Specific PCR-based marker for detection of pathogenic groups of Fusarium oxysporum f. sp. cucumerinum in India

For the detection of Fusarium oxysporum f. sp. cucumerinum pathogenic groups, a specific PCR-based marker was developed. Specific random amplified polymorphic DNA (RAPD) markers which identified in four pathogenic groups I, II, III, and IV were cloned into P^Gem-Teasy vector. Cloned fragments were sequenced, and used for developing sequence characterized amplified regions (SCAR) primers for detection of pathogenic groups. F. oxysporum f. sp. cucumerinum isolates belonging to four pathogenic groups in India, cucumber nonpathogenic F. oxysporum, F. oxysporum f. sp. moniliforme and melonis, Fusarium udum, and isolate of Alternaria sp. were tested using developed specific primers. A single 1.320 kb, 770 bp, 1.119 kb, and 771 bp fragment were amplified from pathogenic group I, II, III, and IV isolates, respectively. Results showed the PCR based marker, which used in this research work, could detect up to 1 ng of fungal genomic DNA. The specific SCAR primers and PCR technique developed in this research easily detect and differentiate isolates of each F. oxysporum f. sp. cucumerinum pathogenic groups.     

Salt tolerance of Capsicum annuum with introduced total DNA from Rhizophora apiculata

Total DNA of Rhizophora apiculata, a salt-resistant seashore plant, was introduced into Capsicum annuum cv. via pollen tubes formed after self-pollination. The transformed progenies showed obvious stronger salt tolerance compared with the control. About 55% of the transgenes can survive, flower, and fruit when grown on the beach and watered directly with seawater. By contrast, no control survived under the same condition. Further analyses revealed a specific protein band of 17.5 kDa on protein SDS-PAGE gel and a specific DNA band of 1.1 kb in RAPD analyses. These results indicate that it is feasible to introduce exogenous DNA through pollen tubes, and that the improved salt tolerance of the progenies of transformed plants is related to genomic change.     

New mutations of locus control region in Saudi sickle patients

Sickle cell anemia (SCA) is a common hematological disease affecting humans. Detection of a single base pair mutation in β-globin gene is an important diagnostic tool for SCA. The aim was to study the molecular survey of locus control regions (LCR) in Saudi patients with sickle cell anemia, and to identify the genetic variables and their clinical manifestations.MethodologyBlood samples from 69 unrelated sickle cell disease patients were obtained from the KKUH, Riyadh between 2017–2019. In this study, the DNA was extracted and PCR was performed. Additional PCR amplifications reactions covering the LCR were performed by using another different set of primers. Seven specific primer pairs were used to amplify seven regions in the locus control region (LCR) of β globin family. The generated fragments were sequenced to identify the possible alterations in this region.ResultsThe results gained from sequencing experiments revealed a wide range of genomic alterations. A total of 69 gene alterations have been recognized in the locus control region;- The first fragment LCR-HS1 shows 20 alterations; The second fragment LCR-HS2 revealed six changes; The third fragment LCR-HS3 shows many changes; The fifth LCR-HS5 region revealed four changes; The sixth fragment LCR-HS6 revealed eight changes; The seventh LCR-HS7 fragment demonstrates ten changes.ConclusionIt clear that this study has successfully identified LCR mutations for random Saudi patients with SCD. The above results should be taken further to set up management strategies to improve outcomes.     

High genetic differentiation revealed by RAPD analysis of narrowly endemic Sinocalycanthus chinensis Cheng et S.Y. Chang,an endangered species of China

Genetic diversity and genetic differentiation of narrowly endemic Sinocalycanthus chinensis Cheng et S.Y. Chang, an endangered species of China, were analyzed using random amplified polymorphic DNA (RAPD) markers. Totally, 165 stable and clearly scored RAPD bands were achieved from 12 primers. The genetic diversity of S. chinensis was high (P% = 68.84; h = 0.2421 ± 0.1978; I = 0.3615 ± 0.2789), whereas that at the population level was relatively low (P% = 14.49; h = 0.0578 ± 0.0167; I = 0.0843 ± 0.0236). High genetic differentiation among populations was detected based on Shannon's information index (0.7668), Nei's gene diversity (0.7613) and AMOVA (0.8183). This might be explained by its survival in refugia during the last glaciation in southeastern China with origin from a widespread continental progenitor. The 10 populations from different geographical sites could be clustered into two groups. Low gene flow due to mixed mating system and anthropogenic activities likely played important roles in shaping the population genetic structure of the species.     

Genotoxicity assessment of ammonia in cultured Oreochromis niloticus using RAPD assay

Limited efforts have been made to study the genotoxic effects of ammonia in cultured OOreochromi niloticus. Therefore, the present study was planned to assess the genotoxic effect of ammonia in cultured O. niloticus using random amplified polymorphic DNA (RAPD) assay. Fish was categorized into four groups. The 1st group exposed to 2.5 mg/L of total ammonia nitrogen (TAN) (0.16 NH₃ mg/L), the 2nd exposed to 5.0 mg/L of total ammonia nitrogen (TAN) (0.32 NH₃ mg/L) and the 3rd exposed to 10.0 mg/L of total ammonia nitrogen (TAN) (0.65 NH₃ mg/L), in addition to control group for the treatment period of 6 days. The results revealed that some genes in O. niloticus are susceptible to DNA disturbances/mutation as a result of exposure to high concentration of ammonia in water, this clearly indicated using RAPD screening assay.     

Determination of the genetic similarities of fingerprints from Escherichia coli O157:H7 isolated from different sources in the North West Province,South Africa using ISR,BOXAIR and REP-PCR analysis

The aim of this study was to determine the genetic relationships of Escherichia coli O157:H7 isolated from pigs, cattle, pork, beef, humans and water samples using REP, ISR and BOXAIR PCR analysis. A total of 94 isolates were subjected to the REP-PCR analysis while 95 were screened for ISR and BOXAIR PCR fingerprints. The band sizes for amplicons from the ISR-PCR analysis ranged from 0.173 kb to 0.878 kb. However, a large proportion of the isolates had four bands ranging from 0.447 kb to 0.878 kb. Cluster analysis of the BOXAIR PCR profiles based on banding patterns revealed seven main clusters. It was identified in the clusters III, IV and VII in the BOXAIR PCR that 17.9%, 16.8% and 18.9%, of E. coli O157:H7 isolates respectively were present from all the animal species, meat and water samples. REP-PCR analysis produced 9 different patterns with bands ranging from 0 to 12 per isolate. The band sizes ranged from 200 bp to 8000 bp. Nine major clusters (I–IX) were identified. From the three different species sampled cluster eight was the largest and a mixed cluster with 23.4% (22/94) of the E. coli O157:H7 isolates. These indicate that food products obtained from supermarkets in the study area are contaminated with E. coli O157:H7.     

Rapid detection of Trichinella spiralis larvae in muscles by loop-mediated isothermal amplification

Trichinella spiralis is a tissue-dwelling nematode parasite. A loop-mediated isothermal amplification (LAMP) assay was developed and validated for the sensitive and rapid detection of T. spiralis larvae in muscle samples. Sixteen sets of primers were designed to recognise distinct sequences of a conserved gene, a 1.6 kb repetitive element of the Trichinella genome. One set of primers was selected as the most appropriate for rapid detection. The specificity and sensitivity of the primers in LAMP reactions for T. spiralis larvae and muscle samples of mice infected with T. spiralis were determined. Another 10 heterologous parasites were selected for specificity assays. The results showed that target DNA was amplified and visualised by monitoring turbidity and adding calcein detection methods within 70 min at an isothermal temperature of 63 °C. The sensitivity of LAMP with the detection limit of 362 fg/μl was >10 times higher than that for PCR. The designed primers had a good specificity. No cross-reactivity was found with the DNA of any other parasites. The assay was able to detect T. spiralis in all mouse muscle samples infected with 10 T. spiralis larvae on day 20 p.i. We believe this is the first report regarding the application of the LAMP assay for detection of T. spiralis larvae in muscle samples from experimentally infected mice. This method demonstrates a potentially valuable means for the direct detection of T. spiralis larvae in meat inspection.     

Characterization of genetic variance within and among five populations of Sperata seenghala (Skyes, 1839) revealed by random amplified polymorphic DNA markers

The genetic diversity among five populations (Bhadbada reservoir, Mohinisagar reservoir, Bansagar reservoir, Bargi reservoir and Gandhisagar reservoir) was revealed using random amplified polymorphic DNA markers. 10 random primers screened, 5 primers revealed various banding patterns and yielded 71 total loci as an average of which 39.60 (55.77%) were polymorphic between the population and 86.84% within the population of Sperata seenghala. Population wise the highest genetic polymorphism was obtained in Bhadbada reservoir as 67.61% whereas the lowest was in Gandhisagar reservoir as 49.30%. However, Analysis of Molecular Variance indicated low genetic diversity (H_pop = 0.0921 ± 0.1249; I = 0.1584 ± 0.1942) in Bansagar reservoir. Relative genetic differentiation (G_ST = 0.3993) and restricted gene flow (N_m = 0.7523) as an average indicated low gene diversity among the fish populations. The un-weighted pair group method with averages (UPGMA) dendrogram showed 05 major clusters, each cluster representing a population. Fish population of Mohinisagar reservoir showed high genetic distance (0.3981) with respective Bargi reservoir population and highest genetic identity (0.8846) reflected between Bansagar and Gandhisagar reservoir. Highest genetic distance between Mohinisagar and Bargi reservoir fish populations shows no significant correlation between genetic and geographical distance of the genotypes collected from different lentic and geographical isolated water bodies. This investigation indicated that lowest genetic diversity existed in different geographic populations of S. seenghala. All the five populations were found to be low in genetic variation, which is useful information for future conservation measures of S. seenghala confined in natural water bodies of Madhya Pradesh.     

Effect of long-term exposure to mobile phone radiation on alpha-Int1 gene sequence of Candida albicans

Over the last decade, communication industries have witnessed a tremendous expansion, while, the biological effects of electromagnetic waves have not been fully elucidated. Current study aimed at evaluating the mutagenic effect of long-term exposure to 900-MHz radiation on alpha-Int1 gene sequences of Candida albicans. A standard 900 MHz radiation generator was used for radiation. 10 ml volumes from a stock suspension of C. albicans were transferred into 10 polystyrene tubes. Five tubes were exposed at 4 °C to a fixed magnitude of radiation with different time periods of 10, 70, 210, 350 and 490 h. The other 5 tubes were kept far enough from radiation. The samples underwent genomic DNA extraction. PCR amplification of alpha-Int1 gene sequence was done using one set of primers. PCR products were resolved using agarose gel electrophoresis and the nucleotide sequences were determined. All samples showed a clear electrophoretic band around 441 bp and further sequencing revealed the amplified DNA segments are related to alpha-Int1 gene of the yeast. No mutations in the gene were seen in radiation exposed samples. Long-term exposure of the yeast to mobile phone radiation under the above mentioned conditions had no mutagenic effect on alpha-Int1 gene sequence.     

Synthetic glycolipids for glioma growth inhibition developed from neurostatin and NF115 compound

Neurostatin, a natural glycosphingolipid, and NF115, a synthetic glycolipid, are inhibitors of glioma growth. While neurostatin shows high inhibitory activity on gliomas its abundance is low in mammalian brain. On the contrary NF115 exhibits less inhibitory activity on gliomas, but could be prepared by chemical synthesis. In this study we describe synthetic compounds, structurally related to NF115, capable of inhibiting glioma growth at low micromolar range. We used DNA microarray technology to compare the genes inhibited in U373-MG human glioma cells after treatment with the natural or synthetic inhibitor. New synthetic compounds were developed to interact with the product of Rho GDP dissociation inhibitor alpha gene, which was repressed in both treatments. Compounds that were inhibitors of glioma cell growth in assays for [3H]-thymidine incorporation were then injected in C6 tumor bearing rats and the tumor size in each animal group were measured. The GC-17, GC-4 and IG-5 are new compounds derived from NF115 and showed high antiproliferative activity on tumor cell lines. The GC-17 compound inhibited U373-MG glioblastoma cells (3.2 μM), the effects was fifty times more potent than NF115, and caused a significant reduction of tumor volume (P < 0.05) when tested in Wistar rats allotransplanted with C6 glioma cells.     

DNA damage induction and antiproliferative activity of vanadium(V) oxido monoperoxido complex containing two bidentate heteroligands

Several peroxidovanadium(V) complexes have been shown as a potent anticancer agents. The aim of this study was to investigate the interaction of monoperoxidovanadium(V) complex Pr₄N[VO(O₂)(ox)(phen)], (Vphen), [phen = 1,10-phenantroline, ox = oxalate(2?) and Pr₄N = tetra(n-propyl)ammonium(1+)] with DNA. UV–Vis spectrophotometry and the alkaline single-cell gel electrophoresis (SCGE, the comet assay) were used to examine the possibility of the vanadium(V) complex to induce changes in DNA. The interaction of Vphen with calf thymus DNA resulted in absorption hyperchromicity in DNA spectrum and shift of the absorption band of DNA to longer wavelengths for the [complex]/[DNA] concentration ratio equals to 4 and after 60 min of incubation. The rise in DNA absorption (by 34%) and bathochromic shift (Δλ_max = 6 nm) are indicative of the interaction between DNA and the complex molecules. DNA strand breaks in cellular DNA were investigated using the comet assay. The human lymphocytes were exposed to various concentrations of Vphen for 30 min. The results revealed that Vphen contributed to the DNA damage expressed as DNA strand breaks in concentration dependent manner. The used concentrations of Vphen (ranging from 0.1 to 100 μmol/L) caused higher DNA damage in lymphocytes compared to untreated cells (from 1.2 times for 0.1 μmol/L to 1.8 times for 100 μmol/L). Vphen was screened for its potential antitumor activity towards murine leukemia cell line L1210. Vphen exhibited significant antiproliferative activity depending on its concentration and time of exposure. The IC₅₀ values were 0.247 μg/mL (0.45 μmol/L) for 24 h, 0.671 μg/mL (1.21 μmol/L) for 48 h and 0.627 μg/mL (1.13 μmol/L) for 72 h.     

Kinesin family member 14 is a candidate prognostic marker for outcome of glioma patients

Background & aim: Human kinesin superfamily proteins (KIFs) are a conserved class of microtubule-dependent molecular motor proteins with adenosine triphosphatase activity and motion characteristics. As a member of KIFs, KIF14 plays an important role in the regulation of cell cycle and mitotic progression. Deregulation of KIF14 has been found in several human malignancies and also has been demonstrated to be involved in tumor progression and related to patient survival. The aim of this study was to investigate the clinicopathological significance of KIF14 expression in glioma. Methods: Real-time quantitative RT-PCR assay was performed to detect KIF14 mRNA expression, and Western blot and immunohistochemistry analyses were performed to detect KIF14 protein expression in human gliomas and non-neoplastic brain tissues, respectively. Then, the association of KIF14 immunostaining with clinicopathological factors and prognosis of glioma patients was also statistically analyzed. Results: KIF14 mRNA and protein expression were respectively increased 5.5- and 4.2-fold on average in glioma tissues relative to non-neoplastic brain tissues (both P < 0.001). Additionally, both KIF14 mRNA and protein expression increased with ascending pathological grade. Then, the high KIF14 immunostaining in glioma tissues was significantly associated with advanced pathological grade (P = 0.008), low Karnofsky performance score (KPS) (P = 0.02), high mitotic index (P = 0.005) and Ki-67 index (P = 0.008). Furthermore, both univariate and multivariate Cox regression analyses determined that KIF14 overexpression effectively predicted decreased overall survival in patients with gliomas. Conclusions: These findings offer the first convinced evidence that KIF14 expression in gliomas is tumor-specific and increased in more aggressive tumors. KIF14 might function as a candidate prognostic marker for human gliomas.     

Xanthan gum production and rheological behavior using different strains of Xanthomonas sp.

The proposal of the present study was to select and carry out the molecular characterization of strains of Xanthomonas sp. in order to correlate with gum production and determine possible genetic alterations during the study. The gums produced were also evaluated rheologically. Ten strains of Xanthomonas were used in the screening and the best ones in terms of productivity were Xanthomonas campestris pv. mangiferaeindicae 1230 (8.93 g/L), X. campestris pv. campestris 254 (9.49 g/L) and X. campestris pv. campestris 1078 (9.67 g/L). The gum produced by X. campestris pv. mangiferaeindicae presented the best apparent viscosity. The results for the profiles of the bands produced by RAPD showed considerable genetic variability amongst the evaluated strains, making not possible to neither group the strains according to pathovar or species, nor correlate the band profile with the productivity obtained. According to the RAPD analysis, no detectable mutations occurred in these bacteria during the study.     

Common gene variants in RAD51, XRCC2 and XPD are not associated with clinical outcome in soft-tissue sarcoma patients

BackgroundDNA repair mechanisms play a major role in cancer risk and progression. Germline variants in DNA repair genes may result in altered gene function and/or activity, thereby causing inter-individual differences in a patient's tumor recurrence capacity. In genes of the DNA repair pathway the gene variants RAD51 rs1801320 G > C, XRCC2 rs3218536 G > A and XPD rs13181 A > C have been previously related to genetic predisposition and prognosis of various cancer entities. In this study we investigated the association between these polymorphisms and time to recurrence (TTR) and overall survival (OS) in soft-tissue sarcoma (STS) patients after curative surgery.MethodsTwo hundred sixty STS patients were included in this retrospective study. Germline DNA was genotyped by 5′-exonuclease (TaqMan) technology. Kaplan Meier curves and multivariate Cox proportional models were calculated for TTR and OS.ResultsA statistically significant association was observed between tumor grade and adjuvant radiotherapy and TTR and between tumor grade and OS. No association was found between RAD51 rs1801320 G > C, XRCC2 rs3218536 G > A and XPD rs13181 A > C and TTR and OS in univariate and multivariate analysis.ConclusionOur results underline a prognostic effect of tumor grade and adjuvant radiotherapy in STS patients but indicate no association between RAD51 rs1801320 G > C, XRCC2 rs3218536 G > A and XPD rs13181 A > C and clinical outcome in STS patients after curative surgery.     

Combined molecular MRI and immuno-spin-trapping for in vivo detection of free radicals in orthotopic mouse GL261 gliomas

Free radicals play a major role in gliomas. By combining immuno-spin-trapping (IST) and molecular magnetic resonance imaging (mMRI), in vivo levels of free radicals were detected within mice bearing orthotopic GL261 gliomas. The nitrone spin trap DMPO (5,5-dimethyl pyrroline N-oxide) was administered prior to injection of an anti-DMPO probe (anti-DMPO antibody covalently bound to a bovine serum albumin (BSA)–Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)–biotin MRI contrast agent) to trap tumor-associated free radicals. mMRI detected the presence of anti-DMPO adducts by either a significant sustained increase (p < 0.001) in MR signal intensity or a significant decrease (p < 0.001) in T₁ relaxation, measured as %T₁ change. In vitro assessment of the anti-DMPO probe indicated a significant decrease (p < 0.0001) in T₁ relaxation in GL261 cells that were oxidatively stressed with hydrogen peroxide, compared to controls. The biotin moiety of the anti-DMPO probe was targeted with fluorescently-labeled streptavidin to locate the anti-DMPO probe in excised brain tissues. As a negative control a non-specific IgG antibody covalently bound to the albumin–Gd-DTPA–biotin construct was used. DMPO adducts were also confirmed in tumor tissue from animals administered DMPO, compared to non-tumor brain tissue. GL261 gliomas were found to have significantly increased malondialdehyde (MDA) protein adducts (p < 0.001) and 3-nitrotyrosine (3-NT) (p < 0.05) compared to normal mouse brain tissue, indicating increased oxidized lipids and proteins, respectively. Co-localization of the anti-DMPO probe with either 3-NT or 4-hydroxynonenal was also observed. This is the first report regarding the detection of in vivo levels of free radicals from a glioma model.     

Decreased expression of microRNA-200b is an independent unfavorable prognostic factor for glioma patients

Background and aimAs a member of the microRNA (miR)-200 family, miR-200b has been recognized as one of the fundamental regulators of epithelial–mesenchymal transition, chemosensitivity, cell proliferation, and cell cycle. Especially in glioma, miR-200b targets the CREB1 gene and suppresses the tumor cell growth in vitro. However, its involvement in human glioma has not yet been determined. The aim of this study was to investigate the clinical significance of miR-200b expression in this disease.MethodsmiR-200b expression in 266 pairs of human gliomas and matched nonneoplastic brain tissues was measured by real-time quantitative RT-PCR assay.ResultsCompared with nonneoplastic brain tissues, the expression level of miR-200b was significantly decreased in glioma tissues (tumor vs. normal: 2.87 ± 2.05 vs. 8.78 ± 2.50, P < 0.001). Of 266 patients with gliomas, 166 (62.41%) were in low miR-200b expression group. In addition, we found that the glioma tissues from high-grade tumors (grade III and IV) had much lower miR-200b expression than glioma tissues from low grade tumors (grade I and II). Moreover, the expression level of miR-200b was positively correlated with Karnofsky performance status (KPS) scores of glioma tissues. The results of a 60-month follow-up in 266 glioma patients further demonstrated that lower miR-200b expression was correlated with worse progression-free survival and overall survival in the patients with grade III and IV gliomas. Both univariate and multivariate analyses revealed that miR-200b was an independent prognostic indicator for glioma.ConclusionThese findings prove that the decreased expression of miR-200b may be associated with malignant tumor progression and poor prognosis in patients with gliomas, suggesting the potential role of miR-200b in glioma management. miR-200b may be a novel and valuable signature for predicting the clinical outcome of patients with gliomas.     

Opisthorchis viverrini and Haplorchis taichui: Development of a multiplex PCR assay for their detection and differentiation using specific primers derived from HAT-RAPD

Specific primers for the detection of Opisthorchis viverrini and Haplorchis taichui were investigated by using the HAT-RAPD PCR method. Fourteen arbitrary primers (Operon Technologies) were performed for the generation of polymorphic DNA profiles. The results showed that a 319 bp fragment generated from the OPA-04 primer was expected to be O. viverrini-specific while a 256 bp fragment generated from the OPP-11 primer was considered to be H. taichui-specific. Based on each sequence data, two pairs of specific primers were designed and sequences of each primer were as follows; H. taichui; Hapt_F5′-GGCCAACGCAATCGTCATCC-3′and Hapt_R1 5′-CTCTCGACCTCCTCTAGAAT-3′ which yielded a 170 bp PCR product. For O. viverrini, OpV-1F: 5′-AATCGGGCTGCATATTGACCGAT-3′ and OpV-1R: 5′-CGGTGTTGCTTATTTTGCAGACAA-3′ which generated a 319 bp PCR product. These specific primers were tested for efficacy and specific detection for all parasites DNA samples. The results showed that 170 and 319 bp specific PCR products were generated as equivalent to positive result in H. taichui and O. viverrini, respectively by having no cross-reaction with any parasites tested. PCR conditions are recommended at 68 °C annealing temperature and with 0.5 mM magnesium chloride (Mg Cl₂). Additionally, specific primers developed in this study were effective to determine the presence of both parasites in fish and snail intermediate hosts, which the DNA of O. viverrini was artificially spiked since it is rarely found in northern Thailand.The H. taichui and O. viverrini-specific primers successfully developed in this study can be use for epidemiological monitoring, preventing management and control programs.     

Complete nucleotide sequence of pGO1, the prototype conjugative plasmid from the staphylococci

In view of its historical significance as the prototype class III plasmid from the staphylococci, and its ongoing importance as a laboratory tool, we have determined the complete nucleotide sequence of pGO1. At exactly 54 kb, pGO1 is 2–4 kb larger than previously reported, and shares extensive (～31–46 kb) regions of near identical DNA sequence with other class III plasmids. In particular, we confirm that pGO1 is almost identical to plasmid pSK41 along the entire length of the latter, but additionally contains a co-integrated copy of plasmid pSK639, which accounts for the difference in size (～8 kb), and the fact that pGO1, but not pSK41, confers resistance to trimethoprim. The pSK639 co-integrant appeared to have undergone mutational inactivation of its mobilization functions, a finding which was confirmed experimentally. Although originally identified through an association with aminoglycoside resistance, the pGO1/pSK41 backbone replicon continues to play a key role in the dissemination of antibiotic resistance determinants in the staphylococci.