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At least 757 records · Page 42Linked to original sources
PubMed2006

Pre-extraction sample handling by automated frozen disruption significantly improves subsequent proteomic analyses.

Here we quantitatively characterize two common homogenization strategies in the analysis of tissue proteomes: classical manual homogenization (MH) and an automated frozen disruption (AFD) technique. In a variety of tissues, many proteins were more efficiently extracted, resolved and detected, with high reproducibility after AFD, amounting to as much as 2% of the total resolved proteome. The benefits of AFD over MH are 2-fold: (1) AFD yields a much more thorough homogenate than MH; and (2) as a deep frozen alternative, AFD maintains a level of biological complexity that is not retained during MH. Thus, AFD coupled with refined 2DE protocols and Sypro Ruby staining yields quantitative proteomic analyses.

Animals↗
PubMed2006

ProteomIQ blue, a potent post-stain for the visualization and subsequent mass spectrometry based identification of fluorescent stained proteins on 2D-gels.

Manual spot excision for protein identification from fluorescent stained two-dimensional (2-D) gels is hard to accomplish. Here, we explore the use of ProteomIQ Blue as a post-stain method for the visualization of fluorescent stained/labeled proteins. We show that ProteomIQ Blue post-staining is almost as sensitive as staining with SYPRO Ruby or cyanine dyes alone. More than 90% of the protein spots that are stained with the fluorescent stains are still detectable with ProteomIQ Blue. In protein identification by mass spectrometry, ProteomIQ Blue post-stained spots provide high sensitivity and high protein sequence coverage of the peptide mass maps in both MALDI-TOF-MS and ESI-MS/MS analyses. In conclusion, post-staining of fluorescent stained gels with ProteomIQ Blue provides a facile and a powerful method to achieve quantitative protein analysis as well as protein identification in the same semianalytical gel without requiring sophisticated/expensive robotic equipment.

Bacterial Proteins↗
PubMed2006

Serial changes in urinary proteome profile of membranous nephropathy: implications for pathophysiology and biomarker discovery.

Membranous nephropathy is one of the most common causes of primary glomerular diseases worldwide. The present study adopted a gel-based proteomics approach to better understand the pathophysiology and define biomarker candidates of human membranous nephropathy using an animal model of passive Heymann nephritis (PHN). Clinical characteristics of Sprague-Dawley rats injected with rabbit anti-Fx1A antiserum mimicked those of human membranous nephropathy. Serial urine samples were collected at Days 0, 10, 20, 30, 40, and 50 after the injection with anti-Fx1A (number of rats = 6; total number of gels = 36). Urinary proteome profiles were examined using 2D-PAGE and SYPRO Ruby staining. Quantitative intensity analysis and ANOVA with Tukey post-hoc multiple comparisons revealed 37 differentially expressed proteins among 6 different time-points. These altered proteins were successfully identified by MALDI-TOF MS and classified into 6 categories: (i) proteins with decreased urinary excretion during PHN; (ii) proteins with increased urinary excretion during PHN; (iii) proteins with increased urinary excretion during PHN, but which finally returned to basal levels; (iv) proteins with increased urinary excretion during PHN, but which finally declined below basal levels; (v) proteins with undetectable levels in the urine during PHN; and (vi) proteins that were detectable in the urine only during PHN. Most of these altered proteins have functional significance in signaling pathways, glomerular trafficking, and controlling the glomerular permeability. The ones in categories (v) and (vi) may serve as biomarkers for detecting or monitoring membranous nephropathy. After normalization of the data with 24-h urine creatinine excretion, changes in 34 of initially 37 differentially expressed proteins remained statistically significant. These data underscore the significant impact of urinary proteomics in unraveling disease pathophysiology and biomarker discovery.

Biomarkers↗
PubMed2000

Photomechanical drug delivery into bacterial biofilms.

PURPOSE: To investigate whether photomechanical waves generated by lasers can increase the permeability of a biofilm of the oral pathogen Actinomyces viscosus. METHODS: Biofilms of Actinomyces viscosus were formed on bovine enamel surfaces. The photomechanical wave was generated by ablation of a target with a Q-switched ruby laser and launched into the biofilm in the presence of 50 microg/ml methylene blue. The penetration depth of methylene blue was measured by confocal scanning laser microscopy. Also, the exposed biofilms were irradiated with light at 666 nm. After illumination, adherent bacteria were scraped and spread over the surfaces of blood agar plates. Survival fractions were calculated by counting bacterial colonies. RESULTS: Confocal scanning laser microscopy revealed that a single photomechanical wave was sufficient to induce a 75% increase in the penetration depth of methylene blue into the biofilm. This significantly increased the concentration of methylene blue in the biofilm enabling its photodestruction. CONCLUSIONS: Photomechanical waves provide a potentially powerful tool for drug delivery that might be utilized for treatment of microbial infections.

Actinomyces viscosus↗
PubMed1999

cDNA sequence and mapping of the mouse Copb gene encoding the beta subunit of the COPI coatomer complex.

COPI-coated vesicles are involved in retrograde-directed selective transport of proteins from the Golgi complex to the endoplasmic reticulum (ER) as well as mediate anterograde transport of cargo proteins within the Golgi or in endosomal trafficking. The COPI protein complex contains an ADP-ribosylation factor (ARF1) and seven coatamer subunits (alpha, beta, beta', gamma, delta, epsilon, zeta-COP). The localization and function of human beta subunit of coatamer (COPB) suggests it is likely a candidate gene of ruby-eye-2 (ru2), which is a mouse model of human Hermansky-Pudlak syndrome characterized by the dysfunction of several subcellular organelles. In this study, we determined the entire coding sequence of mouse (Copb) cDNA by combining an overlapping mouse EST contig with EST walking. beta-COP was found highly conserved in mouse, rat, and human, and it is ubiquitously expressed in mouse. The Copb gene was mapped to mouse Chr 7 at a position of 53.3 cM by radiation hybrid mapping. Our RH mapping data, sequencing of RT-PCR products, and Western blotting exclude the Copb gene as a candidate for ru2.

Amino Acid Sequence↗
PubMed1999

Topical drug delivery in humans with a single photomechanical wave.

PURPOSE: Assess the feasibility of in vivo topical drug delivery in humans with a single photomechanical wave. METHODS: Photomechanical waves were generated with a 23 nsec Q-switched ruby laser. In vivo fluorescence spectroscopy was used as an elegant non-invasive assay of transport of 5-aminolevulinic acid into the skin following the application of a single photomechanical wave. RESULTS: The barrier function of the human stratum corneum in vivo may be modulated by a single (110 nsec) photomechanical compression wave without adversely affecting the viability and structure of the epidermis and dermis. Furthermore, the stratum corneum barrier always recovers within minutes following a photomechanical wave. The application of the photomechanical wave did not cause any pain. The dose delivered across the stratum corneum depends on the peak pressure and has a threshold at approximately 350 bar. A 30% increase in peak pressure, produced a 680% increase in the amount delivered. CONCLUSIONS: Photomechanical waves may have important implications for transcutaneous drug delivery.

Administration, Cutaneous↗
PubMed2004

Identification of endothelial proteins by MALDI-MS using a compact disc microfluidic system.

Vascular endothelial proteins have been analyzed using two-dimensional (2D) gel electrophoresis and subsequent mass spectrometry, with separate methods for the intervening sample preparations. Compact disc (CD) technology was found to be rapid, giving high overall yield both with ordinary Coomassie staining and with Sypro Ruby staining. Combined with automatic in-gel digestion, the CD technology has great capacity for large numbers of protein analysis, although for limited sample numbers, manual methods can give similar sequence coverage. In a test set of 48 samples, 45 proteins were identified using the CD preparation technique, 32 identified with higher sequence coverage using the CD technique, 7 with higher using ZipTips in a robotic workstation, and 5 with higher coverage using dried droplets of unpurified samples. In the process of these methodological comparisons, basic patterns for 116 endothelial proteins were defined, representing 297 separate protein spots on the 2D gels.

Compact Disks↗
PubMed2000

Treatment of dentine hypersensitivity by lasers: a review.

BACKGROUND, AIMS: Since the development of the ruby laser by Maiman in 1960, a variety of papers on potential applications for lasers in dentistry have been published. The purpose of this paper is to summarise laser applications for the treatment of dentine hypersensitivity. The effects of laser on pulp tissue and problems on laser treatment are also reviewed. This article reviews the role of lasers for the treatment of dentine hypersensitivity since 1985, summarises many research reports from the last decade, and surmises what the future may hold for lasers in this treatment. METHOD: To date, 4 kinds of lasers have been used for the treatment of dentine hypersensitivity, and the effectiveness ranged from 5.2 to 100%, which was dependent on the laser type and parameters used. The mechanism involved in laser treatment of dentine hypersensitivity are relatively unknown. RESULTS: These require clarification to result in safely effective treatment optimization. In general, the efficiency for the treatment of dentine hypersensitivity using lasers is higher than other methods, but in severe cases, it is less effective. CONCLUSION: It is necessary to consider the severity of dentine hypersensitivity before laser use.

Dental Pulp↗
PubMed2002

Inducing and probing non-thermal transitions in semiconductors using femtosecond laser pulses.

Soon after it was discovered that intense laser pulses of nanosecond duration from a ruby laser could anneal the lattice of silicon, it was established that this so-called pulsed laser annealing is a thermal process. Although the radiation energy is transferred to the electrons, the electrons transfer their energy to the lattice on the timescale of the excitation. The electrons and the lattice remain in equilibrium and the laser simply 'heats' the solid to the melting temperature within the duration of the laser pulse. For ultrashort laser pulses in the femtosecond regime, however, thermal processes (which take several picoseconds) and equilibrium thermodynamics cannot account for the experimental data. On excitation with femtosecond laser pulses, the electrons and the lattice are driven far out of equilibrium and disordering of the lattice can occur because the interatomic forces are modified due to the excitation of a large (10% or more) fraction of the valence electrons to the conduction band. This review focuses on the nature of the non-thermal transitions in semiconductors under femtosecond laser excitation.

Electrons↗
PubMed1991

Lasers in dentistry.

Clinical lasers are of two types; soft lasers are essentially an aid to healing with relatively few rigorous studies available to support their use. Surgical hard lasers, however, can cut both hard and soft tissues and replace the scalpel and drill in many areas. From initial experiments with the ruby laser most clinicians are using Argon, CO2 and now NdYAG systems. The first dental laser based on a NdYAG engine provides handpieces of similar size to conventional instrumentation and, being fed by a fibre-optic 'cable', has the flexibility for intra-oral use that the CO2 lasers, widely used in oral surgery, lack. Furthermore, extensive clinical investigation has demonstrated their safety in clinical practice and the fact that procedures can usually be performed without a local anaesthetic is obviously seen as a considerable advantage by patients. Sterilising as it cuts, the NdYAG laser promises to find uses not only in caries removal and soft tissue surgery, but also in endodontics and gingival curettage.

Dental Instruments↗
PubMed2004

Proteomic-based identification of haptoglobin-1 precursor as a novel circulating biomarker of ovarian cancer.

Screening for specific biomarkers of early-stage detection of ovarian cancer is a major health priority due to the asymptomatic nature and poor survival characteristic of the disease. We utilised two-dimensional gel electrophoresis (2DE) to identify differentially expressed proteins in the serum of ovarian cancer patients that may be useful as biomarkers of this disease. In this study, 38 ovarian cancer patients at different pathological grades (grade 1 (n=6), grade 2 (n=8) and grade 3 (n=24)) were compared to a control group of eight healthy women. Serum samples were treated with a mixture of Affigel-Blue and protein A (5 : 1) for 1 h to remove high abundance protein (e.g. immunoglobulin and albumin) and were displayed using 11 cm, pH 4-7 isoelectric focusing strips for the first dimension and 10% acrylamide gel electrophoresis for the second dimension. Protein spots were visualised by SYPRO-Ruby staining, imaged by FX-imager and compared and analysed by PDQuest software. A total of 24 serum proteins were differentially expressed in grade 1 (P<0.05), 31 in grade 2 (P<0.05) and 25 in grade 3 (P<0.05) ovarian cancer patients. Six of the protein spots that were significantly upregulated in all groups of ovarian cancer patients were identified by nano-electrospray quadrupole quadrupole time-of-flight mass spectrometry (n-ESIQ(q)TOFMS) and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOFMS) as isoforms of haptoglobin-1 precursor (HAP1), a liver glycoprotein present in human serum. Further identification of the spots at different pathological grades was confirmed by Western blotting using monoclonal antibody against a haptoglobin epitope contained within HAP1. Immunohistochemical localisation of HAP1-like activity was present in malignant ovarian epithelium and stroma but strong immunostaining was present in blood vessels, areas with myxomatous stroma and vascular spaces. No tissue localisation of HAP1-like immunoreactivity was observed in normal ovarian surface epithelium. These data highlight the need to assess circulating concentration of HAP1 in the serum of ovarian cancer patients and evaluate its potential as a biomarker in the early diagnosis of ovarian cancer.

Biomarkers, Tumor↗
PubMed1996

Slurry preparation by high-pressure homogenization for the determination of heavy metals in zoological and botanical certified reference materials and animal feeds by electrothermal atomic absorption spectrometry.

High-pressure homogenization was evaluated for the preparation of slurries suitable for the determination by ETAAS of Cr, Cu, Fe, Mn, Ni and Se in soft organ tissues (liver and kidney), certified reference materials of biological and botanical origin and animal feeds. Frozen fresh organ tissue, (2 g) or certified reference material or dried, ground plant material (0.1 g) was blended, at high speed with 20 ml of ethanol-water (1 + 9 v/v) containing 0.25% m/m of tetramethylammonium hydroxide and the resulting mixture was subjected to homogenization at 38.9 MPa. After four passes through the homogenizer, the resulting solution was suitable for analysis by ETAAS. Capping the flat valve head of the homogenizer with a ruby disc appreciably reduced (but did not eliminate) metal contamination introduced by the processing. Homogenization of botanical reference materials or dried animal feeds resulted in preparations with variable amounts of residual fibres and particulate matter in the resulting suspensions. Nonetheless, all the Cu and Mn and virtually all of the Fe had been transferred to the supernatant fraction and remained with that fraction for at least 10 d. The addition of EDTA to the solvent modestly increased the mobilization of Fe from the matrix but also increased the contamination from the homogenizer. The slopes of the calibration curves generated by the method of standard additions were not significantly different from those of calibration curves generated with aqueous standards in a homogenized blank indicating that there was no significant matrix effect for any of the analytes in the nine reference materials, liver or kidney or the five animal feed samples and that aqueous standards could be used to calibrate the instrumental response.

Animal Feed↗
PubMed2004

Designing molecules for PDT: red light-induced DNA cleavage on disulfide bond activation in a dicopper(II) complex.

The binuclear copper(II) complex [Cu)(RSSR)2](1), where RSSR is a dianionic Schiff base derived from 2-(thioethyl)salicylaldimine having a disulfide bond is prepared, structurally characterized by X-ray crystallography and its photo-induced DNA cleavage activity studied. The Schiff base ligand H2RSSR is also structurally characterized. The crystal structure of shows the discrete dimeric nature of the complex with each metal showing square-planar geometry with a CuN2O2 coordination (Cu...Cu, 5.011(1)A). The tetradentate Schiff base RSSR acts as a linker of two copper centers. The sulfur atoms in the disulfide unit do not show any apparent interaction with the metal ion. Complex 1, which is cleavage inactive in the dark in the presence of reducing agents, shows significant cleavage of supercoiled pUC19 DNA on exposure to UV light of 312 nm or visible light of different wavelengths under aerobic conditions, in the absence of any additives. DNA cleavage data from control experiments reveal involvement of the disulfide unit as a photosensitizer undergoing photo-induced S-S bond cleavage on exposure to UV light and the resulting species activates molecular oxygen to form singlet oxygen (1O2) that causes DNA cleavage following a type-II process. Photo-induced DNA cleavage by 1 on red-light exposure using a CW laser of 632.8 nm or a pulsed ruby laser of 694 nm is proposed to involve sulfide radicals in a type-I process and hydroxyl radicals as the reactive species.

Copper↗
PubMed2005

Red-light photosensitized cleavage of DNA by (l-lysine)(phenanthroline base)copper(II) complexes.

Ternary copper(II) complexes [Cu(l-lys)B(ClO4)](ClO4)(1-4), where B is a heterocyclic base, viz. 2,2'-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 3) and dipyrido[3,2-a:2',3'-c]phenazene (dppz, 4), are prepared and their DNA binding and photo-induced DNA cleavage activity studied (l-lys =l-lysine). Complex 2, structurally characterized by X-ray crystallography, shows a square-pyramidal (4 + 1) coordination geometry in which the N,O-donor l-lysine and N,N-donor heterocyclic base bind at the basal plane and the perchlorate ligand is bonded at the elongated axial site. The crystal structure shows the presence of a pendant cationic amine moiety -(CH2)4NH3+ of l-lysine. The one-electron paramagnetic complexes display a d-d band in the range of 598-762 nm in DMF and exhibit cyclic voltammetric response due to Cu(II)/Cu(I) couple in the range of 0.07 to -0.20 V vs. SCE in DMF-Tris-HCl buffer. The complexes having phenanthroline bases display good binding propensity to the calf thymus DNA giving an order: 4 (dppz) > 3 (dpq) > 2 (phen)>> 1 (bpy). Control cleavage experiments using pUC19 supercoiled DNA and distamycin suggest major groove binding for the dppz and minor groove binding for the other complexes. Complexes 2-4 show efficient DNA cleavage activity on UV (365 nm) or visible light (694 nm ruby laser) irradiation via a mechanistic pathway involving formation of singlet oxygen as the reactive species. The amino acid l-lysine bound to the metal shows photosensitizing effect at red light, while the heterocyclic bases are primarily DNA groove binders. The dpq and dppz ligands display red light-induced photosensitizing effects in copper-bound form.

Animals↗
PubMed2026

A smartphone-integrated plasmonic biosensor for amplification-free detection of African swine fever virus.

African Swine Fever Virus (ASFV) poses a catastrophic threat to global swine production, with recent outbreaks across Europe, Asia, and the Caribbean, significantly elevating the biosecurity risk to the United States' billion-dollar pork industry. Current diagnostic gold standards are laboratory-dependent and introduce critical delays in outbreak response. To address this gap, a plasmonic biosensor based on functionalized gold nanoparticles (GNPs) was developed for the rapid, amplification-free detection of ASFV. GNPs were surface-functionalized with 11-mercaptoundecanoic acid (MUDA) and combined in situ with ASFV-specific oligonucleotide probes targeting a conserved region of the p72 (B646L) gene. The detection mechanism relies on acid-induced aggregation: hybridization of target ASFV DNA to the probe generates a rigid duplex that shields the nanoparticles from acid-induced destabilization, maintaining a ruby-red color, whereas in the absence of target DNA the GNPs aggregate, producing a visible red-to-blue color shift. The optimized plasmonic biosensor demonstrated 100% analytical specificity, with no cross-reactivity against a panel of 19 non-target bacterial genomic DNA samples representative of the swine environment. Detection limits determined by the IUPAC 3&#x3c3; criterion were 285 copies per reaction for Probe 1 and 402 copies per reaction for Probe 2, within the same order of magnitude as the qPCR reference assay run on the same dilution series (approximately 312 copies per reaction) under the experimental conditions used here. A smartphone-based Bio-Analytics App employing an RGB color-conversion algorithm served as a quantitative reader, yielding signal-to-noise ratios (S/N) that strongly correlated with benchtop spectrophotometric readings (A520/A620 ratio, R2 = 0.96) and achieved diagnostic concordance with qPCR binary calls. This platform offers a robust and low-cost (&#x223c;$2 per test), amplification-free approach to ASFV screening with potential for point-of-need deployment, subject to future validation in clinical specimens.

Journal Article↗
PubMed2001

Fluorescent tracer in pilocarpine-treated rats shows widespread aberrant hippocampal neuronal connectivity.

Neuronal fibres of the hippocampal formation of normal and chronic epileptic rats were investigated by fluorescent tracing methods using the pilocarpine model of limbic epilepsy. Two months after onset of spontaneous limbic seizures, hippocampal slices were prepared and maintained in vitro for 10 h. Small crystals of fluorescent dye [fluorescein (fluoro-emerald) and tetramethylrhodamine (fluoro-ruby)] were applied to different hippocampal regions. The main findings were: (i) in control rats there was no supragranular labelling when the mossy fibre tract was stained in stratum radiatum of area CA3. However, in epileptic rats a fibre network in the inner molecular layer of the dentate gyrus was retrogradely labelled; (ii) a retrograde innervation of area CA3 by CA1 pyramidal cells was disclosed by labelling remote CA1 neurons after dye injection into the stratum radiatum of area CA3 in chronic epileptic rats; (iii) labelling of CA1 neurons apart from the injection site within area CA1 was observed in epileptic rats but not in control animals; and (iv), a subicular-hippocampal projection was present in pilocarpine-treated rats when the tracer was injected just below the stratum pyramidale of area CA1. The findings show that fibre rearrangement in distinct regions of the epileptic hippocampal formation can occur as an aftermath of pilocarpine-induced status epilepticus.

Action Potentials↗
PubMed2000

Phacomatosis pigmentovascularis type IIa successfully treated with two types of laser therapy.

We describe a 28-year-old Japanese woman with phacomatosis pigmentovascularis type IIa who was treated by 27 sessions of Q-switched ruby laser irradiation to the site of dermal melanosis on her face and three sessions of dye laser irradiation to the port-wine stain on her left cheek. This is the first report of the successful treatment of a cutaneous lesion in a patient with phacomatosis pigmentovascularis. As the outcome of the treatments was excellent, we conclude that phacomatosis pigmentovascularis type IIa can be treated successfully by the combination of the two types of laser therapy.

Adult↗