A versatile tRNA modification-sensitive northern blot method with enhanced performance

The ~22 mitochondrial and ~45 cytosolic tRNAs contain several dozen different posttranscriptional modified nucleotides such that each carries a unique constellation that complements its function. Many tRNA modifications are linked to altered gene expression and their deficiencies due to mutations in tRNA modification enzymes (TMEs) are responsible for numerous diseases. Easily accessible methods to detect tRNA hypomodifications can facilitate progress in advancing such molecular studies. Our lab developed a northern blot method that can quantify relative levels of base modifications on multiple specific tRNAs ~10 years ago which has been used to characterize four different TME deficiencies and is likely further extendable. The assay method depends on differential annealing efficiency of an DNA-oligo probe to the modified versus unmodified tRNA. The signal of this probe is then normalized by a second probe elsewhere on the same tRNA. This positive hybridization in the absence of modification (PHAM) assay has proven useful for i6A37, t6A37, m3C32 and m2,2G26 in multiple laboratories. Yet, over the years we have observed idiosyncratic inconsistency and variability in the assay. Here we document these for some tRNAs and probes and illustrate principles and practices for improved reliability and uniformity in performance. We provide an overview of the method and illustrate benefits of the improved conditions. This is followed by data that demonstrate quantitative validation of PHAM using a TME deletion control, and that nearby modifications can falsely alter the calculated apparent modification efficiency. Finally, we include a calculator tool for matching probe and hybridization conditions.

Methanogenesis at High Temperature, High Ionic Strength and Low pH in the Volcanic Area of Dallol, Ethiopia

The Dallol geothermal area originated as a result of seismic activity and the presence of a shallow underground volcano, both due to the divergence of two tectonic plates. In its ascent, hot water dissolves and drags away the subsurface salts. The temperature of the water that comes out of the chimneys is higher than 100 °C, with a pH close to zero and high mineral concentration. These factors make Dallol a polyextreme environment. So far, nanohaloarchaeas, present in the salts that form the walls of the chimneys, have been the only living beings reported in this extreme environment. Through the use of complementary techniques: culture in microcosms, methane stable isotope signature and hybridization with specific probes, the methanogenic activity in the Dallol area has been assessed. Methane production in microcosms, positive hybridization with the Methanosarcinales probe and the δ13CCH4-values measured, show the existence of extensive methanogenic activity in the hydrogeothermic Dallol system. A methylotrophic pathway, carried out by Methanohalobium and Methanosarcina-like genera, could be the dominant pathway for methane production in this environment.

FEATURES OF VIOLATIONS OF THE STATE OF THE VAGINAL ECOSYSTEM IN PREGNANT WOMEN WITH BACTERIAL VAGINOSIS

The aim: To assess the condition of the vaginal ecosystem in pregnant women with BV. Materials and methods: The main group consisted of 60 pregnant women with BV in the II trimester. The bacterioscopic examination, of vaginal smears was carried out. DNA diagnostics of the microbial spectrum of vaginal contents was performed. Bacteria with biofilm were visualized by fluorescence hybridization in situ. Results: Biofilms were found in 25 women (41.65%) of the main group, the main component of which was bacteria belonging to the Gardnerella cluster at a concentration of 7.9 ± 0.13 log CFU/ g. Atopobium vagine cluster bacteria gave positive hybridization signals in more than half of the patients and amounted to 6.8 ± 0.15 lg CFU / g. In addition, Snethia spp. was determined as a part of the biofilm at a concentration of 5.8 ± 0.3 lg CFU / g. Conclusions: Thus, the use of the proposed treatment regimen for women with vaginal dysbiosis led to the elimination of pathogenic and conditionally pathogenic microflora. However, the effectiveness of treatment in 5 cases was lower than expected, which indicates the emergence of bacterial resistance.

Sex chromosome composition revealed in Characidium fishes (Characiformes: Crenuchidae) by molecular cytogenetic methods

The W chromosome of the fishes Characidium cf. fasciatum, Characidium sp. and Characidium cf. gomesi is heterochromatic, as is usually seen in most Characidium species. Samples of W-chromatin were collected by mechanical microdissection and amplified by DOP-PCR (degenerate oligonucleotide-primed polymerase chain reaction), to be used as painting probes (DCg and CgW) and for sequence analysis. FISH (fluorescence in situ hybridization) with DCg probe painted the whole W chromosome, the pericentromeric region of Z chromosomes and the terminal region of B chromosomes. DOP-PCR-generated fragments were cloned, sequenced and tested by in situ hybridization, but only CgW4 produced positive hybridization signals. Clone sequence analysis recovered seven distinct sequences, of which six did not reveal any similarity to other known sequences in the GenBank or GIRI databases. Only CgW9 clone sequence was recognized as probably derived from a Helitron-transposon similar to that found in the genome of the zebrafish Danio rerio. Our results show that the composition of Characidium’s W chromosome does seem rich in repetitive sequences as well as other W chromosomes found in several species with a ZW sex-determining mechanism.

Development of Natural Fiber Reinforced Laminated Hybrid Composites

In this study, mechanical properties of composite laminates reinforced with various forms of glass fibers have been investigated. Tensile testing, impact testing and optical microscopy and SEM analysis results were discussed. The results of glass fiber reinforced novel composite material have been compared with the results of a commercial car front bumper material tests performed in same conditions. Study concluds that glass fiber has positive hybridization effect and increased tensile strengths, elastic modules and impact strengths in laminar hybrid composites.

Microbial community analysis of a full-scale membrane bioreactor treating industrial wastewater

A Kubota™ submerged membrane bio-reactor was applied to treat wastewater from a sugar manufacturing industry. To achieve optimal results, fundamental and extended understanding of the microbiology is important. Fluorescence in situ hybridization was used to evaluate the microbial community present. The majority of cells visualized in the sludge flocs by staining with the DNA fluorochrome DAPI, hybridized strongly with a bacterial probe. Probes specific for the alpha-, beta-, and gamma-subclasses of proteobacteria and high G + C Gram positive bacteria were used to characterize the community structures by in situ hybridization. Sampling was carried out over 12 weeks and samples were fixed with 4% paraformaldehyde for gram positive organisms and ice cold ethanol for gram negative organisms. The activated sludge population usually constitutes about 80 to 90% of proteobacteria. However, in this study it was found that a relatively small amount of proteobacteria was present within the system. No positive hybridization signal was observed with any of the applied eubacterial family- level probes.

Towards tracking marine larvae with in situ hybridization

Marine invertebrate larvae represent a transitory, but nonetheless important component of planktonic communities. Assessing their contribution to plankton diversity has been hindered by numerous methodological difficulties, notably at the identification step. For many sessile invertebrates, planktonic larvae also play a crucial role, as they are their sole dispersal vectors. Understanding connectivity patterns among marine populations is fundamental for managing coastal ecosystems and their associated resources. Indirect approaches, relying on population genetics models, have widely contributed to elucidate population structure and gene flow patterns, but show, in some cases, conflicting results with larval dispersal potential. In an attempt to facilitate surveys of larval distributions and abundances, an in situ hybridization on whole larvae method was tested over a range of marine invertebrate larvae collected from environmental plankton samples. Ribosomal RNA-targeted oligonucleotide probes were used for hybridization, followed by a colorimetric reaction allowing signal detection at the light microscope. Promising results were obtained, showing an unambiguous positive hybridization signal with a eukaryotic (positive) probe, but no signal with a negative probe. Using species-specific probes, the method could be applied to resolve key current questions in marine ecology, addressing both wide and fine scales.

Identification of a repetitive sequence belonging to a PPE gene of Mycobacterium tuberculosis and its use in diagnosis of tuberculosis

A repetitive sequence specific to Mycobacterium tuberculosis was isolated from a λgt11 library of M. tuberculosis by DNA–DNA hybridization using genomic DNA of M. tuberculosis as probe followed by subtractive hybridization with a cocktail of other mycobacterial DNA. This led to identification of CD192, a 1291 bp fragment of M. tuberculosis containing repetitive sequences, which produced positive hybridization signals with M. tuberculosis DNA within 30 min. Nucleotide sequencing revealed the presence of several direct and inverted repeats within the 1291 bp fragment that belonged to a PPE family gene (Rv0355) of M. tuberculosis. The use of CD192 as a DNA probe for the identification of M. tuberculosis in culture and clinical samples was investigated. The 1291 bp sequence was present in M. tuberculosis, Mycobacterium bovis and M. bovis BCG, but was not present in many of the other mycobacterial strains tested, including M. tuberculosis H37Ra. More than 300 clinical isolates of M. tuberculosis were probed with CD192, and the presence of the 1291 bp sequence was observed in all the clinical strains, including those lacking IS6110. The sequence displayed RFLP among the clinical isolates. A PCR assay was developed which detected M. tuberculosis with 100 % specificity from specimens of sputum, cerebrospinal fluid and pleural effusion from clinically diagnosed cases of tuberculosis.