Using isobaric tags for relative and absolute quantification (iTRAQ) with digital gene expression (DGE) techniques, a nearly complete catalog of expressed protein and mRNA was acquired. From the catalog, we reliably identified 3172 proteins and 9,865,230 tags mapped to genes, and subsets of 141 proteins and 98 mRNAs, which were differentially expressed between the dl2 mutant and wild type. The correlation analysis between the abundance of differentially expressed mRNA and DEPs  revealed numerous discordant changes in mRNA/protein pairs and only a modest correlation was observed, indicative of divergent regulation of transcription and translational processes. The DEPs were analyzed for their involvement in biological processes and metabolic pathways. Up- or down- regulation of some key proteins confirmed that the physiological process of vascular differentiation is an active process. These key proteins included those not previously reported to be associated with vascular differentiation processes, and included proteins that are involved in the spliceosome pathway. Together, our results show that the developmental and physiological process of the leaf vascular system is a thoroughly regulated and complicated process and this work has identified potential targets for genetic modification that could be used to regulate the development of the leaf vasculature.Leaf vascular system differentiation and venation patterns play a key role in transporting nutrients and maintaining the plant shape, which is an important agronomic trait for improving photosynthetic efficiency. However, there is little knowledge about the regulation of leaf vascular specification and development. Here we utilized the rice midribless mutant ( The plant vascular system performs important functions, such as delivery of nutrients to the various plant tissues, supply of mechanical support, as well as acting as an effective long-distance communication system relating to abiotic and biotic conditions above and below ground  analysis is a new and powerful tool that permits deep coverage of transcriptome profiles and enables the examination of expression changes with a high dynamic range Bentley, . This apdl2 mutant. In an attempt to have a global view of gene activity in the process of leaf vascular differentiation in rice, and probe candidate genes involved in plant vascular differentiation, we examined the abundance of transcripts and proteins by DGE and iTRAQ technology, respectively, and correlated the abundance difference between mRNAs and proteins. The result indicated that the transcriptomic and proteomic expression profiles were poorly correlated, and there is a modest correlation between transcript and protein levels. In addition, according to their functional annotation, we found some differentially expressed proteins (DEPs) which might be involved in leaf vascular patterning and may help to elucidate the drooping leaf phenotype in dl2 mutants. Our analysis also revealed different control mechanisms that dictate gene activity, uncovering several novel aspects in the metabolism of leaf vascular differentiation in rice. This study provides new clues to address the relationship between the abundance of mRNAs and proteins in plant vascular patterning.This study investigates genome-wide expression of the proteome and transcriptome of plant leaf vascular differentiation in the rice midribless dl2 mutant (MT) of rice  and at 30°C. Humidity was controlled at approximately 60%. Both MT and WT were of the same age and grown under the same conditions. The leaves were sampled for analysis from two-month-old rice plants when the leaves were fully expanded and the MT plants displayed a drooping leaf phenotype. Two biological replicates for each experiment were performed for transcriptome and proteome analysis, and each with pooled leaves from 10 independent plants per replicate. In addition, the leaf samples were flash-frozen in liquid nitrogen and preserved at −80°C for subsequent quantitative real-time PCR (qPCR) analysis.The Protein extraction was performed based on a previous report . The trypsin digestion protocol has been previously described in detail , and followed by 11-min gradient with 5–35% buffer B , and then by ramping up to 80% solvent B for 1 min. The absorbance at 214 nm was monitored and a total of 20 fractions were collected, lyophilized and desalted using the Strata X column . Eluents were lyophilized and stored at −80°C before LC-ESI-MS/MS analysis.SCX chromatography was performed on a high performance liquid chromatography system  using a 250 × 4.6 mm (5-μm particle size) ultremex SCX column , based on the following study  and loaded by an autosampler onto a 2 cm C18 trap column. Then, peptides were eluted onto a 10 cm analytical C18 column (inner diameter 75 μm) packed in-house. The samples were loaded at 8 μl/min for 4 min, then the 35 min gradient was run at 300 nL/min starting from 2 to 35% B , followed by 5 min linear gradient to 60%, then, followed by 2 min linear gradient to 80%, and maintenance at 80% B for 4 min, and finally return to 5% in 1 min.Data acquisition was performed with a TripleTOF 5600 System  fitted with a Nanospray III source  and a pulled quartz tip as the emitter , based on a recent report .Raw data files obtained from the TripleTOF 5600 System  were transformed into MGF files using Proteome Discoverer 1.2 (Thermo Fisher Scientific), and the MGF file was searched. Protein identification was performed by using Mascot search engine (version 2.3.02) against a database containing 66,338 rice protein sequences , Oxidation (M), Deamidated (NQ) as the potential variable modifications, and Carbamidomethyl (C), iTRAQ 8 plex (N-term), iTRAQ 8 plex (K) as fixed modifications. The charge states of peptides were set to +2 and +5. Specifically, an automatic decoy database search was carried out in Mascot by choosing the decoy checkbox. To reduce the probability of false peptide characterization, only peptides with scores (≥20) at 99% or greater confidence by Mascot probability analysis were considered as greater than “identity” and counted as identified, and each confident protein identification involved at least one unique peptide. For protein quantification, it was required that a protein contain at least two unique peptides. The quantitative protein ratios were evaluated and normalized by the median ratio in Mascot. We only used ratios with Total RNA was obtained with the RNeasy Plant Mini Kit , and contaminating DNA was digested with the DNA-free Kit , according to the kit instructions. RNA quantity was measured in a NanoDrop ND-1000 spectrophotometer . RNA quality was evaluated with an Agilent Bioanalyzer Model 2100 . mRNA was enriched by using oligo (dT) magnetic beads.DGE library construction and sequencing were carried out at Huada Genomics Co., Ltd., . Briefly, the mRNA was broken into short fragments (about 200 bp) by adding the fragmentation buffer, and then the cDNA was synthesized and single nucleotide A (adenine) was added, which is used for ligation of sequencing adaptors. Finally, the fragments were enriched by PCR amplification, and the enriched library products were used for sequencing analysis via Illumina HiSeq™ 2000.http://rice.plantbiology.msu.edu/). The number of explicit clean tags for each gene was computed and then normalized to TPM  was substituted with the Q-value to determine the threshold of P-value in multiple tests and analysis   analysis was employed to verify the DGE results using the thermocycler CFX1000  with SYBR Premix Ex Taq TMII  kit (Takara). Primers for specific genes were designed using Primer Premier 5.0. qPCR assays were performed in triplicate  with two independent biological replicates, with β- Pfaffl, .http://www.ricedata.cn/gene/). Results were tested for the significance of protein identification and protein abundance variation using the Mascot search engine . A total of 8835 unique peptides and 3172 proteins from the two experiments were characterized with higher than 99% confidence as shown in supplementary data  for measuring DEPs between MT and WT samples. In addition, only proteins which were identified by two or more peptides and had ≥1.6-fold change of the ratios (tags 117/116 and 121/119) were considered to be DEPs.In an attempt to determine DEPs between WT and MT leaf samples, the repeatability of two biological replicates of WT (tags 116 and 117) and MT (tags 121 and 119) was evaluated first  after the comparison (Table http://www.blast2go.org/), respectively. A protein might be associated with or located in one or more cellular components, suggesting that it performs functions in one or more biological process. Among these, following the functional classification with the three unrelated GO ontologies, 82 (58%) are involved in biological processes, 82 (58%) have molecular functions, and 106 (75%) are cellular components  and involved in cell cycle inhibition, apoptosis as a transcription factor  in the comparison, according to the more stringent criteria   showed a significant difference (8) Table . To vali8) Table . The abs8) Table . The tra8) Table . The remQ-value)  . Surprisingly, only two genes overlapped according to the more stringent criteria Figure . We thene) Table  and a pon Figure . When DEn Figure  a correl1 Figure  was obta1 Figure . To reve1 Figure  and mRNAe) Table , and a cd Figure . Similar4 Figure . Generalr = 0.081). This is expected because mRNA levels are not always consistent with protein levels due to post-transcriptional, translational and/or post-translational regulation . mRNA processing, known as pre-mRNA splicing, is performed precisely and efficiently by the spliceosome. The spliceosome assembly and pre-mRNA splicing process requires a large set of proteins and the formation of protein–protein, protein–RNA, and RNA–RNA complexes  showed an 8-fold increase in accumulation levels Table , and itsWT Table . The AraWT Table . We alsoWT Table . ThioredWT Table , and it WT Table . In our To date, differential transcriptomic and proteomic experiments have provided an overview of the accumulation patterns of mRNAs and proteins , and the DEPs may be considered as candidates involved in the physiological process of leaf vascular pattern, but it requires further study.We have previously reported on the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Campylobacter jejuni is known to form biofilms in vitro in food chain-relevant conditions, but the exact roles and composition of the extracellular matrix are still not clear. Extracellular DNA has been found in many bacterial biofilms and can be a major component of the extracellular matrix. Here we show that extracellular DNA is also an important component of the C. jejuni biofilm when attached to stainless steel surfaces, in aerobic conditions and on conditioned surfaces. Degradation of extracellular DNA by exogenous addition of DNase I led to rapid biofilm removal, without loss of C. jejuni viability. Following treatment of a surface with DNase I, C. jejuni was unable to re-establish a biofilm population within 48 h. Similar results were obtained by digesting extracellular DNA with restriction enzymes, suggesting the need for high molecular weight DNA. Addition of C. jejuni genomic DNA containing an antibiotic resistance marker resulted in transfer of the antibiotic resistance marker to susceptible cells in the biofilm, presumably by natural transformation. Taken together, this suggest that eDNA is not only an important component of C. jejuni biofilms and subsequent food chain survival of C. jejuni, but may also contribute to the spread of antimicrobial resistance in C. jejuni. The degradation of extracellular DNA with enzymes such as DNase I is a rapid method to remove C. jejuni biofilms, and is likely to potentiate the activity of antimicrobial treatments and thus synergistically aid disinfection treatments.Biofilms make an important contribution to survival and transmission of bacterial pathogens in the food chain. The human pathogen  Campylobacter jejuni is the most common cause of bacterial foodborne infection within the UK  state  biofilms   at 37°C. For growth on plates, strains were either grown on Brucella agar or BAB with Skirrow antibiotic supplement . Broth culture was carried out in Brucella broth (Becton & Dickinson).2, 10% CO2 and 85% N2). Cells from the Skirrow plate were used to inoculate Brucella broth then grown overnight as a shaking culture . Following overnight growth, cell cultures were adjusted to an A600 of 0.05 in Brucella medium or Brucella medium supplemented with 5% v/v chicken juice. To allow biofilm formation, 1 ml of this solution was added to either a sterile borosilicate glass test tube (Corning) or 3 ml to a six-well polystyrene tissue culture plate (Corning) containing a sterile stainless steel coupon . In each biofilm assay a test tube containing sterile Brucella medium was incubated alongside the C. jejuni containing tubes to ensure sterility was maintained and, following crystal violet staining, to quantification of staining levels where biofilm was not present. Tubes were incubated at 37°C in atmospheric air conditions using an Innova 4230 (New Brunswick Scientific) incubator at 37°C. Unless otherwise stated all biofilms were formed in aerobic conditions at 37°C for 48 h before staining procedures were carried out. For each assay a microaerobic biofilm control was also undertaken, to ensure that oxygen availability does not have a major effect on results and to allow comparison with previous studies , or DNase I (Fermentas), or RNase (QIAGEN) were added to test tubes containing diluted C. jejuni suspension prior to static incubation and then incubated at 37°C for 48 h in aerobic conditions. Equal volumes (4 μl) of the buffers and bovine serum albumin were also added if recommended by the manufacturers. For the assays assessing the time required for DNase I activity, biofilms were allowed to form for 48 h before addition of 4 U/ml v/v DNase I enzyme  and 4 μl of DNase I buffer to the samples, followed by incubation for up to 2 h. During the incubation with enzyme the samples were placed in 37°C, aerobic conditions. All samples were subsequently stained with crystal violet.For DNase I treatments, unless otherwise stated, a volume of 4 μl DNase I enzyme (Fermentas), giving a final concentration within the biofilm of 4 U/ml v/v and 4 μl of DNase I buffer (Fermentas) were added to each test tube, along with 1 ml of diluted cell suspension at either the start of the static incubation or after 12, 24, 36, or 48 h of static incubation. Following treatment, static cultures were placed back in 37°C, aerobic conditions to complete the 48 h incubation before staining with crystal violet to allow biofilm quantification. For restriction enzyme digest of biofilms 4 μl of 10 U/μl 600 of 0.05, or sterile Brucella medium, followed by a further 48 h incubation at 37°C in aerobic conditions. All samples were subsequently stained with crystal violet. In order to ensure consistency between control and treatment samples all tubes were manipulated in exactly the same way, being removed and placed back in the same incubation conditions during each enzyme addition. Heat inactivated DNase I was prepared by incubating an aliquot of DNase I and its buffer at 95°C for 10 min and allowing to cool before addition to the biofilm cultures.For assessment of biofilm regrowth, biofilms were allowed to establish for 48 h followed by a 15 min incubation with DNase I. Tubes were then washed twice with sterile PBS followed by addition of either an equal volume of bacterial culture with an A600 of 0.3. Aliquots were mixed with gel loading buffer (NEB) and added to a 0.9% agarose gel and run at 100 V for 45 min in 0.5% TBE buffer. A 1 kb ladder (NEB) was used for size comparison. Following electrophoresis, nucleic acids were stained using ethidium bromide, and DNA was visualized using a GelVue UV light and documented using a U:Genius gel documentation system (Syngene). The amount of eDNA in planktonic and biofilm fractions was quantified by comparing the intensity of the DNA bands after UV illumination and comparison with the 3 kb marker fragment (125 ng), using ImageJ . Quantification was based on three independent experiments.Following incubation to allow biofilm formation in both aerobic and microaerobic conditions, the supernatant was removed and the tubes were rinsed once with sterile PBS to remove loosely attached bacterial populations. After rinsing and removal of the rinse suspension a second 1 ml volume of sterile PBS was added to each test tube and a sterile cotton wool swab was used to gently swab to walls of the test tube, releasing the biofilm from the walls of the test tube and in to suspension. The resuspended biofilm (PBS containing the loosened biofilm cells) and supernatant  from several biofilm cultures were collected and pooled before diluting to a ABamHI, BlpI, HaeIII, HindIII, MscI, or RsaI, all supplied by NEB), or DNase I , or RNase (QIAGEN) was added to a mixture containing ~500 ng of C. jejuni NCTC 11168 or 81116 genomic DNA, prepared using a commercial kit (QIAGEN) following manufactures guidelines, 1 μl of 10× enzyme buffer (if required), 1 μl of 1 mg/ml BSA (if required) and molecular grade water to a final volume of 10 μl. Samples were incubated for 60 min in a 37°C water bath to allow digestion of the genomic DNA. DNA was visualized using a GelVue UV light and documented using a U:Genius gel documentation system (Syngene).A 1 μl volume of restriction enzyme .500 of the solution measured.This method was carried out as previously described  Brucella agar containing 10 μl/ml chloramphenicol. After 2 days of growth at 37°C in microaerobic conditions, the dilution resulting in two or more colonies was recorded. Cell viability in biofilm assays was assessed upon initial addition of cultures into static culture and following static incubation, prior to crystal violet staining and, where necessary, following the 72 h TTC incubation.Statistical analysis was carried out using GraphPad Prism software. At least three biological replicates  were used to calculate mean and standard deviation. Significance was measured using either a Mann–Whitney test (biofilm formation) or ANOVA (DNA yield).C. jejuni NCTC 11168 and 81116 were generated and used for the investigation of eDNA. Separation of nucleic acids on agarose gels showed the presence of extracellular DNA in both the biofilm and planktonic fractions, independent of whether the biofilm samples were incubated in aerobic or microaerobic conditions . Where genomic DNA had been added to the suspension at the start of static incubation, resistant cells were present in both planktonic  is now frequently used in the treatment of cystic fibrosis (Konstan and Ratjen, P. aeruginosa biofilms has shown that eDNA can not only provide structural stability at early stages of biofilm formation (Whitchurch et al., P. aeruginosa biofilm organization and expansion (Gloag et al., E. coli, Shewanella, and P. aeruginosa when exposed to phosphate and carbon deficient environments (Palchevskiy and Finkel, C. jejuni, the eDNA can contribute to the spread of genetic traits within populations, both in the biofilm and in the planktonic populations. Genetic material can be transferred within the biofilm either by direct cell to cell transmission or uptake of exogenous DNA. Conjugation within biofilms is a well reported phenomenon, with examples reported in mixed species oral biofilm models (Hannan et al., C. jejuni strains NCTC 11168 and 81–176 in microaerobic cultures are able to transfer genetic material between bacterial cells both within biofilms and planktonic suspension (Bae et al., C. jejuni is also able to utilize exogenously added DNA for acquisition of genetic traits. This transfer is also able to occur in aerobic conditions, more closely resembling the conditions C. jejuni encounters while in the food chain.The eDNA within the extracellular matrix appears to have multiple functions, depending on the bacterial species investigated. Previous research in C. jejuni extracellular matrix at all stages of maturation. This is in contrast to P. aeruginosa, which become less susceptible to DNase I treatment as the biofilm matures (Whitchurch et al., C. jejuni biofilm formation, but to date there has been little investigation of the extracellular matrix components themselves. C. jejuni produces a polysaccharide containing β1-3 and/or β1-4 linkages which is reactive to calcofluor white (McLennan et al., C. jejuni biofilms.We demonstrate here that eDNA is an important component of the Neisseria gonorrhoeae (Hamilton et al., P. aeruginosa (Renelli et al., Staphylococcus aureus eDNA can be released via co-ordinated lysis of a subset of the population, controlled by quorum sensing (Mann et al., C. jejuni (He et al., C. jejuni, this will require further investigation. P. aeruginosa biofilms showed higher concentrations of eDNA within the biofilm when cultures were supplemented with salmon sperm DNA (Chiang et al., C. jejuni NCTC 11168 and 81116 are able to utilize exogenous DNA, this does not lead to a net increase in biofilm formation. In contrast, addition of eDNA to C. jejuni 81–176 biofilm cultures led to increased biofilm biomass (Svensson et al., Although eDNA has been shown to be present within the biofilms of many different bacteria, the mechanism of its release into the extracellular milieu is still under investigation. There are two main mechanisms of DNA release; secretion and cell lysis. Secretion of eDNA has been shown in several species, including C. jejuni (Brown et al., L. monocytogenes to stainless steel surfaces is enhanced by surface pre-conditioning with fish and meat emulsions (Gram et al., C. jejuni biofilm formation (Brown et al., C. jejuni biofilms formed on surfaces conditioned with chicken juice, suggesting that DNase I treatment could provide a useful addition to current treatment regimens.Another problem frequently encountered within food processing environments is the presence of food product debris. This presence of this debris on surfaces can lead to surface conditioning and increased bacterial attachment, as observed with chicken juice and It should be noted that we found DNase I treatment had no effect on cell viability, only biofilm shedding. This is as expected since DNase I is only in contact with the DNA of the extracellular matrix, reducing the structural integrity of the colonies forming the biofilm, but is not able to cause a loss of viability in bacterial cells with intact membranes. This means that although the DNase I treatment provides a rapid and effective method of biofilm dispersal it would best be used in combination with antimicrobial treatments, ensuring effective biofilm degradation and bacterial inactivation.C. jejuni biofilm and its degradation results in a reduction of biofilm levels below detection levels (Tresse et al., C. jejuni biofilms with DNase I also prevents re-establishment of biofilms, possibly allowing more efficient antimicrobial treatment. DNase I treatment is effective on food chain relevant surfaces and hence could provide a useful addition to current food chain cleaning regimes.In conclusion, eDNA is an essential component of the HB, MR, RB, and AV designed the study. HB, KH, and MR performed the experimental work and analyzed the data. HB prepared the manuscript, and KH, MR, RB, and AV contributed to the final manuscript.Roy P. Betts is a full time employee of Campden BRI, which provided additional funding for the PhD-studentship of Helen L. Brown. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Medical staff informed NMDOH that the child and family used kajal, a traditional eye cosmetic brought from Afghanistan, their country of origin. Further investigation revealed that patient 1’s brother, aged 4 months (patient 2), also had an elevated BLL of 33.5 μg/dL. Laboratory analysis of kajal used by the family showed a lead content of 54%. These two cases highlight the potential for lead poisoning among refugee populations in the United States and call attention to contaminated consumer products as a source of lead exposure. Physicians who provide health services to refugee and immigrant children should be aware of this potential exposure. Health-care providers who routinely screen refugee and immigrant children for elevated BLLs should consider asking questions about the use of traditional eye cosmetics.Lead is a toxic metal that damages blood cells, the kidneys, the cardiovascular system, and the developing nervous system. The risk for lead exposure causing subsequent cognitive and neurobehavioral deficits is especially high among toddlers because of their hand-to-mouth activities and their higher absorption of ingested lead compared with adults . In Januμg/dL. Two weeks later, confirmatory venous blood lead testing of patients 1 and 2 showed BLLs of 18.9 μg/dL and 33.5 μg/dL, respectively; both results exceeded CDC’s current reference value of 5.0 μg/dL.μg/dL. All three children were asymptomatic, as reported by their physicians. Communication with the family indicated that the cultural practice of applying kajal to the children’s eyelids was intended to promote eye health. Other traditional eye cosmetics  are widely used in Asia, Africa, and the Middle East and have been implicated as sources of lead poisoning  was suspected as a secondary source. Because of the mother’s simultaneous use of kajal during breastfeeding, the children’s physician recommended a blood lead test for her. Venous lead testing of the children’s mother showed a BLL of 6 The kajal and the curry powder were collected and analyzed for lead content using Environmental Protection Agency method 200.8.μg/dL, from 18.9 μg/dL. Patient 2’s venous BLL dropped to 28.2 μg/dL, from 33.5 μg/dL. Four months after discontinuing use of kajal, patient 2’s venous BLL further declined to 22.1 μg/dL and patient 1’s venous BLL declined to 14.7 μg/dL. Monitoring of the children’s BLLs will continue until their BLLs are <10.0 μg/dL for 3 months, in accordance with current state guidelines.§Patients 1 and 2 were found to be healthy, with no apparent developmental delays or medical problems. In accordance with CDC guidelines, follow-up testing was performed on both patients approximately 1 month after the initial confirmatory testing. Patient 1’s venous BLL dropped to 17.7 These cases highlight the risk for pediatric lead exposure among refugee populations and draw attention to the potential exposures to lead-contaminated imported products. Historically, most U.S. childhood lead poisoning cases have been associated with ingestion of chips and dust from lead-based paint sources ,6. Lead Kajal and similar traditional eye cosmetic preparations have been found to contain lead concentrations as high as 70% and have been documented as sources of childhood lead poisoning for >30 years ,3. The cRefugee children have been found to have a higher average BLL at their time of arrival in the United States, as compared with average BLLs measured in U.S. children . EducatiWhat is already known on this topic?Lead poisoning continues to be an important, preventable health problem. The common source of lead exposure in the United States is deteriorating lead-based paint and dust; however, some traditional remedies and cosmetics also contain lead.What is added by this report?μg/dL, respectively. Investigation implicated kajal, a cosmetic imported from Afghanistan, that was applied as a folk remedy to the children’s eyelids. The kajal was found to contain 54% lead.Two male children in New Mexico, aged 20 and 4 months, were found to have elevated blood lead levels of 27.0 and 33.5 What are the implications for public health practice?Health-care providers who provide health services to refugee and immigrant children, even in small communities, should be aware of the unique lead exposure risk factors among this population. Expanding lead screening to all infants and children, and pregnant women, might avoid unintentionally excluding cases. Clinicians and other health-care workers might reduce the risk for lead exposures by discussing lead exposure hazards with their patients, especially with women receiving prenatal care or during early childhood screening programs.Pediatricians and other health-care professionals should incorporate lead screening as part of routine medical evaluation of refugee and immigrant children, specifically, those who are relocating from countries with a documented history of increased lead exposure risks associated with cultural practices or unregulated industrial processes. Health communication addressing potential lead hazards should be provided in a culturally sensitive manner. Clinicians and other health-care workers providing services to refugees and immigrants from Africa, Asia, and the Middle East should be aware of potential sources of lead in these populations and ask about the use of traditional eye cosmetics, especially with women receiving prenatal care or during early childhood screening programs, and questions about the use of folk remedies should be included in lead-exposure risk assessment questionnaires. Additionally, public health education campaigns concerning lead exposure risks and geared to refugee and immigrant populations might increase awareness of lead content in traditional eye cosmetics.


Enzyme replacement therapy (ERT) with laronidase,  is the primary treatment option for patients with attenuated mucopolysaccharidosis type I (MPS I). This study examined the effect of early ERT on clinical manifestations.This multinational, retrospective case series abstracted data from records of 20 patients with Hurler-Scheie syndrome within nine sibships that included older siblings treated with laronidase after the development of significant clinical symptoms, and younger siblings treated before significant symptomatology. Median age at diagnosis was 5.6 and 0.5 years for older and younger siblings, respectively. Median age at ERT initiation was 7.9 and 1.9 years for older and younger siblings, respectively.Improvement or stabilization of somatic signs and symptoms was more notable in younger siblings. Organomegaly present at onset of ERT improved in the majority of both older and younger siblings. Analysis of physician-rated symptom severity demonstrated that cardiac, musculoskeletal, and cognitive symptoms, when absent or mild in younger siblings at ERT initiation, generally did not develop or progress. The majority of older siblings had height/length Z-scores greater than two standard deviations below the mean (less than -2) at both time points. In general, Z-scores for younger siblings were closer to the sex- and age-matched means at follow-up.These findings suggest early initiation of laronidase, prior to the onset of symptoms in patients with attenuated MPS I, can slow or prevent the development of severe clinical manifestations. Mucopolysaccharidosis type I (MPS I) is an autosomal recessive disorder caused by deficient α-L-iduronidase activity, a lysosomal enzyme involved in degradation of the glycosaminoglycans (GAGs) heparan and dermatan sulfate. Enzyme deficiency leads to accumulation of GAGs in multiple tissues resulting in a progressive and multisystem disease. MPS I includes a spectrum of clinical presentations ranging from severe (Hurler syndrome) to attenuated  . PatientTreatment options include hematopoietic stem cell transplant (HSCT) recommended before 2.5 years of age for patients with the most severe phenotype, and enzyme replacement therapy (ERT) with laronidase  indicated as the primary treatment option for patients with attenuated MPS I –6. The lDue to the large phenotypic heterogeneity seen in MPS I, correlating ERT timing to the effect on the clinical course and progression of symptoms can be challenging. However, it is evident from assessing treatment outcomes in siblings with similar genetic backgrounds and expected rates of disease progression that pre-symptomatic initiation of ERT drastically alters the clinical course of MPS I , 10. In Here, we present the largest compilation of sibling data to date for patients with the H/S phenotype. This case series examines data from sibling pairs/sibships that included older siblings treated with laronidase after the development of significant clinical symptoms, and younger siblings, the majority of whom initiated laronidase before significant symptomatology.This multinational, retrospective, chart review case series was conducted at the authors’ institutions. All patients with MPS I diagnosed by enzyme biochemical determination and/or molecular analysis were treated with laronidase according to prescribing information (weekly intravenous infusions of 100 U/kg of body weight). Sibships were identified where younger siblings initiated ERT at an earlier age than older siblings. In general, older siblings initiated treatment with laronidase after the onset of MPS I symptoms while younger siblings initiated ERT before onset of significant physical and clinical symptoms. Written informed consent was provided by the legal guardians/parents of all patients.Treating physicians completed questionnaires assessing signs and symptoms of MPS I prior to and following ERT. Data from patient records, medical histories and neurological, cardiovascular, pulmonary, gastroenterology, musculoskeletal, ophthalmologic, audiologic, and psychomotor developmental examinations were abstracted including:Height/length (absolute); Description of facial features observed during physical examination; Presence of corneal opacity, hearing loss/use of hearing aids, sleep apnea, hernia; Results of pulmonary function tests (PFT); Liver and spleen size determined by palpation and/or imaging studies; Cardiac disease description/severity determined from echocardiogram; Joint and skeletal disease observed during physical examination and/or radiographic imaging; Presence/severity of dysostosis multiplex, joint contractures and affected joints; Motor development assessed during physical examination; Cognitive and language development assessed by formal evaluation or by investigator impression; and Ability to perform activities of daily living (ADL).Data were abstracted at a time point just prior to initiating ERT  and follow-up data were collected after at least 3 years of ERT .The frequency of signs and symptoms at T1 was determined from a maximum value of 14 symptoms that included coarse facial features, corneal clouding, hearing loss, sleep apnea, abnormal PFT, cardiac abnormalities, hepatomegaly, splenomegaly, hernia, dysostosis multiplex, joint contractures, motor developmental delay, language/cognitive delay, and restrictions in ADL. Symptom severity was rated by physicians for corneal opacity, cardiac symptoms, dysostosis multiplex, and joint contractures. Z-scores for height/length were calculated according to National Health and Nutrition Examination Survey and Center for Disease Control Growth Chart Information as revised on June 8, 2000.n = 10) and 0.5 years  for younger siblings (n = 10). All older siblings initiated laronidase by 14 years of age, while younger siblings initiated therapy before 4 years of age (with one exception). Five younger siblings (sibships 1–5a) were diagnosed at less than 1 year of age and initiated treatment by 1.3 years of age. For analysis of responses to treatment, these patients are presented together as Group A. A second group of younger siblings  began laronidase treatment after age 2. Age at diagnosis, treatment, and duration of treatment for each patient, and median data by group are presented in Table Twenty patients within nine sibships (seven sibling pairs and two sets of three siblings) were identified from nine centers in Argentina (two sites), Brazil, France, Spain, Saudi Arabia, UK, and the US (two sites). Median age (range) at diagnosis was 5.6 years 1, 9) for older siblings . In addition to symptoms noted in Fig. The younger sibling was 5 months of age at T1 and already had cardiac symptoms ranging from mild to severe Fig. , and milThe older sibling had significant limitations in ADL, moderate musculoskeletal disease, and moderate to severe cognitive delay and delays in fine and gross motor development when ERT was initiated at 12 years of age. All existing symptoms persisted or progressed in this sibling except for improvement in organomegaly. The younger sibling began ERT at 8 months of age and after 8 years had no restrictions in ADL, no cognitive delays, and only mild musculoskeletal symptoms and mild to moderate ocular disease. Cardiac symptoms remained stable.Siblings had normal fine and gross motor development and were able to walk unassisted prior to the start of ERT. The older sibling also had no cardiac symptoms at T1. Cardiac disease was mild in both siblings at T2; other symptoms stabilized, except for progression of respiratory symptoms. Cognitive delays persisted in the older sibling, and developed in the younger. Both siblings had poor compliance (~60 %) with ERT, which may have impacted the symptom progression.Two siblings initiated ERT before 5 years of age, one prior to the development of any symptoms, and one after mild cardiac symptoms, ocular disease, and organomegaly, and moderate delays in motor skills were apparent. Similar symptoms  were present in the oldest sibling who began ERT at 5.8 years of age. Additionally, this sibling presented with mild joint contractures at this time. At T2, there were improvements in organomegaly and motor function. Pulmonary function was at 30–40 % of normal at follow-up (no information available at T1). Ocular involvement and lower extremity joint symptoms progressed, but cardiac disease remained stable. In the sibling who initiated ERT at 2.5 years, organomegaly and motor function delays improved, cardiac symptoms remained stable, and mild joint contractures developed. The sibling who began ERT at <1 year of age remained healthy, with the exception of mild ocular and cardiac symptoms. Psychomotor development remained normal and there were no limitations in ADL. In both younger siblings, pulmonary function was between 70 and 94 % of normal at T2. In general, the severity of symptoms in the youngest sibling remained milder than those of the other siblings.Two siblings initiated ERT at 11 and 13 years, respectively, and the youngest began ERT at 3 years of age. All three siblings had mild cardiac and musculoskeletal symptoms at T1. Cardiac symptoms remained mild at T2, and joint contractures progressed in all three siblings and were severe at T2. However, dysostosis multiplex progressed only in the older siblings to moderate to severe, and remained mild in the younger sibling. Pulmonary function was 45–50 % of normal in the two older siblings at T2, and 70–80 % of normal in the younger sibling. None of the siblings had restrictions in ADL.Both siblings began ERT prior to 5 years of age and had significant somatic, motor, and cognitive impairments prior to initiating therapy. After 5 years of ERT, cardiac symptoms stabilized, and musculoskeletal symptoms and motor development improved in the older sibling. Cardiac symptoms progressed in the younger sibling but remained mild. Corneal clouding progressed in both siblings. In the younger sibling who began both intensive physiotherapy and speech therapy at the age of 5, motor development remained normal, and cognitive skills and musculoskeletal disease improved.Both siblings had progression of cardiac symptoms at T2. Dysostosis multiplex stabilized in both siblings, and remained milder in the younger sibling. The younger sibling’s joint contractures progressed into the moderate range. While the older sibling had behavioral issues and required an aide for 1-on-1 schooling, the younger sibling had no such issues.Both siblings began ERT at a late age , and had similar symptom profiles at T1, with ocular, respiratory, cardiac, and musculoskeletal involvement. At T2, ocular and cardiac symptoms progressed from mild to moderate/severe in the older sibling. In addition, pulmonary function worsened, and contractures and lower extremity weakness progressed. In contrast, the younger sibling had stabilization of mild ocular symptoms, and cardiac disease remained less severe in the mild to moderate range. Both patients had stabilization of dysostosis multiplex and no progression of motor function delays.All older siblings except those from sibships 2 and 8 had Z-scores greater than two standard deviations below the mean (less than −2). At T2, Z-scores deviated further from the mean in the majority of the older siblings. Although remaining two standard deviations below the mean, in sibship 4, the Z-score improved from −3 to −2 and in sibship 9 improved from −6 to −5.6. In general, Z-scores for younger siblings at T2 were closer to sex- and age-matched means. For 6/9 of the younger siblings, Z-scores ranged from +0.88 to −1.4 at T2, and represented either improvements in height/length or no significant change from T1. For the younger siblings in sibships 6 and 9, the Z-scores at T2 were more negative than at T1 with height/length 2.5 or more standard deviations from the mean. Importantly, in all sibships (with the exception of sibship 6) the heights of the younger siblings at T2 were greater than their older siblings at comparable ages. Height/length Z-scores for all sibships at T1 and T2 are shown in Fig. The data collected from this multinational case series demonstrate the benefit of early ERT initiation in patients with attenuated MPS I. Laronidase is believed to stabilize but not improve cardiac and skeletal/connective tissue disease when pathological changes are already present at the start of treatment, and early ERT may help prevent the onset of disease features . HoweverGrouping sibships based on the age of ERT initiation in the younger sibling provided additional insight into the benefits of early treatment and the development of clinical features. In Group A (younger sibling initiated treatment ≤1.3 years of age), mild to severe symptoms were noted before treatment initiation in older siblings while symptoms were absent to mild in younger siblings. Most striking is the continued absence of clinical symptoms in the younger sibling from sibship 1 after nearly 5 years of ERT contrasted to the stabilization, but not reversal, of symptoms in the older sibling. Prominent differences were seen within other sibships in Group A for corneal opacity, organomegaly, dysostosis multiplex, and joint contractures. The extent of cardiac involvement was variable amongst siblings; however, improvement or stabilization of symptoms was noted following ERT. Younger siblings in Group A also tended to have fewer delays in motor and cognitive function, and less restriction in ADL.For Group B , symptoms present in older siblings at T1 ranged from mild to severe. In younger siblings, symptoms generally ranged from absent to moderate, largely depending on the age treatment was initiated. At T2, symptoms noted in younger siblings at the time of ERT initiation either improved or stabilized, and only one developed symptoms classified as severe . Clinical differences were noted between older and younger siblings in Group B for organomegaly and dysostosis multiplex. Following treatment initiation, older siblings experienced improvements  or stabilization while younger siblings in this group experienced slower and less severe disease progression.Short stature is a key clinical feature of MPS I. The rate of growth in children with MPS I is slower than in the healthy population, and differences in height between healthy and affected children increase with age . Some stAs a retrospective study, our analyses were limited by lack of control over the primary data collection, missing data, and issues with objective data analysis. While we recognize that the assessment of symptoms severity by physicians was subjective and could be influenced by sociocultural differences in medical practice, it is important to note that the patients were followed for most of their lives by the same physicians, who were able to compare symptoms in the siblings for a number of years and through various developmental stages. We believe this factor can limit the bias in assessing severity between siblings. Given the difficulties in conducting randomized controlled trials in the setting of a rare disease, and the geographical dispersion of patients with MPS I, we believe that comparing data between the siblings rather than across patients is an effective retrospective design. While comparing siblings is an insightful model to better understand the benefits of early treatment initiation in MPS I due to similar genetic backgrounds, there are a number of factors to consider. Phenotypic variability resulting from the effects of modifying polymorphisms is known to occur with some mutations , includiCompliance with ERT is important in order to achieve maximal clinical effect . In our These findings support early initiation of ERT with laronidase prior to the onset of symptoms in patients with attenuated MPS I in order to delay disease progression and/or prevent the development of severe clinical manifestations.


Distinguishing acute Q fever from respiratory infections, fever, or hepatitis requires serologic testing or PCR. Differentiating acute Q fever from infections caused by other pathogens is essential. We conducted a retrospective case–control study to evaluate differences in clinical signs, symptoms, and outcomes for 82 patients with acute Q fever and 52 control patients who had pneumonia, fever and lower respiratory tract symptoms, or fever and hepatitis, but had negative serologic results for Q fever. Patients with acute Q fever were younger and had higher C-reactive protein levels but lower leukocyte counts. However, a large overlap was found. In patients with an indication for prophylaxis, chronic Q fever did not develop after patients received prophylaxis but did develop in 50% of patients who did not receive prophylaxis. Differentiating acute Q fever from other respiratory infections, fever, or hepatitis is not possible without serologic testing or PCR. If risk factors for chronic Q fever are present, prophylactic treatment is advised. Coxiella burnetii. During 2007–2010, the southern part of the Netherlands had the largest outbreak of Q fever ever reported  IgM ELISA , which detects IgM against phase II antigens and has a cutoff index of 1.1; a complement fixation assay (CFA) , which detects C. burnetii phase II antigens and shows a positive result if the titer is >1:10; and a Q fever immunofluorescent assay (IFA) for IgG and IgM , which detects IgM and IgG against phase I and phase II antigens and shows a positive result if the titer is >1:16.Serologic analysis was performed for blood samples by using the <3 weeks before presentation; and 1) a positive serum PCR result <21 days of onset of disease; or 2) a negative serum PCR result, but a positive ELISA result for IgM against phase II antigens of C. burnetii and a positive CFA result for immunoglobulins against C. burnetii; or 3) a negative serum PCR result but a positive ELISA result and a positive IFA result for IgM and IgG against phase I and phase II antigens of C. burnetii; or 4) two serum samples tested by CFA or IFA during an interval of >2 weeks that showed seroconversion or a 4-fold increase in titer. On the basis of the algorithm published by the Dutch working group on diagnostics of acute Q fever , moxifloxacin (400 mg 1×/d), or ciprofloxacin (500 mg 2×/d) for 2 test was used. To evaluate the effect of prophylactic treatment, the Barnard exact test was used because this test is more powerful than the Fisher exact test for instances of smaller sample sizes . For analysis of qualitative data, the Pearson’s χA total of 82 patients with acute Q fever who fulfilled inclusion criteria for the study group and 52 patients who fulfilled criteria for the control group were included in the study . PatientOf patients with acute Q fever, 18% had shortness of breath  compared9 cells/L vs. 11.5 × 109 cells/L; p = 0.006). Leukocyte counts remained significantly lower in the first 3 days after presentation (p = 0.006–0.043). At admission to the hospital, no differences were found between the groups for levels of alkaline phosphatase and γ-glutamyl transpeptidase. However, from day 1 onward, levels of alkaline phosphatase and γ-glutamyl transpeptidase were significantly higher in patients with acute Q fever .Patients with acute Q fever had a higher levels of C-reactive protein   and loweC. burnetii was performed for 41 patients in the study group  .Treatment was started before a diagnosis was made. Significantly more patients with acute Q fever started treatment with doxycycline than patients in the control group  . For 8 pHospitalization , admission to an intensive care unit , and need for respiratory support  were less common for the study group than for the control group . Also, dSixteen patients with acute Q fever met the criteria for prophylactic treatment to prevent development of chronic Q fever . IndicatMycoplasma sp. infection and 1 of pneumonia without a known causative agent. Eight patients in the control group died during follow-up. One of them died of a non-Hodgkin lymphoma and 1 of consequences of an Aspergillus sp. infection. For the other 6 patients who died, no detailed information was available.The mortality rate during a 12-month follow-up period was 6% for the study group compared with 19% for the control group (p = 0.02). None of the patients in the study group died during the episode of acute Q fever. Four patients in the study group died because of reasons unrelated to Q fever. One patient died of consequences of an infected vascular prosthesis caused by chronic Q fever, although adequate treatment was started after the diagnosis. In contrast, 2 patients in the control group died during initial hospitalization, 1 of a Pneumocystis jiroveci, Moraxella catarrhalis, Legionnella pneumophila, Chlamydia sp., Haemophilus influenzae (2 patients), Mycoplasma sp. (3 patients), influenza virus and Mycoplasma sp., and Staphylococcus aureus and Streptococcus pneumonia. The remaining 3 patients were given diagnoses of acute myeloid leukemia, non-Hodgkin lymphoma, and restrictive pericarditis.A total of 49 control patients were given a diagnosis of pneumonia; for 38 of these patients, no causative agent was found. For the remaining 11 patients, causative agents were C. burnetii was considered a possible etiologic agent in many patients who came to a hospital. The higher number of patients in the study group can be explained by strict implementation of inclusion criteria for the control group.This retrospective case–control study evaluated differences in clinical signs and symptoms between patients with acute Q fever referred to a hospital and a control group. Because patients in the control group had Q fever included in the differential diagnosis, a selection bias is possible. However, differences were found between the 2 groups. In addition, because of the Q fever outbreak during that time, ,,,,Consistent with findings of earlier studies  score  of unknown origin in the Netherlands depends on the Although changes were made in the national guidelines for treating CAP issued by the Dutch Working Party on Antibiotic Policy in 2011  of these patients, which is slightly higher than expected (,,After having acute Q fever, patients often report long-lasting fatigue, which frequently persists for >6 months. This symptom after acute Q fever has been designated Q fever fatigue syndrome. Our data suggest a prevalence of 11%, which is lower than expected; other studies reported a prevalence of ≈20% worldwide and a higher prevalence in the Netherlands (C. burnetii from other pathogens is not possible without Q fever serologic analysis and PCR in patients coming to a hospital. In disease-endemic areas or in instances in which patients have risk factors for Q fever, suspicion should remain high, and the threshold for performing Q fever serologic analysis and PCR should remain low. Because only 46% of patients received adequate treatment acute Q fever in our study, treatment for acute Q fever should be improved. Furthermore, our findings underline the recommendation that prophylactic treatment should be given to patients with risk factors for developing chronic Q fever. However, more studies are needed to develop uniform guidelines with regard to optimal prophylactic treatment.Although we found some differences in clinical manifestations for patients with acute Q fever coming to a hospital compared with controls, considerable overlap between both groups hamper the use of these variables for clinical differentiation. Differentiating


We have read the case report of Nihon-Yanagi et al. The patient they described developed hepatic granuloma two times and the granulomatous lesion was surrounding metal staples/clips suggesting that the granuloma was due to surgical staples/clips.Hepatic granulomas (HGs) are reported in around 5 % of patient who undergo a liver biopsy and caused by several diseases including sarcoidosis, tuberculosis, hydatid cyst, brucellosis, typhoid fever, chronic hepatitis B and C and primary biliary cirrhosis (PBC). Chronic hepatitis B and C infections are the most common and serious causes of liver damage in patient with renal failure. Their prevalence is a higher than people without renal failure. We have previously reported that the prevalences of HGs in patients with chronic hepatitis B and C are 1.5 and 1.3 % respectively. The described patient was on hemodialysis for 12 years. The other causes of HG seem excluded; however hepatitis B and C infections and PBC should have been tested and excluded before ascribing the HGs to surgical staples/clipping material. Dear Sir,We have read the case report of Nihon-Yanagi et al.  with greHGs are reported in around 5 % of patient who undergo a liver biopsy and caused by several diseases including sarcoidosis, tuberculosis, hydatid cyst, brucellosis, typhoid fever, chronic hepatitis B and C, and primary biliary cirrhosis (PBC) , 3. ChroAnother cause of HBs is PBC. It is an immune-mediated cholestatic liver disease characterized by destruction of cholangiocytes. The histology includes a portal tract inflammatory infiltrate composed of plasma cells, mononuclear cells, and neutrophils . NoncaseIn chronic liver disease and PBC, the described granulomas are microscopic granulomas and the ones in the presented case are macroscopic granulomas. However it is not known whether the causes of microscopic granulomas may also cause macroscopic granulomas or contribute to the development of macroscopic granulomas of any other causes.The other causes of HG seem excluded; however hepatitis B and C infections and PBC should have been tested and excluded before ascribing the HGs to surgical staples/clipping material.


Antioxidant biocomposites have been successfully developed from cellulose acetate, eco-friendly triethyl citrate plasticizer and onion extract as a source of natural antioxidants. First, an onion extraction process was optimized to obtain the extract with highest antioxidant power. Extracts under absolute ethanol and ethanol 85% were the extracts with the highest antioxidant activity, which were the characterized through different methods, DPPH  and ABTS ), that measure radical scavenger activity, and polyphenolic and flavonoid content. Afterwards, the extract was incorporated in cellulose acetate as polymer matrix owing to develop an active material intended to oxidative sensitive food products packaging. Different concentrations of onion extract and plasticizer were statistically studied by using response surface methodology in order to analyze the influence of both factors on the release of active compounds and therefore the antioxidant activity of these materials. Active food packaging appears as a promising technology with increasing applications due to its advantages over traditional methods on food preservation. Active packaging (AP) is defined as a package system that deliberately incorporates components that release or absorb substances into or from the packaged food or the environment surrounding the food to extend the shelf life or to maintain or improve the condition of the packaged food (Regulation CE No. 450/2009). Antioxidant active packaging is one of the most interesting components of AP because oxidation is one of the main problems on the preservation of oxygen sensitive food products, and recently the demand for natural antioxidant active packaging is increasing enormously due to its advantages compared with the addition of antioxidants directly to the food ,2,3. NowAllium cepa) are botanically included in the Lilliaceae and, after tomatoes, are the second most horticultural vegetable [In vitro studies have shown that flavonoids compounds may have positive effects against inhibition oxidation of biomolecules by its antioxidative potential, anti-inflammatory, anti-allergic, and antifungal effect [Therefore, the aim of this study was the development of an antioxidant active packaging based on acetate cellulose, as biodegradable polymer matrix, and red onion extract, as a source of antioxidant compounds. Onions  the scavenging of undesirable compounds that are involved in any step of the oxidation process, such as oxygen, radical oxidative species or metal ion from the headspace or from the food; and (ii) the technologies based on the release of antioxidants to the food. The release of the active agent is dependent on several factors, such as polymer type and food, and concentrations of the bioactive agents, among others. In this case, the design of active packages must involve the positive interaction between package and packaged food or headspace atmosphere ,18. TherSeveral workers have used surface response for designing food formulations and processing and optimization of different method ,24,25. RConsidering that the several parameters employed during film production influence film properties, the present study aimed to determine the optimal formulation by using response surface methodology in order to obtain films with activity antioxidant and evaluate kinetic release behavior of these materials.ca. 30.000) was supplied by Aldrich. Reagent-grade absolute ethanol, quercetin dihydrate, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, 2,2ʹ-azinobis(3-ethylbenzothiazoline-6-sulphonate) (ABTS), Folin Ciocalteau phenol reagent, anhydrous sodium carbonate, gallic acid, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), and triethyl citrate (>99%) (TEC) were purchased from Sigma Aldrich Quimica S.A. . Red onions were bought at a local supermarket in Santiago, Chile. Owing to avoiding differences in batches, the starting materials were all bought at the same time.Cellulose acetate  methods, which are often used because they are simple, inexpensive and robust techniques.s-triazine (TPTZ) to a colored product, so, in combination with other methods, it can be very useful in distinguishing dominant mechanisms with different antioxidants [FRAP assay is a total electron transfer based method and measures reduction of ferric 2,4,6-tripyridyl-oxidants . DPPH an•+ radical cationic solution were produced by reacting 7 mM ABTS in water with 2.45 mM potassium persulfate (K2S2O8) and then stored in darkness at room temperature for 16 h. ABTS•+ radical solution was diluted to give an absorbance value of 1 at 734 nm. All experiments were performed in triplicate. In both assays, the percentage inhibition values were calculated using this Equation (1):sample represents the sample absorbance and Acontrol the control sample absorbance . To standardize the results, scavenging activities of the DPPH· and ABTS•+ radicals were expressed as Trolox equivalents using a calibrated curve of Trolox concentration versus I (%).DPPH· radical-scavenging activity of onion extracts was evaluated according to the method described by Okada and Okada with some modifications . About 5et al. (2011) studies [3 solution (20 mmol/L) mixed with 25 mL of an acetate buffer . The reaction mixture was stored at room temperature for 30 min and the absorbance was measured at 593 nm. Results were expressed as mg Trolox/100 g sample.FRAP assay was measured using 150 μL of onion extract in accordance with Lu  studies . The FRAet al. [2 solution was added, and after 5 min, 0.3 mL 10% AlCl3 was added. After 6 min, 2 mL 1 M NaOH solution was added and the total volume was made up to 10 mL with distilled water. The solution was well mixed and the absorbance was measured at a 510 nm. Total flavonoid content was expressed as mg quercetin/100 g fresh sample.Total flavonoid content was measured by the aluminum chloride colorimetric assay, following the method described by Albishi et al. . One milIn order to obtain an extract with higher concentration to be incorporated for film development, onion was extracted with ethanol (3 g:20 mL) at 40 °C during 3 h. Solution was filtered and concentrated to a final concentration of 600 mg onion/ml extract.Films were obtained by a solution–extension–evaporation process (“casting”), containing cellulose acetate, triethyl citrate, TEC, as plasticizer and onion extract. Then, 2.5 g of polymer was dissolved in 50 mL acetone at 50 °C under stirring. As Finally, films were stored at room temperature in vacuum packages until further analysis. The total antioxidant activity of the films was determined by extractions in ethanol using ultrasonic at room temperature. Resulting film extracts were analyzed using DPPH method. Blank samples (films without OE incorporated) were also analyzed.2 piece of each plastic sample and 5 mL of the simulant (area-to-volume ratio 6 dm2/L) were placed in tubes. Periodically, vials were opened and the concentration of the antioxidants in the aqueous simulants was analyzed by indirect measurement through DPPH scavenging activity assay (explained at versus I (%).Release studies of the active compounds from the films were carried out by determining the migration from the polymer into 10% ethanol solution, as aqueous food simulant, at 40 °C. Double-sided total immersion migration tests were performed as follows: a 3 cmFisher’s least significant difference (LSD) procedure was used to evaluate statistically significant differences at the 95.0% confidence level, between the samples extracted with different solvents.i.e., antiradical activity and total polyphenol). A Central Composite Rotatable Design (CCRD) including 18 experiments formed by 4 central points and 4 (k = 1.41421) axial points to 22 full factorial design was used. Two levels for each variable were selected: plasticizer: 20%–50% respect to polymer weight, and onion extract: 5%–10% respect to polymer weight. Total phenolic content (TPC) and antioxidant capacity  were chosen as the response variables. The behavior of the response surface was fitted to a second order polynomial model, according to Equation (2):Y is the calculated response by the model; β0 is a constant intercept; βi is lineal; and βii and βij are squared and interaction coefficient, respectively. Optimization procedures were performed using Statgraphics Centurión XV . The quality of the fit of the polynomial model equation was evaluated by adjusted coefficients of determination (R2adj), standard error of estimation and lack of fit, according to Myers and Montgomery (2002) [Response surface methodology (RSM) was used in this experiment to study the effects of plasticizer and onion extract concentration on the antioxidant activity of film material (y (2002) .The range and coded level of the red onion extract concentration and plasticizer concentration as process variables studied are listed in p = 0.0213).First, multiple extractions under different solvents were carried out in order to select the best solvent to extract most antioxidant compounds. In general, the antioxidant activity varied in the red onion extracts and ranged from 6.5 to 9.7 mg Trolox/100 g of sample material, according the solvent used for the extraction. The data for DPPH radical-scavenging activity values are shown in Once 85% ethanol and absolute ethanol were selected as the two best solvents, these two extracts were analyzed through other methods. It is recommended to use various methods for evaluating the antioxidant capacity of complex heterogeneous systems like foods, as no general standardized protocol is currently available due to the dependence of the antioxidant activity on the chemical structure, mechanisms and antioxidant’s chemical environment .Extracts were evaluated through the following assays: DPPH and ABTS to measure radical-scavenging activity, and FRAP to measure ferric reducing power of extracts. Polyphenolic and flavonoid content were also determined through Folin Cicolteau and aluminum chloride colorimetric assay, respectively.As et al. [et al. [These results were in accordance with those of Kahkonen et al.  who dete [et al. , who det [et al. ,33.Response surface methodology was carried out owing to estimate the optimal formulation of the films with the incorporation of red onion extract obtained. A central composite design was employed to evaluate the DPPH radical-scavenging activity and Total Phenol after the formulation of different films. The statistical combination of the independent variables in actual values along with the predicted and experimental response was presented in Y) and Total phenols as a function of red onion concentration extract (X1) and plasticizer concentration (X2). In both cases, there was no significant (p > 0.05) lack-of-fit, which indicated that the response models were accurately fitted for predicting the responses as function of independent variables studied  and (4) represents DPPH  affected by the concentration of plasticizer and red onion extract  for each response variable, which is given in  extract . It is mR2aj) indicated that the accuracy of the model obtained from the ANOVA was quite good; whose values were 0.90 and 0.87 for total phenol and DPPH activity, respectively. These results suggest that the models fitted for the antioxidant activity and total phenols are suitable (significant and predictive) and lead to significant regression, low residual values, no lack of fit, and satisfactory coefficients of determination  surface plots to better visualize the relationship between the plasticizer and onion extract processing conditions as independent variables and antioxidant activity and total phenol as response variables . GraphicFinally, regarding to the results of blank samples, there were no antioxidant activity observed.Release of active agents from developed materials to an aqueous food simulant was kinetically monitored though migration assays. Antioxidant active agents released from PLA based films were indirectly measured as antioxidant activity of food simulant through DPPH method because it very well known that antioxidant activity is completely proportional to antioxidant release kinetics ,7,35,36.The extent of release at equilibrium depends on the compatibility of the migrant with food simulant and the polymeric film. The higher the solubility in the simulant, the higher the release expected. Although some studies have shown release of active compounds increased with plasticizer concentration, in this work, as Controlling OE and plasticizer concentrations, different release rates and extents can be achieved, which is incredibly interesting for future food conservation based in active food packaging.p > 0.05), thereby influencing the release of the antioxidant agent from the film formulated. Nevertheless, plasticizer had no significant effect on the release rate, but films with higher plasticizer concentration showed faster release due to higher mobility of the polymer chains.It was possible to obtain biodegradable materials with antioxidant capacity for future oxygen sensitive food products conservation. A natural extract obtained from onions was successfully obtained and incorporated in cellulose acetate and the effect of onion extract and plasticizer concentration was studied through response surface methodology. The independent variables studied were plasticizer and onion extract concentrations. The results indicated that onion concentration had a significant effect on the DPPH radical-scavenging activity and total phenol (


Indeed, few data for active microbial biomass (AMB) exist with which to compare model output. Here, we tested the hypothesis that differences in soil microbial respiration rates across various environmental conditions are more closely related to differences in AMB  than in TMB. We measured basal respiration (SBR) of soil incubated for a week at two temperatures (24 and 33°C) and two moisture levels (10 and 20% soil dry weight [SDW]), and then determined TMB, AMB, microbial specific growth rate, and the lag time before microbial growth (tlag) using the Substrate-Induced Growth Response (SIGR) method. As expected, SBR was more strongly correlated with AMB than with TMB. This relationship indicated that each g active biomass C contributed ~0.04 g CO2-C h−1 of SBR. TMB responded very little to short-term changes in temperature and soil moisture and did not explain differences in SBR among the treatments. Maximum specific growth rate did not respond to environmental conditions, suggesting that the dominant microbial populations remained similar. However, warmer temperatures and increased soil moisture both reduced tlag, indicating that favorable abiotic conditions activated soil microorganisms. We conclude that soil respiratory responses to short-term changes in environmental conditions are better explained by changes in AMB than in TMB. These results suggest that decomposition models that explicitly represent microbial carbon pools should take into account the active microbial pool, and researchers should be cautious in comparing modeled microbial pool sizes with measurements of TMB.Heterotrophic respiration contributes a substantial fraction of the carbon flux from soil to atmosphere, and responds strongly to environmental conditions. However, the mechanisms through which short-term changes in environmental conditions affect microbial respiration still remain unclear. Microorganisms cope with adverse environmental conditions by transitioning into and out of dormancy, a state in which they minimize rates of metabolism and respiration. These transitions are poorly characterized in soil and are generally omitted from decomposition models. Most current approaches to model microbial control over soil CO If they have represented microbes at all, decomposition models have most commonly represented microbial biomass as a single pool .Microbial respiration responds rapidly to changing environmental conditions, strongly influencing soil carbon cycling, and its feedbacks to climate change   that help shed light on the mechanisms by which microbes influence soil CO2 production.To quantify the importance of active and dormant microbial pools in explaining soil respiratory responses to abiotic factors, we incubated soil at different temperature and moisture levels for a week and subsequently analyzed the correlation of SBR with TMB and AMB. Since, (1) only active microorganisms are able to drive soil biogeochemical processes and (2) abiotic factors can cause microorganism to be activated or inactivated without necessarily altering TMB, we hypothesized that soil respiratory responses to changing environmental conditions would be better explained by changes in AMB than in TMB. We used a kinetic approach based on the Substrate Induced Growth Response (SIGR) technique  from the top 0–15 cm layer  in a deciduous forest at Purdue University's Ross Biological Reserve, Indiana, USA , in May 2014. The soil is classified as (2–6% slope) Russell (Alfisol) silt loam , prepared a composite sample by sieving the soil through a 2 mm mesh, and adjusted the soil moisture content to 10% (moisture deficit) and 20%  of soil dry weight (SDW). Finally, we placed 25 g of soil (dry weight) in 0.26 L septum-capped glass jars (microcosms) and stored them at 24 and 33°C for 1 week .4) SO4, 2.25 mg K2HPO4, and 3.62 mg MgSO4 per gram of soil  afterwards. We measured CO2 concentration by withdrawing 5 mL of gas from the microcosm headspace using a syringe and injecting it into an infrared gas analyzer . To avoid negative pressure, we opened the jars at the end of each measurement , aerated the headspace until the CO2 concentration in the microcosms equilibrated with ambient air, closed the jars, and withdrew the initial gas sample  of the following measurement.After soil samples had been incubated for 1 week at 24 and 33°C, (hereafter unheated and heated) and 10 and 20% SDW soil moisture (hereafter dry and wet) conditions, we indirectly measured TMB and AMB  using soil respiratory responses (SIGR) to glucose and mineral nutrient inputs  is soil respiration rate at time t (in mg C g−1 h−1), V is the volume of the microcosm headspace, C1– C0 is CO2 concentration change in mg L−1, W is the dry mass of the soil , and t is the time between the first (C0) and the second (C1) CO2 concentration measurements .We calculated soil respiration rates as in Speratti and Whalen :Rs(t)=Vtlag, we used the model proposed by Panikov and Sizova . μ is defined as the slope of the growth curve at its inflection point when nutritional and environmental conditions are optimum . To avoid violating this assumption, only specific growth rates (μ) were calculated for different treatments. AMB was estimated based on substrate-induced exponential growth curves. Differences in exponential curves reflect differences in AMB, caused by the treatments, at the time point immediately before substrate addition.To estimate TMB, AMB, microbial specific growth rate, and d Sizova  =Rum Pirt, . At non-um Pirt, . It is ium Pirt,  during t(Rs) during the lag and exponential phases that followed substrate amendment. We omitted the first 3 h of measurements from the analysis to exclude the transient effects of mixing and preparing the soil on soil respiration rates  microbial biomass, and the denominator accounts for total maintenance respiration.tlag as in Blagodatskaya et al.  function in R (version 3.1.1). We used R2 as a measure of goodness of fit.We estimated SBR as the mean of the eight soil respiration measurements taken in the 4 h prior to substrate amendment. For the CO10-transformed AMB for statistical analyses, but for clarity we present the untransformed data in the text and figures. All values are means of three replicates per treatment.To estimate the significance of the differences in SBR and microbial parameters between treatments, we applied a two-way ANOVA using the aov function in R (version 3.1.1). When significant differences were found, we conducted post-hoc pairwise comparisons using the TukeyHSD function . To meet assumptions regarding the normality of residuals and homogeneity of variances we logWe calculated the percentage of the variance explained by each independent factor by dividing the sum of squares of each factor (from the ANOVAs mentioned above) by the total sum of squares  = 145.51, P < 0.001; Figure tlag by 43% in dry treatments and by 79% in wet soils. In contrast, μ did not respond to temperature and soil moisture Supplementary Tables R2 = 0.14, F = 1.6, P = 0.235].AMB responses to warming and soil moisture were strongly negatively correlated with 2-C g−1 soil h−1 per μg active biomass C g−1 soil.As we expected, soil respiration rates across the temperature and soil moisture treatments were strongly correlated with AMB, but not with TMB Figure . The cortlag) than did soil moisture . As we predicted, SBR was more strongly correlated with AMB than with TMB. An increase in AMB after warming and wetting with no significant change in TMB suggests a quick shift of dormant microorganisms to an active physiological state, which corresponded to the increase in respiration. Given that the specific growth rate is an intrinsic feature of the microbial population . The combined analysis of the proportion of variances explained by abiotic factors and the relationships between SBR and total and active MB suggests that SBR responses to temperature are strongly linked to changes in AMB. The activation of dormant microorganisms under warmed conditions raises the question of how and why environmental factors trigger activation of different microbial taxa. While we cannot directly address these questions here, we speculate that the temperature of heated soils may have been closer to the optimum for microbial processes than that of unheated soils. On the other hand, moisture explained a larger portion of the variation in SBR than in AMB. This could be explained in part by the fact that AMB, but not SBR, was insensitive to moisture under unheated conditions. This suggests that the importance of microbial physiology for explaining soil carbon processes could vary across moisture  decreased TMB but increased AMB, suggesting that part of the dead biomass was re-utilized by the active microbial fraction. By recycling energy and nutrients from dead biomass, microorganisms in the more favorable conditions likely remained in an active state, which reduced total biomass, but maximized active biomass and led to the highest fraction of active biomass (5.0 ± 0.2% of TMB) in the warm and wet treatment. Rapid declines in TMB after re-moistening of pre-conditioned soil have been detected under laboratory conditions without substrate addition  could strongly affect biogeochemical processes such as carbon cycling. Transitions between active and dormant physiological states have been incorporated into some dynamic microbial community models to simulate community responses to environmental changes, such as drying-rewetting cycles Konopka, . However2-C h−1. In a pasture topsoil (0–15 cm) from Pergamino, Argentina, this relationship varied from 0.01 to 0.18 g CO2-C h−1 per g active biomass C, depending on the availability of labile C . The CO2 released from 15-cm deep cores of California grassland soils in the first 2 h after a rewetting event ranged from 8 to 33 mmol m−2, depending on the precipitation pattern preceding the rewetting event. This peak in soil respiration was strongly linked  to increases in the abundance of potentially active bacteria  could underestimate future soil CO2 emissions.While we observed a strong response of AMB to temperature (e.g., Figure We conclude that soil respiratory responses to short-term changes in environmental conditions are better explained by changes in the active fraction of the soil microbial pool than by changes in TMB. Based on these results, we suggest that decomposition models that explicitly represent microbial processes should take into account the active microbial pool, and recommend researchers be cautious when comparing modeled microbial pool sizes with measurements of microbial biomass.AS substantially contributed to the conception, design, and critical review of the work, as well as to the acquisition, analysis, and interpretation of the data. EB substantially contributed to the analysis and interpretation of the data, as well to the critical review of the work. JD substantially contributed to the conception, design, and critical review of the work, as well as to the analysis, and interpretation of the data.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In a few days, glomeruli reach complete development, with a structure that is similar to that of the mammalian counterpart, showing a fenestrated endothelium and a basement membrane covered by the multiple ramifications of mature podocytes. As a further advantage, zebrafish embryos are permeable to low molecular compounds, and this explains their extensive use in drug efficacy and toxicity experiments. Here we show that low concentrations of adriamycin (i.e. 10 and 20 µM), when dissolved in the medium of zebrafish embryos at 9 hours post-fertilization and removed after 48 hours (57 hpf), alter the development of podocytes with subsequent functional impairment, demonstrated by onset of pericardial edema and reduction of expression of the podocyte proteins nephrin and wt1. Podocyte damage is morphologically confirmed by electron microscopy and functionally supported by increased clearance of microinjected 70 kDa fluorescent dextran. Importantly, besides pericardial edema and glomerular damage, which persist and worsen after adriamycin removal from the medium, larvae exposed to adriamycin 10 and 20 µM do not show any myocardiocyte alterations nor vascular changes. The only extra-renal effect is a transient delay of cartilage formation that rapidly recovers once adriamycin is removed. In summary, this low dose adriamycin model can be applied to analyze podocyte developmental defects, such as those observed in congenital nephrotic syndrome, and can be taken in consideration for pharmacological studies of severe early podocyte injury.The zebrafish pronephros is gaining popularity in the nephrology community, because embryos are easy to cultivate in multiwell plates, allowing large number of experiments to be conducted in an  Damage to the podocyte, the highly ramified cell that surrounds and completely covers with primary and secondary processes the basement membrane of the glomerular capillary, accounts for the majority of proteinuric diseases of the kidney, and is prominent in nephrotic syndrome due to primary or secondary causes Despite novel insights gained in the last decade on specific molecules and pathways involved in the pathogenesis of proteinuric diseases, there is still the need of more complete understanding of the events leading to podocyte damage and of models to be utilized for drug screening to translate research results into valuable therapies.Danio rerio) pronephros constitutes a simple and accessible system to study in vivo numerous aspects of renal cell biology. The transparency of zebrafish larvae and the 48 h time-frame of complete pronephros maturation makes it feasible to analyze glomerular and tubular development and study numerous aspects of cell differentiation in an in vivo setting In this framework, the zebrafish .Zebrafish were raised and maintained as previously described All experimentations in the zebrafish Lab  conform to the ITA and EU guidelines on research practice (European directory 2007/526/CE). The zebrafish experimentation and protocols were approved by the Italian Ministry of Health and ASL Varese (Prot. N. 014AVB0020033). When appropriate, anesthesia was induced by 1∶20 to 1∶100 dilution of 4 mg/ml Tricaine  and live fish were positioned in methylcellulose for microscope observation.For the Palb test a committee of Italian Health Ministry approved the experimental protocol  in compliance with Italian regulation (D.L.vo 116/92).The Palb test was used to preliminarily assess which concentration of adriamycin could actively modify the permeability of isolated rat glomeruli. The technique is extensively in use in our laboratory Dose-dependent and time-dependent studies were performed by adding 5 to 40 µM adriamycin to the E3 medium of 3 to 10 hours post fertilization (hpf) old zebrafish embryos. The substance was left in the medium for 48 h, then animals were washed several times with E3 medium and were immersed in medium without adriamycin until the end of the experiments. A total number of 340 animals were used in these studies.Body length was determined using digital photographs and applying the length measurement tool of Sigma Scan Pro software (Jandel Scientific Softaware).Pericardial edema was estimated using the area measurement tool of the same software to assess the cross-sectional pericardial area and the total body area. Results, obtained from 45 adriamycin-treated embryos and normalized to those obtained from 45 untreated controls, were expressed as cardiac/body ratio.All pictures were taken at the same resolution and magnification with the live fish positioned in a lateral orientation.D. rerio, are constantly surrounded by very low osmolar medium. Therefore, the ability to exclude water is essential to survival and depends on gills, kidney, and skin, which all contribute to maintain proper internal volume and osmolarity. In consideration of this behavior, formation of edema could be due either to alteration of kidney function and/or to malfunction of other water barriers Freshwater fish, such as Danio rerio nephrin 5′-CAG TCA CAG GCC TTA ACC CTT CAA-3′ (forward) and 5′-CGA GGC GTT GAT AAG CTC TCT GCT-3′(reverse); Danio rerio wt1a 5′-TGC TGA TCC TCC TTC TAG CC-3′ (forward) and 5′-GAA CGG AGG AGT GTG TTG TG-3′ (reverse); Danio rerio wt1b 5′-AGC CAA CCA AGG ATG TTC AG-3′ (forward) and 5′-GAA TGC CAT TAA AGT AGT TCC TC-3′ (reverse) and Danio rerio β-actin (house-keeping gene) 5′-CAG CAA GCA GGA GTA CGA TGA GT-3′ (foward) and 5′-TTG AAT CTC ATT GCT AGG CCA TT-3′ (reverse).Total RNA was extracted from 60 early larvae exposed to adriamycin and 60 controls at 72 and 120 hpf, using Trizol Reagent , and RNA concentration was determined by Nanoquant . One microgram of total RNA was reverse transcribed using MMLV reverse transcriptase (Applied Biosystems). The following oligonucleotides were applied as forward and reverse primers for quantitative Real Time RT-PCR (QRT-PCR): For light microscopy, 40 embryos per group and time point were fixed in 4% buffered paraformaldehyde, dehydrated, and paraffin-embedded. 2–3 µm thick sections were cut by Microm HM450 sliding microtome and stained with hematoxylin eosin.For transmission electron microscopy, conducted on 20 animals per group and time point, fixation was performed in a mixture of paraformaldehyde, glutaraldehyde, and phosphate buffer, and samples were embedded in resin.For whole mount actin staining, 20 embryos per group and time point were fixed overnight at 4°C in 4% phosphate-buffered paraformaldehyde, then permeabilized at 4°C in PBS containing 2% Triton-X100, and incubated in FITC-phalloidin (Sigma) for 3 hours at room temperature.A qualitative assay was employed to investigate glomerular blood filtration, as described by Hentschel et al. The heartbeat frequency was determined by direct visual counting of ventricle beating using a stereomicroscope (Leica). Movies of the beating heart were recorded using an inverted microscope (Leica) connected to a videocamera, and capturing two full heart beats per second.For cartilage staining, 20 animals per group and time point were prepared by fixation in 4% phosphate-buffered paraformaldehyde and maintained in methanol at 20°C until use. Then, staining was performed by Alcian blue solution .At 72 hpf, 40 embryos per group were fixed in 4% paraformaldehyde for 2 hours at room temperature and stained for assessment of endogenous alkaline phosphate activity, as described Data are presented as mean ± standard error. Comparison between groups of data was performed by one-way analysis of variance (ANOVA).The Palb test demonstrated that adriamycin was able to increase permeability of isolated rat glomeruli starting from a concentration of 10 µM. The highest permeability values were obtained at concentrations of 20, 30 and 40 µM .In order to establish the age of animals to be used in our experiments, 5 to 40 µM adriamycin was added to the E3 medium at different time points and washed out after 48 hours. By these experiments we observed that the earliest exposure (at 1–3 hpf) to any concentration of adriamycin caused severe developmental changes followed by death .Instead, when adriamycin was given at 9 hpf, embryos displayed normal development until hatching . NoteworConsistently, at 72 hpf the cardiac/body ratio was increased as a consequence of any adriamycin dosage (5–40 µM), and a significant difference versus untreated embryos was reached by dosages from 10 to 40 µM .Treated embryos also showed a moderate reduction of length with dosages of 20–40 µM . In addiTo assess if pericardial edema was due to glomerular damage or to alteration of other water barriers, such as gills and skin, we performed a mannitol test. Mannitol does not readily cross between biological compartments and its addition increases the osmolarity of water. Therefore, by abolishing the osmotic gradient between interior body fluids and water environment, mannitol is able to reduce pericardial edema when it is due to skin or gills failure To further prove glomerular involvement, we analyzed the expression of specific glomerular markers, namely wt1a, wt1b and nephrin. In keeping with previous results Adriamycin-treated animals displayed a significant decrease of expression of glomerular markers at 72 hpf , supportThe histological studies at 72 hpf control larvae showed completely formed glomeruli, but light microscopy was insufficient to reveal glomerular damage , that waAs reported Larvae exposed to 20 and 30 µM adriamycin had globally decreased fluorescence. FITC-labeled dextran accumulated in the pericardial area, due to edema ; consequLarvae exposed to adriamycin 10 and 20 µM were followed up to 120 hpf. In these larvae, pericardial edema progressively increased  and was In parallel severe podocyte damage was detected by TEM, with large areas of fusion, thinning, and microvillous degeneration of the foot processes, which were randomly attached to the glomerular basement membrane , all feaAdriamycin is known to induce cardiomyopathy in humans Therefore, in our model it was essential to identify a dosage of adriamycin causing podocyte defects without cardiac side effects.At 72 hpf the majority of animals given 30 (60% of larvae) and 40 µM (90% of larvae) adriamycin had a cardiac phenotype. Changes included heart malformation and decrease of the heart rate . InsteadTo analyze other potential damaging effects of adriamycin in zebrafish embryos, we studied vessel, cartilage, and muscle formation.Zebrafish angiogenesis starts at 24 hpf and is completed by 72 hpf. Subintestinal vessels (SIVs) are generally utilized in this type of studies because of their accessibility and relatively simple anatomy.We found normal SIV formation after 10 and 20 µM adriamycin, whereas at higher concentrations, particularly 40 µM, we observed the appearance of abnormal spikes projecting from the subintestinal vessel basket .In addition, adriamycin showed dose-dependent effects on the craniofacial cartilage, whose development was delayed . NoteworInstead, adriamycin did not influence muscle development, that was similar to controls for any adriamycin dosage during the whole observation period .Our results indicate that addition of adriamycin 10 and 20 µM to the medium of zebrafish embryos at 9 hpf, followed by its removal after 48 h, induces selective and progressive podocyte changes and a measurable alteration of permeability of the glomerular barrier.2 of body surface. The drug has multiple side effects, such as nephrotoxicity, cardiotoxicity, and neurotoxicity.Adriamycin is a cytotoxic anthracycline antibiotic used in antimitotic chemotherapy. In humans, it is infused intravenously to treat neoplastic diseases, such as acute leukemia, multiple myeloma, Hodgkin's and non-Hodgkin's lymphomas, and several sarcomas and carcinomas. The most common dosage is 60 to 75 mg/mAdriamycin is utilized by the nephrology research community primarily because it causes podocyte foot process effacement and nephrotic syndrome in rodents The primordial zebrafish pronephron appears at 19 hpf, during early somitogenesis, as a mass of intermediate mesoderm that lies under the second and third somite in vitro (10–60 µg/ml) and utilized in rodents (150 mg/kg).Among other molecules known to be harmful to podocytes, puromycin aminonucleoside has been applied to zebrafish embryos alb on isolated rat glomeruli.Instead, our protocol applies the same dosages of adriamycin which are utilized on cultured podocytes Confirming data from the literature While adriamycin toxicity has been mostly examined in adult zebrafish, there are not many studies on developmental toxicity. A recent publication Albeit in these experiments adriamycin was added later and left in the medium longer than in our protocol, the data are concordant with our observations.Adriamycin 30 and 40 µM administration caused relevant systemic effects in the majority of larvae. Instead, addition of 10–20 µM adriamycin determined almost exclusively glomerular effects and were characterized by a very low mortality rate in absence of major teratogenic effects. The main extraglomerular consequence we observed with these dosages was a delay of cartilage development, that rapidly reversed after elimination of the drug from the medium.Instead, pericardial edema persisted and worsened after adriamycin removal, allowing further analysis of podocyte defective development. We performed different experiments to prove the prominent glomerular involvement caused by our experimental protocol, and results concordantly excluded the involvement of other water barriers and confirmed the altered morphology of podocytes, the increased clearance of fluorescent dextran, and the diminished expression of podocyte markers.Wt1 and nephrin are essential regulators of nephrogenesis in zebrafish, particularly of glomerular structure formation. In contrast to other animals, fish possesses two wt1 paralogs, and during the first 24 hours of development they are expressed in an overlapping, though not identical, spatial and temporal pattern; after completion of pronephros differentiation, expression of wt1a and wt1b is limited to the podocytes In our protocol, adriamycin decreased the expression of these genes, and expression levels remained lower than controls after removal from the medium.In conclusion, we have established that low concentrations of adriamycin specifically target the pronephron structure, when dissolved in the medium at 9 hpf. Main advantages of this model are the feasibility and high reproducibility, making it prospectively useful to analyze podocyte development and to test drug efficacy in a very severe condition of podocyte developmental damage.Movie S1Recording of heart beating in untreated larvae at 72 hpf.(ZIP)Click here for additional data file.Movie S2Recording of heart beating in larvae exposed to 20 µM adriamycin at 72 hpf. There are no appreciable differences as compared to untreated larvae.(ZIP)Click here for additional data file.Movie S3Recording of heart beating in larvae exposed to 40 µM adriamycin at 72 hpf. Distortion of the heart shape and change of contractility are clearly detectable.(ZIP)Click here for additional data file.