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Background: The optimal therapy for deep burn wounds is based on the early debridement of necrotic tissue followed by wound coverage to avoid a systemic inflammatory response and optimize scar-free healing. The outcomes are affected by available resources and underlying patient factors, which represent challenges in burn care and suboptimal outcomes. In this study, we aimed to determine optimal burn-wound management using enzymatic debridement (NexoBrid™, MediWound Germany GmbH, Rüsselsheim, Germany) and intact fish skin (Kerecis® Omega3 Wound, Isafjordur, Iceland). Methods: In this retrospective case series, 12 patients with superficial or deep dermal burn wounds were treated with enzymatic debridement followed by fish skin, Suprathel® (PolyMedics Innovations GmbH, Denkendorf, Germany), or a split-thickness skin graft (STSG). Patients’ outcomes regarding healing and scar quality were collected objectively and subjectively for 12 months after the burn injury. Results: Wounds treated with fish skin demonstrated accelerated wound healing, a significantly higher water-storage capacity, and better pain relief. Furthermore, improved functional and cosmetic outcomes, such as elasticity, skin thickness, and pigmentation, were demonstrated. The pain and itch expressed as POSAS scores (Patient and Observer Scar Assessment Scale) for fish skin decreased compared to those for wounds managed with an STSG or Suprathel. Importantly, fish skin-treated wounds had significantly improved sebum production and skin elasticity than those treated with Suprathel but showed no significant superiority compared to STSG-treated wounds. Conclusions: Enzymatic debridement in combination with intact fish skin grafts resulted in the faster healing of burn wounds and better functional and aesthetic outcomes than split-thickness skin grafts and Suprathel treatment.

Background and Aims Much literature evidence points towards an essential role of mitochondrial quality control mechanisms in acute kidney injury progression. Renal ischemic-reperfusion injury (IRI) results in metabolic adaptation of proximal tubule epithelial cells, a site of high mitochondrial turnover (1). Moreover, exacerbation of renal injury has been demonstrated following IRI in both PTEN-induced kinase 1 (PINK1) knockout and Parkin knockout mice (2). The identification of a single point mutation (m.547A>T) within the mitochondrial genome of a large pedigree of patients with maternally-inherited tubulointerstitial kidney disease, highlights the importance of mitochondrial integrity in human renal pathophysiology (3). USP30 is a mitochondrial associated deubiquitylating enzyme that acts on the mitochondrial import receptor subunit TOM20 to repress PINK1/Parkin-mediated mitophagy and modulate mitochondrial protein transport (4,5). Within kidney, USP30 expression is predominantly tubular and accordingly, USP30 inhibition may provide a mechanism to protect against IRI. MTX008 is a selective small molecule inhibitor of USP30. Prophylactic administration of MTX008 15 mg/kg (p.o.) BID has shown robust and reproducible efficacy in a mouse model of IRI-induced kidney fibrosis (6). Here we present data showing that dosing MTX008 15 mg/kg (p.o.) BID in a therapeutic paradigm, also results in efficacy in the same IRI-induced kidney fibrosis model. Method On Day 0 (zero) C57BL/6 mice were anaesthetized, and their left renal pedicle was clamped transiently for 45 min, causing circulatory arrest in the kidney with consecutive IRI. Mice were then administered either vehicle or MTX008 15 mg/kg (p.o.) BID for 21 days, with first treatment starting five hours following IRI surgery (i.e. therapeutics dosing). Mice were monitored and kidneys harvested at Day +21. Kidney sections were quantitatively assessed for relative cellular morphology, fibrosis and macrophage infiltration using blinded histological scoring methods. Results Body weight was similar between groups and remained constant throughout the observation periods. Masson trichrome staining revealed significantly less tubular atrophy in MTX008 treated animals on Day +21. Similarly, fibronectin expression and macrophage infiltration in the cortex was also significantly reduced in MTX008 treated mice on Day +21. Conclusion MTX008, a novel selective small molecule inhibitor of USP30 has shown efficacy in a model of IR-induced CKD when dosed therapeutically. Initiating treatment after the establishment of IRI has shown significant benefits towards reduced progression to CKD with less tubular atrophy and renal fibrosis. Mission Therapeutics is investigating MTX008 in a variety of preclinical renal injury models with a view to developing this novel molecule towards the clinic. Therapeutic dosing opens a new and attractive opportunity for the treatment of AKI and sets our UPS30 inhibitor apart from other molecules currently in development.

Background and Aims Diclofenac is frequently used for pain control. In a previous study, we showed that already a single oral dose of diclofenac could reduce renal perfusion in healthy individuals. To investigate the influence of oral diclofenac administration on renal inflammation in the setting of pre-existing renal damage, we used a mouse model of subclinical acute kidney injury (AKI) induced by renal ischemia-reperfusion injury (IRI) followed by diclofenac administration. Method Male CD1 mice (7-8 weeks old) underwent unilateral renal pedicle clamping for 15min to induce subclinical AKI. After reperfusion mice received a single oral dose of 100 or 200mg/kg diclofenac via oral gavage. Vehicle treated mice with unilateral IRI served as control. At day 1, mice were placed into metabolic cages to collect urine. Histology was performed on day 1 and 14 for renal morphology. Inflammation and fibrosis were investigated by immunohistochemistry and qPCR. Results Diclofenac treated mice showed reduced urine production. Morphologically, signs of AKI were more pronounced in diclofenac treated kidneys which also showed more Cox-2 positive tubuli in the cortex. On mRNA expression level the pro-inflammatory markers IL-6 and CXCL2, the chemoattractant for neutrophils, were elevated in the diclofenac group. Early upregulation of the pro-fibrotic markers CTGF and PAI-1 was detected already on d1 after IRI in the diclofenac group and tubular atrophy was pronounced after two weeks. Conclusion Already, a single oral dose of diclofenac causes aggravation of renal inflammation and progressive renal fibrosis in the setting of pre-existing subclinical acute kidney injury.

This photovoice project was undertaken by the Family Advisory Committee of the Canada Fetal Alcohol Spectrum Disorder (FASD) Research Network to describe the experiences and the challenges faced by those supporting loved ones with FASD. Photovoice is a method of bringing forth the voice of those who are typically marginalized. It is a process by which people can act as recorders and potential catalysts for social action and change. The Family Advisory Committee chose the photovoice technique to communicate both their experiential knowledge and the research knowledge on both the challenges and rewards in supporting individuals with FASD. This photovoice project highlights several themes that are consistent with the results of caregiver research and provide a glimpse into the experience of those supporting loved ones with FASD. Through this type of knowledge translation the Family Advisory Committee hopes to enhance policy maker and service provider understanding of the necessity of providing support and services not only to the individual with FASD but to those who support them as well.

Rational design and elaborate modular construction of interfacial architectures in which molecular transport is mediated by responsive/adaptive nanostructures has become a growing and fertile field of research in supramolecular materials chemistry. This work presents, for the first time, the use of PNIPAM-capped mesoporous silica thin films as thermosensitive cation-selective mesochannels. Thus far, this feature has only been observed in thermosensitive biological channels. The interfacial architecture created here accomplishes its specific functions through the concerted or simultaneous action of spatially addressed subunits with temperature response and ion exclusion capabilities. The thermo-perm-selectivity effect stems from the synergistic interplay between the pH-dependent electrostatic characteristics of the silica scaffold and the thermo-controlled steric effects introduced by the capping brush layer. It is hoped that the ?nanoarchitectonic? approach presented here will provide new routes toward the generation of heterosupramolecular nanosystems displaying addressable transport properties similar to those encountered in biological ion channels. Fil: Schmidt, Sonja. Universität Göttingen; Alemania Fil: Alberti, Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentina Fil: Vana, Philipp. Universität Göttingen; Alemania Fil: Soler Illia, Galo Juan de Avila Arturo. Universidad Nacional de San Martin. Instituto de Nanosistemas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Azzaroni, Omar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentina

How rainfall infiltration rate and soil hydrological characteristics develop over time under forests of different ages in temperate regions is poorly understood. In this study, infiltration rate and soil hydrological characteristics were investigated under forests of different ages and under grassland. Soil hydraulic characteristics were measured at different scales under a 250-year-old grazed grassland (GL), 6-year-old (6yr) and 48-year-old (48yr) Scots pine (Pinus sylvestris) plantations, remnant 300-year-old individual Scots pine (OT) and a 4000-year-old Caledonian Forest (AF). In situ field-saturated hydraulic conductivity (Kfs) was measured, and visible root:soil area was estimated from soil pits. Macroporosity, pore structure and macropore connectivity were estimated from X-ray tomography of soil cores, and from water-release characteristics. At all scales, the median values for Kfs, root fraction, macroporosity and connectivity values tended to AF > OT > 48yr > GL > 6yr, indicating that infiltration rates and water storage increased with forest age. The remnant Caledonian Forest had a huge range of Kfs (12 to >4922 mm h−1), with maximum Kfs values 7 to 15 times larger than those of 48-year-old Scots pine plantation, suggesting that undisturbed old forests, with high rainfall and minimal evapotranspiration in winter, may act as important areas for water storage and sinks for storm rainfall to infiltrate and transport to deeper soil layers via preferential flow. The importance of the development of soil hydrological characteristics under different aged forests is discussed.

The multicomponent Passerini reaction of aldehydes, carboxylic acids, and isocyanides is used to produce a series of novel reactive (meth)acrylic and styrenic monomers carrying pendant double bond, (trimethylsilyl protected) triple bond, diene, acetate, or pentafluorophenyl functionality. Dichloromethane and water were compared as solvents in the synthesis of 15 different monomers, with water resulting in significantly higher, up to quantitative, isolated yields with minimal purification. Characterization by 1H, 13C, and 19F NMR spectroscopy, FT-IR spectroscopy and mass spectrometry confirmed the synthesis and high purity of the functional α-acyloxycarboxamide products. The monomers are shown to be well suited for the RAFT-synthesis of well-defined homopolymers, statistical copolymers with methyl methacrylate, poly(ethylene glycol) methyl ether methacrylate, and styrene, statistical copolymers produced from two different Passerini-derived monomers, and AB diblock copolymers. SEC-measured polydispersities were generally low, ĐM ≤ 1.29, and 1H NMR spectroscopy confirmed copolymer molar compositions in good agreement with comonomer feed ratios. We expect this synthetic strategy to provide access to a wide range of novel multifunctional materials and demonstrate preliminary postpolymerization modification of a polystyrene derivative by cleavage of its pendent acetate groups and coupling of the dye Methyl Red to the resulting alcohol groups.

The morphology of roots and root systems influences the efficiency by which plants acquire nutrients and water, anchor themselves and provide stability to the surrounding soil. Plant genotype and the biotic and abiotic environment significantly influence root morphology, growth and ultimately crop yield. The challenge for researchers interested in phenotyping root systems is, therefore, not just to measure roots and link their phenotype to the plant genotype, but also to understand how the growth of roots is influenced by their environment. This review discusses progress in quantifying root system parameters (e.g. in terms of size, shape and dynamics) using imaging and image analysis technologies and also discusses their potential for providing a better understanding of root:soil interactions. Significant progress has been made in image acquisition techniques, however trade-offs exist between sample throughput, sample size, image resolution and information gained. All of these factors impact on downstream image analysis processes. While there have been significant advances in computation power, limitations still exist in statistical processes involved in image analysis. Utilizing and combining different imaging systems, integrating measurements and image analysis where possible, and amalgamating data will allow researchers to gain a better understanding of root:soil interactions.

Graphical abstract, Highlights • Functional cloning identifies a Leishmania-specific 58 kD antimony resistance marker, ARM58. • ARM58 overexpression confers antimony resistance. • A neighbouring, structurally related gene lacks antimony resistance function. • ARM58 comprises 4 related domains of unknown function, classified as DUF1935. • Cosmid-based functional cloning is feasible in L. braziliensis in spite of RNAi., Protozoa of the Leishmania genus cause a variety of disease forms that rank at the top of the list of neglected tropical diseases. Anti-leishmanial drugs based on pentavalent antimony have been the mainstay of therapy for over 60 years and resistance against them is increasingly encountered in the field. The biochemical basis for this is poorly understood and likely diverse. No stringent correlation between genetic markers and antimony resistance has so far been shown, prompting us to use a functional cloning approach to identify markers of resistance. Using gene libraries derived from drug-resistant and drug-sensitive Leishmania braziliensis clinical isolates in a functional cloning strategy, we repeatedly selected one gene locus located on chromosome 20 whose amplification confers increased antimony (III) resistance in vitro to an otherwise sensitive L. braziliensis clone. The gene responsible for the effect encodes a previously hypothetical protein that we dubbed LbrARM58. It comprises four repeats of a domain of unknown function, DUF1935, one of them harbouring a potential trans-membrane domain. The gene is so far unique to the Leishmania genus, while a structurally related gene without antimony resistance functionality is also found in Trypanosoma spp. Overexpression of LbrARM58 also confers antimony resistance to promastigotes and intracellular amastigotes of the related species Leishmania infantum, indicating a conserved function in Old World and New World Leishmania species. Our results also show that in spite of their RNAi system, L. braziliensis promastigotes can serve as acceptor cells for episomally propagated cosmid libraries, at least for the initial stages of functional cloning efforts.

Over the last decade, X-ray computed tomography (CT) has been used increasingly to characterise the microscale architecture of soils. As a result significant progress has been made in the acquisition and interpretation of X-ray CT data, as well as in the thresholding of 3D greyscale CT images in order to produce binary (black and white) ones. Nevertheless, sizeable uncertainties persist, in particular concerning optimal instrumental settings used to generate the greyscale images. In this context, the key aim of this study is to investigate in detail the effect of scanning resolution and reconstruction settings such as noise reduction and 32-bit to 8-bit mapping interval on the 3D X-ray CT imaging of soil structure and the impact on the performance of thresholding methods. To assess the quality of the X-ray CT greyscale images, measures of contrast, noise and sharpness are proposed and tested on a series of images of five different soil samples. At the same time, performance of four segmentation algorithms, i.e., three methods recently developed to deal specifically with soil samples and Otsu's method as a benchmark, was evaluated using functional measures of 3D binary images, including Minkowski functionals and surface pore connected fraction. Results of these analyses suggest that the acquisition and reconstruction parameters investigated significantly affect the quality of soil images, and the subsequent thresholding process. In particular, it was found that thresholding by any of the four methods is greatly affected by the quality of image sharpness, which for soil images appears to be mainly controlled by the scanning resolution. As a result, it is concluded that no matter what reconstruction resolution is required in a study, in order to allow an accurate identification of the pore space, the sample should always be scanned at the highest resolution permitted by the scanning instrument and the sample size. Results also suggest that the three segmentation methods recently developed for soil images thresholding are robust to different levels of noise as well as the choice of the 32-bit mapping interval, as long as lower and upper interval limits for mapping are chosen within suitable boundaries.

We investigated interacting effects of matric potential and soil strength on root elongation of maize and lupin, and relations between root elongation rates and the length of bare (hairless) root apex. Root elongation rates and the length of bare root apex were determined for maize and lupin seedlings in sandy loam soil of various matric potentials (−0.01 to −1.6 MPa) and bulk densities (0.9 to 1.5 Mg m−3). Root elongation rates slowed with both decreasing matric potential and increasing penetrometer resistance. Root elongation of maize slowed to 10 % of the unimpeded rate when penetrometer resistance increased to 2 MPa, whereas lupin elongated at about 40 % of the unimpeded rate. Maize root elongation rate was more sensitive to changes in matric potential in loosely packed soil (penetrometer resistances

Adequate contact with the soil is essential for water and nutrient adsorption by plant roots, but the determination of root–soil contact is a challenging task because it is difficult to visualize roots in situ and quantify their interactions with the soil at the scale of micrometres. A method to determine root–soil contact using X-ray microtomography was developed. Contact areas were determined from 3D volumetric images using segmentation and iso-surface determination tools. The accuracy of the method was tested with physical model systems of contact between two objects (phantoms). Volumes, surface areas and contact areas calculated from the measured phantoms were compared with those estimated from image analysis. The volume was accurate to within 0.3%, the surface area to within 2–4%, and the contact area to within 2.5%. Maize and lupin roots were grown in soil ( 70 µm) of the soil sieved to different aggregate fractions was calculated from binarized data. Root-soil contact was greater in soil than in vermiculite and increased with decreasing aggregate or particle size. The differences in root–soil contact could not be explained solely by the decrease in porosity with decreasing aggregate size but may also result from changes in particle and aggregate packing around the root.

Intestinal celiac disease (CD) is triggered by peptic-tryptic digest of gluten, known as Frazer's Fraction (FF), in genetically predisposed individuals. Here, we investigate the immediate effects of FF on the actin cytoskeleton and the subsequent trafficking of actin-dependent and actin-independent proteins in COS-1 cells. Morphological alterations in the actin filaments were revealed concomitant with a drastic reduction in immunoprecipitated actin from cells incubated with FF. These alterations elicit impaired protein trafficking of intestinal sucrase–isomaltase, a glycoprotein that follows an actin-dependent vesicular transport to the cell surface. However, the actin-independent transport of intestinal lactase phlorizin hydrolase remains unaffected. Moreover, the morphological alteration in actin is induced by direct interaction of this protein with gliadin peptides carrying the QQQPFP epitope revealed by co-immunoprecipitation utilizing a monoclonal anti-gliadin antibody. Finally, stimulation of cells with FF directly influences the binding of actin to Arp2. Altogether, our data demonstrate that FF directly interacts with actin and alters the integrity of the actin cytoskeleton thus leading to an impaired trafficking of intestinal proteins that depend on an intact actin network. This direct interaction could be related to the endocytic segregation of gliadin peptides as well as the delayed endocytic vesicle trafficking and maturation in gliadin-positive intestinal epithelial cells and opens new insights into the pathogenesis of CD.

Summary This article presents an integrative approach to calculating the weight of potential biowaste and collected biowaste materials, as the basis for a life-cycle assessment (LCA) of biowaste management. Biowaste contains kitchen and garden (yard) waste of households. This approach could be used for waste management planning and for the implementation of biowaste schemes. Case studies and examples in the literature are analyzed to model the mass of the flow of biowaste. This article defines relevant operands, presents the main assumptions, and describes the calculation principles. Spatial aspects and the uncertainties related to the inclusion of this aspect are explicitly considered in the calculation of the weight of the potential biowaste. We also present the calculation principles for obtaining the weight of (1) biowaste used in home composting, (2) the organic portion of residual waste, (3) biowaste separately collected by a bring system, and (4) biowaste separately collected by curbside collection (known in some areas as kerbside collection). By choosing the biowaste potential in kilograms per capita year (kg/cap yr) as the functional unit, previously ignored options within the biowaste system could be assessed. For example, widening the system boundaries allows LCA studies to assess the contribution of private and public transport of waste to ecological impact categories. It allows examining the effects of supporting home composting through financial incentives and the introduction of a separate collection system. This study focuses on the comparison of different collection types and on the characteristics of the area under investigation. It also incorporates the behavior of the inhabitants of households and includes a sensitivity analysis of relevant operands. This approach is being included in an LCA assessing biowaste management options.

We treated 10 children with X-linked SCID (SCID-X1) using gammaretrovirus-mediated gene transfer. Those with sufficient follow-up were found to have recovered substantial immunity in the absence of any serious adverse events up to 5 years after treatment. To determine the influence of vector integration on lymphoid reconstitution, we compared retroviral integration sites (RISs) from peripheral blood CD3+ T lymphocytes of 5 patients taken between 9 and 30 months after transplantation with transduced CD34+ progenitor cells derived from 1 further patient and 1 healthy donor. Integration occurred preferentially in gene regions on either side of transcription start sites, was clustered, and correlated with the expression level in CD34+ progenitors during transduction. In contrast to those in CD34+ cells, RISs recovered from engrafted CD3+ T cells were significantly overrepresented within or near genes encoding proteins with kinase or transferase activity or involved in phosphorus metabolism. Although gross patterns of gene expression were unchanged in transduced cells, the divergence of RIS target frequency between transduced progenitor cells and post-thymic T lymphocytes indicates that vector integration influences cell survival, engraftment, or proliferation.

Modelling of environmental impacts from the application of treated organic municipal solid waste (MSW) in agriculture differs widely between different models for environmental assessment of waste systems. In this comparative study five models were examined concerning quantification and impact assessment of environmental effects from land application of treated organic MSW: DST (Decision Support Tool, USA), IWM (Integrated Waste Management, UK), THE IFEU PROJECT (Germany), ORWARE (ORganic WAste REsearch, Sweden) and EASEWASTE (Environmental Assessment of Solid Waste Systems and Technologies, Denmark). DST and IWM are life cycle inventory (LCI) models, thus not performing actual impact assessment. The DST model includes only one water emission (biological oxygen demand) from compost leaching in the results and IWM considers only air emissions from avoided production of commercial fertilizers. THE IFEU PROJECT, ORWARE and EASEWASTE are life cycle assessment (LCA) models containing more detailed land application modules. A case study estimating the environmental impacts from land application of 1 ton of composted source sorted organic household waste was performed to compare the results from the different models and investigate the origin of any difference in type or magnitude of the results. The contributions from the LCI models were limited and did not depend on waste composition or local agricultural conditions. The three LCA models use the same overall approach for quantifying the impacts of the system. However, due to slightly different assumptions, quantification methods and environmental impact assessment, the obtained results varied clearly between the models. Furthermore, local conditions (e.g. soil type, farm type, climate and legal regulation) and waste composition strongly influenced the results of the environmental assessment.

Eph receptors are a unique family of receptor tyrosine kinases (RTK) that play critical roles in embryonic patterning, neuronal targeting, and vascular development during embryogenesis. In adults, Eph RTKs and their ligands, the ephrins, are frequently overexpressed in a variety of cancers and tumor cell lines, including breast, prostate, non-small cell lung and colon cancers, melanomas, and neuroblastomas. Unlike traditional oncogenes that often function only in tumor cells, recent data show that Eph receptors mediate cell-cell interaction both in tumor cells and in tumor microenvironment, namely the tumor stroma and tumor vasculature. As such, Eph RTKs represent attractive potential targets for drug design, as targeting these molecules could attack several aspects of tumor progression simultaneously. This review will focus on recent advances in dissecting the role of Eph RTKs in tumor cells, tumor angiogenesis, and possible contribution to trafficking of inflammatory cells in cancer.

Measurements in soils have been traditionally used to demonstrate that soil architecture is one of the key drivers of soil processes. Major advances in the use of X-ray Computed Tomography (CT) afford significant insight into the pore geometry of soils, but until recently no experimental techniques were available to reproduce this complexity in microcosms. This article describes a 3D additive manufacturing technology that can print physical structures with pore geometries reflecting those of soils. The process enables printing of replicated structures, and the printing materials are suitable to study fungal growth. This technology is argued to open up a wealth of opportunities for soil biological studies.

International audience; Soil respiration represents the second largest CO 2 flux from terrestrial ecosystems to the atmosphere , and a small rise could significantly contribute to further increase in atmospheric CO 2. Unfortunately, the extent of this effect cannot be quantified reliably, and the outcomes of experiments designed to study soil respiration remain notoriously unpredictable. In this context, the mathematical simulations described in this article suggest that assumptions of linearity and presumed irrelevance of micro-scale heterogeneity, commonly made in quantitative models of microbial growth in subsurface environments and used in carbon stock models, do not appear warranted. Results indicate that microbial growth is non-linear and, at given average nutrient concentrations, strongly dependent on the microscale distribution of both nutrients and microbes. These observations have far-reaching consequences, in terms of both experiments and theory. They indicate that traditional, macroscopic soil measurements are inadequate to predict microbial responses, in particular to rising temperature conditions, and that an explicit account is required of microscale heterogeneity. Furthermore , models should evolve beyond traditional, but overly simplistic, assumptions of lineari-ty of microbial responses to bulk nutrient concentrations. The development of a new generation of models along these lines, and in particular incorporating upscaled information about microscale processes, will undoubtedly be challenging, but appears to be key to understanding the extent to which soil carbon mineralization could further accelerate climate change.

Summary Background Because worldwide tuberculosis (TB) control had never been assessed, WHO set up a surveillance and monitoring project in 1995. The objectives were to assess the performance of national TB programmes; to assess the extent of implementation of the WHO strategy of TB control; and to attempt a comparison between regions that had adopted the WHO strategy and those that had not. Methods In June, 1996, we sent data-collection forms requesting information on national TB programmes' control policies, 1995 case notifications, and 1994 treatment results to 216 countries, areas, and territories. We assessed the performance of national TB programmes by comparing case notifications with estimated incidence and by outcome of treatment in cohorts of patients. We also investigated worldwide treatment success and case detection among sputumsmear-positive patients. Findings 180 (83%) of the 216 countries, areas, and territories surveyed replied to WHO (98% of the worldwide population). In 1995, the WHO control strategy had been implemented in 75 countries, and in 39 of these implementation was countrywide. Up to 23% of the worldwide population lived in regions where the strategy was available. In 1995, 3 297 688 cases of TB (all types) were reported, of which 1 161 411 (35%) were sputum-smear positive. 54% of all reported cases in countries that used the WHO strategy were sputumsmear positive, compared with 30% in other countries. The worldwide case-detection rate of new sputum-smearpositive cases was 35%. 92% of cases registered for treatment in 1994 in regions that used WHO strategy were assessed for outcome and 76% were treated successfully, compared with 54% and 42%, respectively, in regions that had not implemented the WHO strategy. Among cases reported worldwide in 1994, the documented treatment-success rate was 43% Interpretation National TB programmes that have adopted the WHO TB control strategy achieve higher cure rates, but their impact on TB is modest on a worldwide scale. Wider continuous coverage with the WHO strateg is needed for effective worldwide TB control.

WOS:000324901700014; International audience; Soil microbial communities live in the soil pore network and therefore the access they have to organic substrate, oxygen and water depends on how this network is structured. In this experimental study, the relationship between soil structure and soil organic matter dynamics was investigated by measuring the kinetics of organic carbon decomposition in samples that varied in the physical structure of the soil pore network. Soil cores with different structures (undisturbed, sieved and disaggregated by agitation in water), but with the same bulk density, were incubated and the mineralisation of native and added (C-13-labelled substrates, fructose and vanillin) organic carbon was measured for a period of 127 days. The incubation was commenced after the immediate effects of the perturbations caused by the soil structural treatments had dissipated. At the end of the incubation, the microbial biomass and microbial community structure were determined. It was found that the respiration kinetics were not related to soil structural parameters, which were determined using X-ray micro-CT. The structure treatments had no significant effect on the mineralisation of soil or added organic carbon, despite the significantly different physical environments. However, the microbial community structure of the undisturbed and dispersed samples were significantly different. These results indicate that neither decomposer access to organic substrate nor the environmental conditions in which decomposition occurred were impaired by the soil structure treatments and suggest that the controls on carbon dynamics may take place at scales below those that were manipulated in this experiment. (C) 2013 Elsevier Masson SAS. All rights reserved.

Lidocaine is the most commonly chosen prokinetic for treating postoperative ileus in horses, a motility disorder associated with ischaemia-reperfusion injury of intestinal tissues. Despite the frequent use of lidocaine, the mechanism underlying its prokinetic effects is still unclear. Previous studies suggested that lidocaine altered cell membrane characteristics of smooth muscle cells. Therefore, the present study aimed to elucidate effects of lidocaine administration on characteristics of detergent-resistant membranes in equine jejunal smooth muscle. Lidocaine administration caused significant redistribution of flotillin-2, a protein marker of detergent-resistant membranes, in fractions of sucrose-density-gradients obtained from ischaemia-reperfusion injured smooth muscle solubilised with Triton X-100. It was concluded that lidocaine induced disruption of detergent-resistant membranes which might affect ion channel activity and therefore enhance smooth muscle contractility.

Background In areas with low caries prevalence, indices are needed for caries detection, which can also be used to identify initial lesions. Aim The aim of this study was to assess the caries prevalence among 12-year-olds using ICDAS criteria and to investigate the influence of independent variables on the findings. Design The study was conducted in two regions of Germany. In Region 1, children received regular school-based prophylaxis, including fluoride varnish 2×/yr. In Region 2, there was no use of fluoride varnish in schools. Information on different factors influencing the outcome variable of caries experience was collected using structured questionnaires. DF-S values were calculated at different ICDAS cut-off points. To compare the mean caries scores of the subgroups, nonparametric tests were performed. Variables associated with caries were included in a binary logistic regression analysis. Results At D1–6FS and D1+2FS level, the differences between the regions were statistically significant (P = 0.005 and P = 0.01, respectively). Regression analysis identified the variables ‘use of fluoridated toothpaste’, ‘fissure sealants’, and ‘ethnic origin’ as factors significant to the prevention of caries at various stages. Conclusion In a population with low caries prevalence, significant differences between subgroups could only be found when initial lesions were included.

Sustainable intensification is seen as the main route for meeting the world’s increasing demands for food and fibre. As demands mount for greater efficiency in the use of resources to achieve this goal, so the focus on roots and rootstocks and their role in acquiring water and nutrients, and overcoming pests and pathogens, is increasing. The purpose of this review is to explore some of the ways in which understanding root systems and their interactions with soils could contribute to the development of more sustainable systems of intensive production. Physical interactions with soil particles limit root growth if soils are dense, but root–soil contact is essential for optimal growth and uptake of water and nutrients. X-ray microtomography demonstrated that maize roots elongated more rapidly with increasing root–soil contact, as long as mechanical impedance was not limiting root elongation, while lupin was less sensitive to changes in root–soil contact. In addition to selecting for root architecture and rhizosphere properties, the growth of many plants in cultivated systems is profoundly affected by selection of an appropriate rootstock. Several mechanisms for scion control by rootstocks have been suggested, but the causal signals are still uncertain and may differ between crop species. Linkage map locations for quantitative trait loci for disease resistance and other traits of interest in rootstock breeding are becoming available. Designing root systems and rootstocks for specific environments is becoming a feasible target.

Prostate-specific membrane antigen (PSMA) is a type-II membrane glycoprotein that was initially identified in LNCaP cells. It is expressed at elevated levels in prostate cancer. In view of the correlation between the expression levels of PSMA and disease grade and stage, PSMA is considered to be one of the most promising biomarkers in the diagnosis and treatment of prostate cancer. In LNCaP cells PSMA undergoes internalization via clathrin-coated pits followed by accumulation in the endosomes. PSMA associates with different types of detergent-resistant membranes (DRMs) along the secretory pathway. Its mature form is mainly insoluble in Lubrol WX, but does not associate with Triton X-100-DRMs. To understand the mechanism of PSMA internalization we investigated its association during internalization with DRMs. For this purpose, internalization was induced by antibody cross-linking. We demonstrate at the biochemical and cell biological levels that: [i] exclusively homodimers of PSMA are associated with Lubrol WX-DRMs, [ii] antibody-induced cross-linking of PSMA molecules results in a time-dependent partitioning into another DRMs type, namely Triton X-100-DRMs, and [iii] concomitant with its association with Triton-X-100-DRMs internalization of PSMA occurs along tubulin filaments. In a previous work (Colombatti et al. (2009) PLoS One 4: e4608) we demonstrated that the small GTPases RAS and RAC1 and the MAPKs p38 and ERK1/2 are activated during antibody cross-linking. As downstream effects of this activation we observed a strong induction of NF-kB associated with an increased expression of IL-6 and CCL5 genes and that IL-6 and CCL5 enhanced the proliferative potential of LNCaP cells synergistically. These observations together with findings reported here hypothesize a fundamental role of DRMs during activation of PSMA as platforms for trafficking, endocytosis and signalling. Understanding these mechanisms constitutes an essential prerequisite for utilization of PSMA as a therapeutically suitable target in prostate cancer.

BACKGROUND Prostate-specific membrane antigen (PSMA) is a promising biomarker in the diagnosis of prostate cancer and a potential target for antibody-based therapeutic strategies. We isolated the canine PSMA cDNA and investigated the cellular and biochemical characteristics of the recombinant protein as a potential target for animal preclinical studies of antibody based-therapies. METHODS Canine PSMA cDNA was isolated by PCR, cloned into expression vectors and transfected into COS-1 and MDCK cells. The biosynthesis and glycosylation of the recombinant protein were investigated in pulse-chase experiments, the cellular localization by confocal laser microscopy, the mode of association of PSMA with the membrane with solubilization in different detergents and its quaternary structure in sucrose-density gradients. RESULTS Canine PSMA shows 91% amino acid homology to human PSMA, whereby the major difference is a longer cytoplasmic tail of canine PSMA compared to its human counterpart. Canine PSMA is trafficked efficiently along the secretory pathway, undergoes homodimerization when it acquires complex glycosylated mature form. It associates with detergent-resistant membranes, which act as platforms along its intracellular trafficking. Confocal analysis revealed canine PSMA at the cell surface, Golgi, and the endoplasmic reticulum. A similar distribution is revealed for human PSMA, yet with reduced cell surface levels. CONCLUSIONS The cloning, expression, biosynthesis, processing and localization of canine PSMA in mammalian cells is described. We demonstrate that canine PSMA reveals similar characteristics to human PSMA rendering this protein useful as a translational model for investigations of prostate cancer as well as a suitable antigen for targeted therapy studies in dogs. Prostate 73: 642–650, 2013. © 2012 Wiley Periodicals, Inc.

Functional intestinal epithelium relies on complete polarization of enterocytes marked by the formation of microvilli and the accurate trafficking of glycoproteins to relevant membrane domains. Numerous transport pathways warrant the unique structural identity and protein/lipid composition of the brush border membrane. Annexin II (Ca(2+)-dependent lipid-binding protein) is an important component of one of the apical protein transport machineries, which involves detergent-resistant membranes and the actin cytoskeleton. Here, we investigate in intestinal Caco-2 cells the contribution of annexin II to the sorting and transport of brush border hydrolases and role in intestinal cell polarity. Downregulation of annexin II in Caco-2-A4 cell line results in a severe reduction of the levels of the brush border membrane resident enzyme sucrase isomaltase (SI) as well as structural components such as ezrin. This reduction is accompanied by a redistribution of these proteins to intracellular compartments and a striking morphological transition of Caco-2 cells to rudimentary epithelial cells that are characterized by an almost flat apical membrane with sparse and short microvilli. Concomitant with this alteration is the redistribution of the intermediate filament protein keratin 19 to the intracellular membranes in Caco-2-A4 cells. Interestingly, keratin 19 interacts with annexin II in wild type Caco-2 cells and this interaction occurs exclusively in lipid rafts. Our findings suggest a role for annexin II and K19 in differentiation and polarization of intestinal cells.

Previous observations of retroviral vector induced oncogenesis in preclinical models and in a clinical SCID-X1 gene therapy study have prompted large-scale integration site analysis. In vitro and in vivo studies indicate that lentiviral vectors may lower the risk of insertional mutagenesis compared to common retroviral vectors. Several studies could show that integration site distribution is vector dependent (MLV, HIV, SIV, ASLV) and the type of LTR may play a crucial role. Here, we have analyzed the possible influence of distinct internal promoters and transgenes on integration site distribution and clone survival.

Previous studies have demonstrated leukemic complications in mice after high-copy retroviral gene transfer of the multidrug resistance 1 (MDR1) cDNA, encoding a membrane-located efflux pump expressed in hematopoietic stem cells. In contrast, no such complications or MDR1-associated alterations of hematopoiesis were observed in numerous other studies exploring MDR1 gene transfer into cell lines, mice, dogs, nonhuman primates, and human subjects. Here, we show that leukemias associated with retroviral expression of MDR1 depend on high vector dose, and involve the selection of clones with combinatorial insertional mutagenesis of proto-oncogenes or other signaling genes. Compared with insertion patterns in normal long-term repopulating hematopoietic cells, such hits were overrepresented in leukemic clones, pointing to a causal role. A similar constellation of insertion sites was also observed in a leukemia arising after high-copy retroviral gene transfer of a fluorescent protein. Spectral karyotyping demonstrated additional chromosomal translocations in a subset of cases, indicative of secondary genetic instability. We also show that insertional mutants can be amplified in vitro prior to transplantation. On the basis of these findings, we suggest the use of preclinical dose-escalation studies to define a therapeutic index for retroviral transgene delivery.

Reliable techniques for screening large numbers of plants for root traits are still being developed, but include aeroponic, hydroponic and agar plate systems. Coupled with digital cameras and image analysis software, these systems permit the rapid measurement of root numbers, length and diameter in moderate (typically

The need for safer gene therapy vectors was highlighted by the occurrence of three cases of retroviral vector-induced leukemia in children after the cure of severe combined immunodeficiency by gene therapy. These severe adverse events enhanced the development of new gene therapy vector systems, which aim to reduce influence of insertions on integrity and expression of genomic host DNA. Lentiviral vectors with self-inactivating (SIN) long terminal repeats (LTRs) have no LTR enhancer activity and are not expected to produce insertional gene activation. In our study we analyzed the integrations sites of three different lentiviral SIN-HIV-based- vectors. These vectors differed in their internal elements (Promotor, Transgene, WPRE) which allowed us to investigate whether internal elements are influencing target site selection and clone survival. A total of 1422 integration sites were analyzed at three different time points (1 day, 30 days, 60 days) after transduction of identical HeLa cells by LAM- PCR. Our results showed similar gene involvement and chromosomal distribution of integration sites for all vector types analyzed, independent of vector composition. 62–63% of the integrations were detected in Refseq genes, 82–86% were found within Refseq genes and their surrounding 10kb. Surprisingly, 271 of 1422 integration sites were clustered as common integration sites (CIS). Computer simulations allowed us to show that this high number of CIS was significantly different from a modeled random distribution of integration sites. Gene ontology analysis showed no difference in significantly overrepresented gene categories between the distinct vector types. Interestingly, we detected a time dependent increase or decrease in significance. Specific gene categories like phosphorylation, protein kinase or ATP binding activity increased in significance from freshly transduced cells (1 day) to 30 days. This observation was even more pronounced at the latest time point analyzed (60 days). Other gene categories showed the exactly opposite effect. Gene ontology analysis of freshly transduced cells showed a significant overrepresentation of genes involved in cell cycle regulation, whereas the analysis of the later time points did not show overrepresentation of this gene category. These results suggest that lentiviral SIN-HIV-based vectors may induce clonal selection in vitro independently of internal vector elements. The character of such effects as well as any putative relevance of such genotoxicity for the in vivo situation will have to be investigated in detail for the role of individual vector/cell type configurations.

Successful gene therapy trials of ADA-SCID and SCID-X1 demonstrated the curative potential of oncoretroviral gene transfer. Integration of the retroviral vectors used in these studies has been thought to be a random process but severe side effects in gene therapy and in vitro studies revealed preferred insertion of these vectors mainly around transcription start sites. In SCID patients proliferation advantage of gene corrected cells was one reason for the success of the trials, whereas in the most recent chronic granulomatous disease (CGD) gene therapy trial corrected cells do not have any selective advantage therefore the two patients received mild busulfan treatment before transplantation. High efficiency transduction and conditioning have helped in the successful correction of the patients. Peripheral blood granulocytes show a stable expression (>10%) of the transgene (gp91phox) in patient 1 (15 months post treatment) as well as in patient 2 (11 months post treatment). We reasoned that, unlike T cells, which have the capability to proliferate independent of their bone marrow progenitors, granulocytes more directly reflect the influence of retrovirus insertion, and should therefore allow to closely monitor clonal fate in vivo and its potential relation to vector insertion. To study the clonality of the corrected myelopoiesis, the long term activity of individual cell clones, and the distribution of integration sites in active cells we carried out high sensitive LAM-PCR. The highly polyclonal composition of transduced cells forming myelopoiesis caused the sustained expression of gp91phox. Individual clones carrying the transgene could be detected at multiple time points. To define whether corrected cells have a proliferation advantage due to their vector integration we started large-scale sequencing and mapping of involved insertion sites. We here present >700 unique mappable integration sites of the two treated patients. The distribution of the SFFV based retroviral vector integration sites in this trial turned non random 5 months after transplantation. Corrected long-term myelopoiesis expanded 3- to 5- fold in the two patients due to activating common integration sites (CIS) in the zinc finger transcription factor homologs MDS1/EVI1, PRDM16, or in SETBP1, suggesting that these genes influence regulation of normal long-term hematopoiesis in humans. Our data indicate that the therapeutic benefit in this trial was activated through insertional side effects, therefore our findings have important implications in novel gene therapy approaches.

Retrovirus gene transfer into hematopoietic stem cells is an effective treatment of X-linked severe combined immunodeficiency (SCID-X1). Nevertheless, the recent occurrence of T ALL like disease in three SCID-X1 patients led to intensive studies on vector biosafety with focus on insertional mutagenesis. Large-scale mapping of retrovirus integration sites in human cell lines revealed preferential vector integration into genomic coding regions, in particular near transcription start sites. We hypothesized that in clinical applications, the distribution of insertion sites may be influenced by additional factors, including the gene expression profile of the target cell population and the selection of hematopoietic cells capable of engrafting and proliferating in vivo. In this trial, six patients have been treated successfully since 2001 using a GALV-pseudotyped MLV-based vector carrying the therapeutic common gamma chain gene for transduction of autologous CD34+ cells. The patients did not receive any conditioning therapy before transplantation. Using a high-volume integration site sequencing approach, we have charcterized more than 400 unique integration sites derived from the patients. So far, no vector integration in the LMO2 oncogene could be detected in this trial, and the patients do not reveal any other evidence for malignancy or clonal deformation of their stem cell compartment. We could show that integration of the mammalian gammaretroviral vector in this gene therapy trial is not random. Integration of the vector happens generally within or close to gene coding regions, preferential surrounding the transcription start site. Moreover, we found common integration sites (CIS) in RefSeq gene regions. The targeted RefSeq genes were classified according to the Gene Ontology database. Our data strengthen the hypothesis that curative gene therapeutic treatment requires a sustained polyclonal contribution of ex vivo manipulated stem and progenitor cells. The distribution of insertions into the vicinity genes and common integration sites indicates that the observed distribution may also have been influenced by the biological effects these insertions may have caused in their target cells.

The integration of a retroviral vector as used in hematopoietic gene therapy trials produces a transition sequence from the vector DNA into the genomic DNA and may thus serve as a stable molecular marker unique for each cell clone. High sensitive linear amplification mediated PCR (LAM-PCR) allows the detection of specific retroviral integration sites. Thus it is possible to determine the clonal composition of the hematopoietic system in vivo (1). We could show that the hematopoietic repopulation in human SCID-X1 patients was derived from various, long-lived progenitor cell clones indicating retroviral transduction into pluripotent cells (manuscript submitted). In two cases of lymphoproliferative disorder after successful SCID-X1 gene therapy integration of the retroviral vector into the LMO-2 oncogene was probable the main reason for malignancy (2). The distribution of integration sites over the whole genome, the potential preference for integration at certain loci and which cells receive genetic correction and engraftment are therefore of considerable interest. Recently, another gene therapy trial has successfully corrected 4 infants suffering from SCID-X1. A GALV-pseudotyped MLV-based vector carrying the therapeutic common gamma chain gene was used for transduction of autologous CD34+ cells. The patients did not receive any conditioning therapy before transplantation. We analyzed lymphoid and myeloid DNA from all patients. The transduction efficiency of T lymphocytes and myeloid cells was up to 100 and 1 %, respectively. In vivo clonality analysis of CD3+ cells showed a polyclonal composition 1 to 2 years after transplantation. The myeloid repopulation also consisted of various different clones. These data may indicate transduction of pluripotent and long term active stem or progenitor cells. We here report on more than 300 sequenced integration sites of the patients, whereas 250 sequences could be assigned unequivocally to a unique locus. So far, no vector integration in the LMO-2 oncogene could be detected in this trial, and the patients do not reveal any other evidence for malignancy or clonal deformation of their stem cell compartment. We could show that integration of the mammalian gammaretroviral vector in this gene therapy trial is not random. Integration of the vector happens generally within or close to specific regions of genes. We found common integration sites (CIS) in RefSeq gene regions. The targeted RefSeq genes were classified according to the Gene Ontology database. Our data strongly support the presumption that curative gene therapeutic treatment requires a sustained polyclonal contribution of ex vivo manipulated stem and progenitor cells and provide an important insight into the integration manner of GALV-pseudotyped MLV-based vectors.

As recent case reports have suggested that proto-oncogene activation by replication-deficient retroviral vectors may trigger leukemia in mice and men, there is an urgent need for models allowing predictive investigations into the pathomechanisms of this complication. Previously, we have shown that dose-escalated replication-defective retrovirus vectors (such as HaMDR1 or SF91dsRedpre) may trigger combinatorial leukemia-initiating events in single cells, with a probability that allows prospective studies in simple mouse models (C57Bl/6). Meanwhile, we have performed molecular analyses for 5 different leukemias, harboring 6 to 12 vector insertions according to Southern blot. 34 insertions recovered from these clones by LM-PCR and LAM-PCR showed that 4 leukemias had combinatorial hits in at least one established proto-oncogene (class 1 genes: Hivep1, Mllt3, Fli1, HoxA7, Brd2, Evi1, Csfr3, Bcl11a, Ski) with at least one hit in other signaling genes (class 2 genes). One leukemia for which 2 insertions remain to be identified showed at least 3 class 2 hits, so far without a class 1 event. All leukemias presented with hits in genes involved in cytoplasmic signal cascades along with genes contributing to nuclear regulatory networks. Some of these leukemias had a normal karyotype. A comparison with 62 insertion sites from healthy control animals (determined in peripheral blood DNA harvested 7 months after transplantation) revealed a significant enrichment of class 1 hits in leukemias (14-fold over expected and 3-fold over control). Healthy animals still showed 6 hits (~10%) in class 1 genes, obviously not being sufficient for leukemia induction. Three of these occurred upstream of Evi1, one upstream of Nfic, one upstream of Pdclg2, and one in Bcl11a. Interestingly, upstream vector insertion was preferred in class 1 gene hits. Overall, insertions recovered from both normal and leukemic samples revealed an overrepresentation of hits within (or upstream of) known or predicted genes (89 and 90%, respectively). In line with the data of a related murine marking study (Kustikova, Fehse et al., ASH 2004), our study strongly suggests that single hits in class 1 or class 2 genes are not sufficient to induce leukemia in mice. However, certain combinations of such events in single cells may well be leukemogenic with a given latency. Our findings establish a powerful approach for both oncogene discovery and safety validation in gene therapy.