Use of the Verrucomicrobia-specific probe EUB338-III and fluorescent in situ hybridization for detection of "Candidatus Xiphinematobacter" cells in nematode hosts

Fluorescent in situ hybridization with a 16S rRNA probe specific for Verrucomicrobia was used to (i) confirm the division-level identity of and (ii) study the behavior of the obligate intracellular verrucomicrobium "Candidatus Xiphinematobacter" in its nematode hosts. Endosymbionts in the egg move to the pole where the gut primordium arises; hence, they populate the intestinal epithelia of juvenile worms. During the host's molt to adult female, the endosymbionts concentrate around the developing ovaries to occupy the ovarian wall. Some bacteria are enclosed in the ripening oocytes for vertical transmission. Verrucomicrobia in males stay outside the testes because the tiny spermatozoids are not suitable for transmission of cytoplasmic bacteria.     

Miniaturized all fiber probe for optical coherence tomography and pH detection of biological tissue

We present a novel all-fiber probe with 710-μm outside diameter for combined optical coherence tomography and pH detection. In cancer surgery, a significant challenge is how to completely remove the malignant tumor without cutting too much normal tissue. The difference between cancer tissue and normal tissue not only lies in morphology and structure but also in tissue pH, where malignant tissue has a lower pH. This dual-modality probe combined optical coherence tomography and pH detection of biological tissue, is expected to determine whether the tissue is cancerous quickly and accurately. The probe utilizes a typical three-segment structure (double-clad fiber - no-core fiber - graded-index fiber). We obtained a lateral resolution of ~10.6 μm, a working distance of ~506 μm and a pH measurement accuracy of 0.01 pH unit for the probe. The performance of the all-fiber probe was verified through an ex vivo experiment using the porcine brain specimen.     

The development of 1,3-diphenylisobenzofuran as a highly selective probe for the detection and quantitative determination of hydrogen peroxide

1,3-Diphenylisobenzofuran (DPBF) has been developed as a selective probe for the detection and quantitative determination of hydrogen peroxide in samples containing different reactive nitrogen and oxygen species (RNOS). DPBF is a fluorescent probe which, for almost 20 years, was believed to react in a highly specific manner toward some reactive oxygen species (ROS) such as singlet oxygen and hydroxy, alkyloxy or alkylperoxy radicals. Under the action of these individuals DPBF has been rapidly transformed to 1,2-dibenzoylbenzene (DBB). In order to check if DPBF can act as a unique indicator of the total amount of different RNOS, as well as oxidative stress caused by an overproduction of these individuals, a series of experiments was carried out, in which DPBF reacted with peroxynitrite anion, superoxide anion, hydrogen peroxide, hypochlorite anion, and anions commonly present under biological conditions, namely nitrite and nitrate. In all cases, except for hydrogen peroxide, the product of the reaction is DBB. Only under the action of H₂O₂ 9-hydroxyanthracen-10(9H)-one (oxanthrone) is formed. This product has been identified with the use of fluorescence spectroscopy, NMR spectroscopy, high performance liquid chromatography coupled with mass spectrometry, infrared spectroscopy, elemental analysis, and cyclic voltammetry (CV). A linear relationship was found between a decrease in the fluorescence intensity of DPBF and the concentration of hydrogen peroxide in the range of concentrations of 0.196–3.941?mM. DPBF responds to hydrogen peroxide in a very specific way with the limits of detection and quantitation of 88 and 122.8?μM, respectively. The kinetics of the reaction between DBBF and H₂O₂ was also studied.     

Species-specific DNA probe and development of a quantitative PCR assay for the detection of Morganella morganii

Aims: To develop a SYBR Green quantitative PCR assay (qPCR) for the specific detection of Morganella morganii, a fish pathogen responsible for the Histamine Fish Poisoning. Methods and Results: A new primer set, amplifying a 179‐bp fragment of the 16S rRNA gene, was selected for specificity, and 14 M. morganii strains and 32 non‐Morganella strains were evaluated. The melting temperature of 84°C was consistently specific for the amplicon. Two standard curves were constructed: the minimum detection sensitivity was 0·563 pg of pure DNA, corresponding to DNA extracted from nine cells of M. morganii. The qPCR assay was evaluated in experiments with seeded fish samples, and the regression coefficient values were calculated. Conclusions: A highly specific and rapid assay was developed for the detection of M. morganii in tuna fish samples. Significance and Impact of the Study: This method represents the first study about the quantification of pathogenic M. morganii in fish products. This approach can be utilized to prevent the presence of this undesirable species in the food chain.     

Evaluation of a digoxigenin-labelled probe for detection of Aeromonas spp.

An outer membrane protein-based Digoxigenin (DIG)-labelled DNA probe was developed for the specific detection of Aeromonas sp. from food/environmental/clinical samples. Dot blot reaction answered for all the Aeromonas isolates and was negative for Escherichia coli , Pseudomonas sp., Klebsiella sp., Vibrio parahaemolyticus , V. harveyi , V. alginolyticus , V. vulnificus . Edwardsiella tarda and Staphylococcus sp. As this protein is highly conserved in various Aeromonas species, the probe has the potential for use as a rapid and reliable diagnostic tool.     

The development and evaluation of monoclonal antibodies for the detection of polycyclic aromatic hydrocarbons

A highly sensitive enzyme-linked immunosorbent assay (ELISA) for the detection of 3- to 5-ring polycyclic aromatic hydrocarbons (PAHs) has been developed. A functional derivative of dibenzothiophene was synthesized and covalently linked to carrier proteins that were used to produce monoclonal antibodies (mAbs). During the conjugation step, the conjugation efficiency was improved by the presence of 25% N,N-dimethylformamide (DMF). Antibodies were selected based on a competitive inhibition assay to isolate those with the highest sensitivity for free PAHs. When using the mAb in an ELISA format, free PAHs were detected at a concentration as low as 0.1 μg/L (0.1 ppb) in aqueous samples.     

An optimized probe set for the detection of small interchromosomal aberrations by use of 24-color FISH

The rapid spread of the use of new 24-color karyotyping techniques has preceded their standardization. This is best documented by the fact that the exact resolution limits have not yet been defined. Indeed, it is shown here that a substantial proportion of interchromosomal aberrations will be missed by all multicolor karyotyping systems currently in use. We demonstrate that both the sensitivity and the specificity of 24-color karyotyping critically depend on the fluorochrome composition of chromosomes involved in an interchromosomal rearrangement. As a solution, we introduce a conceptual change in probe labeling. Seven-fluorochrome sets that overcome many of the current limitations are described, and examples of their applications are shown. The criteria presented here for an optimized probe-set design and for the estimation of resolution limits should have important consequences for pre- and postnatal diagnostics and for research applications.     

A comprehensive assessment of the musculoskeletal system: The CAMS-Knee data set

Combined knowledge of the functional kinematics and kinetics of the human body is critical for understanding a wide range of biomechanical processes including musculoskeletal adaptation, injury mechanics, and orthopaedic treatment outcome, but also for validation of musculoskeletal models. Until now, however, no datasets that include internal loading conditions (kinetics), synchronized with advanced kinematic analyses in multiple subjects have been available. Our goal was to provide such datasets and thereby foster a new understanding of how in vivo knee joint movement and contact forces are interlinked – and thereby impact biomechanical interpretation of any new knee replacement design. In this collaborative study, we have created unique kinematic and kinetic datasets of the lower limb musculoskeletal system for worldwide dissemination by assessing a unique cohort of 6 subjects with instrumented knee implants (Charité – Universitätsmedizin Berlin) synchronized with a moving fluoroscope (ETH Zürich) and other measurement techniques (including whole body kinematics, ground reaction forces, video data, and electromyography data) for multiple complete cycles of 5 activities of daily living. Maximal tibio-femoral joint contact forces during walking (mean peak 2.74 BW), sit-to-stand (2.73 BW), stand-to-sit (2.57 BW), squats (2.64 BW), stair descent (3.38 BW), and ramp descent (3.39 BW) were observed. Internal rotation of the tibia ranged from 3° external to 9.3° internal. The greatest range of anterio-posterior translation was measured during stair descent (medial 9.3 ± 1.0 mm, lateral 7.5 ± 1.6 mm), and the lowest during stand-to-sit (medial 4.5 ± 1.1 mm, lateral 3.7 ± 1.4 mm). The complete and comprehensive datasets will soon be made available online for public use in biomechanical and orthopaedic research and development.     

Leishmania donovani: development and characterisation of a kinetoplast DNA probe and its use in the detection of parasites

The polymerase chain reaction is used increasingly widely for the diagnosis of both cutaneous and visceral leishmaniasis and for the identification of asymptomatic carriers in the population in endemic disease areas. The use of complex-specific hybridisation probes in conjunction with the polymerase chain reaction increases the specificity as well as the sensitivity of the diagnostic procedure as it discriminates between different infecting Leishmania species. A minicircle kinetoplast DNA probe, B4 Rsa, which hybridizes to all members of the Leishmania (L.) donovani complex has been identified and characterised. It is a segment of a minicircle highly conserved in Bangladeshi and Indian L. (L.) donovani isolates.     

Pentaprobe: a comprehensive sequence for the one-step detection of DNA-binding activities

The rapid increase in the number of novel proteins identified in genome projects necessitates simple and rapid methods for assigning function. We describe a strategy for determining whether novel proteins possess typical sequence-specific DNA-binding activity. Many proteins bind recognition sequences of 5 bp or less. Given that there are 4⁵ possible 5 bp sites, one might expect the length of sequence required to cover all possibilities would be 4⁵ × 5 or 5120 nt. But by allowing overlaps, utilising both strands and using a computer algorithm to generate the minimum sequence, we find the length required is only 516 base pairs. We generated this sequence as six overlapping double-stranded oligonucleotides, termed pentaprobe, and used it in gel retardation experiments to assess DNA binding by both known and putative DNA-binding proteins from several protein families. We have confirmed binding by the zinc finger proteins BKLF, Eos and Pegasus, the Ets domain protein PU.1 and the treble clef N- and C-terminal fingers of GATA-1. We also showed that the N-terminal zinc finger domain of FOG-1 does not behave as a typical DNA-binding domain. Our results suggest that pentaprobe, and related sequences such as hexaprobe, represent useful tools for probing protein function.     

G-Quadruplex structure: a target for anticancer therapy and a probe for detection of potassium

G-Quadruplexes are four-stranded DNA structures that play important regulatory roles in the maintenance of telomere length by inhibiting telomerase activity. Telomeres are specialized functional DNA-protein structures consisting of a variable number of tandem G-rich repeats together with a group of specific proteins. Telomere losses during cell replication are compensated by telomerase, which adds telomeric repeats onto the chromosome ends in the presence of its substrate--the 3 -overhang. Recently, quadruplexes have been considered as a potential therapeutic target for human cancer because they can inhibit telomerase activity, and some quadruplex-interacting drugs can induce senescence and apoptosis of cancer cells. In addition, due to the potassium preference to the other cations, especially sodium ions, quadruplexes have been suggested for developing potassium detection probes with higher sensitivity and selectivity. This review will illustrate these two aspects to provide further understanding of G-quadruplex structures.