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In the present study, a protocol for Agrobacterium tumefaciens-mediated transformation has been optimized for Woodfordia fruticosa (L.) Kurz. Precultured axenic leaf segments were co-cultivated with A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301 with β-glucuronidase (uidA) containing intron as the reporter gene and hygromycin phosphotransferase (hpt) as a selectable marker gene. After 3 days of co-cultivation, leaf segments were cultured on MS medium containing Thidiazuron (TDZ 4.54 μM) and Indole-3-acetic acid IAA (1.14 μM) + 20 mg/l hygromycin + 200 mg/l cefotaxime (PTSM1) for 4 weeks (includes a single subculture onto the same medium at a 2 week interval). They were subsequently cultured for 3 weeks on MS medium containing Thidiazuron (TDZ 4.54 μM) and Indole-3-acetic acid IAA (1.14 μM) + 25 mg/l hygromycin + 100 mg/l cefotaxime (PTSM2) medium for further development and shoot elongation. The hygromycin resistant shoots were rooted on a rooting medium (PTRM) containing half strength MS medium + 4.90 μM IBA + 25 mg/l hygromycin. A highest transformation efficiency of 44.5% with a mean number of 2.6 transgenic shoots per explant was achieved. Successful transformation was confirmed by the histochemical GUS activity of the regenerated shoots, PCR and RT-PCR analysis using respective primers. Southern blot analysis revealed that the hpt gene integrated into the genome of transgenic W. fruticosa. Establishment of genetic transformation protocol may facilitate the improvement of this medicinal plant in terms of enhancement of secondary metabolites.

Trichomoniasis is the most common nonviral sexually transmitted disease; it is caused by Trichomonas vaginalis and seriously threatens human reproductive health. Telomeres are specialised DNA-protein complexes at the ends of chromosomes that have a protective function. The aim of the present study was to identify and characterise the telomeric DNA of T. vaginalis-which has not been previously reported-by multiple molecular methods including sequencing, the Bal nuclease (BAL) 31 nuclease assay, fluorescence in situ hybridisation (FISH), and Southern blotting. We found numerous repeated units of TTTTAGGG in T. vaginalis genomic DNA digested with S1 nuclease in combination with XbaI restriction enzyme. The (TTTTAGGG) n tandem repeats were also highly sensitive to BAL 31 exonuclease digestion. We confirmed that the (TTTTAGGG) n repeats were located at the end of T. vaginalis chromosomes by FISH. Restriction enzyme digestion combined with Southern blotting using a digoxigenin-labelled (TTTTAGGG) 5 probe showed that the T. vaginalis telomeric DNA length varied from 1.0 to 1.5 kb. This is the first report on the telomeric DNA sequence of T. vaginalis which includes the length and distribution on chromosomes; our findings lay a foundation for further study on telomere maintenance mechanisms in T. vaginalis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

An intronic GGGGCC hexanucleotide repeat expansion in the chromosome 9 open reading frame 72 (C9orf72) gene was considered as the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia in Caucasian populations. Using repeat-primed polymerase chain reaction analysis and Southern blotting methods, we assessed the frequency and size of hexanucleotide repeat expansion in a cohort of 918 sporadic ALS (SALS) patients and 632 control individuals of Han Chinese origin. We identified 8 (0.87%) of the SALS patients and none of control individuals as carriers of C9orf72 expansions with 700-3500 repeats. A comprehensive neuropsychological battery was conducted on 4 expansion-positive ALS patients, where 3 patients were found to have cognitive impairment. All expansion-positive patients were genotyped for the previously reported 20 single-nucleotide polymorphism (SNP) risk haplotypes on chromosome 9p21. Among them, 13 SNP risk haplotypes were shared in all expansion carriers, suggesting a common founder from European ancestry. Further meta-analysis demonstrated that the intermediate expansion size with 24-30 repeats, rare in both patients and controls, were significantly associated with the risk for ALS. To our knowledge, this is the first study to identify a proportion of Chinese SALS patients carrying this pathologic expansion of up to ∼3500 repeats and to completely elaborate the 20-SNP risk haplotypes in Chinese expansion-positive patients, providing indispensable evidence for the origin, geographical range, and population prevalence of the C9orf72-associated ALS., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Histomonas meleagridis, a protozoan parasite that can infect gallinaceous birds, affects mainly the liver and caeca of infected birds. As a consequence of the recent ban of chemotherapeuticals in Europe and the USA, histomonosis gained somewhat more attention due to its re-emergence and the fact that there is no effective treatment available. Therefore, special attention is now also given towards the diagnosis and the control of the disease. In the actual study we report the development of highly specific and sensitive real-time PCR methods for detection and quantification of the parasite, based on two protein coding genes, Fe-hydrogenase (FeHYD) and rpb1. Both genes seem to be in a single copy in H. meleagridis as shown by southern blotting and absolute quantification using real-time PCRs on samples containing a known amount of the parasite. The real-time PCR assays based on FeHYD and rpb1 genes were found to be an efficient method for the quantification and detection of H. meleagridis in in vitro grown cultures, tissues of infected birds and in faecal samples. Both real-time PCRs were able to detect up to a single cell in in vitro cultures of H. meleagridis and in fecal samples spiked with H. meleagridis. Finally, qPCR assays were shown to be highly specific for H. meleagridis as samples containing either of the two H. meleagridis genotypes were positive, whereas samples containing other protozoa such as Tetratrichomonas gallinarum, Trichomonas gallinae, Simplicimonas sp., Tritrichomonas sp., Parahistomonas wenrichi, Dientamoebidae sp. and Blastocystis sp. were all negative., (Copyright © 2015 Elsevier B.V. All rights reserved.)

An intronic G(4)C(2) hexanucleotide repeat expansion in C9ORF72 is a major cause of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Several mechanisms including RNA toxicity, repeat-associated non-AUG translation mediated dipeptide protein aggregates, and haploinsufficiency of C9orf72 have been implicated in the molecular pathogenesis of this disorder. The aims of this study were to compare the use of two different Southern blot probes for detection of repeat expansions in an amyotrophic lateral sclerosis and frontotemporal lobar degeneration pathological cohort and to determine the levels of C9orf72 transcript variants and protein isoforms in patients versus control subjects. Our Southern blot studies identified smaller repeat expansions (250-1800 bp) that were only detectable with the flanking probe highlighting the potential for divergent results using different Southern blotting protocols that could complicate genotype-phenotype correlation studies. Further, we characterize a new C9orf72 antibody and show for the first time decreased C9orf72 protein levels in the frontal cortex from patients with a pathological hexanucleotide repeat expansion. These data suggest that a reduction in C9orf72 protein may be a consequence of the disease., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)