In CKD, the risk of kidney failure and death depends on the severity of proteinuria, which correlates with the extent of podocyte loss and glomerular scarring. We investigated whether proteinuria contributes directly to progressive glomerulosclerosis through the suppression of podocyte regeneration and found that individual components of proteinuria exert distinct effects on renal progenitor survival and differentiation toward a podocyte lineage. In particular, albumin prevented podocyte differentiation from human renal progenitors in vitro by sequestering retinoic acid, thus impairing retinoic acid response element (RARE)-mediated transcription of podocyte-specific genes. In mice with Adriamycin nephropathy, a model of human FSGS, blocking endogenous retinoic acid synthesis increased proteinuria and exacerbated glomerulosclerosis. This effect was related to a reduction in podocyte number, as validated through genetic podocyte labeling in NPHS2.Cre;mT/mG transgenic mice. In RARE-lacZ transgenic mice, albuminuria reduced retinoic acid bioavailability and impaired RARE activation in renal progenitors, inhibiting their differentiation into podocytes. Treatment with retinoic acid restored RARE activity and induced the expression of podocyte markers in renal progenitors, decreasing proteinuria and increasing podocyte number, as demonstrated in serial biopsy specimens. These results suggest that albumin loss through the damaged filtration barrier impairs podocyte regeneration by sequestering retinoic acid and promotes the generation of FSGS lesions. Our findings may explain why reducing proteinuria delays CKD progression and provide a biologic rationale for the clinical use of pharmacologic modulators to induce regression of glomerular diseases.

Summary 1. The breeding behaviour of the white‐clawed crayfish, Austropotamobius pallipes, which is in decline throughout Europe, was analysed. 2. Observations and experiments focused on: (i) several aspects of mating behaviour, showing the primary role of the female during courtship, the patterns of mature male receptivity and the possible existence of a mating stimulus produced by either a mating pair or a receptive female; (ii) the potential for mate choice either by males (males were not choosy, mating with the first receptive female they met) or by females (that rejected the smallest males and males with only one cheliped) and (iii) intermale fighting before copulation (larger males copulated more often, with the exception of some ’sneakers‘). 3. Sperm competition occurred when a new male was presented with a female after a previous male‘s spermatophore had been deposited; the new male cleaned the rival male‘s spermatophore by feeding on it before copulation. This behaviour has also been observed in some pseudoscorpions, millipedes and collembolans, but always in cases where the spermatophore is deposited on the substratum. Introduction

We analyzed spatial behavior in the white-clawed crayfish, Austropotamobius pallipes, inhabiting a stream in Tuscany (Italy). Our study suggests an overall complex movement pattern , where nomadic movements are intercalated by stationary phases. There was a low rate of recapture within the stretch of stream inspected, suggesting either a tendency of the species to disperse or a mortality rate caused by predators or the loss of tags with molts. However, one part of the population showed a conservative use of space, as indicated by 1) more extensive movement during a 24-h cycle than during the one-year cycle, 2) a weak tendency to return to the "home" pool if released at a distance of 50 m, and 3) an equal distribution of up-and downstream movements. No correlation was found between either 1) the displacement from the point of first capture and the length of t i e the specimen was followed through recaptures or 2) the number of captures and the maximum distance traveled.

A method for the determination of the organic acids directly in the urine employing derivatization with trimethyloxonium tetrafluoroborate as a methylating agent and sequential extraction by head space and direct immersion/solid phase microextraction is reported. Furoic acid, hippuric acid, methylhippuric acid, mandelic acid, phenylglyoxylic acid and trans, trans muconic acid contained in urine and proposed by the American Conference of Governmental Industrial Hygienists as biological exposure indices were determined after a fast and economically convenient preparation step and sensitive gas chromatography-ion trap-mass spectrometry/tandem mass spectrometry analysis. Urine is rather a complex sample and hence the acquisition method required specific GC-MS instrumentation capable of supporting the changeover, fully automated during a single chromatographic separation, from mass to tandem mass spectrometry and both chemical and electron ionization modes. The automation of the analytical method provides a number of advantages, including reduced analysis time for both routine analysis and method development, and greater reproducibility. The equilibrium and kinetics of this substances vs head space/direct immersion-solid phase microextraction were investigated and evaluated theoretically.

Background: Adenosine deaminase (ADA)-severe combined immunodeficiency (SCID) is caused by genetic variants that disrupt the function of ADA. In its early-onset form, it is rapidly fatal to infants. Delayed or late-onset ADA-SCID is characterized by insidious progressive immunodeficiency that leads to permanent organ damage or death. Quantification of T-cell receptor excision circles (TRECs) or tandem mass spectrometry (tandem-MS) analysis of dried blood spots (DBSs) collected at birth can identify newborns with early-onset ADA-SCID and are used in screening programs. However, it is not clear whether these analyses can identify newborns who will have delayed or late-onset ADA-SCID before symptoms appear. Objective: We performed a retrospective study to evaluate whether tandem-MS and quantitative TREC analyses of DBSs could identify newborns who had delayed-onset ADA-SCID later in life. Methods: We tested stored DBSs collected at birth from 3 patients with delayed-onset ADA-SCID using tandem-MS (PCT EP2010/070517) to evaluate levels of adenosine and 29-deoxyadenosine and real-time PCR to quantify TREC levels. We also analyzed DBSs from 3 newborns with early-onset ADA-SCID and 2 healthy newborn carriers of ADA deficiency. Results: The DBSs taken at birth from the 3 patients with delayed-onset ADA-SCID had adenosine levels of 10, 25,

a b s t r a c tErtapenem (Invanz ® ) is a newly developed carbapenem ␤-lactam antimicrobial agent. The drug usage in pediatric age needs an accurate drug monitoring for effective patient management. The aim of this study was to evaluate the use of dried blood spot (DBS) specimens to measure ertapenem concentration during treatment. The analysis was performed by UPLC-MS/MS operating in multiple reaction monitoring (MRM) mode. The calibration curve in matrix was linear in the concentration range of 0.5-100 mg/L with correlation coefficient value higher than 0.997. Performance parameters of this method like lower limit of detection (LLOD, 0.2 mg/L), lower limit of quantification (LLOQ, 0.5 mg/L), matrix effect (20%), intra-and inter-day imprecision (CV within than 15%) and accuracy (between 94 and 155%) of drug concentrations have been evaluated. The drug stability at different temperatures was tested for one month, to evaluate the risks of sample delivery at different climatic conditions.The reported method allows now ertapenem analysis and offers many advantages for patients including the possibility of collecting samples at home.This new assay is both precise and accurate and is especially suitable for therapeutic drug monitoring and pharmacokinetic studies in neonates in whom obtaining larger blood samples is not convenient or possible.

a b s t r a c tLinezolid is a new drug from the oxazolidinone class of antibiotics used against mycobacteria and multidrug resistant (MDR) Gram-positive bacterial infections, which may are also glycopeptide-resistant. The drug usage in pediatric age needs an accurate drug monitoring for effective patient management. The aim of this study was to evaluate the use of dried blood spot (DBS) specimens to determinate linezolid levels during treatment. Advantages of DBS include short collection time, low invasiveness, ease and low cost of sample collection, transport and storage. The analysis was performed in LC-MS/MS operating in positive ion mode and multiple reaction monitoring (MRM) mode. The calibration curve in matrix was linear in the concentration range of 1-100 mg/L with correlation coefficient value of 0.9987. Intraday and interday coefficients of variation were within 3.6% and 13.0%, respectively. We also tested the thermal and temporal drug stability in dried blood spots at four different temperatures to evaluate the risks of sample delivery in different conditions. The short term stability studies showed that linezolid concentration remained stable for at least one month under all the conditions tested.This new assay has favorable characteristics being highly precise and accurate and allows a fast linezolid analysis with a total run time 22 min long, in gradient analysis. Concentration data for plasma and DBS samples from patients after treatment were compared showing a good correlation.Correlation between DBS data and serum samples measured by HPLC-UV was satisfactory. The benefit for patients is the ability to monitor the treatment with a simple and convenient sample collection at home.

Tyrosinemia type I is a genetic disorder characterized by accumulation in the blood and urine of the toxic metabolite succinylacetone (SUAC), not detectable in healthy samples. In many countries, newborns are screened for tyrosinemia type I using tyrosine as a primary marker. Unfortunately, tyrosine accumulation may take longer to occur and it may be not obvious when specimens are collected, in the first few days of life, as for newborn screening. In 2008, we reported changes to simultaneously measure acylcarnitines, amino acids, and SUAC during expanded newborn screening. We established the usefulness of this method after identifying a first asymptomatic newborn affected by tyrosinemia type I. Now we report a second infant with positive SUAC screening result (14.1 mmol/L, n.v.<2) and normal tyrosine concentration (74 mmol/L; n.v.<250). We also performed molecular analysis of FAH gene in both patients after diagnosis at newborn screening. They had consanguineous parents and were both homozygous for two known disease-causing mutations of the FAH gene. The outcome of patients detected in the MS/MS screening is significantly favorable. We also report our results of newborn screening for tyrosinemia type I before and after inclusion of SUAC as a primary marker for this disease.