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DNA extraction and purification are routine processes in mostplant genetic transformation laboratories. Although there are differentcommercial kits that allow accurate DNA purification, the total cost ofbuying multiple sets of these kits can be spectacular. We use spincolumn and laboratory solutions to develop a simple method of DNApurification that can meet different research needs. This method is usedto extract DNA from the leaves of Brassica and genetically modifiedplants and also bacteria; extract plasmid DNA from E. coli orAgrobacterium tumefaciens; purify the DNA fragments of PCRproducts and the resulting fragments of digestion with restrictionendonuclease. DNA concentration of optical density (OD) value wascalculated at 260 nm wavelength and OD260 / OD280 ratios were usedto determine DNA quality. The quantity and quality of DNA obtainedby this method was similar to that of isolated DNA using commercialKits. In comparison, it has been shown that this method allowsobtaining DNA from different sources with similar quantity and purityand low costs.

Background: Polymorphisms of long noncoding RNAs are lately documented as hazardous factors for the development of numerous tumors. Furthermore, the evaluation of noncoding RNAs has emerged as a novel detector of breast cancer patients. We aimed to genotype the HOXA transcript at the distal tip (HOTTIP) rs1859168 and assess its relationship with the levels of the serum HOTTIP and its target miR-615-3p in patients with breast cancer (BC). Methods: One hundred and fifty-one patients with BC, 139 patients with fibroadenoma (FA), and 143 healthy participants were incorporated into the current study. The genotyping of rs1859168 and the measurements of the HOTTIP and miR-615-3p levels were assessed using quantitative real-time PCR. Results: We revealed a significant association between each of the CC genotypes, C allele, dominant and recessive models, and the increased risk of BC (p = 0.013, p &lt, 0.001, p &lt, 0.001, and p &lt, 0.001, respectively) relative to the healthy controls. Similarly, the CC genotype, C allele, and recessive model were observed to be related to the increased incidence of BC with respect to FA (p &lt, 0.001 for all). A significant upregulation of HOTTIP and a marked decrease of miR-615-3p were verified in patients with BC compared to each of the healthy individuals, patients with FA, and the non-BC group (healthy subjects + FA) (p &lt, 0.001 for all). A significant negative correlation was demonstrated between the expression of HOTTIP and miR-615-3p in the serum of patients with BC. The HOTTIP expression was upregulated, while that of miR-615-3p was downregulated in patients with BC who carried the CC genotype with respect to those who carried the AA or AC genotypes (p &lt, 0.05 for all). Conclusions: The genetic variants of rs1859168 are linked to an increased susceptibility to BC. Moreover, HOTTIP and miR-615-3p may be used as novel indicators and targets for the treatment of patients with BC.

Genetic variants in microRNAs (miRNAs) can alter the miRNAs expression and/or function, accordingly, affecting the related biological pathways and disease risk. Dysregulation of miR-155 and miR-146a expression levels has been well-described in viral hepatitis B (HBV). In the current study, we aimed to assess rs767649 T/A and rs57095329 A/G polymorphisms in miR-155, and miR-146a genes, respectively, as risk factors for Chronic HBV (CHBV) in the Egyptian population. Also, we aimed to do in silico analysis to investigate the molecules that primarily target these miRNAs. One hundred patients diagnosed as CHBV and one hundred age and sex-matched controls with evidence of past HBV infection were genotyped for miR-155 (rs767649) and miR-146a (rs57095329) using real-time polymerase chain reaction. The rs767649 AT and AA genotypes in CHBV patients confer four folds and ten folds risk respectively, as compared to control subjects [(AOR = 4.245 (95%CI 2.009–8.970), pppp = 0.004] and [AOR = 5.604 (95%CI 2.157–14.563), p

Background Neonatal sepsis is a serious condition. Recent clinical studies have indicated that microRNAs (miRNAs) are key players in the pathogenesis of sepsis, which could be used as biomarkers for this condition. Patients and methods A total of 90 neonates with sepsis and 90 healthy neonates were enrolled in this study. qRT-PCR was performed to measure the expression levels of serum miR-34a-5p and miR-199a-3p. Results miR-34a-5p and miR-199a-3p serum levels were significantly reduced in neonates with sepsis compared with those in healthy neonates (P = 0.006 and P = 0.001, respectively). Significant correlations of miR-34a-5p and miR-199a-3p with each of TLC, RDW, RBS, and C-reactive protein (CRP) as well as SNAPII were observed, indicating their associations with the severity of neonatal sepsis. Conclusion miR-34a-5p and miR-199a-3p may be useful as novel biomarkers in neonatal sepsis and may provide a new direction for its treatment.

Open reading frame (ORF) of Putative En/Spm transposon in Brassica potentially helps to understand the relationship between various eukaryotic transposable elements. The current study was initially conducted to isolate and analyze the putative En/Spm-related transposon gene from Brassica juncea. PCR products (750 bp) from B. juncea (accession PI 649105 and PI 271442) were cloned, sequenced, and analyzed. Results of BLAST showed identical sequences between two accessions with 100% similarity. The amplified DNA transposon and conserved domain compared to the GenBank database to evaluate the genetic diversity relationships. Sequence of this putative transposon gene from B. juncae was a 98% similar to B. rapa subsp pekinensis at the nucleotide level, and 94% with the En/Spm-related transposon protein of B. oleracea at the amino acid level. Conserved domain architecture was related to transposase_21_pfam0299 and transposase family tnp2 and had a relationship with space outside the cell structure and/or to space outside the plasma membrane.

Twenty strains of four Trichoderma species (Trichoderma harzianum, Trichoderma viride, Trichoderma koningii and Trichoderma asperellum) were evaluated for its potential to control the root-knot nematode Meloidogyne javanica. Culture filtrates from Trichoderma strains were tested in 24-well tissue culture plates for effects on Meloidogyne javanica. Chitwood egg hatch and mobility of hatched second-stage juveniles (J2) were evaluated, all the twenty Trichoderma strains showed the ability to colonize M. javanica separated eggs and second-stage juveniles (J2) in sterile in vitro assays. T. asperellum possess the strongest egg-parasitic ability and very effective against 2nd stage larvae of M. javanica. In this investigation, randomly amplified polymorphic DNA (RAPD) markers was used to estimate the genetic variations between four strains of Trichoderma asperellum (KC898190, KC898191, KC898192 and KC898193) which were previously isolated from the rhizospheres of different plants growing in Fayoum Governorate, Egypt as a new strain of T. asperellum in Egypt. RAPD assay using 6 random primers identified T. asperellum strains with 5 specific unique markers.

Colorectal cancer (CRC) represented the second cause of mortality among cancer patients. Long noncoding RNAs and microRNAs (miRNAs) serve as noninvasive biomarkers for CRC surveillance and introduce new therapeutic approaches. LINC00657 and miR-106a expression levels play a pivotal role in CRC. This study included 190 Egyptian subjects, and the expression levels of LINC00657 and miR-106a in serum were measured by using quantitative real-time polymerase chain reaction. We found that upregulation of LINC00657 and downregulation of miR-106a are significantly associated with the development of CRC. Also, a positive correlation was detected between their serum levels. In addition, serum LINC00657 can distinguish adenomatous polyposis (AP) patients and/or ulcerative colitis (UC) patients from controls. Also the miRNA-106a expression level discriminates AP but not UC from healthy individuals. Our study cited new diagnostic biomarkers for CRC, AP, and UC among Egyptians in addition to be noninvasive screening tools for CRC in both healthy subjects and those having precancerous lesions. © 2019 IUBMB Life, 71(9):1322-1335, 2019.

Background: Preeclampsia is a severe pregnancy-related disease mainly in developing countries. Non-coding RNAs are very important factors in regulating the expression of certain genes and are involved in pathogenesis of numerous diseases. Recently, non coding RNA biomarkers are used as diagnostic tools for different disorders such as PE.Methods: lnc RNA HULC expression levels was measured in serum collected from 22 patients with PE and 23 healthy pregnancies, using real-time PCR.Results: HULC expression was elevated reaching significant levels (p < 0.001with 2.16 fold change) in patients compared with control group. HULC was markedly increased in both severe and mild preeclamptic subgroups when compared to healthy controls (p

Long non-coding RNAs (lncRNAs) play an important role in gene regulation and show greater tissue specificity and complexity of biological functions. There is on-going research in their contribution in autoimmune diseases like multiple sclerosis (MS). Our study aimed at the evaluation of serum levels of lncRNAs, MALAT1 and lnc-DC in MS patients and the investigation of the association between these lncRNAs and the disease activity. Serum from 45 MS patients and 45 healthy controls was separated. MALAT1 and lnc-DC expression levels were assayed by qRT-PCR. MALAT1 and lnc-DC were significantly increased in MS patients (P=0.004 and P=0.006, respectively) in comparison with controls. There was a significant increase in expression of MALAT1 in secondary progressive MS (SPMS) subgroup compared with controls (P

Hydrolytic enzymes producing Trichoderma species have long been recognized as an agent for controlling plant diseases caused by various phytopathogenic fungi. This study aims to isolate and characterize of new bio fungicides from Egyptian soils and assess of their antagonistic activity against some pathogenic fungi (Fusarium semitectum and Alternaria alternata). Four isolates of the Trichoderma asperellum were isolated from rhizosphere soil of different host plants collected from Fayoum governorate, Egypt. The isolates were characterized according to morphological characterization, microscopic observations and confirmed by sequencing of the ITS region of 18S rRNA. Trichoderma asperellum isolates were evaluated for their potential to antagonize the plant pathogenic fungi (F. semitectum and A. alternata) in vitro using the dual culture technique. Four out of twenty Trichoderma isolates (20%) were identified as T. asperellum based on morphological characteristics and confirmed by sequencing of ITS region of 18SrRNA. The four selected T. asperellum isolates (Tas1, Tas2, Tas3 and Tas4) were screened for their ability to produce chitinase on solid agar medium using bromocresol purple for developing the clear zone around colonies, and characterized due to its antagonistic effect against mycelial growth of pathogenic fungi. These results indicate that molecular systematic studies based on the sequence of ITS region are important for confirmation of phenotypic characterization of Trichoderma isolates. To the best of our knowledge, there is no information on the occurrence of T. asperellum in Egypt and this is the first report of the occurrence and isolation of T. asperellum from Egyptian soils.

Plant growth promoting is a multitask phenomenon. The bacteria achieve this by suppressing plant pathogens, production of plant-growth regulators, fixing atmospheric nitrogen, and solubilizing phosphate and micronutrients. Therefore, the present study aimed to examine the facultative oligotrophic bacteria isolates as biocontrol and plant growth promoting agents. The test isolates (FNS1 and FNS2) possessed Plant Growth Promoting Rhizobacteria (PGPR) (abilities including Indole Acetic Acid (IAA) production, Salicylic acid (SA) production, zinc and phosphate solubilization, N2-fixation, cellulase and chitinase production) and potency against pathogenic fungi causing damping-off disease (Pythium ultimum and Rhizoctonia solani). Isolates were tested against high temperature, pH and salt tolerant characters which prevail in this region. Molecular identification by 16s rDNA and nucleotide sequencing was made for the test isolates. Snap bean (Phasolius vulgaris L. cv, Xera) plants were inoculated with bacterial isolates (FNS1 and FNS2) in presence of 1/3 Nitrogen (N)-recommended dose. Inoculated plants increased significantly in N-content and plants dry weight. The antagonizer strains together enhanced the level of defense principles like Peroxidase (PO), Polyphenol Oxidase (PPO) and Total phenol content of plants. Damping-off disease suppression was expressed as there was increase in the percentage of survived plants which were inoculated when compared with uninoculated plants. These results indicate that Bacillus thioparans and Stenotrophomonas maltophilia have growth promoting potential and can be tested at field level for investment as bioinoculant.