Small-molecule-mediated OGG1 inhibition attenuates pulmonary inflammation and lung fibrosis in a murine lung fibrosis model.

Interstitial lung diseases such as idiopathic pulmonary fibrosis (IPF) are caused by persistent micro-injuries to alveolar epithelial tissues accompanied by aberrant repair processes. IPF is currently treated with pirfenidone and nintedanib, compounds which slow the rate of disease progression but fail to target underlying pathophysiological mechanisms. The DNA repair protein 8-oxoguanine DNA glycosylase-1 (OGG1) has significant roles in the modulation of inflammation and metabolic syndromes. Currently, no pharmaceutical solutions targeting OGG1 have been utilized in the treatment of IPF. In this study we show Ogg1-targeting siRNA mitigates bleomycin-induced pulmonary fibrosis in male mice, highlighting OGG1 as a tractable target in lung fibrosis. The small molecule OGG1 inhibitor, TH5487, decreases myofibroblast transition and associated pro-fibrotic gene expressions in fibroblast cells. In addition, TH5487 decreases levels of pro-inflammatory mediators, inflammatory cell infiltration, and lung remodeling in a murine model of bleomycin-induced pulmonary fibrosis conducted in male C57BL6/J mice. OGG1 and SMAD7 interact to induce fibroblast proliferation and differentiation and display roles in fibrotic murine and IPF patient lung tissue. Taken together, these data suggest that TH5487 is a potentially clinically relevant treatment for IPF but further study in human trials is required.

Effective protein extraction combined with data independent acquisition analysis reveals a comprehensive and quantifiable insight into the proteomes of articular cartilage and subchondral bone.

OBJECTIVE: The objectives of this study was to establish a sensitive and reproducible method to map the cartilage and subchondral bone proteomes in quantitative terms, and mine the proteomes for proteins of particular interest in the pathogenesis of osteoarthritis (OA). The horse was used as a model animal. DESIGN: Protein was extracted from articular cartilage and subchondral bone samples from three horses in triplicate by pressure cycling technology or ultrasonication. Digested proteins were analysed by data independent acquisition based mass spectrometry. Data was processed using a pre-established spectral library as reference database (FDR 1%). RESULTS: We identified to our knowledge the hitherto most comprehensive quantitative cartilage (1758 proteins) and subchondral bone (1482 proteins) proteomes in all species presented to date. Both extraction methods were sensitive and reproducible and the high consistency of the identified proteomes (>97% overlap) indicated that both methods preserved the diversity among the extracted proteins. Proteome mining revealed a substantial number of quantifiable cartilage and bone matrix proteins and proteins involved in osteogenesis and bone remodeling, including ACAN, BGN, PRELP, FMOD, COMP, ACP5, BMP3, BMP6, BGLAP, TGFB1, IGF1, ALP, MMP3, and collagens. A number of proteins, including COMP and TNN, were identified in different protein isoforms with potential unique biological roles. CONCLUSION: We have successfully developed two sensitive and reproducible non-species specific workflows enabling a comprehensive quantitative insight into the proteomes of cartilage and subchondral bone. This facilitates the prospect of investigating the molecular events at the osteochondral unit in the pathogenesis of OA in future projects.

Guided bone regeneration using individualized ceramic sheets.

Guided bone regeneration (GBR) describes the use of membranes to regenerate bony defects. A membrane for GBR needs to be biocompatible, cell-occlusive, non-toxic, and mouldable, and possess space-maintaining properties including stability. The purpose of this pilot study was to describe a new method of GBR using individualized ceramic sheets to perfect bone regeneration prior to implant placement; bone regeneration was assessed using traditional histology and three-dimensional (3D) volumetric changes in the bone and soft tissue. Three patients were included. After full-thickness flap reflection, the individualized ceramic sheets were fixed. The sites were left to heal for 7 months. All patients were evaluated preoperatively and at 7 months postoperative using cone beam computed tomography and 3D optical equipment. Samples of the regenerated bone and soft tissue were collected and analyzed. The bone regenerated in the entire interior volume of all sheets. Bone biopsies revealed newly formed trabecular bone with a lamellar structure. Soft tissue biopsies showed connective tissue with no signs of an inflammatory response. This was considered to be newly formed periosteum. Thus ceramic individualized sheets can be used to regenerate large volumes of bone in both vertical and horizontal directions independent of the bone defect and with good biological acceptance of the material.

Targeted mass spectrometry analysis of neutrophil-derived proteins released during sepsis progression.

Early diagnosis of severe infectious diseases is essential for timely implementation of lifesaving therapies. In a search for novel biomarkers in sepsis diagnosis we focused on polymorphonuclear neutrophils (PMNs). Notably, PMNs have their protein cargo readily stored in granules and following systemic stimulation, an immediate increase of neutrophil-borne proteins can be observed into the circulation of sepsis patients. We applied a combination of mass spectrometry (MS) based approaches, LC-MS/MS and selected reaction monitoring (SRM), to characterise and quantify the neutrophil proteome in healthy or disease conditions. With this approach we identified a neutrophil-derived protein abundance pattern in blood plasma consisting of 20 proteins that can be used as a protein signature for severe infectious diseases. Our results also show that SRM is highly sensitive, specific, and reproducible and, thus, a promising technology to study a complex, dynamic and multifactorial disease such as sepsis.

Intrinsically conducting polymer nanowires for biosensing.

Nanomaterials are commonly exploited to increase the sensitivity of sensors. Conductive polymers are emerging as promising sensing materials as they are easy to functionalize with the appropriate sensing probes, and also act as signal transducers. By constraining the material into one dimensional nanowires, extraordinary sensitivity is achieved. This review deals with the fabrication of these electrically conductive polymer nanowire (ECPNW) sensors and their use for detecting nucleic acid sequences, proteins and pathogens.

Large area protein nanopatterning for biological applications.

Large area nanopatterns of functional proteins are demonstrated. A new approach to analyze atomic force microscopy height histograms is used to quantify protein and antibody binding to nanoscale patches. Arrays of nanopatches, each containing less than 40 laminin molecules, are shown to be highly functional binding close to 1 monoclonal anti-laminin IgG (site by IKVAV sequence) or 3-4 polyclonal anti-laminin IgG's per surface bound laminin. Complementary quartz crystal microbalance measurements indicate higher functionality at nanopatches than on homogeneous surfaces.

A novel tool for high-resolution transmission electron microscopy of intact interfaces between bone and metallic implants.

A key feature in the understanding of the mechanisms of integration versus rejection of implanted materials is a deepened understanding of the elemental and molecular compositions of the interface zone between the surface of the synthetic man-made material and the biological components of tissue. Intact interfaces between metallic implants and tissues have not been able to image and analyse on the ultrastructural level with the common transmission electron microscopy (TEM) sample preparation techniques. By using focused ion beam microscopy for site-specific preparation of TEM samples, intact interfaces between metal implants and calcified tissue were imaged for the first time. The interface's elemental and crystallographic compositions were determined using energy dispersive X-ray mapping and electron diffraction. The developed technique fulfills a long-sought-for demand to correlate the surface properties of implanted metal prostheses with the fine structure and composition of preserved interfaces with tissues.

Pancytopenia induced by low-dose methotrexate. A study of the cases reported to the Finnish Adverse Drug Reaction Register From 1991 to 1999.

OBJECTIVE: To study cases of low-dose methotrexate-induced pancytopenia with special reference to clinical outcome and factors predisposing to bone marrow suppression. METHODS: Patient files of 14 cases of methotrexate-induced pancytopenia reported to the National Agency for Medicines in Finland from 1991 to 1999 were reviewed. A review of four additional cases was included. RESULTS: Of the 18 patients (median age 72 years), 12 had rheumatoid arthritis, one psoriatic arthritis, five psoriasis without arthritis, and one pemphigus erythematosus. Major co-morbidity was recorded in 12 patients, and 16 patients used significant concomitant drugs. Eight patients had a mildly or moderately elevated serum creatinine concentration. In every patient the occurrence of cytopenia was abrupt. Eight patients (44%) died, and the most frequent cause of death was infection. CONCLUSIONS: Our data show that methotrexate-induced pancytopenia is associated with high mortality especially in cases with significant co-morbidity and concomitant medications.

Antimycobacterial flavones from Haplopappus sonorensis.

Crude extracts of Haplopappus sonorensis (A. Gray) S.F. Blake (Asteraceae), showed activity against Mycobacterium tuberculosis H(37)Rv. By assay-guided fractionation, 5-hydroxy-3,7,4'-trimethoxyflavone (1). 5,7-dihydroxy-3,4'-dimethoxyflavone (2). and 5,4'-dihydroxy-3,7-dimethoxyflavone (3). were identified as the antimycobacterial principles. Compound 2 was the most active compound.

Quality assessment of 26,304 herniorrhaphies in Denmark: a prospective nationwide study.

BACKGROUND: Groin hernia repair is one of the most frequent operations, but there is no consensus about surgical or anaesthetic technique. Furthermore, no nationwide studies have been done. Our aim was to investigate outcome results of groin hernia surgery to improve quality of treatment. METHODS: We prospectively recorded 26304 groin hernia repairs done in Denmark from Jan 1, 1998, to June 30, 2000, in a nationwide Danish hernia database. FINDINGS: 93% of all groin herniorrhaphies done in Denmark in the 30 months of the study were recorded in the database. Kaplan-Meier estimates of reoperation rates 30 months after anterior mesh repair and laparoscopic repair were significantly lower than after sutured posterior wall repairs in primary inguinal hernia (2.2% and 2.6% vs 4.4%; p<0.0001). Reoperation rates were also lower with anterior mesh repair (6.1%; p<0.0001) and laparoscopic repair (3.4%; p<0.0001) than with sutured posterior wall repair (10.6%) after recurrent hernia. Use of Lichtenstein mesh repair increased from 33% in January, 1998, to 62% in June, 2000, whereas use of laparoscopic repair remained constant at about 5%. Kaplan-Meier estimates of reoperation rates were 2.8% in the first 15 months and 1.6% in the second (p=0.03). For elective repairs, only 59% of patients were treated on an outpatient basis, and only 18% had local anaesthesia. INTERPRETATION: Mesh repairs have a lower reoperation rate than conventional open repairs. Systematic prospective recording of treatment and outcome variables in a national clinical database improved the overall quality of surgical care. However, there is a large potential for cost savings and more efficient patient care with extended use of mesh techniques, outpatient surgery, and local anaesthesia.

Intramolecular homolytic substitution at selenium: synthesis of novel selenium-containing vitamin E analogues.

Treatment of 1-(benzylselenenyl)-5-butyl-5-nonanol (10) with oxalyl chloride followed by the sodium salt of N-hydroxypyridine-2-thione afforded the corresponding pyridine-2-thione-N-oxycarbonyl (PTOC) oxalate ester which was not isolated but immediately heated to provide 2,2-dibutylselenane (7). This transformation presumably involves a tertiary alkyl radical that undergoes intramolecular homolytic substitution at selenium with loss of the benzyl radical to provide the selenium-containing ring system (7). A similar protocol, when applied to 1-(2-benzylselenenyl-5-methoxyphenyl)-3-methyl-3-heptanol (18) and 1-(2-benzylselenenyl-5-methoxyphenyl)-3,7,11,15-tetramethyl-3-hexadecanol (19), followed by deprotection, afforded the selenium-containing alpha-tocopherol analogues 4 and 1f, respectively, in moderate yields. To the best of our knowledge, these transformations represent the first examples of tertiary radicals involved in homolytic substitution chemistry at selenium.

The antioxidant profile of 2,3-dihydrobenzo[b]furan-5-ol and its 1-thio, 1-seleno, and 1-telluro analogues.

A novel synthesis of 2,3-dihydrobenzo[b]thiophene-5-ol based on intramolecular homolytic substitution on sulfur was reported. The "antioxidant profile" of the series of 2,3-dihydrobenzo[b]furan-5-ol (2a) its 1-thio (2b), 1-seleno (2c) and 1-telluro (2d) analogues was determined by studies of redox properties, the capacity to inhibit stimulated lipid peroxidation, the reactivity toward tert-butoxyl radicals, the ability to catalyze decomposition of hydrogen peroxide in the presence of glutathione, and the inhibiting effect on stimulated peroxidation in liver microsomes. The one-electron reduction potentials of the aroxyl radicals corresponding to compounds 2a-2d, E degrees (ArO(*)/ArO(-)) were 0.49, 0.49, 0.49, and 0.52 V vs NHE, respectively, as determined by pulse radiolysis. With increasing chalcogen substitution the compounds become slightly more acidic (pK(a) = 10.6, 10.0, 9.9, and 9.5, respectively, for compounds 2a-2d). By using Hess' law, the homolytic O-H bond dissociation enthalpies of compounds 2a-2d (340, 337, 336, and 337 kJ mol(-)(1), respectively) were calculated. The reduction potentials for the proton coupled oxidation of compounds 2a-2d (ArOH --> ArO(*) + H(+)) as determined by cyclic voltammetry in acetonitrile were 1.35 (irreversible), 1.35 (quasireversible) 1.13 (reversible), and 0.74 (reversible) V vs NHE, respectively. As judged by the inhibited rates of peroxidation, R(inh), in a water/chlorobenzene two-phase lipid peroxidation system containing N-acetylcysteine as a thiol-reducing agent in the aqueous phase, the antioxidant capacity increases (2d > 2c = 2b > 2a) as one traverses the group of chalcogens. Whereas the times of inhibition, T(inh), were slightly reduced for the oxygen (2a) and sulfur (2b) derivatives in the absence of the thiol-reducing agent, they were drastically reduced for the selenium (2c) and tellurium (2d) derivatives. This seems to indicate that the organochalcogen compounds are continuously regenerated at the lipid aqueous interphase and that regeneration is much more efficient for the selenium and tellurium compounds. The absolute rate constants for the oxidation of compounds 2a-2b by the tert-butoxyl radical in acetonitrile/di-tert-butyl peroxide (10/1) were the same-2 x 10(8) M(-)(1) s(-)(1). Whereas the oxygen, sulfur, and selenium derivatives 2a-2c were essentially void of any glutathione peroxidase-like activity, the organotellurium compound 2d accelerated the initial reduction of hydrogen peroxide, tert-butyl hydroperoxide, and cumene hydroperoxide in the presence of glutathione 100, 333, and 213 times, respectively, as compared to the spontaneous reaction. Compounds 2a-2d were assessed for their capacity to inhibit lipid peroxidation in liver microsomes stimulated by Fe(II)/ADP/ascorbate. Whereas the oxygen, sulfur, and selenium compounds showed weak inhibiting activity (IC(50) values of approximately 250, 25, and 13 microM, respectively), the organotellurium compound 2d was a potent inhibitor with an IC(50) value of 0.13 microM.

A proteomic approach to mimic fibrosis disease evolvement by an in vitro cell line.

Subepithelial fibrosis in asthma involves an increase in the thickening of the lamina reticularis and is due to increased deposition of collagen I, III and V, and fibronectin. The cause of the thickening of the reticular layer is not known in detail, however, it is proposed to be caused by bronchial myofibroblasts. The transformation of fibroblasts to myofibroblasts may be contributed by inflammatory cytokines. In this paper we have studied and compared in vivo tissue material with a human fibroblast target cell. A normal primary fetal fibroblast cell line and HFL-1 (human fibroblast lurg cells) were used as a comparison between fibroblasts from human central biopsies regarding morphology and cell proliferation. Both cell morphology and cell proliferation rate was similar between the different set of cell cultures. Furthermore, it could be concluded that fibroblasts cultures from patients with asthma were surrounded by more extracellular matrix molecules compared to the primary cell line HFL-1, which may mimic the in vivo situation during formation of fibrosis. We wanted to investigate if differential protein display by two-dimensional (2-D) gel electrophoresis and subsequent protein identification by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry could reveal proteins induced by cytokine stimulation that can be correlated to the transformation of normal human fetal lungs cells into a more myofibroblast like phenotype. After stimulation with transforming growth factor-beta (TGF-beta) several myofibroblast markers were found to be regulated. Especially cytoskeletal and cytoskeletal-associated proteins like actin isoforms and tropomyosin, proteins that are responsible for contraction as well as transportation of extra cellular matrix proteins, which are overproduced in the formation of fibrosis. These results indicate that TGF-beta, which is increased in a fibrotic process, participates in the transformation of fibroblasts to myofibroblasts.

Proteomics -- the protein expression technology to study connective tissue biology.

During the formation of peribronchial fibrosis in asthma, remodeling of connective tissue is due to an increase in deposition of extracellular matrix components like that of specific types of collagens and proteoglycans. By taking bronchial biopsies, we were able to isolate cell cultures derived from asthmatic patients and healthy volunteers, which provides a good model system to study differences regarding cell morphology and key connective tissue proteins in the remodeling process. Proteomics, utilizing two-dimensional electrophoresis and modern image analysis systems have made it possible to study protein expression and regulation of proteins in biological systems. By using this powerful tool, it is possible to quantitatively study protein regulation and to obtain increased knowledge about the mechanism behind the inflammatory process and formation of peribronchial fibrosis. We have optimized a proteomic protocol enabling detailed investigation of the protein expression pattern in human lung cells. An increased expression pattern was obtained, whereby 20 protein spots could be detected by image analysis in the <45 kDa region. Out of these, specific regulations of four spots were found by quantitative image analysis and spots of interest were identified by MALDI TOF-MS. This protocol enables us to study 1000--2000 proteins simultaneously and the possibility to correlate protein expression to the physiological status of the cell culture investigated. We have found that two proteins, actin and tropomyosin, are increased in expression due to transforming growth factor-beta stimulation. These proteins are correlated to the transformation of normal fibroblasts to myofibroblasts which are involved in the remodeling processes observed in asthma.

Secondary metabolites characteristic of Penicillium citrinum, Penicillium steckii and related species.

Two new carboxylic acids, tanzawaic acid E (1) and F (2) in addition to the unknown benzopyran 3,7-dimethyl-1,8-dihydroxy-6-methoxy-isochroman (3), and the known mycotoxin 3,7-dimethyl-8-hydroxy-6-methoxyisochroman (4) were produced by a marine-derived strain of Penicillium steckii isolated from an unidentified tunicate. The carboxylic acids and the benzopyran were identified on the basis of mass spectrometry, and one and two dimensional NMR spectroscopic techniques. The structures 1 and 2 resemble tanzawaic acid A-D, previously isolated from Penicillium citrinum. Screening of isolates of species related to P. citrinum and P. steckii showed that P. citrinum (25 isolates) consistently produced citrinin and tanzawaic acid A, P. steckii (18 isolates) produced isochroman toxins (except 2) and tanzawaic acid E, P. sizovae consistently produced tanzawaic acid A, P. corylophilum (10 isolates) produced citreoisocoumarinol and P. sumatrense (15 isolates) always produced curvularin.

Calafianin, a bromotyrosine derivative from the marine sponge Aplysina gerardogreeni.

Calafianin (1) and two known compounds, aerothionin and (3, 5-dibromo-2-hydroxy-4-methoxyphenyl)acetic acid, were isolated from the marine sponge Aplysina gerardogreeni. The structure of 1 was determined by NMR analysis and mass spectrometry.

Unguisins A and B: new cyclic peptides from the marine-derived fungus emericella unguis

Unguisin A (1) and B (2), the first cyclic heptapeptides containing GABA in the ring, were isolated from a marine-derived strain of Emericella unguis. The chemical structures of 1 and 2 were elucidated by extensive 2D NMR techniques, and the stereochemistry of the individual amino acids was determined using Marfey's method.

Heparan sulfate upregulates platelet-derived growth factor receptors on human lung fibroblasts.

Heparan sulfate is a molecule that possesses a large structural variability and which has been shown to inhibit the proliferation of fibroblasts in vitro. The aim of this study was to determine whether the anti-proliferative effects of heparan sulfate were exerted by regulation of the activity of the platelet-derived growth factor and/or of the platelet-derived growth factor receptors. Both l-iduronate-rich, anti-proliferative and the l-iduronate-poor, non-anti-proliferative heparan sulfate species, were incubated with confluent human embryonic lung fibroblasts for 24 h. The mRNA levels for PDGF-AA, PDGF-BB, and their receptors were measured. Binding studies were performed with [125I]-PDGF-BB and [125I]-EGF for 2 h at 4 degreesC in cultures preincubated with both types of heparan sulfate for 24 h. In separate experiments, cultures were incubated together with heparan sulfate and [125I]-PDGF-BB for 2 h at 4 degreesC. Increases of two- to threefold in the mRNA levels for both the alpha- and the beta-receptors of PDGF was obtained after treatment with both types of heparan sulfate, whereas the mRNA levels of both the PDGF-AA and the PDGF-BB were essentially unaffected. A sixfold increase in binding was only noted for [125I]-PDGF-BB in cultures pre-treated with the anti-proliferative heparan sulfate for 24 h, whereas no effect was noted with use of the non-anti-proliferative heparan sulfate. Incubating the [125I]-PDGF-BB and the anti-proliferative heparan sulfate together for 2 h resulted in a smaller, threefold increase in binding. This indicates that the anti-proliferative heparan sulfate both stabilizes and increases expression of the PDGF receptors. To investigate whether the increased number of PDGF receptors could affect cell activity, cells were preincubated with anti-proliferative heparan sulfate and then treated with PDGF-BB. This resulted in an increase in mitogenicity compared to cells treated only with PDGF-BB. Neither an increase in binding for [125I-EGF] nor an increase in the mitogenic response of EGF could be observed in cultures pre-treated with the anti-proliferative heparan sulfate. The results indicate that the extracellular matrix itself may regulate important biological phenomena such as cell proliferation and matrix production through affecting the expression of receptors of PDGF, which initiate both stimulatory and inhibitory signals.

Recording properties and biocompatibility of chronically implanted polymer-based intrafascicular electrodes.

We implanted polymer-based longitudinal intrafascicular electrodes (polyLIFEs) in feline dorsal rootlets acutely and for periods of two to six months to evaluate their electrical properties and biocompatibility. A total of 38 implanted electrodes were analyzed. Some 25 of the 38 electrodes were implanted with an insulative flexible polymer cuff, which was required for recording of afferent activity in situ. Electrode impedances remained stable for the duration of the experiments. The distributions of axons were measured at three levels of the implanted rootlets: the implant level, 1-2 mm proximal to the implant with respect to the cell body, and 1-2 mm distal to the implant with respect to the cell body. Similar measurements were made in five samples of fascicles neighboring an implant and six samples of control tissue from animals in which no implants were placed. The polyLIFEs demonstrated a high degree of biocompatibility, as no adverse effects on axon size were observed in either the implanted fascicle or neighboring neural tissue. However, the insulative cuffs were found to be a source of compression, resulting in necrosis of the neural tissue.

Propagation velocity of muscle action potentials in the growing normal child.

Power spectrum analysis of myoelectric signals has been used to measure the propagation velocity in a large number of voluntary action potentials in biceps and brachioradialis muscles of normal children, 0-20 years old. The method used can be applied to both single motor units signals and interference electromyogram. From measurements of single motor units, the distribution of potential velocities in the muscle can be obtained, which makes conclusive statements on the development of the velocity during childhood more reliable. The propagation velocities are found to increase with age, body height, and muscle diameter. The dependencies on these size parameters are close to that of the square root function, in accordance with electrophysical theory.