Influence of biochar particle size on biota responses.

Despite the increasing interest for biochar as a soil amendment, a knowledge gap remains on its impacts on non-target soil and aquatic species. We hypothesised that biochar particle size and application rate can play a role in the toxicity to biota. Pine woodchip biochar was incorporated in a clean soil at three particle size classes: small (<0.5 mm), medium (1-2 mm), and large (<4 mm), and at two concentrations: 1% and 6% w/w. A laboratory screening with earthworm Eisenia andrei avoidance behaviour bioassay was carried out to test the most adequate application rates, particle sizes and soil-biochar pre-incubation period. Thereafter, a 28-day greenhouse microcosm experiment was conducted as an ecologically more representative approach. Survival, vertical distribution and weight changes of E. andrei, and bait-lamina consumption were recorded. Soil leachates from the microcosms were collected to evaluate their impact on Daphnia magna immobilisation and Vibrio fischeri (Microtox®) bioluminescence. A feeding experiment with E. andrei was also performed to address earthworm weight changes and to conduct a screening of PAH-type metabolites in their tissue. The 6% <0.5 mm treatment pre-incubated for 96 h induced significant avoidance of the earthworms. Significantly lower bait-lamina consumption was observed in microcosms for the 6% <0.5 mm treatment. Moreover, particle size was a statistically significant factor regarding the loss of weight in the feeding experiment and higher concentration of naphthalene-type metabolites detected in E. andrei tissue, when exposed to <0.5 mm biochar particles. The leachates had no adverse effects on the aquatic species. The results suggest that particles <0.5 mm of pine woodchip biochar can pose sub-lethal effects on soil biota.

A new gene, encoding an anion transporter, is mutated in sialic acid storage diseases.

Sialic acid storage diseases (SASD, MIM 269920) are autosomal recessive neurodegenerative disorders that may present as a severe infantile form (ISSD) or a slowly progressive adult form, which is prevalent in Finland (Salla disease). The main symptoms are hypotonia, cerebellar ataxia and mental retardation; visceromegaly and coarse features are also present in infantile cases. Progressive cerebellar atrophy and dysmyelination have been documented by magnetic resonance imaging (ref. 4). Enlarged lysosomes are seen on electron microscopic studies and patients excrete large amounts of free sialic acid in urine. A H+/anionic sugar symporter mechanism for sialic acid and glucuronic acid is impaired in lysosomal membranes from Salla and ISSD patients. The locus for Salla disease was assigned to a region of approximately 200 kb on chromosome 6q14-q15 in a linkage study using Finnish families. Salla disease and ISSD were further shown to be allelic disorders. A physical map with P1 and PAC clones was constructed to cover the 200-kb area flanked by the loci D6S280 and D6S1622, providing the basis for precise physical positioning of the gene. Here we describe a new gene, SLC17A5 (also known as AST), encoding a protein (sialin) with a predicted transport function that belongs to a family of anion/cation symporters (ACS). We found a homozygous SLC17A5 mutation (R39C) in five Finnish patients with Salla disease and six different SLC17A5 mutations in six ISSD patients of different ethnic origins. Our observations suggest that mutations in SLC17A5 are the primary cause of lysosomal sialic acid storage diseases.

Hemoglobin precipitation greatly improves 4-methylumbelliferone-based diagnostic assays for lysosomal storage diseases in dried blood spots.

Derivatives of 4-methylumbelliferone (4MU) are favorite substrates for the measurement of lysosomal enzyme activities in a wide variety of cell and tissue specimens. Hydrolysis of these artificial substrates at acidic pH leads to the formation of 4-methylumbelliferone, which is highly fluorescent at a pH above 10. When used for the assay of enzyme activities in dried blood spots the light emission signal can be very low due to the small sample size so that the patient and control ranges are not widely separated. We have investigated the hypothesis that quenching of the fluorescence by hemoglobin leads to appreciable loss of signal and we show that the precipitation of hemoglobin with trichloroacetic acid prior to the measurement of 4-methylumbelliferone increases the height of the output signal up to eight fold. The modified method provides a clear separation of patients' and controls' ranges for ten different lysosomal enzyme assays in dried blood spots, and approaches the conventional leukocyte assays in outcome quality.

Can straw-biochar mulching mitigate erosion of wildfire-degraded soils under extreme rainfall?

High severity wildfires cause a drastic alteration of soil carbon cycling - both oxidising and thermally altering soil organic matter (SOM) - and usually are followed by strong runoff and erosion events. To restore wildfire-degraded soils, SOM needs to be rebuilt while soil erosion is prevented. Post-fire straw mulching has been shown to mitigate soil erosion by providing a protective cover against rainsplash. However, SOM takes many decades or centuries to rebuilt naturally. Biochar, co-applied with straw to the soil surface can replace the SOM of the O-horizon, while the stabilised soil - by straw mulching - may gain in SOM naturally and by downward movement of biochar. We conducted a field study to test if straw-only and straw-biochar co-application could restore soils degraded by wildfire in one high burn severity (HBS) and one moderate burn severity (MBS) study area in southern Portugal and Spain, respectively, by monitoring erosion and SOM for the most intense rainfall period of the first post-fire year. Burned sites were characterized for soil and sediment physical properties, TOC content, SOM quality by thermogravimetry (DTG) and nuclear magnetic resonance (NMR 13C) spectroscopy. Straw-biochar mulching significantly reduced soil erosion by 76% and 65% in the HBS and MBS sites, respectively, in both cases similar to the erosion reduction by straw-only mulching. DTG and NMR 13C indicated that a relatively small proportion of the biochar eroded, i.e. 0.7%, indicating that co-application of straw with biochar may help restore the SOC lost in the wildfire in the medium term. The amount of SOM eroded was lower with straw-biochar mulching than in the untreated plots for both study areas. Straw-biochar mulching mitigates erosion of wildfire-degraded soils under extreme rainfall, while a relatively small proportion of the biochar is lost by erosion. Future studies need to monitor medium term effects.

Spatial distribution of pyrogenic carbon in Iberian topsoils estimated by chemometric analysis of infrared spectra.

Understanding the global carbon (C) cycle is critical to accurately model feedbacks between climate and soil. Thus, many climate change studies focused on soil organic carbon (SOC) stock changes. Pyrogenic carbon (PyC) is one of the most stable fractions of soil organic matter (SOM). Accurate maps based on measured PyC contents are required to facilitate future soil management decisions and soil-climate feedback modelling. However, consistent measurements that cover large areas are rare. Therefore, this study aimed to map the PyC content and stock of the Iberian Peninsula, which covers contrasting climatic zones and has long-term data on wildfire occurrence. A partial least square (PLS) regression using the mid-infrared spectra (1800-400 cm-1) was applied to a dataset composed of 2961 soil samples from the Iberian component of the LUCAS 2009 database. The values of PyC for LUCAS points were modelled to obtain a map of topsoil PyC by a random forest (RF) approach using 36 auxiliary variables. The results were validated through comparison with documented historical wildfire activity and anthropogenic energy production. A strong relationship was found between these sources and the distribution of PyC. Our study estimates that the accumulated PyC in Iberian Peninsula soils comprises between 3.09 and 20.39% of total organic carbon (TOC) in the topsoil. Forests have higher PyC contents than grasslands, followed by agricultural soils. The incidence of recurrent wildfires also has a notable influence on PyC contents. This study shows the potential of estimating PyC with a single, rapid, low cost, chemometric method using new or archived soil spectra, and has the ability to improve soil-climate feedback modelling. It also offers a possible tool for measuring, reporting and verifying soil C stocks, which is likely to be important moving forward if soils are used as sinks for C sequestration.

Cerebellar ataxia with elevated cerebrospinal free sialic acid (CAFSA).

In order to identify new metabolic abnormalities in patients with complex neurodegenerative disorders of unknown aetiology, we performed high resolution in vitro proton nuclear magnetic resonance spectroscopy on patient cerebrospinal fluid (CSF) samples. We identified five adult patients, including two sisters, with significantly elevated free sialic acid in the CSF compared to both the cohort of patients with diseases of unknown aetiology (n = 144; P < 0.001) and a control group of patients with well-defined diseases (n = 91; P < 0.001). All five patients displayed cerebellar ataxia, with peripheral neuropathy and cognitive decline or noteworthy behavioural changes. Cerebral MRI showed mild to moderate cerebellar atrophy (5/5) as well as white matter abnormalities in the cerebellum including the peridentate region (4/5), and at the periventricular level (3/5). Two-dimensional gel analyses revealed significant hyposialylation of transferrin in CSF of all patients compared to age-matched controls (P < 0.001)--a finding not present in the CSF of patients with Salla disease, the most common free sialic acid storage disorder. Free sialic acid content was normal in patients' urine and cultured fibroblasts as were plasma glycosylation patterns of transferrin. Analysis of the ganglioside profile in peripheral nerve biopsies of two out of five patients was also normal. Sequencing of four candidate genes in the free sialic acid biosynthetic pathway did not reveal any mutation. We therefore identified a new free sialic acid syndrome in which cerebellar ataxia is the leading symptom. The term CAFSA is suggested (cerebellar ataxia with free sialic acid).

Evaluation of Diabetes Care parameters in capillary blood collected with a novel sampling device.

The aim of this study was to determine whether the Hem-Col method of obtaining and storing blood is an acceptable alternative to venepuncture for measuring Diabetes Care parameters. Design and methods : Hem-Col is a novel blood collection device that is designed to collect capillary blood drawn with a finger prick. Hem-Col is a microtube containing an anticoagulant and a preservation buffer to enhance analyte stability in whole blood. The Diabetes Care parameters cholesterol, creatinine, HbA1c, high density lipoprotein (HDL) cholesterol, low density lipoprotein (LDL) cholesterol, and triglycerides were measured both in blood/plasma collected via Hem-Col and blood/plasma collected with venepuncture. The results were compared to assess the agreement between the two methods. Results : HbA1c shows agreement after storage for up to 120 hours at temperatures ranging from 4 to 37 °C. Cholesterol, HDL cholesterol, LDL cholesterol, triglycerides and creatinine can be measured after 120 hours of storage in Hem-Col buffer, if high temperatures are avoided, and with the use of correction factors or adaptations to reported reference intervals. Conclusion : Hem-Col is suitable for the measurement of HbA1c after storage for up to 120 hours at temperatures ranging from 4 to 37 °C. Cholesterol, creatinine, HDL cholesterol, LDL cholesterol and triglycerides can be measured after 120 hours of storage in Hem-Col buffer, if high temperatures are avoided. Further studies are required to determine whether Hem-Col can replace the venepuncture for the Diabetes Care parameters.

Methods for a prompt and reliable laboratory diagnosis of Pompe disease: report from an international consensus meeting.

Pompe disease is an autosomal recessive disorder of glycogen metabolism caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). It presents at any age, with variable rates of progression ranging from a rapidly progressive course, often fatal by one-year of age, to a more slowly, but nevertheless relentlessly progressive course, resulting in significant morbidity and premature mortality. In infants, early initiation of enzyme replacement therapy is needed to gain the maximum therapeutic benefit, underscoring the need for early diagnosis. Several new methods for measuring GAA activity have been developed. The Pompe Disease Diagnostic Working Group met to review data generated using the new methods, and to establish a consensus regarding the application of the methods for the laboratory diagnosis of Pompe disease. Skin fibroblasts and muscle biopsy have traditionally been the samples of choice for measuring GAA activity. However, new methods using blood samples are rapidly becoming adopted because of their speed and convenience. Measuring GAA activity in blood samples should be performed under acidic conditions (pH 3.8-4.0), using up to 2 mM of the synthetic substrate 4-methylumbelliferyl-alpha-D-glucoside or glycogen (50 mg/mL), in the presence of acarbose (3-9 microM) to inhibit the isoenzyme maltase-glucoamylase. The activity of a reference enzyme should also be measured to confirm the quality of the sample. A second test should be done to support the diagnosis of Pompe disease until a program for external quality assurance and proficiency testing of the enzymatic diagnosis in blood is established.

Soil monitoring in Europe: a review of existing systems and requirements for harmonisation.

Official frameworks for soil monitoring exist in most member states of the European Union. However, the uniformity of methodologies and the scope of actual monitoring are variable between national systems. This review identifies the differences between existing systems, and describes options for harmonising soil monitoring in the Member States and some neighbouring countries of the European Union. The present geographical coverage is uneven between and within countries. In general, national and regional networks are much denser in northern and eastern regions than in southern Europe. The median coverage in the 50 km x 50 km EMEP cells applied all over the European Union, is 300 km(2) for one monitoring site. Achieving such minimum density for the European Union would require 4100 new sites, mainly located in southern countries (Italy, Spain, Greece), parts of Poland, Germany, the Baltic countries, Norway, Finland and France. Options are discussed for harmonisation of site density, considering various risk area and soil quality indicator requirements.

Sialic acid storage diseases. A multiple lysosomal transport defect for acidic monosaccharides.

A defective efflux of free sialic acid from the lysosomal compartment has been found in the clinically heterogeneous group of sialic acid storage disorders. Using radiolabeled sialic acid (NeuAc) as a substrate, we have recently detected and characterized a proton-driven carrier for sialic acid in the lysosomal membrane from rat liver. This carrier also recognizes and transports other acidic monosaccharides, among which are uronic acids. If no alternative routes of glucuronic acid transport exist, the disposal of uronic acids can be affected in the sialic acid storage disorders. In this study we excluded the existence of more than one acidic monosaccharide carrier by measuring uptake kinetics of labeled glucuronic acid [( 3H]GlcAc) in rat lysosomal membrane vesicles. [3H]GlcAc uptake was carrier-mediated with an affinity constant of transport (Kt) of 0.3 mM and the transport could be cis-inhibited or trans-stimulated to the same extent by sialic acid or glucuronic acid. Human lysosomal membrane vesicles isolated from cultured fibroblasts showed the existence of a similar proton-driven transporter with the same properties as the rat liver system (Kt of [3H]GlcAc uptake 0.28 mM). Uptake studies with [3H]NeuAc and [3H]GlcAc in resealed lysosome membrane vesicles from cultured fibroblasts of patients with different clinical presentation of sialic acid storage showed defective carrier-mediated transport for both sugars. Further evidence that the defective transport of acidic sugars represents the primary genetic defect in sialic acid storage diseases was provided by the observation of reduced, half-normal transport rates in lymphoblast-derived lysosomal membrane vesicles from five unrelated obligate heterozygotes. This study reports the first observation of a human lysosomal transport defect for multiple physiological compounds.

Isolation and structural characterization of sialic acid-containing storage material from mucolipidosis I (sialidosis) fibroblasts.

Sialic acid-containing storage material was isolated from cultured human mucolipidosis I (sialidosis) fibroblasts by gel permeation chromatography on Bio-Gel P-6 followed by medium-pressure anion-exchange chromatography on Mono Q. The structure determination of the isolated sialyloligosaccharides was carried out by 500-MHz 1H-NMR spectroscopy in conjunction with sugar analysis and analytical HPLC. The storage material showed completely sialylated mono-, di- and triantennary N-glycosidic N-acetyllactosamine oligosaccharides having the Man beta 1----4GlcNAc sequence at the reducing end in common. Heterogeneity occurred with respect to the linkages between terminal sialic acid and the penultimate galactose residues (alpha 2----3/alpha 2----6). It turned out that all the identified carbohydrate chains are consistent with the neuraminidase deficiency.

Transport of organic anions by the lysosomal sialic acid transporter: a functional approach towards the gene for sialic acid storage disease.

Transport of sialic acid through the lysosomal membrane is defective in the human sialic acid storage disease. The mammalian sialic acid carrier has a wide substrate specificity for acidic monosaccharides. Recently, we showed that also non-sugar monocarboxylates like L-lactate are substrates for the carrier. Here we report that other organic anions, which are substrates for carriers belonging to several anion transporter families, are recognized by the sialic acid transporter. Hence, the mammalian system reveals once more novel aspects of solute transport, including sugars and a wide array of non-sugar compounds, apparently unique to this system. These data suggest that the search for the sialic acid storage disease gene can be initiated by a functional selection of genes from a limited number of anion transporter families. Among these, candidates will be identified by mapping to the known sialic acid storage disease locus.

Photoaffinity labeling of the lysosomal neuraminidase from bovine testis.

ASA-NeuAc2en, a photoreactive arylazide derivative of sialic acid, is shown to be a powerful competitive inhibitor of lysosomal neuraminidase from bovine testis (Ki approximately 21 microM). Photoaffinity labeling and partial purification of preparations containing this lysosomal neuraminidase activity result in specifically and non-specifically labeled polypeptides. Only labeling in a 55 kDa polypeptide is found to be specific, since it could be prevented by the competitive neuraminidase inhibitor NeuAc2en. We conclude that the 55 kDa polypeptide in the bovine testis beta-galactosidase/neuraminidase/protective protein complex contains the catalytic site of neuraminidase.

Characterization of a heavy metal ion transporter in the lysosomal membrane.

Lysosomes are thought to play a role in various aspects of heavy metal metabolism. In the present study we demonstrate for the first time the presence of a heavy metal ion transport protein in the lysosomal membrane. Uptake of radioactive silver both in highly purified lysosomal membrane vesicles and in purified intact lysosomes showed the typical kinetics of a carrier-mediated process. This transport was stimulated by ATP hydrolysis, and showed specificity for Ag+, Cu2+, and Cd2+. All biochemical properties of this lysosomal metal ion transporter could classify it as a heavy metal transporting P-type ATPase. Long Evans Cinnamon (LEC) rats, an animal model for the copper transport disorder Wilson disease, showed normal lysosomal silver transport.

Progesterone receptor isoform expression in human meningiomas.

The majority of meningiomas express the progesterone receptor (PR), and therefore meningiomas are considered to be progesterone-responsive. In addition, an association has been reported between PR and prognosis. At least two PR isoforms exist, PR-B (116--120 kDa) and PR-A (81 kDa), each of which are likely to have different biological functions. Knowledge of the differential expression of both isoforms is necessary to understand the effects of progesterone on meningioma growth. Therefore, in this study, PR-A and PR-B expression levels were determined in 61 human meningiomas by immunoblotting. Total PR expression levels were determined with a ligand binding assay (LBA) (total PR(LBA)). Both PR isoforms and an additional PR 78 kDa protein (PR-78) were expressed in the meningiomas. Meningiomas expressing more PR-A than PR-B had significantly higher total PR(LBA) levels (P<0.001). The PR-78 band intensity was negatively associated with that of PR-B (r(s)=-0.76, P<0.0001). PR-78 may represent an endogenous degradation product, but a similar regulation pathway in the biogenesis of both PR-B and PR-78 is not excluded. Meningiomas contain both PR isoforms, but in highly variable ratios and this variability may have some biological significance. Most meningiomas express more PR-A than PR-B. Therefore in meningioma, assuming that PR-B is more transcriptionally active than PR-A, progesterone responsiveness could be based on transrepression rather than on transactivation of target genes, and progesterone blockade may only be effective in certain subsets of meningiomas.

The eyes of man as a photic bisensor system.

Non-patterned photic stimulation of the eyes of human subjects was obtained by homogeneous illumination of the closed eyes. The subjects perceived a difference between the stimulation intensity of the left and of the right eye as a field of view consisting of a bright and a dark half, adjoined across an indeterminate vertical midline. It is not clear which parts of the visual system mediate the residual perceptual functions that survive this stimulatory reduction of man's eyes to a photic bisensor system. It is argued that animals with side-set eyes might use this perceptual ability for photic orientation in an anisotropic light field.

Occurrence, regulation, and significance of progesterone receptors in human meningioma.

The abundant expression of progesterone receptors (PR) in human meningiomas is well established. It is unknown, however, how PR expression is regulated, especially since estrogen receptors (ER) are virtually absent in these tumors. At the mRNA level, ER splice variants occur in meningioma but these appear not to be involved in the apparently autonomous PR expression. In an earlier study, because other ER-inducible proteins were either not expressed at all (pS2) or were expressed at a very low level compared to their expression in breast cancer (Cathepsin-D), the authors have postulated that the autonomous PR expression in meningioma is PR promoter-related rather than ER-related and have studied PR expression in cultured meningioma cells. PR levels appeared to decrease rapidly in vitro in monolayer as well as in three dimensional spheroid cultures. Culture conditions thus are not yet sufficient for the quantitative evaluation of PR expression. To evaluate whether PR deterioration is associated with cell turnover (meningiomas grow much faster in vitro than in vivo), the relationship between expression of the apoptotic proteins Bcl-2 and Bax and PR expression was investigated. Bcl-2 expression was found to be highest in meningioma with low PR levels, and in breast cancer tissue with high PR levels. Bax expression was not related to PR expression in any of the two tissues. Given the potential benefit of antiprogestin treatment and the occurrence in meningiomas of a protein capable of binding to the estrogen-responsive element, the expression of PR in meningioma remains a fascinating phenomenon which requires further investigation.

TP53 mutations in human meningiomas.

Overexpression of p53 has been reported to play a role in the development of neoplasms of the central nervous system. Meningiomas are generally benign intracranial tumors originating from the meninges. Overexpression of the p53 protein in meningiomas and an association with histological type and recurrence has been reported. Mutation of the TP53 gene leads to a more stable p53 protein in quantities high enough for detection by immunohistochemistry. In the search for these mutations the core domain of the TP53 gene of meningiomas has been analyzed. Only a very low incidence of mutations was reported. The apparent discordance between overexpression of p53 protein and TP53 gene mutations may be explained by mutations located outside the core domain. This issue was addressed in the present study. All 11 exons of 17 meningiomas were analyzed for DNA alterations by PCR single-strand conformation polymorphism (PCR-SSCP) analysis with subsequent sequencing. PCR-SSCP analysis showed a various number of band shifts and nucleotide alterations, caused either by alterations in the flanking introns or common polymorphisms (codon 36 and 72). The allele frequencies of the polymorphisms found in this small population of tumors resemble the frequencies reported in the literature. In addition, three nucleotide changes located in introns 2, 3 and 7 were found in 11, 3 and 4, respectively, of 17 specimens. Based on this study and on reports by others we conclude that it is not very likely that TP53 mutations are involved in the etiology of meningiomas.

Adult type GMl-gangliosidosis: a complementation study on somatic cell hybrids.

Two adult siblings with progressive pyramidal and extrapyramidal lesions, and generalized muscle atrophy had a profound deficiency of beta-galactosidase in all the cells and body fluids examined. Neuraminidase activity was normal in fibroblasts. The fused fibroblasts of infantile GMl-gangliosidosis and each of these adult patients had beta-galactosidase activity as expected for the average value in a mixture of equal numbers of parental cells. However, there was a remarkable increase in the activity of beta-galactosidase when the cells from each of these cases were fused with those from the beta-galactosidase-deficient adult with cherry-red spots, cerebellar ataxia, myoclonus and neuraminidase deficiency in fibroblasts. It was concluded that the two siblings represent a new genetic variant (adult type) of GMl-gangliosidosis.

[A woman suffering from systemic lupus erythematosus and acquired thrombotic thrombocytopenic purpura]. / Een vrouw met lupus erythematodes disseminatus en verworven trombotische trombocytopenische purpura.

A 41-year-old woman with known systemic lupus erythematosus (SLE) was diagnosed with thrombotic thrombocytopaenic purpura (TTP). At the time of admission she was suffering from petechia, purpura and had neurological symptoms. At first a relapse of the SLE was suspected. Additional laboratory findings demonstrated haemolytic anaemia, thrombocytopaenia and high levels of fragmentocytes. After multiple plasmapheresis treatments and immunosuppressive therapy she recovered. TTP can be differentiated from other thrombotic microangiopathic syndromes by its normal levels of prothrombin time, partially activated thromboplastin time (APTT), fibrinogen and direct Coombs-test. Further investigation is needed to confirm the diagnosis by determination of the activity of Von Willebrand factor cleaving protease ADAMTS-13. In this patient, the ADAMTS-13 activity returned after plasmapheresis. This case demonstrates the importance of fast and appropriate laboratory testing in order to diagnose TTP quickly.