FKRP-dependent glycosylation of fibronectin regulates muscle pathology in muscular dystrophy.

The muscular dystrophies encompass a broad range of pathologies with varied clinical outcomes. In the case of patients carrying defects in fukutin-related protein (FKRP), these diverse pathologies arise from mutations within the same gene. This is surprising as FKRP is a glycosyltransferase, whose only identified function is to transfer ribitol-5-phosphate to α-dystroglycan (α-DG). Although this modification is critical for extracellular matrix attachment, α-DG's glycosylation status relates poorly to disease severity, suggesting the existence of unidentified FKRP targets. Here we reveal that FKRP directs sialylation of fibronectin, a process essential for collagen recruitment to the muscle basement membrane. Thus, our results reveal that FKRP simultaneously regulates the two major muscle-ECM linkages essential for fibre survival, and establishes a new disease axis for the muscular dystrophies.

Cellular rescue in a zebrafish model of congenital muscular dystrophy type 1A.

Laminins comprise structural components of basement membranes, critical in the regulation of differentiation, survival and migration of a diverse range of cell types, including skeletal muscle. Mutations in one muscle enriched Laminin isoform, Laminin alpha2 (Lama2), results in the most common form of congenital muscular dystrophy, congenital muscular dystrophy type 1A (MDC1A). However, the exact cellular mechanism by which Laminin loss results in the pathological spectrum associated with MDC1A remains elusive. Here we show, via live tracking of individual muscle fibres, that dystrophic myofibres in the zebrafish model of MDC1A maintain sarcolemmal integrity and undergo dynamic remodelling behaviours post detachment, including focal sarcolemmal reattachment, cell extension and hyper-fusion with surrounding myoblasts. These observations imply the existence of a window of therapeutic opportunity, where detached cells may be "re-functionalised" prior to their delayed entry into the cell death program, a process we show can be achieved by muscle specific or systemic Laminin delivery. We further reveal that Laminin also acts as a pro-regenerative factor that stimulates muscle stem cell-mediated repair in lama2-deficient animals in vivo. The potential multi-mode of action of Laminin replacement therapy suggests it may provide a potent therapeutic axis for the treatment for MDC1A.

Fusion of Magnetic Resonance Imaging and Real-Time Elastography to Visualize Prostate Cancer: A Prospective Analysis using Whole Mount Sections after Radical Prostatectomy.

PURPOSE: To determine whether the fusion of multiparametric magnetic resonance imaging (MRI) with transrectal real-time elastography (RTE) improves the visualization of PCa lesions compared to MRI alone. MATERIALS AND METHODS: In a prospective setting, 45 patients with biopsy-proven PCa received prostate MRI prior to radical prostatectomy (RP). T2 and diffusion-weighted imaging (T2WI/DW-MRI) and, if applicable, dynamic contrast-enhanced sequences (T2WI/DW/DCE-MRI) were used to perform MRI/RTE fusion. The probability of PCa on MRI was graded according to the PI-RADS score for 12 different prostate sectors per patient. MRI images were fused with RTE to stratify suspicious from non-suspicious sectors. Imaging results were compared to whole mount sections using nonparametrical receiver operating characteristic curves and the area under these curves (AUC). RESULTS: 41 of 45 patients were eligible for final analyses. Histopathology confirmed PCa in 261 (53%) of 492 prostate sectors. MRI alone provided an AUC of 0.62 (T2WI/DW-MRI) and 0.65 (T2WI/DW/DCE-MRI) to predict PCa and was meaningfully enhanced to 0.75 (T2WI/DW-MRI) and 0.74 (T2WI/DW/DCE-MRI) using MRI/RTE fusion. Sole MRI showed a sensitivity and specificity of 57.9% and 61% with the best results for ventral prostate sectors whereas RTE was superior in dorsal and apical sectors. MRI/RTE fusion improved sensitivity and specificity to 65.9% and 75.3%, respectively. Additional use of DCE sequences showed a sensitivity and specificity of 65% and 55.7% for MRI and 72.1% and 66% for MRI/RTE fusion. CONCLUSION: MRI/RTE fusion provides improved PCa visualization by combining the strength of both imaging techniques in regard to prostate zonal anatomy and thereby might improve future biopsy-guided PCa detection.

[Right lower quadrant abdominal pain--the usual suspects? Diagnosis and therapy of a symptomatic mesenteric cyst]. / Rechtsseitige Unterbauchschmerzen--die üblichen Verdächtigen? Diagnose und Therapie einer symptomatischen Mesenterialzyste.

HISTORY AND CLINICAL FINDINGS: A 17-year-old male sought medical attention for right lower abdominal pain. At clinical examination an abdominal mass in the right lower abdominal quadrant was accompanied by pain and tenderness in this very region. The febrile patient (temperature axillary: 37.5 °C, rectal: 38.6 °C) was in reduced general health. INVESTIGATIONS: Except for an elevated C-reactive protein (CRP: 14.3 mg/dl, normal: < 0.5) laboratory tests were inconspicuous. Abdominal ultrasound showed a hypoechogenic/anechogenic septated mass measuring 7.2 × 10.4 × 15 cm as well as small amounts of fluid. Abdominal computed tomography confirmed these findings. Radiographically there was evidence for appendicitis. TREATMENT AND COURSE: Empiric antibiotic therapy was immediately commenced. Within 48 hours laparotomy was performed showing an abdominal mass which seemed to infiltrate the transverse colon, the ileocolic artery and the mesenteric root. No signs of appendicitis were found. A radical resection of the abdominal mass was performed meeting current standards of oncologic surgery. The postoperative course was favourable. Histopathological investigation showed a mesenteric cyst incorporating a hematoma and tissue with signs of chronic inflammation and granulation. CONCLUSION: Mesenteric cysts are rare pathologies occurring most frequently during childhood. They may become clinically overt in case of infection, haemorrhage or secondary intestinal obstruction. In the presented case presented hematoma formation and chronic inflammation had presumably induced peritonitis and clinical signs of appendicitis. Abdominal ultrasound and computed tomography facilitated diagnostic work-up. The patient thereby benefited from a scheduled laparotomy allowing a complete recovery.

Advanced oxidation processes: mechanistic aspects.

The reactive intermediate in Advanced Oxidation Processes (AOPs) is the *OH radical. It may be generated by various approaches such as the Fenton reaction (Fe2+/H2O2), photo-Fenton reaction (Fe3+/H2O2/hnu), UV/H2O2, peroxone reaction (O3/H2O2), O3/UV, O3/activated carbon, O3/dissolved organic carbon (DOC) of water matrix, ionizing radiation, vacuum UV, and ultrasound. The underlying reactions and *OH formation efficiencies are discussed. The key reactions of *OH radicals also addressed in this review.

The basics of oxidants in water treatment. Part A: OH radical reactions.

The Advanced Oxidation Processes (AOPs) are based on the reactions of the highly reactive *OH radicals. The formation of *OH by the various AOPs and their ensuing reactions are reviewed.

Elimination of the musk fragrances galaxolide and tonalide from wastewater by ozonation and concomitant stripping.

Ozone reacts with the musk fragrances tonalide and galaxolide with rate constants of 8 M(-1)s(-1) and 140 M(-1)s(-1), respectively. In wastewater, ozone eliminates only the more reactive compound, galaxolide, in competition with its reaction with the wastewater matrix. As both compounds are also stripped in a bubble column, tonalide is also eliminated to some extent.

p53 deficiency rescues apoptosis and differentiation of multiple cell types in zebrafish flathead mutants deficient for zygotic DNA polymerase delta1.

Cell culture work has identified the tumor suppressor p53 as a component of the S-phase checkpoint control system, while in vivo studies of this role of p53 in whole-vertebrate systems were limited. Here, we describe zebrafish mutants in the DNA polymerase delta catalytic subunit 1, based on the positional cloning of the flathead (fla) gene. fla mutants display specific defects in late proliferative zones, such as eyes, brain and cartilaginous elements of the visceral head skeleton, where cells display compromised DNA replication, followed by apoptosis, and partial or complete loss of affected tissues. Antisense-mediated knockdown of p53 in fla mutants leads to a striking rescue of all phenotypic traits, including completion of replication, survival of cells, and normal differentiation and tissue formation. This indicates that under replication-compromised conditions, the p53 branch of the S-phase checkpoint is responsible for eliminating stalled cells that, given more time, would have otherwise finished their normal developmental program.

The acanthocephalan Paratenuisentis ambiguus as a sensitive indicator of the precious metals Pt and Rh from automobile catalytic converters.

Recent studies revealed that intestinal acanthocephalans of fish can accumulate heavy metals to concentrations orders of magnitude higher than those in the host tissues or the aquatic environment. This significant heavy metal accumulation by acanthocephalans, even surpassing that of established free living accumulation bioindicators, encouraged us to study the bioavailability of the platinum-group-metals (PGM) Pt and Rh for parasites. These precious metals are used in catalytic converters of cars for exhaust gas purification in Europe since the early 1980s. In addition to the beneficial effect in reducing the emission of CHx, CO and NOx of cars there is an increasing emission of these metals. However, it still remains unclear if these elements become accumulated in the biosphere and whether they affect the health of organisms. The present study reveals that in European eels (Anguilla anguilla) naturally infected with the eoacanthocephalan parasite Paratenuisentis ambiguus and experimentally exposed to ground catalytic converter material, the parasites take up and accumulate the catalytic active metals Pt and Rh whereas in the examined host tissues we found no metal uptake. Compared with the PGM concentrations in the water the worms contained 1600 times higher Rh and 50 times higher Pt concentrations. Thus, the parasites can be used as sentinel organisms reflecting even very low levels of precious metals.

Physiological changes in skin barrier function in relation to occlusion level, exposure time and climatic conditions.

Skin occlusion is a condition encountered with various articles as part of everyday life and resulting changes in skin barrier physiology often remain unnoticed. In the present study we aimed to understand the impact of absorbent feminine hygiene articles, one vapor-permeable and one vapor-impermeable, on skin hydration in response to exposure time and different environmental climatic conditions. Using a forearm model, volunteers were patched with moistened vapor-permeable and -impermeable articles in parallel for 1, 3, and 6 h and under different climatic conditions (i.e. 20 degrees C/30% relative humidity, 25 degrees C/50% relative humidity, 30 degrees C/ 75% relative humidity). The physiological changes in the skin barrier function were measured via skin hydration, evaporation of superficial water (skin surface water loss, SSWL) and relative humidity in the microclimate between skin and occlusive article (RH(mc)). The results show that skin hydration, SSWL, and RH(mc) under a vapor-permeable article are reduced versus the vapor-impermeable article for all exposure times at 25 degrees C and 50% relative humidity. SSWL and RH(mc) decrease from their 1-hour peak values with increasing exposure time, while skin hydration decreases only after 3 h of exposure. Lower environmental temperature (20 degrees C) and lower relative humidity (30%) have little impact on the reduction of SSWL and RH(mc,) but more so on the reduction of skin hydration. Higher temperature (30 degrees C) and higher relative humidity (75%) increase RH(mc) and skin hydration under both vapor-permeable and -impermeable articles while SSWL is reduced under the vapor-impermeable article under these conditions. In conclusion, vapor-permeability is the key factor for physiological changes in the barrier function of the skin under occlusion, exposure time and climatic conditions being modulating factors. These findings have been integrated into a model of skin hydration under occlusion in the context of absorbent hygiene articles. While current vapor-impermeable articles are effective in reducing the excessive moisture on the skin due to bodily discharge, vapor permeability adds a further measurable benefit in reducing skin overhydration.

Fenton chemistry of 1,3-dimethyluracil.

Hydroxyl radicals were generated in the Fenton reaction at pH 4 (Fe(2+) + H(2)O(2) --> Fe(3+) + .OH + OH-, k approximately equal to 60 L mol(-1) s(-1)) and by pulse radiolysis (for the determination of kinetic data). They react rapidly with 1,3-dimethyluracil, 1,3-DMU (k = 6 x 10(9) L mol(-1) s(-1)). With H(2)O(2) in excess and in the absence of O(2), 1,3-DMU consumption is 3.3 mol per mol Fe(2+). 1,3-DMUglycol is the major product (2.95 mol per mol Fe(2+)). Dimers, prominent products of .OH-induced reactions in the absence of Fe(2+)/Fe(3+) (Al-Sheikhly, M.; von Sonntag, C. Z. Naturforsch. 1983, 31b, 1622) are not formed. Addition of .OH to the C(5)-C(6) double bond of 1,3-DMU yields reducing C(6)-yl 1 and oxidizing C(5)-yl radicals 2 in a 4:1 ratio. The yield of reducing radicals was determined with tetranitromethane by following the buildup of nitroform anion. Reaction of 1 with Fe(3+) that builds up during the reaction or with H(2)O(2) gives rise to a short-chain reaction that is terminated by the reaction of Fe(2+) with 2, which re-forms 1,3-DMU. In the presence of O(2), 1.1 mol of 1,3-DMU and 0.6 mol of O(2) are consumed per mol Fe(2+) while 0.16 mol of 1,3-DMU-glycol and 0.17 mol of organic hydroperoxides (besides further unidentified products) are formed. In the presence of O(2), 1 and 2 are rapidly converted into the corresponding peroxyl radicals (k = 9.1 x 10(8) L mol(-1) s(-1)). Their bimolecular decay (2k = 1.1 x 10(9) L mol(-1) s(-1)) yields approximately 22% HO(2)./O(2).(-) in the course of fragmentation reactions involving the C(5)-C(6) bond. Reduction of Fe(3+) by O(2).(-) leads to an increase in .OH production that is partially offset by a consumption of Fe(2+) in its reaction with the peroxyl radicals (formation of organic hydroperoxides, k approximately 3 x 10(5) L mol(-1) s(-1); value derived by computer simulation).

The importance of mesomerism in the termination of alpha-carboxymethyl radicals from aqueous malonic and acetic acids.

In the dioxygen-free aqueous malonic and acetic acid systems. alpha-carboxymethyl radicals are produced through hydrogen abstraction from the parent compound by radiolytically generated OH radicals. H abstraction from the CO2-/CO2H group followed by decarboxylation is a process of small importance (< or = 5%). The alpha-carboxymethyl radicals terminate by recombination. Two types of recombination product are observed which are characterised by the formation of a C-C linkage or a C-O linkage. alpha-Carboxymethyl radicals are mesomeric systems. Their mesomeric state depends on the state of protonation and determines the proportion of the C-C- versus C-O-linked dehydrodimers they produce.

A common carbanion intermediate in the recombination and proton-catalysed disproportionation of the carboxyl radical anion, CO2*-, in aqueous solution.

The carboxyl radical anion, CO2*- was produced by the reactions of OH radicals with either CO or formic acid in aqueous solution. The pKa(*CO2H) was determined by pulse radiolysis with conductometric detection at pH approximately equals 2.3. The bimolecular decay rate constant of CO2*- (2k approximately equals 1.4 x 10(9) dm3mol(-1)s(-1)) was found to be independent of pH in the range 3-8 at constant ionic strength. The yields of the products of the bimolecular decay of the carboxyl radicals, CO2 and the oxalate anion were found to depend strongly on the pH of the solution with an inflection point at pH 3.8. This pH dependence is explained by assuming a head-to-tail recombination of the CO2*- radicals followed by either rearrangement to oxalate or a protonation of the adduct, which subsequently leads to the formation of CO2 and formate. The recombination of CO2*- to give oxalate directly is estimated to have a contribution of <25%.

Pulse radiolysis of the DNA-binding bisbenzimidazole derivatives Hoechst 33258 and 33342 in aqueous solutions.

PURPOSE: The DNA-minor-groove-ligands bisbenzimidazoles Hoechst 33258 and 33342 have been reported to protect against radiation-induced DNA-strand breakage. In order to elucidate the mechanisms of protection by these DNA-binding compounds, pulse radiolysis studies on the reactions of the OH radical, the solvated electron and the H atom with Hoechst as well as OH-radical-induced nucleotide radical quenching by free Hoechst (model level) was investigated. MATERIALS AND METHODS: The pulse radiolysis of Hoechst 33258 and 33342 was studied in N2O and N2O/O2-(4:1)-saturated aqueous solutions in the absence and presence of azide and bromide ions and nucleotides. RESULTS: In a fully scavenged system (3 x 10(-2) mol x dm(-3) t-butanol, N2O/O2-saturated), a transient is formed which in the presence of phosphate buffer is no longer observed. This is assigned metastable quinonoid forms of Hoechst (lambdamax(Hoechst) = 340; lambdamax(transient) = 370 nm) which is generated in protonation/ deprotonation reactions by H+/OH- formed during the pulse. To prevent their formation 10(-3) mol x dm(-3) phosphate buffer was added in all other experiments. The transient spectra formed upon OH-radical attack (k=9 x 10(9) dm3 x mol(-1) x s(-1)) indicate that a major part of the primary OH-adduct radicals undergo rapid transformation (k approximately 5 x 10(5) x s(-1)), attributed to water elimination yielding an N-centered radical. This intermediate, also generated by N3. (k = 4 x 10(9) dm3 mol(-1) x s(-1)), subsequently complexes with a Hoechst molecule [k = 8 x 10(8) dm3 x mol(-1) x s(-1) epsilon(440 nm) = 1.4 x 10(4) dm3 mol(-1) x cm(-1)]. The N-centered radical does not react with O2 (k < 5 x 10(5) dm3 mol(-1) x s(-1)), but reacts readily with the superoxide radical (k= 1.0 x 10(9) dm3 x mol(-1) x s(-1)). Hoechst reacts with the peroxyl radicals derived from uridine (k approximately 5 x 10(6) dm3 x mol(-1) x s(-1)) or 5'-UMP (k approximately 1 x 10(7) dm3 mol(-1) x (s-1)), but not with the less oxidizing (e.g. methylperoxyl radical) yielding intermediates whose spectral properties are similar to those of the N-centered radical. However, they decay at a much lower rate (2k approximately 1 x 10(8) dm3 mol(-1) x s(-1)) than the N-centered radicals generated by N3. (2k= 1.1 x 10(9) dm3 x mol(-1) s(-1)), and it has been suggested that these peroxyl radicals form adducts rather than undergoing electron transfer. The H atom (k= 7 x 10(9) dm3 x mol(-1) x s(-1)) and the solvated electron (k= 2.3 x 10(10) dm3 x mol(-1) x s(-1)) yield, albeit noticeably different, H-adduct radicals which also strongly absorb in the 440 nm region. The reduction potential of Hoechst 33258 has been determined electrochemically at 0.84-0.90 V vs. NHE at pH 6.8. CONCLUSION: Hoechst reacts fast only with strongly oxidizing radicals by electron transfer (e.g. with the adenine-and guanine-derived heteroatom-centered radicals), but also more slowly with nucleo-base-derived peroxyl radicals, here albeit via addition. This may have important implications with regard to its protection owing to DNA-radical quenching under oxic vs. anoxic conditions.

Free-radical-induced chain breakage and depolymerization of poly(methacrylic acid): equilibrium polymerization in aqueous solution at room temperature.

Hydroxyl radicals, generated by ionizing radiation in N2O saturated aqueous solutions, abstract H atoms from poly(methacrylic acid) at the methyl and methylene groups, and radicals 1 and 2 are formed, respectively. The reactions of the poly(methacrylic acid) radicals were investigated by pulse radiolysis (using optical and conductometric detection), EPR, product analysis, and kinetic simulations. The conductometric detection allowed us to measure the rate of chain scission and monomer release. Under conditions in which the polymer is largely deprotonated, the primary radical 1 abstracts a hydrogen (k= 3.5 x 10(2)s(-1)) from the methylene group, and this yields the more stable secondary radical 2. This radical undergoes chain scission by beta-fragmentation (k= 1.8 s(-1)), and the terminal (end-of-chain) radical 3 is formed. The polymer radicals terminate only slowly (2k= 80 dm3mol(-1)s(-1)). This allows effective depolymerization (depropagation) to take place (k=0.1 s(-1)). The yield of monomer release is higher than the original radical yield by up to two orders of magnitude. Once monomer is formed, it reacts with 3 (propagation, k= 15 dm3mol(-1)s(-1)), and a situation close to an equilibrium radical polymerization is approached. From these data, the equilibrium monomer concentration is calculated at 6.7 x 10(-3) mol dm(-3) at room temperature. The standard entropy of propagation is estimated at -185 to -150 J mol(-1)K(-1). Because the monomer reaches concentrations in the millimolar range, the *OH radicals increasingly react with monomers (results in oligomerization) rather than with the polymer. This effect is reflected by, for example, a lowering of chain-scission yields upon prolonged irradiation. In acid solutions, the decay of the polymer radicals becomes much faster (estimated at about 10(7)dm3mol(-1)s(-1) at pH3.5), and monomer release is no longer observed.

Bleomycin versus OH-radical-induced malonaldehydic-product formation in DNA.

PURPOSE: To compare the actions of bleomycin and ionizing radiation on DNA regarding the formation of malonaldehyde-like products. MATERIALS AND METHODS: Calf thymus DNA was treated with iron/bleomycin or gamma-radiation at pH 7. Products were analysed by HPLC. The thiobarbituric-acid reactivity of the samples was determined directly or after HPLC by post-column derivatization. ESI mass spectra were taken on-line following HPLC. RESULTS: Malonaldehyde and malonaldehyde-like products as detected by the sensitive 2-thiobarbituric acid (TBA) assay are formed in gamma-irradiated DNA and thymidine solutions as well as upon treatment of DNA with bleomycin/iron. In gamma-irradiated DNA solutions in the presence of oxygen, no base propenals were detected, and the major TBA-active product was malonaldehyde. In the gamma-radiolysis of thymidine, thymine propenal was formed only in traces (not more than 0.07 per cent of the OH-radical yield). Malonaldehyde was practically absent after treatment with bleomycin; three other TBA-active products were seen by HPLC which have been identified as the cytosine, thymine, and adenine propenals. Guanine propenal was not detected under our conditions. CONCLUSIONS: The absence of these base propenals upon gamma-radiolysis implies that although the initiating step of OH-radical and bleomycin action [i.e. H-abstraction at C(4')] may be the same, the bleomycin-iron complex must participate in subsequent steps en route to the base propenals. It is proposed that the bleomycin pathway may involve the interaction of the C(4')-peroxyl radical with the 'spent' bleomycin-iron complex by ligand exchange, under formation of a bleomycin-iron-peroxyl-radical complex, Blm(Fe4+,*OOR), which then decomposes by heterolysis into the alkoxy cation precursor +OR of the base propenal and reconstitution of the bleomycin-iron complex Blm(Fe,O)3+, i.e. gives rise to base propenal formation without the involvement of a C(4')-hydroperoxide.

OH-radical formation by ultrasound in aqueous solution--Part II: Terephthalate and Fricke dosimetry and the influence of various conditions on the sonolytic yield.

Terephthalate and Fricke dosimetry have been carried out to determine the sonolytic energy yields of the OH free radical and of its recombination product H2O2 in aqueous solutions under various operating conditions (nature of operating gas, power, frequency, temperature). For example, in the sonolysis of Ar-saturated terephthalate solutions at room temperature, a frequency of 321 kHz, and a power of 170 W kg-1, the total yield [G(.OH) + 2 G(H2O2)], equals 16 x 10(-10) mol J-1. This represents the total of .OH that reach the liquid phase from gas phase of the cavitating bubble. The higher the solute concentration, the lower the H2O2 production as more of the OH free radicals are scavenged, in competition with their recombination. Fricke dosimetry, in the absence and presence of Cu2+ ions, shows that the yield of H atom reaching the liquid phase is much lower, with G(H.) of the order of 3 x 10(-10) mol J-1. These sonolytic yields are smaller in solutions that are at the point of gas saturation, and increase to an optimum as the initial sonication-induced degassing and effervescence subsides. The probing of the sonic field has shown that the rate of sonolytic free-radical formation may vary across the sonicated volume depending on frequency and power input.