic characteristics is experimentally determined as a time-dependent function expressing the amplification of the stress relaxation. The variation of this experimental function provides a measure of the environmental degradation rate. Where real service environment conditions can be reliably simulated in the laboratory, it is possible to generate master curves that include environmental degradation effect and hence predict the durability of the fiber reinforced plastics under environmental degradation.

Keywords: environmental effects, fiber reinforced plastics durability, prediction, stress effect

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Authors: Swarkar Sharma, Ekta Rai, Ankit Mahajan, Parvinder Kumar, Manoj K Dhar, Sushil Razdan, Kumarasamy Thangaraj, Carol Wise, Shiro Ikegawa M.D., K.K. Pandita M.D.

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Rare disorders are poorly understood hence, remain uncharacterized or patients are misdiagnosed and get poor medical attention. A rare mysterious skeletal disorder that remained unidentified for decades and rendered many people physically challenged and disabled for life has been reported in an isolated remote village ‘Arai’ of Poonch district of Jammu and Kashmir. This village is located deep in mountains and the population residing in the region is highly consanguineous. In our survey of the region, 70 affected people were reported, showing similar phenotype, in the village with a population of approximately 5000 individuals. We were able to collect samples from two multi generational extended families from the village. Through Whole Exome sequencing (WES), we identified a rare variation NM_003880.3:c.156C>A NP_003871.1:p.Cys52Ter, which results in introduction of premature stop codon in WISP3 gene. We found this variation perfectly segregating with the disease in one of the family. However, this variation was absent in other family. Interestingly, a novel splice site mutation at position c.643+1G>A of WISP3 gene, perfectly segregating with the disease was observed in the second family. Thus, exploiting WES and putting different evidences together (familial histories and genetic data, clinical features, radiological and biochemical tests and findings), the disease has finally been diagnosed as a very rare recessive hereditary skeletal disease “Progressive Pseudorheumatoid Arthropathy of Childhood” (PPAC) also known as “Spondyloepiphyseal Dysplasia Tarda with Progressive Arthropathy” (SEDT-PA). This genetic characterization and identification of the disease causing mutations will aid in genetic counseling, critically required to curb this rare disorder and to prevent its appearance in future generations in the population. Further, understanding of the role of WISP3 gene the biological pathways should help in developing treatment for the disorder.

Keywords: whole exome sequencing, Next Generation Sequencing, rare disorders

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Authors: Hongmeng Su, Luyu Zhao, Yanyan Qian, Hong Fan

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Aim: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors that endanger human health seriously. RNA methylation, especially N6-methyladenosine (m⁶A) and 5-methylcytosine (m⁵C), a crucial epigenetic transcriptional regulatory mechanism, plays an important role in tumorigenesis, progression and prognosis. This research aims to systematically evaluate the prognostic value of m⁶A and m⁵C modulators in HCC patients. Methods: Twenty-four modulators of m⁶A and m⁵C were candidates to analyze their expression level and their contribution to predict the prognosis of HCC. Consensus clustering analysis was applied to classify HCC patients. Cox and LASSO regression were used to construct the risk model. According to the risk score, HCC patients were divided into high-risk and low/medium-risk groups. The clinical pathology factors of HCC patients were analyzed by univariate and multivariate Cox regression analysis. Results: The HCC patients were classified into 2 clusters with significant differences in overall survival and clinical characteristics. Nine-gene risk model was constructed including METTL3, VIRMA, YTHDF1, YTHDF2, NOP2, NSUN4, NSUN5, DNMT3A and ALYREF. It was indicated that the risk score could serve as an independent prognostic factor for patients with HCC. Conclusion: This study constructed a Nine-gene risk model by modulators of m⁶A and m⁵C and investigated its effect on the clinical prognosis of HCC. This model may provide important consideration for the therapeutic strategy and prognosis evaluation analysis of patients with HCC.

Keywords: hepatocellular carcinoma, m⁶A, m⁵C, prognosis, RNA methylation

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Authors: Karthik Mittal

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This study performs an unsupervised machine learning analysis to find clusters of related SNPs which highlight biological pathways that are important for the biological mechanisms of breast cancer. Studying genetic variations in isolation is illogical because these genetic variations are known to modulate protein production and function; the downstream effects of these modifications on biological outcomes are highly interconnected. After extracting the SNPs and their effect on different types of breast cancer using the MRBase library, two unsupervised machine learning clustering algorithms were implemented on the genetic variants: a k-means clustering algorithm and a hierarchical clustering algorithm; furthermore, principal component analysis was executed to visually represent the data. These algorithms specifically used the SNP’s beta value on the three different types of breast cancer tested in this project (estrogen-receptor positive breast cancer, estrogen-receptor negative breast cancer, and breast cancer in general) to perform this clustering. Two significant genetic pathways validated the clustering produced by this project: the MAPK signaling pathway and the connection between the BRCA2 gene and the ESR1 gene. This study provides the first proof of concept showing the importance of unsupervised machine learning in interpreting GWAS summary statistics.

Keywords: breast cancer, computational biology, unsupervised machine learning, k-means, PCA

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Authors: M. Vivodík, Ž. Balážová, Z. Gálová

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The aim of this work was to detect genetic variability among the set of 40 castor genotypes using 8 RAPD markers. Amplification of genomic DNA of 40 genotypes, using RAPD analysis, yielded in 66 fragments, with an average of 8.25 polymorphic fragments per primer. Number of amplified fragments ranged from 3 to 13, with the size of amplicons ranging from 100 to 1200 bp. Values of the polymorphic information content (PIC) value ranged from 0.556 to 0.895 with an average of 0.784 and diversity index (DI) value ranged from 0.621 to 0.896 with an average of 0.798. The dendrogram based on hierarchical cluster analysis using UPGMA algorithm was prepared and analyzed genotypes were grouped into two main clusters and only two genotypes could not be distinguished. Knowledge on the genetic diversity of castor can be used for future breeding programs for increased oil production for industrial uses.

Keywords: dendrogram, polymorphism, RAPD technique, Ricinus communis L.

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Authors: David Sarpong, Waleed Khursheed, Ernest Danquah, Erin Murphy

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Shigella is a pathogenic bacterium responsible for shigellosis, a severe diarrheal disease that claims the lives of immunocompromised individuals worldwide. To develop therapeutics against this disease, an understanding of the molecular mechanisms underlying the pathogen’s physiology is crucial. Small non-coding RNAs (sRNAs) have emerged as important regulators of bacterial physiology, including as components of toxin-antitoxin systems. In this study, we investigated the role of RyfA in S. flexneri physiology and virulence. RyfA, originally identified as an sRNA in Escherichia coli, is conserved within the Enterobacteriaceae family, including Shigella. Whereas two copies of ryfA are present in S. dysenteriae, all other Shigella species contain only one copy of the gene. Additionally, we identified a putative open reading frame within the RyfA transcript, suggesting that it may be a dual-functioning gene encoding a small protein in addition to its sRNA function. To study ryfA in vitro, we cloned the gene into an inducible plasmid and observed the effect on bacterial growth. Here, we report that RyfA production inhibits the growth of S. flexneri, and this inhibition is dependent on the contained open reading frame. In-silico analyses have revealed the presence of two divergently transcribed sRNAs, RyfB1 and RyfB2, which share nucleotide complementarity with RyfA and thus are predicted to function as anti-toxins. Our data demonstrate that RyfB2 has a stronger antitoxin effect than RyfB1. This regulatory pattern suggests a novel form of a toxin-antitoxin system in which the activity of a single toxin is inhibited to varying degrees by two sRNA antitoxins. Studies are ongoing to investigate the regulatory mechanism(s) of the antitoxin genes, as well as the downstream targets and mechanism of growth inhibition by the RyfA toxin. This study offers distinct insights into the regulatory mechanisms underlying Shigella physiology and may inform the development of new anti-Shigella therapeutics.

Keywords: sRNA, shigella, toxin-antitoxin, Type 1 toxin antitoxin

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Authors: Amani Ashari, Julia Omar, Arif Hashim, Shahrul Hamid

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Apolipoprotein E (APOE) gene polymorphism has influence on serum lipids which relates to cardiovascular risk. The purpose of this study was to determine the frequency distribution of APOE alleles among Malaysian Type 2 Diabetes Mellitus (DM) patients with and without coronary artery disease (CAD) and their association with serum lipid profiles. A total of 115 patients were recruited in which 78 patients had Type 2 DM without CAD and 37 patients had Type 2 DM with CAD. The APOE polymorphism was detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The APOE ɛ3 allele was the most common one in both groups. There was no significant association between the APOE genotypes and the CAD status in Type 2 DM using Pearson χ² test. Further analysis indicated there were no significant differences in all lipid parameters between E2, E3 and E4 subgroups in both groups. The study showed that the E4 allele carriers of Type 2 DM with CAD patients had higher LDL-C level and lower HDL-C level compared to the other allele carriers. However, analyses showed these levels were not statistically different. The study also showed that the Type 2 DM with CAD group with E2 allele had higher triglyceride (TG). In conclusion, further study with larger sample size is needed to confirm role of E4 as a marker of CAD among Type 2 DM patients in Malaysian population.

Keywords: Apolipoprotein E, diabetes mellitus, cardiovascular disease, lipids

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Authors: S. J. Sirima, C. Thirawan, R.Puntharikorn, K. Ungsumalin, J. Kaemwich

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Acinetobacter spp. is a gram negative bacterium causing the high incidence of multi-drug resistance in patients admitted to an intensive care unit. A hundred isolates of Imipenem-resistant Acinetobacter spp. isolated from patients admitted at tertiary health care center, Northeastern region, Ubon Ratchathani, Thailand, were subjected to modified Hodge test and combined disc test in order to evaluate the production of carbapenemases. The results revealed that about 35% of isolates were found to be carbapenemases producers. In addition, multiplex polymerase chain reactions were performed to detect blaOXA-like genes. It showed that 92% of isolates possess blaOXA-51-like and blaOXA-23-like genes. However, blaOXA-58-like gene was detected in only 8 isolates. No detection of blaOXA-24-like gene was observed in all isolates. In conclusion, an ability to produce carbepenemases would be an important mechanism of multi-drug resistance among clinical isolates of Acinetobacter spp. at tertiary health care center, Northeastern region, Ubon Ratchathani, Thailand. Furthermore, it was likely that the class D carbapenemases genes, blaOXA-51-like and blaOXA-23-like, might contribute to imipenem-resistance exhibiting among isolates.

Keywords: Acinetobacter spp., blaOXA-like genes, carbapenemases, tertiary health care center

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Authors: Komal Vig, Syamala Soumyakrishnan, Yadav Baral

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Bypass surgery, using the autologous vein has been one of the most effective treatments for cardiovascular diseases (CVD). More recently tissue engineering including engineered vascular grafts to synthesize blood vessels is gaining usage. Dacron and ePTFE has been employed for vascular grafts, however, these does not work well for small diameter grafts (<6 mm) due to intimal hyperplasia and thrombosis. In the present study PTFE was treated with LTP to improve the endothelialization of intimal surface of graft. Scaffolds were also modified with polyvinylpyrrolidone coated silver nanoparticles (Ag-PVP) and the antimicrobial peptides, p753 and p359. Human umbilical vein endothelial cells (HUVEC) were plated on the developed scaffolds and cell proliferation was determined by the MTT assay. Cells attachment on scaffolds was visualized by microscopy. mRNA expressions levels of different cell markers were investigated using quantitative real-time PCR (qPCR). X ray photoelectron spectroscopic confirmed the introduction of oxygenated functionalities from LTP air plasma. Microscopic and MTT assays indicated increase in cell viability in LTP treated scaffolds. Gene expression studies shows enhanced expression of cell adhesion marker Integrin- α 5 gene after LTP treatment. The KB test displayed a zone of inhibition for Ag-PVP, p753 and p359 of 19mm, 14mm, and 12mm respectively. To determine toxicity of antimicrobial agents to cells, MTT Assay was performed using HEK293 cells. MTT Assay exhibited that Ag-PVP and the peptides were non-toxic to cells at 100μg/mL and 50μg/mL, respectively. Live/dead analysis and plate count of treated bacteria exhibited bacterial inhibition on develop scaffold compared to non-treated scaffold. SEM was performed to analyze the structural changes of bacteria after treatment with antimicrobial agents. Gene expression studies were conducted on RNA from bacteria treated with Ag-PVP and peptides using qRT-PCR. Based on our initial results, more scaffolds alternatives will be developed and investigated for cell growth and vascularization studies.

Keywords: low temperature plasma, vascular graft, HUVEC cells, antimicrobial

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Authors: Saima Saleem, Zubair Abbasi, Abdul Hameed, Mansoor Ahmed Khan, Navid Rashid Qureshi, Abid Azhar

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Oral Squamous Cell Carcinoma (OSCC) is the leading cause of death in the developing countries like Pakistan. This problem aggravates because of the excessive use of available chewing products. In spite of widespread information on their use and purported legislations against their use the Pakistani markets are classical examples of selling chewable carcinogenic mutagens. Reported studies indicated that these products are rich in reactive oxygen species (ROS) and polyphenols. TP53 gene is involved in the suppression of tumor. It has been reported that somatic mutations caused by TP53 gene are the foundation of the cancer. This study aims to find the loss of TP53 functions due to mutation/polymorphism caused by genomic alteration and interaction with tobacco and its related ingredients. Total 260 tissues and blood specimens were collected from OSCC patients and compared with age and sex matched controls. Mutations in exons 2-11 of TP53 were examined by PCR-SSCP. Samples showing mobility shift were directly sequenced. Two mutations were found in exon 4 at nucleotide position 108 and 215 and one in exon 7 at nucleotide position 719 of the coding sequences in patient’s tumor samples. These results show that substitution of proline with arginine at codon 72 and serine with threonine at codon 240 of p53 protein. These polymorphic changes, found in tumor samples of OSCC, could be involved in loss of heterozygocity and apoptotic activity in the binding domain of TP53. The model of the mutated TP53 gene elaborated a nonfunctional unfolded p53 protein, suggesting an important role of these mutations in p53 protein inactivation and malfunction. This nonfunctional 3D model also indicates that exogenous tobacco related carcinogens may act as DNA-damaging agents affecting the structure of DNA. The interpretations could be helpful in establishing the pathways responsible for tumor formation in OSCC patients.

Keywords: TP53 mutation/polymorphism, OSCC, PCR-SSCP, direct DNA sequencing, 3D structure

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Authors: Batul Kagalwala

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The nervous system is made up of complex delicate structures such as the spinal cord, peripheral nerves and the brain. These are prone to various types of injury ranging from neurodegenerative diseases to trauma leading to diseases like Parkinson's, Alzheimer's, multiple sclerosis, amyotrophic lateral sclerosis (ALS), multiple system atrophy etc. Unfortunately, because of the complicated structure of nervous system, spontaneous regeneration, repair and healing is seldom seen due to which brain damage, peripheral nerve damage and paralysis from spinal cord injury are often permanent and incapacitating. Hence, innovative and standardized approach is required for advance treatment of neurological injury. Nigella sativa (N. sativa), an annual flowering plant native to regions of southern Europe and Asia; has been suggested to have neuroprotective and anti-seizures properties. Neuroregeneration is found to occur in damaged cells when treated using extract of N. sativa. Due to its proven health benefits, lots of experiments are being conducted to extract all the benefits from the plant. The flowers are delicate and are usually pale blue and white in color with small black seeds. These seeds are the source of active components such as 30–40% fixed oils, 0.5–1.5% essential oils, pharmacologically active components containing thymoquinone (TQ), ditimoquinone (DTQ) and nigellin. In traditional medicine, this herb was identified to have healing properties and was extensively used Middle East and Far East for treating diseases such as head ache, back pain, asthma, infections, dysentery, hypertension, obesity and gastrointestinal problems. Literature studies have confirmed the extract of N. sativa seeds and TQ have inhibitory effects on inducible nitric oxide synthase and production of nitric oxide as well as anti-inflammatory and anticancer activities. Experimental investigation will be conducted to understand which ingredient of N. sativa causes neuroregeneration and roots to its healing property. An aqueous/ alcoholic extract of N. sativa will be made. Seed oil is also found to have used by researchers to prepare such extracts. For the alcoholic extracts, the seeds need to be powdered and soaked in alcohol for a period of time and the alcohol must be evaporated using rotary evaporator. For aqueous extracts, the powder must be dissolved in distilled water to obtain a pure extract. The mobile phase will be the extract while the suitable stationary phase (substance that is a good adsorbent e.g. silica gels, alumina, cellulose etc.) will be selected. Different ingredients of N. sativa will be separated using High Performance Liquid Chromatography (HPLC) for treating damaged cells. Damaged brain cells will be treated individually and in different combinations of 2 or 3 compounds for different intervals of time. The most suitable compound or a combination of compounds for the regeneration of cells will be determined using DOE methodology. Later the gene will also be determined and using Polymerase Chain Reaction (PCR) it will be replicated in a plasmid vector. This plasmid vector shall be inserted in the brain of the organism used and replicated within. The gene insertion can also be done by the gene gun method. The gene in question can be coated on a micro bullet of tungsten and bombarded in the area of interest and gene replication and coding shall be studied. Investigation on whether the gene replicates in the organism or not will be examined.

Keywords: black cumin, brain cells, damage, extract, neuroregeneration, PCR, plasmids, vectors

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Authors: Ashraf Y. Z. Khalifa, Mohammed A. Almalki

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Medicago sativa (Alfalfa) is an important forage crop legume worldwide including Saudia Arabia due to its high nutritive value. Soil bacteria exist in root or root-nodules of Medicago sativa in either symbiotic relationships or in associations. The aim of the present study was to isolate and characterize endophytic bacteria that live in association with non-nodulated roots of Medicago sativa growing in Al-Ahsaa region, Saudia Arabia. Several bacterial strains were isolated from sterilized roots of Medicago sativa. Strains were characterized using 16S rRNA gene sequences, phylogenetic relationships analysis, morphological and biochemical characteristics. The strains utilized 50% (10 out of 20) of the different chemical substrates contained in the API20E strip. In general, many strains had the ability to ferment/oxidise all the carbohydrate tested except for rhamnose and the polyol carbohydrate, inositol. Comparative sequence analysis of the 16S rDNA gene indicated that the strains were closely related to the genus Bacillus. Furthermore, the growth parameters of Vigna sinensis were enhanced upon single-inoculation of the isolated strains, compared to the uninoculated control plants. The results highlighted that the root-nodules of Medicago sativa harbor non-nodulating bacterial strains that could have significant agricultural applications.

Keywords: Medicago sativa, endophytic bacteria, Pisum sativum, Vigna sinensis

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Authors: Michael Aupetit, Mahmoud Al-ismail, Khaled Mohamed

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Cancer is a major public health problem all over the world. Breast cancer has the highest incidence rate over all cancers for women in Qatar making its study a top priority of the country. Human cancer is a dynamic disease that develops over an extended period through the accumulation of a series of genetic alterations. A Darwinian process drives the tumor cells toward higher malignancy growing the branches of a progression tree in the space of genes expression. Although it is not possible to track these genetic alterations dynamically for one patient, it is possible to reconstruct the progression tree from the aggregation of thousands of tumor cells’ genetic profiles from thousands of different patients at different stages of the disease. Analyzing the progression tree is a way to detect pivotal molecular events that drive the malignant evolution and to provide a guide for the development of cancer diagnostics, prognostics and targeted therapeutics. In this work we present the development of a Visual Analytic web platform CanVis enabling users to upload gene-expression data and analyze their progression tree. The server computes the progression tree based on state-of-the-art techniques and allows an interactive visual exploration of this tree and the gene-expression data along its branching structure helping to discover potential driver genes.

Keywords: breast cancer, progression tree, visual analytics, web platform

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Authors: Min-Shiuan Liou, Yi Shan Yang, Yang-Zhan Huang, Chia-Wen Hsieh

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A high speed growing and butanol-tolerant Clostridium acetobutylicum HOL1 mutant was screened throughout continuous adaption culture with C. acetobutylicum ATCC 824. The HOL1 strain can grow well in 10 g/L butanol contained CGM medium and can produce about 12.8 g /L butanol during 24 hrs. The C. acetobutylicum HOL1 strain was able to produce 166 mM butanol with 21 mM acetone at pH 4.8, resulting in a butanol selectivity (a molar ratio of butanol to total solvents) of 0.79, which is much higher than that (0.6) of the wild-type strain C. acetobutylicum ATCC 824. The acetate and butyrate accumulation were not observed during fermentation of the HOL1 strain. A hyper-butanol producing C. acetobutylicum HOL1 (pBPHS-3), which was created to overexpress the Bacillus psychrosaccharolyticus originated specific heat-shock protein gene, hspX, from a clostridial phosphotransbutyrylase promoter, was studied for its potential to produce a high titer of butanol. Overexpression of hspX resulted in increased final butanol yield 47% and 30% higher than those of the the ATCC824 and the HOL1 strains, respectively. The remarkable high-speed growth and butanol tolerance of strain HOL1 (pBPHS-3) demonstrates that overexpression of heterogeneous stress protein-encoding gene, hspX, could help C. acetobutylicum to effectively produce a high concentration of butanol.

Keywords: Clostridium acetobutylicum, butanol, heat-shock protein, resistance

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Authors: Ankit Tyagi, Reet Kamal Tiwari, Naveen James

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Landslide is the major geo-environmental problem of Himalaya because of high ridges, steep slopes, deep valleys, and complex system of streams. They are mainly triggered by rainfall and earthquake and causing severe damage to life and property. In Uttarakhand, the Tehri reservoir rim area, which is situated in the lesser Himalaya of Garhwal hills, was selected for landslide hazard zonation (LHZ). The study utilized different types of data, including geological maps, topographic maps from the survey of India, Landsat 8, and Cartosat DEM data. This paper presents the use of a weighted overlay method in LHZ using fourteen causative factors. The various data layers generated and co-registered were slope, aspect, relative relief, soil cover, intensity of rainfall, seismic ground shaking, seismic amplification at surface level, lithology, land use/land cover (LULC), normalized difference vegetation index (NDVI), topographic wetness index (TWI), stream power index (SPI), drainage buffer and reservoir buffer. Seismic analysis is performed using peak horizontal acceleration (PHA) intensity and amplification factors in the evaluation of the landslide hazard index (LHI). Several digital image processing techniques such as topographic correction, NDVI, and supervised classification were widely used in the process of terrain factor extraction. Lithological features, LULC, drainage pattern, lineaments, and structural features are extracted using digital image processing techniques. Colour, tones, topography, and stream drainage pattern from the imageries are used to analyse geological features. Slope map, aspect map, relative relief are created by using Cartosat DEM data. DEM data is also used for the detailed drainage analysis, which includes TWI, SPI, drainage buffer, and reservoir buffer. In the weighted overlay method, the comparative importance of several causative factors obtained from experience. In this method, after multiplying the influence factor with the corresponding rating of a particular class, it is reclassified, and the LHZ map is prepared. Further, based on the land-use map developed from remote sensing images, a landslide vulnerability study for the study area is carried out and presented in this paper.

Keywords: weighted overlay method, GIS, landslide hazard zonation, remote sensing

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Authors: Roudabeh Vakil Monfared, Farhad Mashayekhi

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Breast cancer is the most common malignancy type among women with about 1 million new cases each year. The immune system plays an important role in the breast cancer development. OX40L (also known as TNFSF4), a membrane protein, which is a member of the tumor necrosis factor super family binds to its receptor OX40 and this co-stimulation has a crucial role in T-cell proliferation, survival and cytokine release. Due to the importance of the T-cells in anti-tumor activities of OX40L we studied the association of rs3850641 (T→C) polymorphism of OX40L gene with breast cancer. The study included 123 women with breast cancer and 126 healthy volunteers with no signs of cancer. Genomic DNA was extracted from blood leucocytes. Genotype and allele frequencies were determined in patients and control cases with the method of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and the analysis was performed by Med Calc. The prevalence of genotype frequencies of TT, CT and CC were 60.9%, 30.08% and 8.9 % in patients with breast cancer and 74.6 %, 18.25 % and 7.14 % in healthy volunteers while the T and C allelic frequency was 76.01% and 23.98 % in patients and 83.73% and 16.26% in healthy controls. Respectively Statistical analysis has shown no significant difference from the comparison of either genotype (P=0.06). According to these results, the rs3850641 SNP has no association with the susceptibility of breast cancer in a population in northern Iran. However, further studies in larger populations including other genetic and environmental factors are required to achieve conclusion.

Keywords: OX40L, gene, polymorphism, breast cancer

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Authors: Natalia Koltovaya, Nadezhda Zhuchkina, Ksenia Lyubimova

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We study the biological effects induced by ionizing radiation in view of therapeutic exposure and the idea of space flights beyond Earth's magnetosphere. In particular, we examine the differences between base pair substitution induction by ionizing radiation in model haploid and diploid yeast Saccharomyces cerevisiae cells. Such mutations are difficult to study in higher eukaryotic systems. In our research, we have used a collection of six isogenic trp5-strains and 14 isogenic haploid and diploid cyc1-strains that are specific markers of all possible base-pair substitutions. These strains differ from each other only in single base substitutions within codon-50 of the trp5 gene or codon-22 of the cyc1 gene. Different mutation spectra for two different haploid genetic trp5- and cyc1-assays and different mutation spectra for the same genetic cyc1-system in cells with different ploidy — haploid and diploid — have been obtained. It was linear function for dose-dependence in haploid and exponential in diploid cells. We suggest that the differences between haploid yeast strains reflect the dependence on the sequence context, while the differences between haploid and diploid strains reflect the different molecular mechanisms of mutations.

Keywords: base pair substitutions, γ-rays, haploid and diploid cells, yeast Saccharomyces cerevisiae

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Authors: Chang-Hui Shen, Michelle Esposito, Andrew J. Shen, Michael Adejokun, Diana Laterman

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The packaging of DNA into nucleosomes is critical to genomic compaction, yet it can leave gene promoters inaccessible to activator proteins or transcription machinery and thus prevents transcriptional initiation. Both chromatin remodelers and histone acetylases (HATs) are the two main transcription co-activators that can reconfigure chromatin structure for transcriptional activation. Ino80p is the core component of the INO80 remodeling complex. Recently, it was shown that Ino80p dissociates from the yeast INO1 promoter after induction. However, when certain HATs were deleted or mutated, Ino80p accumulated at the promoters during gene activation. This suggests a link between HATs’ presence and Ino80p’s dissociation. However, it has yet to be demonstrated that Ino80p can be acetylated. To determine if Ino80p can be acetylated, wild-type Saccharomyces cerevisiae cells carrying Ino80p engineered with a double FLAG tag (MATa INO80-FLAG his3∆200 leu2∆0 met15∆0 trp1∆63 ura3∆0) were grown to mid log phase, as were non-tagged wild type (WT) (MATa his3∆200 leu2∆0 met15∆0 trp1∆63 ura3∆0) and ino80∆ (MATa ino80∆::TRP1 his3∆200 leu2∆0 met15∆0 trp1∆63 ura3∆0) cells as controls. Cells were harvested, and the cell lysates were subjected to immunoprecipitation (IP) with α-FLAG resin to isolate Ino80p. These eluted IP samples were subjected to SDS-PAGE and Western blot analysis. Subsequently, the blots were probed with the α-FLAG and α-acetyl lysine antibodies, respectively. For the blot probed with α-FLAG, one prominent band was shown in the INO80-FLAG cells, but no band was detected in the IP samples from the WT and ino80∆ cells. For the blot probed with the α-acetyl lysine antibody, we detected acetylated Ino80p in the INO80-FLAG strain while no bands were observed in the control strains. As such, our results showed that Ino80p can be acetylated. This acetylation can explain the co-activator’s recruitment patterns observed in current gene activation models. In yeast INO1, it has been shown that Ino80p is recruited to the promoter during repression, and then dissociates from the promoter once de-repression begins. Histone acetylases, on the other hand, have the opposite pattern of recruitment, as they have an increased presence at the promoter as INO1 de-repression commences. This Ino80p recruitment pattern significantly changes when HAT mutant strains are studied. It was observed that instead of dissociating, Ino80p accumulates at the promoter in the absence of functional HATs, such as Gcn5p or Esa1p, under de-repressing processes. As such, Ino80p acetylation may be required for its proper dissociation from the promoters. The remodelers’ dissociation mechanism may also have a wide range of implications with respect to transcriptional initiation, elongation, or even repression as it allows for increased spatial access to the promoter for the various transcription factors and regulators that need to bind in that region. Our findings here suggest a previously uncharacterized interaction between Ino80p and other co-activators recruited to promoters. As such, further analysis of Ino80p acetylation not only will provide insight into the role of epigenetic modifications in transcriptional activation, but also gives insight into the interactions occurring between co-activators at gene promoters during gene regulation.

Keywords: acetylation, chromatin remodeler, epigenetic modification, Ino80p

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Authors: Abiodun Amusan, Olugbenga Akinola, Kazeem Akano, María Hernández-Castañeda, Jenna Dick, Akintunde Sowunmi, Geoffrey Hart, Grace Gbotosho

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Introduction: Artemisinin-based Combination Therapy (ACTs) is the cornerstone malaria treatment option in most malaria-endemic countries. Unfortunately, the malaria control effort is constantly being threatened by resistance of Plasmodium falciparum to ACTs. The recent evidence of artemisinin resistance in East Africa and its possibility of spreading to other African regions portends an imminent health catastrophe. This study aimed at evaluating the occurrence, prevalence, and influence of artemisinin-resistance markers on treatment outcomes in Ibadan before and after post-adoption of artemisinin combination therapy (ACTs) in Nigeria in 2005. Method: The study involved day zero dry blood spot (DBS) obtained from malaria patients during retrospective (2000-2005) and prospective (2021) studies. A cohort in the prospective study received oral dihydroartemisinin-piperaquine and underwent a 42-day follow-up to observe treatment outcomes. Genomic DNA was extracted from the DBS samples using a QIAamp blood extraction kit. Fragments of P. falciparum kelch13 (Pfkelch13), P. falciparum coronin (Pfcoronin), P. falciparum multidrug resistance 2 (PfMDR2), and P. falciparum chloroquine resistance transporter (PfCRT) genes were amplified and sequenced on a sanger sequencing platform to identify artemisinin resistance-associated mutations. Mutations were identified by aligning sequenced data with reference sequences obtained from the National Center for Biotechnology Information. Data were analyzed using descriptive statistics and student t-tests. Results: Mean parasite clearance time (PCT) and fever clearance time (FCT) were 2.1 ± 0.6 days (95% CI: 1.97-2.24) and 1.3 ± 0.7 days (95% CI: 1.1-1.6) respectively. Four mutations, K189T [34/53(64.2%)], R255K [2/53(3.8%)], K189N [1/53(1.9%)] and N217H [1/53(1.9%)] were identified within the N-terminal (Coiled-coil containing) domain of Pfkelch13. No artemisinin resistance-associated mutation usually found within the β-propeller domain of the Pfkelch13 gene was found in these analyzed samples. However, K189T and R255K mutations showed a significant correlation with longer parasite clearance time in the patients (P<0.002). The observed Pfkelch13 gene changes did not influence the baseline mean parasitemia (P = 0.44). P76S [17/100 (17%)] and V62M [1/100 (1%)] changes were identified in the Pfcoronin gene fragment without any influence on the parasitological parameters. No change was observed in the PfMDR2 gene, while no artemisinin resistance-associated mutation was found in the PfCRT gene. Furthermore, a sample each in the retrospective study contained the Pfkelch13 K189T and Pfcoronin P76S mutations. Conclusion: The study revealed absence of genetic-based evidence of artemisinin resistance in the study population at the time of study. The high frequency of K189T Pfkelch13 mutation and its correlation with increased parasite clearance time in this study may depict geographical variation of resistance mediators and imminent artemisinin resistance, respectively. The study also revealed an inherent potential of parasites to harbour drug-resistant genotypes before the introduction of ACTs in Nigeria.

Keywords: artemisinin resistance, plasmodium falciparum, Pfkelch13 mutations, Pfcoronin

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Authors: Leila Graini

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In this paper, we demonstrate basic all-optical functions for 2R regeneration (Re-amplification and Re-shaping) based on self-similar spectral broadening in low normal dispersion and highly nonlinear fiber (ND-HNLF) to regenerate the signal through optical filtering including the transfer function characteristics, and output extinction ratio. Our approach of all-optical 2R regeneration is based on those of Mamyshev. The numerical study reveals the self-similar spectral broadening very effective for 2R all-optical regeneration; the proposed design presents high stability compared to a conventional regenerator using SPM broadening with reduction of the intensity fluctuations and improvement of the extinction ratio.

Keywords: all-optical function, 2R optical regeneration, self-similar broadening, Mamyshev regenerator

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Authors: Sdiqeh Jalali, M. R. Khazdair

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Liver X receptors (LXR) have an essential role in the regulation of cholesterol metabolism, and their activation increase ABCG5 and ABCG8 genes expression for the improvement of cholesterol excretion from the body during reverse cholesterol transport (RCT). The aim of this study was to investigate the effects of high-intensity interval (HIT) and low intensity continuous (LIT) trainings on gene expression of these substances after a high-fat diet in Wistar rats. Materials and Methods: Fifteen male Wistar rats were divided into 3 groups: control group (n = 5), HIT exercise group (n = 5) and LIT exercise group (n = 5). All groups used a high-fat diet for 13 weeks, and the HIT and LIT groups performed the specific training program. The expression of LXRβ, ABCG5, and ABCG8 genes was measured after the training period. Findings: Data analysis showed significantly higher levels of LXRβ, ABCG5, and ABCG8 gene expression in HIT and LIT groups compared to the control group (P ≤ 0.05). Conclusion: HIT and LIT trainings after a high-fat diet have beneficial effects on RCT that prevent heart attack. Also, HIT training may have a greater effect on cholesterol excretion during the reverse cholesterol transport mechanism than LIT.

Keywords: liver X receptor, atherosclerosis, interval training, endurance training

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Authors: Sabah Ben Fredj Melki, Yves Brygoo, Ahmed Mliki

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A 700 pb PCR-derived DNA fragment was isolated from Aspergillus carbonarius, Aspergillus niger, and Aspergillus tubingensis using degenerated primers (LC1-LC2c) and two newly designed primer pairs (KSLB-LC6) for Aspergillus niger and (AFl1F-LC2) for Aspergillus tubingensis developed for the acyl transferase (AT) and the KS domains of fungal PKSs. DNA from the most of black Aspergillus species currently recognized was tested. Herein, we report on the identification and characterisation of a part of the novel putative OTA-polyketide synthase gene in A. carbonarius “ACPks”, A. niger “ANPks” and A. tubingenis “ATPks”. The sequences were aligned and analyzed using phylogenetic methods. Primers used in this study showed general applicability and other Aspergillus species belonging to section Nigri were successfully amplified especially in A. niger and A. tubingenis. The predicted amino acid sequences “ACPks” displayed 66 to 81% similarities to different polyketide synthase genes while “ANPks” similarities varied from 68 to 71% and “ATPks” were from 81 to 97%. The AT and the KS domains appeared to be specific for a particular type of fungal PKSs and were related to PKSs involved in different mycotoxin biosynthesis pathways, including ochratoxin A. The sequences presented in this work have a high utility for the discovery of novel fungal PKS gene clusters.

Keywords: Pks genes, OTA Biosynthesis, Aspergillus Nigri, sequence analysis

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Authors: Ksenija Taški-Ajdukovic, Nevena Nagl, Živko Ćurčić, Dario Danojević

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Information about genetic diversity of sugar beet parental populations is of a great importance for hybrid breeding programs. The aim of this research was to evaluate genetic diversity among and within populations and lines of diploid sugar beet pollinators, by using SSR markers. As plant material were used eight pollinators originating from three USDA-ARS breeding programs and four pollinators from Institute of Field and Vegetable Crops, Novi Sad. Depending on the presence of self-fertility gene, the pollinators were divided into three groups: autofertile (inbred lines), autosterile (open-pollinating populations), and group with partial presence of autofertility gene. A total of 40 SSR primers were screened, out of which 34 were selected for the analysis of genetic diversity. A total of 129 different alleles were obtained with mean value 3.2 alleles per SSR primer. According to the results of genetic variability assessment the number and percentage of polymorphic loci was the maximal in pollinators NS1 and tester cms2 while effective number of alleles, expected heterozygosis and Shannon’s index was highest in pollinator EL0204. Analysis of molecular variance (AMOVA) showed that 77.34% of the total genetic variation was attributed to intra-varietal variance. Correspondence analysis results were very similar to grouping by neighbor-joining algorithm. Number of groups was smaller by one, because correspondence analysis merged IFVCNS pollinators with CZ25 into one group. Pollinators FC220, FC221 and C 51 were in the next group, while self-fertile pollinators CR10 and C930-35 from USDA-Salinas were separated. On another branch were self-sterile pollinators ЕL0204 and ЕL53 from USDA-East Lansing. Sterile testers cms1 and cms2 formed separate group. The presented results confirmed that SSR analysis can be successfully used in estimation of genetic diversity within and among sugar beet populations. Since the tested pollinator differed considering the presence of self-fertility gene, their heterozygosity differed as well. It was lower in genotypes with fixed self-fertility genes. Since the most of tested populations were open-pollinated, which rarely self-pollinate, high variability within the populations was expected. Cluster analysis grouped populations according to their origin.

Keywords: auto fertility, genetic diversity, pollinator, SSR, sugar beet

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Authors: Karthick Dharmalingam, Stalin Ramakrishnan, Haseena Banu Hedayathullah Khan, Sachidanandanam Thiruvaiyaru Panchanadham, Shanthi Palanivelu

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Background: Breast cancer is arising as the most dreadful cancer affecting women worldwide. Hence, there arises a need to search and test for new drugs. Herbal formulations used in Siddha preparations are proved to be effective against various types of cancer. They also offer advantage through synergistic amplification and diminish any possible adverse effects. Tridham (TD) is a herbal formulation prepared in our laboratory consisting of Terminalia chebula, Elaeocarpus ganitrus and Prosopis cineraria in a definite ratio and has been used for the treatment of mammary carcinoma. Objective: To study the restorative effect of Tridham and penta galloyl glucose (a component of TD) on DMBA induced mammary carcinoma in female Sprague Dawley rats. Materials and Methods: Rats were divided into seven groups of six animals each. Group I (Control) received corn oil. Group II– mammary carcinoma was induced by DMBA dissolved in corn oil single dose orally. Group III and Group IV were induced with DMBA and subsequently treated with Tridham and penta galloyl glucose, respectively for 48 days. Group V was treated with DMBA and subsequently with a standard drug, cyclophosphamide. Group VI and Group VII were given Tridham and penta galloyl glucose alone, respectively for 48 days. After the experimental period, the animals were sacrificed by cervical decapitation. The mammary gland tissue was excised and levels of antioxidants were determined by biochemical assay. p53 and PCNA expression were accessed using immunohistochemistry. Nrf-2, Cox-2 and caspase-3 protein expression were studied by Western Blotting analysis. p21, Bcl-2, Bax, Bad and caspase-8 gene expression were studied by RT-PCR. Results: Histopathological studies confirmed induction of mammary carcinoma in DMBA induced rats and treatment with TD and PGG resulted in regression of tumour. The levels of enzymic and non-enzymic antioxidants were decreased in DMBA induced rats when compared to control rats. The levels of cell cycle inhibitory markers and apoptotic markers were decreased in DMBA induced rats when compared to control rats. These parameters were restored to near normal levels on treatment with Tridham and PGG. Conclusion: The results of the present study indicate the antineoplastic effect of Tridham and PGG are exerted through the modulation of antioxidant status and expression of cell cycle regulatory markers as well as apoptotic markers. Acknowledgment: Financial assistance provided in the form of ICMR-SRF by Indian Council of Medical Research (ICMR), India is gratefully acknowledged here.

Keywords: antioxidants, Mammary carcinoma, pentaGalloyl glucose, Tridham

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Authors: Ricardo de Oliveira Orsi, Aline Astolfi, Daniel Diego Mendes, Isabella Cristina de Castro Lippi, Jaine da Luz Scheffer, Yan Souza Lima, Juliana Lunardi, Giovanna do Padro Ribeiro, Samir Moura Kadri

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NECTAR Brazilian group (Center of Education, Science, and Technology in Rational Beekeeping) conducted studies on the pesticides honey bees effects using the transcriptome sequencing (RNA-Seq) analyzes for gene expression studies. In this way, we analyzed the effects of Pyraclostrobin and Fipronil on the honey bees with 21 old-days (forager) in laboratory conditions. For this, frames containing sealed brood were removed from the beehives and maintenance on the stove (32°C and 75% humidity) until the bees were born. So, newly emerged workers were marked on the pronotum with a non-toxic pen and reintroduced into their original hives. After 21 days, 120 marked bees were collected with an entomological forces and immediately stored in Petri dishes, perforated to ensure ventilation, and kept fasted for 3 hours. These honeybees were exposed to food contaminated or not with the sublethal dose of Pyraclostrobin (850 ppb/bee) or Fipronil (2.5 ppb/bee). After four hours of exposure, 15 bees from each treatment were referred to transcriptome analysis. Total RNA analysis was extracted from the brain pools (03 brains per pool) using the TRIzol® reagent protocol according to the manufacturer's instructions. cDNA libraries were constructed, and the FASTQC program was used to check adapter content and assess the quality of raw reads. Differential expression analysis was performed with the DESeq2 package. Genes that had an adjusted value of less than 0.05 were considered to be significantly up-regulated. Regarding the Pyraclostrobin, alterations were observed in the pattern of 17 gene related to of antioxidant system, cellular respiration, glucose metabolism, and regulation of juvenile hormone and the hormone insulin. Glyphosate altered the 10 gene related to the digestive system, exoskeleton composition, vitamin E transport, and antioxidant system. The results indicate that the necessity of studies using the sublethal doses to evaluate the pesticides uses and risks on crops and its effects on the honey bees.

Keywords: beekeeping, honey bees, pesticides, transcriptome

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Authors: T. J. Jemi Jeya, V. Sriram, V. Sundar

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Caisson breakwaters are gravity structures resting on the seabed and piercing the free surface sunk in coastal waters to break the energy in the waves and protect the water area behind them by creating tranquil conditions on its lee side for the purpose of berthing of vessels. A number of formula and methodologies have been proposed for calculating the forces on caissons due to waves, most of which being evolved through intensive laboratory and field measurements. The reflection of waves from such breakwaters often generates clapotis, leading to an amplification of waves in its vicinity. This result in increased pressures and forces, forcing researchers to modify its seaside shape as well as placing dissipaters in the form of screens. Apart from the above aspects, this paper also discusses the other important phenomena, like overtopping that dictates the stability of caisson breakwaters.

Keywords: caisson breakwater, Jarlan type breakwater, screens, circular breakwater

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Authors: Orapan Paecharoenchai, Tanasait Ngawhirunpat, Theerasak Rojanarata, Auayporn Apirakaramwong, Praneet Opanasopit

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Cationic niosomes formulated with Span20, cholesterol and novel synthesized spermine-cationic lipids (2-hydrocarbon tail and 4- hydrocarbon tail) in a molar ratio of 2.5:2.5:1 can mediate high gene transfection in vitro. However, the uptake mechanisms of these systems are not well clarified. In the present study, effect of endocytic inhibitors on the transfection efficiency of niosomes/DNA complexes was determined on a human cervical carcinoma cell line (HeLa cells) using the inhibitors of macropinocytosis (wortmannin), clathrin- and caveolae-mediated endocytosis (methyl-β-cyclodextrin), clathrin-mediated endocytosis (chlorpromazine), caveolae-mediated endocytosis (genistein and filipin), cytosolic transfer (ammonium chloride) and microtubules polymerization (nocodazole). The transfection of niosomes with 2-hydrocarbon tail lipid was blocked by nocodazole, genistein, ammonium chloride and filipin, respectively, whereas, the transfection of niosomes with 4-hydrocarbon tail lipid was blocked by nocodazole, genistein, ammonium chloride, methyl-β-cyclodextrin and filipin, respectively. It can be concluded that these niosomes/DNA complexes were internalized predominantly by endocytosis via clathrin and caveolae-independent pathway.

Keywords: cellular internalization, cationic niosomes, gene carriers, spermine-cationic lipids

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Authors: K. H. Reeta, Bhavana Prasher, Mitali Mukerji, Dhwani Dholakia, Sangeeta Khanna, Archana Vats, Shivam Pandey, Sandeep Seth, Subir Kumar Maulik

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Introduction Research has demonstrated a connection between coronary artery disease (CAD) and genetics. We did a deep literature mining using both bioinformatics and manual efforts to identify the susceptible polymorphisms in coronary artery disease. Further, the study sought to validate these findings in an Asian population. Methodology In first phase, we used an automated pipeline which organizes and presents structured information on SNPs, Population and Diseases. The information was obtained by applying Natural Language Processing (NLP) techniques to approximately 28 million PubMed abstracts. To accomplish this, we utilized Python scripts to extract and curate disease-related data, filter out false positives, and categorize them into 24 hierarchical groups using named Entity Recognition (NER) algorithms. From the extensive research conducted, a total of 466 unique PubMed Identifiers (PMIDs) and 694 Single Nucleotide Polymorphisms (SNPs) related to coronary artery disease (CAD) were identified. To refine the selection process, a thorough manual examination of all the studies was carried out. Specifically, SNPs that demonstrated susceptibility to CAD and exhibited a positive Odds Ratio (OR) were selected, and a final pool of 324 SNPs was compiled. The next phase involved validating the identified SNPs in DNA samples of 96 CAD patients and 37 healthy controls from Indian population using Global Screening Array. ResultsThe results exhibited out of 324, only 108 SNPs were expressed, further 4 SNPs showed significant difference of minor allele frequency in cases and controls. These were rs187238 of IL-18 gene, rs731236 of VDR gene, rs11556218 of IL16 gene and rs5882 of CETP gene. Prior researches have reported association of these SNPs with various pathways like endothelial damage, susceptibility of vitamin D receptor (VDR) polymorphisms, and reduction of HDL-cholesterol levels, ultimately leading to the development of CAD. Among these, only rs731236 had been studied in Indian population and that too in diabetes and vitamin D deficiency. For the first time, these SNPs were reported to be associated with CAD in Indian population. Conclusion: This pool of 324 SNP s is a unique kind of resource that can help to uncover risk associations in CAD. Here, we validated in Indian population. Further, validation in different populations may offer valuable insights and contribute to the development of a screening tool and may help in enabling the implementation of primary prevention strategies targeted at the vulnerable population.

Keywords: coronary artery disease, single nucleotide polymorphism, susceptible SNP, bioinformatics

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Authors: Emad Heydarnia, Aref Sepasi, Nika Asefi, Sara Khakshournia, Javad Mohammadnejad

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Background: Despite breakthrough therapeutics in breast cancer, it is one of the main causes of mortality among women worldwide. Thus, drug therapies for treating breast cancer have recently been developed by scientists. Metformin and Sorafenib are well-known therapeutic in breast cancer. In the present study, we combined Sorafenib and PCL-sorafenib with metformin to improve drug absorption and promote therapeutic efficiency. Methods: The MCF-7 cells were treated with Metformin, Sorafenib, or PCL-sorafenib. The growth inhibitory effect of these drugs and cell viability were assessed using MTT and flow cytometry assays, respectively. The expression of targeted genes involved in cell proliferation, signaling, and the cell cycle was measured by Real-time PCR. Results: The results showed that MCF-7 cells treated with Metformin/Sorafenib and PCL-sorafenib/Metformin co-treatment contributed to 50% viability compared to untreated group. Moreover, PI and Annexin V staining tests showed that the cells viability for Metformin/Sorafenib and PCL-sorafenib/Metformin was 38% and 17%, respectively. Furthermore, Sorafenib/Metformin and PCL-sorafenib/Metformin leads to p53 gene expression increase by which they can increase ROS, thereby decreasing GPX4 gene expression. In addition, they affected the expression of BCL2, and BAX genes and altered the cell cycle. Conclusion: Together, the combination of PCL-sorafenib/Metformin and Sorafenib/Metformin increased Sorafenib absorption at lower doses and also leads to apoptosis and oxidative stress increases in MCF-7 cells.

Keywords: breast cancer, metformin, nanotechnology, sorafenib

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Authors: Gourav Jain, Sameer Dixit, Surjeet Kumar Arya, Praveen C. Verma

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Plant cell wall plays a major role in defence mechanism against biotic and abiotic stress as it constitutes the physical barrier between the microenvironment and internal component of the cell. It is a complex structure composed of mostly carbohydrates among which cellulose and hemicelluloses are most abundant that is embedded in a matrix of pectins and proteins. Multiple enzymes have been reported which plays a vital role in cell wall modification, Pectin Methylesterase (PME) is one of them which catalyses the demethylesterification of homogalacturonans component of pectin which releases acidic pectin and methanol. As emitted methanol is toxic to the insect pest, we use PME gene for the better methanol production. In the current study we showed overexpression of PME gene isolated from Withania somnifera under the insect inducible promoter causes enhancement of methanol production at the time of insect feeds to plants, and that provides better insect resistance property. We found that the 85-90% mortality causes by transgenic tobacco in both chewing (Spodoptera litura larvae and Helicoverpa armigera) and sap-sucking (Aphid, mealybug, and whitefly) pest. The methanol content and emission level were also enhanced by 10-15 folds at different inducible time point interval (15min, 30min, 45min, 60min) which would be analysed by Purpald/Alcohol Oxidase method.

Keywords: methanol, Pectin methylesterase, inducible promoters, Purpald/Alcohol oxidase

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