Antiplatelet agents, such as aspirin and P2Y12 inhibitors, are essential in the secondary prevention of cardiovascular disease . The effBlood used was drawn from healthy donors free from antiplatelet medication. Light transmission aggregometry (LTA) was used to determine the optimal concentration of MRS2179. Platelet aggregation was induced by the addition of incremental concentrations of ADP. The optimal concentration of MRS2179 to inhibit ADP induced aggregation was 20µM. Thrombus formation in vivo occurs due to the tethering, adhesion and translocation of platelets to von Willebrand Factor (vWF) under arterial shear conditions . To testThe results of this study demonstrate that a concentration of 20µM of MRS2179 effectively inhibits aggregation. In 13 normal donors 20µM either completely inhibited ADP induced aggregation or enhanced platelet disaggregation (p<0.05). In preliminary experiments from 3 normal donors assayed there were no significant changes in most of the parameters measured in the dynamic assay. However, platelet translocation velocity in the presence of the P2Y1 antagonist was significantly increased (p<0.05).Selective inhibition of the P2Y1 surface receptor results in a significant decrease in aggregation in the presence of an agonist. Preliminary data using a novel dynamic assay of platelet function suggests that P2Y1 inhibition may be of therapeutic value.


To the Editor: The Global Polio Laboratory Network (GPLN) and the World Health Organization’s Polio Eradication Initiative (GPEI) accord high priority to detecting all vaccine-derived polioviruses (VDPVs) because they are neurovirulent and have the potential to cause outbreaks of poliomyelitis and establish poliovirus circulation. In patients with immunodeficiency diseases, persistent infections may become established with live oral poliovirus vaccine (OPV) and develop into VDPVs (>6 nucleotide substitutions (Sabin2) or ≥10 nucleotide substitutions (Sabin1 and Sabin3) in the VP1 region. The VDPV assays were found to be 100% specific for all 3 poliovirus types, 100% sensitive for Sabin1 and Sabin3, and 76% sensitive for Sabin2 —that is, withdrawal of Sabin2—beginning in April 2016  on January 2008. Sabin3 was isolated from stool sample 1 (R46064), collected 11 days after onset of paralysis. Stool sample 2, collected on the 13th day after paralysis, was negative for virus. R46064 was reported as “Sabin-like” by the intratypic differentiation tests  used in the GPLN in 2008  Table. SR46064 produced Sabin3-like results in the VDPV screening assay. R46064 gave false-negative test results because the isolate had a Sabin3 vaccine sequence in the regions corresponding to the probe and primers of the VDPV assay  Figure 2Type 2 VDPV was found in an immunocompetent girl, 3.5 years of age, in March 2014. Sabin2 was isolated from 2 stool samples collected 5 and 8 days after onset of AFP. Sabin2, isolated from stool sample 1 (R93150), was amplified in the VDPV screening assay (reported as Sabin2-like); the isolate from stool sample 2 (R93152) failed to become amplified (reported for sequencing).VP1 sequencing of R93152 showed 6 nt substitutions; therefore, it was reported as VDPV2. R93150 was also sequenced to find out whether it contained the Sabin2 homotypic mixture. VP1 sequence of R93150 showed 6 nt substitutions and no evidence of mixed bases. Substitution was not found at VP1 nt 427/428, the main target of the VDPV screening assay.Complete genome sequence analysis revealed that both isolates contained reversion of the major attenuating site at nt 481 in the 5′ UTR. The genomes of both isolates showed no recombination. Both isolates showed 15 nt substitutions in the capsid region, when compared with Sabin2  Table; oThe occurrence of VDPV2 and VDPV3 as described above may be rare. However, GPLN laboratories are unlikely to detect VDPV strains that produce false-negative results in the VDPV screening assay. False negatives are of greatest concern to the GPEI because they could impede timely detection of VDPV infections (Nucleotide substitutions in the 5′UTR and capsid region of type 2 and type 3 vaccine-derived polioviruses (VDPV), amino acid changes at neutralizing antigenic sites, and nucleotide sequence alignment of VDPV2 and VDPV3 with corresponding Sabin vaccine virus sequences.


The associated region contains a previously identified glaucoma gene, ADAMTS10, which was subjected to mutation screening in the coding regions. A fully segregating missense mutation (p.A387T) in exon 9 was found in 14 cases and 572 unaffected NEs (pFisher = 3.5×10−27) with a high carrier frequency (25.3%). The mutation interrupts a highly conserved residue in the metalloprotease domain of ADAMTS10, likely affecting its functional capacity. Our study identifies the genetic cause of primary glaucoma in NEs and enables the development of a genetic test for breeding purposes. This study establishes also a new spontaneous canine model for glaucoma research to study the ADAMTS10 biology in optical neuropathy.Primary glaucoma is one of the most common causes of irreversible blindness both in humans and in dogs. Glaucoma is an optic neuropathy affecting the retinal ganglion cells and optic nerve, and elevated intraocular pressure is commonly associated with the disease. Glaucoma is broadly classified into primary open angle (POAG), primary closed angle (PCAG) and primary congenital glaucoma (PCG). Human glaucomas are genetically heterogeneous and multiple loci have been identified. Glaucoma affects several dog breeds but only three loci and one gene have been implicated so far. We have investigated the genetics of primary glaucoma in the Norwegian Elkhound (NE). We established a small pedigree around the affected NEs collected from Finland, US and UK and performed a genome-wide association study with 9 cases and 8 controls to map the glaucoma gene to 750 kb region on canine chromosome 20 (p Glaucoma is an optic neuropathy affecting the retinal ganglion cells and optic nerve. Elevated intraocular pressure (IOP) is commonly associated with the disease. However, normal tension glaucoma is diagnosed as well contactin 4 (CNTN4) myocilin (MYOC)neurotrohin 4 (NTF4)optineurin (OPTN)WD repeat domain 36 (WDR36)cytochrome P450 1B1 (CYP1B1)latent transforming growth factor beta binding protein 2 (LTBP2)ATP-binding cassette, sub-family C (CFTR/MRP), member 5 (ABCC5) has been associated with PCAG In human, several loci In dogs, POAG and PCAG have been described in several breeds ADAM metallopeptidase with thrombospondin type 1 motif, 10 (ADAMTS10), has been found in a research colony of Beagles with POAG has been suggested as causative Despite the presence of glaucoma in many breeds the genetic etiology of glaucoma is almost completely unknown in dogs. A novel locus was mapped in Dandie Dinmont Terriers In addition to Beagles, Norwegian Elkhounds are also affected with POAG ADAMTS10 gene.We aimed to find the genetic cause of primary glaucoma in NEs in this study to better understand the molecular pathogenesis, to establish a large, spontaneous canine model for POAG research, and to develop a genetic test for breeding purposes. We discovered a novel missense mutation in the Blood samples from 596 NEs, including 16 glaucoma affected dogs from Finland, Norway, Sweden, United Kingdom and United States were collected to the canine DNA bank at the University of Helsinki, Finland with owneŕs consent and under the permission of the Animal Ethical Committee of County Administrative Board of Southern Finland (ESAVI/6054/04.10.03/2012). All affected dogs were examined by a certified veterinary ophthalmologist. The clinical diagnoses indicated bilateral primary glaucoma with significantly elevated IOP, between 25–86 mmHg , and no detectable underlying cause. In addition, various degrees of optic nerve atrophy and cupping were reported. In addition, a progressive vision loss was detected in the affected dogs leading to complete blindness. Other, secondary clinical signs included lens luxation, corneal stromal edema, Haabs striae, keratitis, vitreal syneresis and retinal degeneration. The average age at the time of diagnosis was 6.5 years.Genomic DNA was extracted from EDTA blood using Chemagic Magnetic Separation Module I (MSM I)  according to the manufacturer’s instructions. DNA from buccal swabs  was extracted using QIAamp DNA Mini Kit (Qiagen).http://www.ncbi.nlm.nih.gov/projects/SNP/).A genome-wide association study (GWAS) was performed using Illumina’s CanineHD BeadChip array  with 9 cases and 8 controls Fig. 1. ADAMTS10, were first sequenced in four NE cases, in four NE controls (unaffected >8 years) and in one unaffected Rough Collie samples. The identified candidate mutation was then validated in 596 NEs, including 7 additional cases. In addition, the mutation was studied in 71 dogs from 17 other breeds affected with POAG, PCAG or PLD and in 115 unaffected dogs from 6 breeds (Table S1).The coding regions of the best candidate gene in the associated region, Table S2) were designed for the ADAMTS10 gene to amplify the coding regions and splice sites by standard PCR (Table S2). PCRs were carried out in 12 µl reactions consisting of 1.2 U Biotools DNA Polymerase , 1.5 mM MgCl2 , 200 µM dNTPs , 1 x Biotools PCR buffer without MgCl2 , 0.83 µM forward and reverse primer  and 10 ng template genomic DNA. Reaction mixtures were subjected to a thermal cycling program of 95°C for 10 min, followed by 35 cycles of 95°C for 30 s, 30 s at the annealing temperature and 72°C for 60 s and a final elongation stage of 72°C for 10 min. ExoSap purified PCR fragments were Sanger sequenced in our core facility the FIMM Technology Center using ABI 3730xl DNA analyzer . Sequence data analysis was performed using the Sequencher software . Build 3.1 of the canine genome reference sequence was applied in the study.Primer pairs  , but it did not affect the result as two mixed model approaches (GenABEL and GAPIT) that better control for population stratification, gave the same results (data not shown).We established a global sample cohort including glaucoma affected (n = 16) and unaffected NEs (>570) to map the disease locus, to identify the causative gene and to validate the segregation of the mutation. We performed a GWAS in a small pedigree of 9 cases and 8 controls. Statistical analyses revealed a 750 kb locus on CFA20 with 23 most highly associated SNPs between 53070684 bp to 53816416 bp (phttp://www.genopro.com) suggested a recessive mode of inheritance as the affected dogs are born to unaffected parents and there are multiple affected littermates in some litters.A pedigree constructed around the affected dogs using GenoPro genealogy software (ADAMTS10 (Table S3). Only one out of the five synonymous variants, p.P171P, co-segregated with the non-synonymous variant. However, it was not suspected to be in the exonic enhancer region when analyzed by the ESE-finder Fig. S1) in the metalloprotease domain of ADAMTS10 protein . The breed-specificity of the p.A387T mutation was indicated by excluding it from 71 glaucoma or PLD affected dogs from 17 breeds and from 115 unaffected dogs from six breeds.To gather further evidence for the p.A387T segregation, we genotyped seven additional NE primary glaucoma cases and four obligatory carriers Fig. 1 aADAMT10 gene in the NEs affected with primary glaucoma using genome wide association analysis and candidate sequencing strategies. We mapped the disease to a known canine POAG locus including the ADAMTS10 candidate gene and subsequently identified a missense mutation in the exon 9 of the ADAMTS10 gene. This is consistent with the previously reported POAG phenotype of primary glaucoma in this breed Our study has identified a novel recessive mutation in the β), which regulates the collagen turnover ADAMTS10 is a secreted glycoprotein The members of the ADAMTS family share the same structural organization including catalytic metalloprotease domain, followed by a disintegrin-like, and cysteine-rich domains, a thrombospondin repeat and a spacer region Fig. 3C.βADAMTS10 interacts with fibrillin-1 and localized to fibrillin-1 microfibril bundles ADAMTS10 gene has been previously associated with POAG in a research colony of Beagles. The Beagle mutation is positioned in the cysteine rich domain and is hypothesized to disrupt protein folding leading to instability In dogs, a missense mutation (p.G661R) in the β, which is known to be elevated in the aqueous humor of glaucoma patients ADAMTS10 and its pathway. Secondary lens luxation diagnosed in the affected NEs may be due to abnormal fibrillin-1 functions as the ADAMTS10 and fibrillin-1 interaction may be impaired causing disruption of the lens ligaments.The NE mutation is different from the Beagles and may result in a different pathogenesis. NE mutation changes a highly conserved residue in the metalloprotease domain, which plays a role in the remodeling of the connective tissue Fig. 3C ADAMTS10 in human WMS patients Dagoneau et al. identified three causative mutations in the metalloprotease domain of ADAMTS10ADAMTS17fibrillin 1 (FBN1) ADAMTS17-mutant dogs ADAMTS10-mutant Beagles ADAMTS10 and ADAMTS17 genes. Further studies are warranted to investigate whether the observed breed- and species–specific differences are due to alternate mutations in the metalloproteinase domains or possible other factors.Mutations in ADAMTS10. This study implicates the significant role of ADAMTS10 in canine POAG by identifying the second mutation in the same gene in dogs. Our affected dogs establish a new model to study ADAMTS10 biology in the microfibrillin theory of the glaucoma In summary, we have discovered the genetic cause of primary glaucoma in the NEs by identifying a missense mutation in Figure S1ADAMTS10 protein alignments. ADAMTS10 sequence alignment between different species. The mutation is located in a highly conserved region across 75 species. The arrow marks the mutated alanine residue.(ZIP)Click here for additional data file.Figure S2Q-Q plots. Identity-by-state (IBS) clustering and CMH meta-analysis (PLINK) were used to adjust for population stratification (A). A mild population stratification was identified in the study cohort by genome wide IBS clustering (adjusted genomic inflation factor λ = 1.1) (B).(TIF)Click here for additional data file.Table S1Dogs affected with glaucoma of PLD (71 dogs in 17 breeds) and healthy dog (115 dogs in six breeds) were sequenced for the ADAMTS10 mutation.(XLSX)Click here for additional data file.Table S2Primers used for PCR amplification and sequencing of canine ADAMTS10 coding regions.(XLSX)Click here for additional data file.Table S3A total of 10 variants were in the coding and splice site regions of ADAMTS10.(XLSX)Click here for additional data file.


An MRI audit substudy was conducted of patients who underwent an MRI prior to treatment in Australia and New Zealand as part of the international PETACC-6 trial in locally advanced rectal cancer.82 patients of the 127 Australasian patients from 15 centres had rectal MRI scans reviewed for technique, data included in reports and comparison of reports with blinded central reporting by 2 experienced radiologists.82% performed minimum T2 sagittal and T2 axial oblique sequences. The high resolution T2 sequence parameters varied significantly with only 33% obtaining a voxel size of ≤1.3. The rate of inclusion of relevant findings in the reports was; T3 distance in mm 21%, N stage 84%, CRM status 72%, EMVI status 29% and distance from the puborectalis sling 17%. 31% of reports included all of; T stage with T3 substage, N stage and CRM involvement. 17% of reports included these 3 findings and EMVI. Eleven reports used a template with 82% of these including the first 3 findings. The agreement with central reporters was T stage 76%, N stage 70%, CRM status 57% and EMVI 16%.There is significant variation in scan quality and low rates of inclusion of all clinically relevant findings in rectal MRI reports reviewed for this audit. The authors recommend adoption of routine sequences and template reports to improve scan technique and report accuracy in rectal cancer MRI staging scans across Australia and New Zealand.


The result showed that the btwd1 is positioned between SNP marks 7HL_6335336 and 7_249275418 with a genetic distance of 0.9 cM and 0.7 cM on chromosome 7H, respectively. The SNP-based high-density genetic map developed and the dwarfing gene btwd1 mapped in this study provide critical information for position cloning of the btwd1 gene and molecular breeding of barley.A high-density linkage map is a valuable tool for functional genomics and breeding. A newly developed sequence-based marker technology, restriction site associated DNA (RAD) sequencing, has been proven to be powerful for the rapid discovery and genotyping of genome-wide single nucleotide polymorphism (SNP) markers and for the high-density genetic map construction. The objective of this research was to construct a high-density genetic map of barley using RAD sequencing. 1894 high-quality SNP markers were developed and mapped onto all seven chromosomes together with 68 SSR markers. These 1962 markers constituted a total genetic length of 1375.8 cM and an average of 0.7 cM between adjacent loci. The number of markers within each linkage group ranged from 209 to 396. The new recessive dwarfing gene  Hordeum vulgare L.) is an important model crop for plant breeding and genetics because it is diploid and has a long history of genetics research. With the development of molecular markers, high density linkage maps of the barley have been constructed using multiple populations since the 21st century. Varshney et al.et al.et al.SNPs are the most abundant type of genetic markers and ideal for studying the inheritance of genomic regions2Restriction-site Associated DNA (RAD) markers detect genetic variation adjacent to restriction enzyme cleavage sites across a target genome1uzu, sdw1 and denso have successfully been used in barley breeding program until nowuzu, sdw1 and denso genes are located on chromosome 3HL89111213Dwarfism is a valuable trait in crop breeding, because it increases lodging resistance and decreases damages due to wind and rain7889btwd1 mapped on chromosome 7H. The dwarfing gene btwd1 is non-allelic with the uzu and sdw1, which have widely been used in China. The chromosome location of btwd1 is different from those of the uzu, sdw1/denso, br1, br2 genes and QTL PH-7btwd1 gene has been successfully applied in breeding program by Huazhong Agricultural University. In 2011, new barley cultivar Huadamai 9 was registered in Hubei Province with Huaai 11 as male parent. In 2014, new barley cultivar Huadamai 10 was registered in Anhui, Henan and Hubei Provinces with Huaai 11 as male parent. Both cultivars have high yield and good quality.Huaai 11 is a new source of dwarfism, and consisted of desirable agronomic traits such as shortened stature and early maturitybtwd1 dwarfing gene using a double haploid population derived from a cross of Huadamai 6 and Huaai 11. This information could be useful for position cloning of this gene and developing new varieties for plant breeders using MAS.In order to efficiently use this new germplasm for barley breeding program, we have constructed a high-density genetic map based on SNP genotyping and further mapping of 2. Height of plants before ripening was measured in the field from soil surface to top of the main culm (with the spike). The height was calculated as the mean of twelve plantsA DH population including 122 lines was developed from a cross between a common feed barley cultivar Huadamai 6 and Huaai 11 using anther culture in this study. The DH population and parents were planted on the Experimental Farm of Huazhong Agricultural University, Wuhan, China. The field trials were conducted following a randomized complete block design with three replications in 2006–2008. Each of the DH and parental lines were grown in three rows in a plot of 0.6 × 1.5 mThe young leaves from each doubled-haploid (DH) lines and parents were collected and frozen for DNA extraction. The CTAB method was used to extract genomic DNA from about 0.6–1.0 g tissues of each accession8btwd1 were from Ren et al.Frozen DNA samples were sent to the Personal Biotechnology Co., Ltd. . The libraries were quantified using Qubit fluorometer (Invitrogen), Agilent 2100 (Agilent Technologies) and real-time quantitative PCR, and then submitted for sequencing on the Illumina HiSeq2000 platform. Each marker was required to have an allele present in at least 85% of DH individuals. Marker genotypes not meeting the minimum thresholds were scored as missing data. The information of SSR markers was as previously describedBefore constructing genetic maps, SNPs were filtered by excluding those had poor quality data. Low quality SNP included those with NormR results <0.2 and SNP with large numbers of missing values (15% or more). Markers showing identical segregation patterns were also excluded, with one marker per co-segregating group retained. The DH population, consisting of 122 individuals, was utilized to construct a genetic map. The input datasets were constructed from 1,894 genotyped SNP markers and 68 SSR markers. The program Joinmap 4.0 was used to calculate the marker order and genetic distance1838,268 polymorphic SNPs were detected between the parental lines Huadamai 6 and Huaai 11, of which 10,367 polymorphic SNPs were detected in the mapping population. All 10,367 polymorphic SNP markers showed segregation within the DH population. Low quality SNP markers excluded as a result of filtering processes comprised of 5,375 markers with inconsistent parental scores, monomorphic and more than 15% missing data. The remaining 4992 polymorphic SNP markers and 153 of the obtained before polymorphic SSR marker met the requirements for use in the construction of a genetic map. After removing some of the co-segregated markers and non-linked markers, the remaining 1894 SNP markers and 68 SSR markers were used to create 7 high-density genetic linkage groups. This high-density genetic linkage map was generated from 1,962 markers covering seven linkage groups with a total map distance of 1,375.8 cM. The averaged distance between two positions across the whole map was 0.7 cM. The number of markers on different chromosomes ranged from 209 on 1H to 396 on 7H. The genetic distance on different chromosome ranged from 145.0 cM on 1H to 230.9 cM on 3H .btwd1, and Huadamai 6 was a two-row common feed barley cultivarbtwd1 controlling plant height in Huaai 11 was positioned between SNP marks 7HL_6335336 and 7_249275418 with genetic distance of 0.9 cM and 0.7 cM on chromosome 7HL, respectively sdw3 gene (2HS)uzu gene (3HL)8122932sdw1/denso geng (3HL)8142934br2 (4H)836ari-e gene (5HL)br1 gene (7HS)836229383922939412283942433839404345383922938394243btwd1 is a novel gene.We have used these SNP markers to map the dwarfing gene btwd1 in Huaai 11 has widely been used in China, and provides barley breeders with a new source for barley genetic improvement. In order to efficiently use this new germplasm Huaai 11 for barley breeding program, we will verify the identified SNP markers associated with btwd1 in the lines developed from Huaai 11. We have also constructed a large F2 population including 16000 lines derived from a cross of Huadamai 6 and Huaai 11. We will fine map dwarfing gene btwd1 using this large F2 population and the SNP markers. In near future, we will clone the dwarf gene btwd1 and verify its function.Utilization of dwarfing genes in barley breeding programs has greatly increased barley yields, particularly in Asia and Europe8btwd1 in barley breeding.In conclusion, we have constructed a high-density barley linkage map using SNP markers derived from the DH population RAD sequencing. Our study provides a valuable genetic resource for molecular markers, map-based gene cloning, MAS and the sequence assembly of the barley reference genome. At the same time, the linked SNP markers identified in the present study can provide a useful marker-assisted selection tool to transfer the dwarfing gene How to cite this article: Ren, X. et al. SNP-based high density genetic map and mapping of btwd1 dwarfing gene in barley. Sci. Rep.6, 31741; doi: 10.1038/srep31741 (2016).


Scientific Reports5: Article number: 1409710.1038/srep14097; published online: 09162015; updated: 03212016This Article contains an error in the order of the Figures. Figure 6 and Figure 7 were published as Figure 7 and Figure 6 respectively. The correct Figures 6 and 7 appear below as


The objective of this article is to report our first experience of CT guided percutaneous thoracic biopsy and to demonstrate the accuracy and safety of this procedure. This was a retrospective study of 28 CT-Guided Percutaneous Needle Biopsies of the Chest performed on 24 patients between November 2014 and April 2015. Diagnosis was achieved in 18 patients (75%), negative results were found in 3 patients . Biopsy was repeated in these cases with two positive results. Complications were seen in 7 patients (29%), Hemoptysis in 5 patients (20%), Pneumothorax in 1 patient  and vaso-vagal shock in 1 patient . CT Guided Percutaneous Needle Biopsy of the Chest is a safe, minimally invasive procedure with high sensitivity, specificity and accuracy for diagnosis of lung lesions. CT Guided Percutaneous Needle Biopsy of the Chest is a minimally invasive procedure frequently indicated for the diagnosis of lung lesions . This te24 patients were included to this retrospective study; they were admitted to the Thoracic surgery department between October 2014 and April 2015. These patients underwent lung biopsies with a core needle biopsy device 16G or 18G) under CT guidance . All patients with peripheral masses suspicious for malignancy and without suspicion of vascular disease were included in this study. The patients were admitted one day before the procedure to the thoracic surgery department and a coagulation test was done . One patient was on oral anticoagulants for atrial fibrillation (Coumadine*), the treatment was replaced with heparin 5 days before the procedure [G or 18G 24 patients  were included in our study. The patients’ mean age was 62 years (43 - 90 years). 22 patients were active smokers. Mean lesion size was 43 mm and the median traversed parenchymal distance was 20 mm. 21 patients had peripheral lung lesions whereas 3 had central lesions. Lesions were solid in 10 patients, solid and necrotic in 11 patients and purely necrotic in 3 patients. Emphysema was seen in 10 patients and pneumothorax was sustained in 2 patients. A total of 27 biopsies were performed in 24 patients. Histopathology results were positive in 18 of 24 patients and negative in 3 patients who underwent a second biopsy. All had malignant lesions. 7 patients in our study developed complications : Hemorrhage in 5 patients « 20, 8% » (3 mild and 1 moderate); small volume pneumothorax in 1 patient « 4,2% »; vasovagal shock in 1 patient « 4,2% ». Alveolar hemorrhage and pneumothorax resolved spontaneously with no need to proceed to other interventions. One patient who received anticoagulation for atrial fibrillation underwent biopsy. Their Warfarin was stopped 5 days before the procedure and replaced with heparin [In our retrospective study on CT Guided Percutaneous Needle Biopsy, diagnostic accuracy rate was 75%. Non diagnostic biopsy rate was 12,5%. Complications were essentially alveolar hemorrhage 20, 8%) and pneumothorax  in a smaller proportion. Spontaneous reabsorption of alveolar hemorrhage and pneumothorax was seen in all patients with no need to proceed to other interventions such as chest drain insertion. Percutaneous CT-guided core needle biopsy is a safe procedure with a high accuracy for diagnosis of lung malignancies [% and pneIn conclusion, CT-guided percutaneous needle biopsy is a safe procedure with a high diagnostic accuracy rate in the diagnosis of malignant lung lesions.Percutaneous thoracic CT guided biopsy is an accurate procedure for diagnosis of lung malignancies;Major complications are pneumothorax and alveolar hemorrhage;CT is a very helpful tool to localize the lesion and achieve the procedure.Percutaneous thoracic CT guided biopsy with 18 G co axial needle is a very safe procedure with lesser complication rate;CT protocol used in this study limits the radiation exposure of patients;The procedure can also be achieved in emphysematous patients without increasing complication rate.


Formyl peptide-receptor 1 and 2 (FPR1 and FPR2) in mice were identified as receptors with contrary affinity for the PAMP fMLF. Formyl-methionyl-leucyl-phenylalanine is either part of the bacterial membrane and is secreted by the mitochondria of eukaryotic ceslls during apoptosis. Furthermore FPR1 and 2 are described as highly relevant factors for the chemotaxis of immune cells. Their role during the acute liver injury has not been investigated yet.-/-), FPR2 (mFPR2-/-) and wild type (WT) mice were challenged with LPS i.p. for 3 h and 6 h. Liver and serum were sampled for further analysis.Constitutive knockout mice for FPR1 (mFPR1-/- compared to wild type and mFPR2-/- mice. After 6 h, IL-6, TNF-α and CXCL1 were significantly higher in mice lacking mFPR1 or 2. Consistent to these findings the numbers of CD11b+ and Ly6G+ immune cells were altered in the livers. The analysis of TLR2 and TLR4 revealed time and genotype specific changes in theirs gene expression. Additionally, the liver in mFPR1- and mFPR2-deficient mice seem to be more susceptible to apoptosis by showing a significant higher number of TUNEL+-cells in the liver than WT-mice and displayed less Ki67-positive nuclei in the liver.Liver transaminases were elevated in all mice 3 h and 6 h post LPS stimulation. Gene expression analysis displayed a reduced expression of the pro-inflammatory cytokines IL-6 and CXCL1 after 3 h in the mFPR1The results suggest a prominent role of FPRs in the regulation of the hepatic inflammatory response after LPS induced liver injury. Deletion of mFPR1 or mFPR2 leads to deregulation of the inflammatory response compared to WT mice, associated with more severe liver injury represented by higher levels of transaminases, apoptotic cells and a reduced regenerative capacity. The inflammatory response after a liver injury is important for the induction of liver regeneration These prior findings suggest a differential role of FPR in the recruitment of the different leucocyte subtypes and who might have different functions divided in between tissue resident and towards injury site recruited cells -/--/-E. coli LPS  and kept for 3 h and 6 h. The mFPR1 Mice were a kind gift from Dr. Philip Murphy of the National Institute for Allergy and Infectious Diseases, NIH Five 8-10 weeks old wild type, mFPR1Mice were sacrificed 3 h and 6 h post LPS-stimulation, blood and liver were removed and preserved for biochemical and immunohistological assays. After stimulation the mice were kept in SII-long-cages with access to food and water ad libitum. Blood was taken retroorbital before sacrifice of the mice. The serum was separated by centrifugation and stored at −20°C until measurement.All experiments were performed in accordance to the German protection of animals act and with permission of the authority of the federal state North Rhine Westphalia. The study protocol was approved by the institutional animal care and use committee , Duesseldorf, Reference number: 84-02.04.2013.A246).Cryopreserved liver tissue was homogenized and RNA was extracted by using the Nucleospin RNA-II Kit . Afterwards 400 ng of total RNA was converted into cDNA by using the Omniscript reverse transcriptase kit . All proceedings were performed according to manufacturers' guidelines.The gene expression analysis was performed using an ABI 7500 Real-Time PCR . Genexpression analysis for the murine (ms) genes IL-6, TNF-α, CXCL1, TLR2, TLR4, mFPR1, mFPR2 and mFPR3 were performed. The Primers for mFPR1 and mFPR2 were published previously +- or Ly6G+-cells per view field.Formalin fixated paraffin embedded (FFPE) liver samples were cut into 5 µm strong sections and stained for CD11b  and Ly6G . The primary antibody was used in a dilution of 1∶100 and a species specific secondary antibody with a HRP-conjugate was diluted 1∶500 to detect the primary antibody. Visualization was performed using 3,3′-diaminobenzidine tetrahydrochloride (DAB) . Antigen retrieval was executed according to manufacturer's instruction. Nuclei were counterstained with Haematoxylin . For each individual animal/genotype 7 pictures were taken in 100 and 200 fold magnification using an Olympus BX51. The 100 fold magnification was used for overview whereas the 200 fold magnification pictures were used for the detailed analysis and counting of the total CD11b+-cells in the BDL-model, cryosections of liver tissue were made and stained with a CD11b-antibody  used in a 1∶200 dilution. Visualization was performed using an anti-rat ALEXAFLUOR546 . Fluorescent microscopy pictures were taken by an AxioImager Z1 .For the immunofluorescent staining of CD11bFFPE-liver tissue was cut as described above in 5 µm strong sections. The TUNEL-staining was made according to the manufacturers' instruction . Visualization was performed using DAB and Nuclei counterstaining was made with Haematoxylin .The analysis of the ubiquitous cell cycle marker Ki67 was performed using the rat-anti mouse Ki67  in a 1∶50 dilution. The detection was conducted with a secondary, HRP-conjugated anti-rat antibody in a 1∶300 dilution. The visualization was performed with DAB . Nuclei counterstaining was performed using Haematoxylin .The datasets were analyzed using the Studentś T-test and p-values ≤0.05 were regarded as significant and indicated in the respective graph.-/-, mFPR2-/- mice displayed no initial differences among the different genotypes. After LPS stimulation for 3 h the transaminases were significantly increased in mFPR2-/- mice (86 U/L) in comparison to wild type mice (66 U/L), whereas the mFPR1-/- mice displayed elevation after 3 h LPS administration, but did not reach a level of significant difference (63 U/L). The analysis of the 6 h time point displayed that transaminases were significantly upregulated in the serum derived from mFPR1-/-(296 U/L) or mFPR2-/-(291 U/L) mice compared to control (76 U/L) mice after LPS-treatment  and aspartate-aminotransferase (AST) of WT, mFPR1reatment . The hisreatment .-/- and the mFPR2-/- mice displayed lower number of CD11b+) and the Ly6G+-cells ). Also significantly lower were the CD11b+-cells ) and the number of Ly6g+-cells ) in the mFPR2-/- mice in comparison to WT mice. At the later time point, 6 h after intraperitoneal application of LPS (+-cell infiltrates per view field in comparison to wild type mice (96±6). Likewise to the CD11b+-cells a higher presence of Ly6G+-cells was found in the livers of mFPR1-/— (160±24) and mFPR2-/—mice  were significantly lower . Taken together different degrees of inflammation occurred in regard to the deficiency of either mFPR1 or mFPR2 as wells as in regard to the temporal progression of inflammation.In order to get a better overview of the different immune cell subtypes infiltrating into the liver, we performed immunohistological straining for the surface markers CD11b and Ly6G. These markers identify two relevant cells types involved in promotion of liver injury. CD11b is known as a marker for activated monocytes whereas Ly6G is known to detect neutrophil granulocytes specifically. The immunohistological analysis of CD11b and Ly6G displays a strong presence of both cell types at 3 h as well as 6 h post LPS-treatment. At the time point 3 h after LPS the mFPR1of CD11b+ and Ly6G whereas the mFPR2-/- mice had a significantly higher expression compared to WT-mice (p<0.01). At 6 h post LPS injection the expression of IL-6 was significantly lower in the wild type animals compared to mFPR1 (p<0.05) and mFPR2-deficient mice (p<0.05). The expression of TNF-α .The quantitative analysis of TLR4 and TLR2 expression displayed a highly different pattern. Toll-like receptor 2 gene expression is strongly elevated in all mice strains which underwent LPS-stimulation at time point 3 h. The differences in gene expression are significant between mFPR2periment . The anaperiment  showed aTo understand whether livers lacking mFPR1 and mFPR2 were more susceptible to pro-apoptotic signalling, a TUNEL-assay was performed .+-cells in mFPR1 (1.4%) and mFPR2 deficient mice (2.03%) compared to WT mice (1.2%). At 3 h only mFPR2-/- mice had a significantly higher number of TUNEL+-cells detectable in the liver. At the 6 h time point the amount of TUNEL+-cells was elevated in all mice strains. However, mFPR1-/- mice (2.53%) and mFPR2-/- mice (2.91%) had both significantly more apoptotic cells detectable in comparison to WT mice (1.28%) 6 h after injection of LPS . The mFPR2-/- mice itself displayed also a non-significant higher tendency for the number of TUNEL+-cells in comparison to the mFPR1-/- mice at 3 h and 6 h post LPS administration.The analysis of the 3 h time point revealed a higher percentage of TUNELn of LPS   ese mice  during t+-nuclei in the liver of either mFPR1 or mFPR2-deficient mice in comparison to wild type mice  and mFPR2-/-  displayed a significantly lower number of proliferative cells compared to WT mice  . At 6 h post LPS administration the differences between WT  and mFPR1-/-  were still present but did not reached a level of significance. The significant lower proliferation of mFPR-deficient mice  compared to WT mice was still present at this time point (p<0.05).Liver regeneration is a response to compensate the loss of cellular mass after an injury. To understand whether mFPR1 and mFPR2 might have an impact on liver regeneration we analysed the proliferative capacity of livers lacking either mFPR1 or mFPR2. For this purpose, the ubiquitous cell cycle marker Ki67 was stained at 0 h, 3 h and 6 h post LPS stimulation. The analysis of the staining revealed a lower number of Ki67ype mice . At 3 h Formyl peptide receptors 1 and 2 are known to function as important mediators of chemotaxis of hematopoietic cells -/- mice. The mFPR2-/- mice displayed a significant higher level of ALT in the serum. At the later 6 h time point, both, mFPR1-/- and mFPR2-/-, displayed a significantly higher Level of ALT. For AST a slightly different pattern appears. Wild type mice had the highest levels of AST detectable in the serum compared to mFPR1 and mFPR2-deficient mice. 6 h post LPS the mFPR1 and mFPR2 knockout mice displayed significant higher levels of AST in the serum. These findings support a protective role for formyl peptide receptors during progression of LPS induced liver injury. The histological analysis after LPS-stimulation revealed a differential recruitment of immune cells in a time and genotype dependent manner. The cytokine IL-6 is not only known as a recruiting molecule for immune cells. It is described as one of the main drivers of hepatoprotection during liver injury -/- and FPR2-/- mice 6 h after LPS stimulation. Interestingly mFPR2-/- mice showed a lower tendency regarding the number of Ly6G+-cells visible per view field. The closest explanation for this phenomenon is the link to the neutrophil recruiting cytokine CXCL1 (CXCL1) which showed the same tendency at least on mRNA level at 3 h and at 6 h post LPS-stimulation. Differential roles for mFPR1 and mFPR2 regarding immune cell homing is not excluded for granulocytes and supported by the literature We compared the effect of mFPR1 and mFPR2 deficiency after LPS-stimulation, mimicking a bacterial mediated liver injury. The initial analysis of the transaminases AS and ALT  from the-/- mice was the highest of all mice strains used in this study. The anti-apoptotic capabilities of FPR2/FPRL1 in primary human neutrophils are controversially discussed. Previous observations by Nagaoka et al. revealed that FPR2 is protective together with the P2X7 receptor -/- and mFPR2-/- mice in comparison to WT-mice. Furthermore these findings suggest a specific anti-apoptotic signalling of mFPR2 towards TNF-α induced pro-apoptotic signalling. Stimulation experiments using a combination of fMLF and pharmacological inhibitors for p38 and MEK resulted in reduced chemotaxis, adhesion and release of superoxide by neutrophils A further finding of the regulation of the anti-inflammatory response is visible for other PAMP-receptors such as TLR4 and TLR2 . The ana2+-Signalling into the focus of attention and suggests an involvement of Ca2+ induced signalling in the mediation of cell cycle progression The investigation of liver proliferation to compensate the loss of liver mass, displayed an impairment of regenerative capacity in mFPR1 and mFPR2-deficient mice. Both genotypes showed a lower proliferation at 3 h and 6 h post LPS-induced liver injury, suggesting critical involvement of mFPR1 and mFPR2 in liver regeneration. Recent studies of liver regeneration showed that other member of the GPCR family especially the cannabinoid type 1 receptors support these findings. Furthermore, it puts the CaThe analysis of the mRNA expression of mFPR1-3 in the BDL model shows an increase of mFPR1 and mFPR3 gene expression in concordance with the number of infiltrating monocytes in the liver . This fiThe consequences of FPR deficiency in an acute model of liver injury seem to be a loss of anti-apoptotic and anti-inflammatory capabilities. Our findings suggest a pro-survival, anti-inflammatory role during an acute LPS-induced liver injury. This is in concordance with previous findings suggesting a positive role for FPR during the phase of acute injury Furthermore, the role of FPRs in progression of chronic inflammatory liver diseases is not understood and ought to be investigated. Based on recent investigations FPR expression could also be shown on natural killer cells Taken together both receptors are important to maintain a functional response to LPS induced liver injury. Furthermore our data suggests that mFPR1 and mFPR2 might also be involved in processes such as liver regeneration and might also have relevance not only during the acute liver injury, but also during chronic liver injury. Further experiments will provide prove to this hypothesis.Figure S1Immunofluorescent staining for CD11b reveals an increase over time after BDL. CD11b+-cells were visualized using Alexa546. Nuclei were counterstained using DAPI.(TIF)Click here for additional data file.


DPY19L2 whole coding sequence. Furthermore we showed that the DPY19L2 protein is located in the inner nuclear membrane of spermatids during spermiogenesis and that it is necessary to anchor the acrosome to the nucleus thus performing a function similar to that realized by Sun proteins within the LINC-complex (Linker of Nucleoskeleton and Cytoskeleton). SUN1 was described to be necessary for gametogenesis and was shown to interact with the telomeres. It is therefore possible that Dpy19l2 could also interact, directly or indirectly, with the DNA and modulate gene expression during spermatogenesis.Globozoospermia is a male infertility phenotype characterized by the presence in the ejaculate of near 100% acrosomeless round-headed spermatozoa with normal chromosomal content. Following intracytoplasmic sperm injection (ICSI) these spermatozoa give a poor fertilization rate and embryonic development. We showed previously that most patients have a 200 kb homozygous deletion, which includes Dpy19l2 knock out and wild type mice in order to identify a potential deregulation of transcripts that could explain the poor fertilization potential of Dpy19l2 mutated spermatozoa.In this study, we compared the transcriptome of testes from DPY19L2 knock out and wild type mice. The transcriptome was carried out using GeneChip® Mouse Exon 1.0 ST Arrays. The biological processes and molecular functions of the differentially regulated genes were analyzed with the PANTHER software.RNA was extracted from testes from Dpy19l2) were down-regulated. These genes were found to be involved in DNA/RNA binding, structural organization, transport and catalytic activity.A total of 76 genes were deregulated, 70 were up-regulated and 6  contains supplementary material, which is available to authorized users. A recent study supported by the World Health Organization indicates than in 2010, an estimated 48.5 million couples worldwide were unable to have a child after five years I is known to play an important role in male fertility. [Ca2+]I signaling is the primary regulator of sperm flagellum beating and calcium intracellular rise is known to be essential for the acrosome reaction [3 in mouse sperm [Dpy19l2 KO mice two calcium binding proteins are up-regulated : Caps2 and Sgk3  during spermiogenesis. We also observed that several genes encoding proteins involved in transports, and in particular Abca1, involved in the cholesterol efflux, were deregulated. This could also contribute to the poor fertilization potential of the round-headed spermatozoa. Secondary anomalies stemming from the morphological abnormalities of the sperm could also lead to a wide range of protein deregulation as exemplified by the absence of PLCzeta. A proteomic analysis of these deregulations could permit to have a functional view of the extent of the molecular anomalies present in Dpy19l2 KO mice. Further work will permit a better comprehension of molecular mechanism involved in spermatogenesis and in the physiopathology of globozoospermia.We showed that Dpy19l2Additional file 1: Table S1: RNA quantification. (DOC 27 KB)Additional file 2: Table S2: Ratios of transcripts values measured in Dpy19l2 WT and KO mice. (XLS 66 KB)Additional file 3: Table S3: PANTHER output of all deregulated genes in Dpy19l2 KO mice. (XLS 60 KB)


Recent evidences suggest an association between unresponsive chronic daily headache (CDH) and idiopathic intracranial hypertension without papilledema (IIHWOP). Diagnosis can be challenging. The CSF opening pressure (OP) cutoff value greater than 200 or 250 mmH2O is debated as the role of transverse sinus stenosis (TSS).To investigate the frequency of IIHWOP and TSS in adult patients with refractory CDH.In a prospective study, patients with refractory CDH were consecutively enrolled. Each participant underwent ophthalmologic evaluation and Optical Coherence Tomography to rule out the presence of papilledema; cerebral MR venography (MRV) to detect TSS; and a lumbar puncture in the lateral decubitus position to measure OP.Among the 23 consecutive patients enrolled, 19  completed the study, 4 female patients dropped out. None of the 19 patients had papilledema. We found a TSS in 11 of 19 cases (58%): bilateral in 3 patients and unilateral in 8 cases. All of 19 cases displayed OP lower than 250 mmH2O (range 102-245) and normal CSF composition. We found a OP greater than 200 mmH2O only in four patients  (17%): two of them achieved an improvement of headache intensity and frequency after 12-18 ml CSF withdrawal; one of them had bilateral TSS.Our preliminary data suggest that IIHOWP may be an underestimated condition. We suggest to keep the 200 mm H2O cutoff value at least until the role of venous stenosis will be clarified.No conflict of interest.


Being widely used NPs, their toxicity assessment studies help in understanding the adverse effects to the humans. It is likely that NPs exposure to the humans can be through different routes but will finally reach the liver. Therefore, an attempt was made to explore the genotoxicity of TiO2 NPs on human liver cells (HepG2).TiO2 NPs were characterized by transmission electron microscopy (TEM) for their primary size, shape and dynamic light scattering for their size, size distribution and zeta potential in culture medium. Cellular uptake of TiO2 NPs in HepG2 cells was detected using flow cytometry method. Moreover, ultrathin sections of cells were analysed using TEM to visualise the internalisation of TiO2 NPs. The genotoxic potential of TiO2 NPs was assessed by micronucleus assay using the conventional and flow cytometry methods.TiO2 NPs revealed a mean diameter size of 30-70 nm and DLS measurements showed a mean hydrodynamic diameter and zeta potential of 192.5 ± 10 nm and -11.4 ± 1.2 mV, respectively. The electron microscopy and flow cytometry studies for particle internalisation showed a significant cellular uptake of TiO2 NPs in the human liver cells (HepG2). A significant (p < 0.05) induction in micronucleus formation was observed at 20 µg/ml of TiO2 NPs exposure when compared to control cells. However further treatment to HepG2 with higher concentrations (40 and 80 µg/ml) showed a decrease in micronucleus formation than 20 µg/ml.TEM analysis of TiO2 NPs than control.In contrary, the flow cytometric results exhibited a significant concentration dependent induction of micronucleus in HepG2 cells exposed to all concentrations  of TiO2 NPs on prepared slides, which hinders the counting of micronucleus . However, in the flow cytometry analysis, these nanoparticles do not interfere with the optics. Hence, it is proposed that in the case of NPs treatment, flow cytometry based micronucleus assay should be used instead of the conventional method.The difference in the micronucleus frequency obtained from conventional and flow cytometry methods in HepG2 may be due to the accumulation of TiO


Acetanaerobacterium and Ruminococcus with relatively high abundance. The characterization of the microbial community corroborated the digestion performance affected at the agitated condition, where lower methane yield and delayed methane production rate were observed. This was further verified by the accumulation of propionic acid in the agitated digester.To investigate the distribution and dynamics of microbial community in anaerobic digestion at agitated and non-agitated condition, 454 pyrosequencing of 16s rRNA was conducted. It revealed the distinct community compositions between the two digesters and their progressive shifting over time. Methanogens and syntrophic bacteria were found much less abundant in the agitated digester, which was mainly attributed to the presence of bacterial genera  The divergent effect of agitation on anaerobic digestion has been reported by some studies, while most of which investigated the conventional physiochemical propertiesClassically, environmental microbial communities are analyzed by construction of 16S rRNA clone libraries and the subsequent sequencing of individual clones. The approach, termed Sanger sequencing, has been applied in Tian et al (2013) to compare the microbial community structures for anaerobic digestion at agitated and non-agitated condition, led to the identification of some major microbial phylotypes in bacteria or archaea domain. Due to the fact that a few numbers of clones can be affordably sequenced, Sanger method has its limitation in revealing the whole complexity of microbial communities and is unlikely to adequately represent the genetic diversity. New development of high-throughput next-generation sequencing technologies (NGS), such as 454 barcoded pyrosequencing, not only eliminates the laborious step of preparing clone libraries, but also makes large scale environmental sequencing cost effective and keeps the bias small 4 production, soluble Chemical Oxygen Demand (sCOD) and Volatile Organic Acids (VOAs) profiles.There were limitations in the previous study Two digesters of 5 L, named digester 1 and 2, were built by modifying Pyrex glass jars −1day−1. Digester 1 and 2 were then emptied and washed and residual substrates were discarded. In trial 2, both digesters were fed with fresh 0.3 kg (wet weight) of washed sugar beet tailings and re-inoculated with approximate 3 L of its own sludge liquor recovered from trial 1 (Sludge liquors from digester 1 and 2 were not mixed). Trial 2 ended at day 14 (or day 32 cumulatively) when the gas production from both digesters was less than 0.05 L@STP L−1day−1.Digester 1 was operated under non-agitated condition. Two kilograms of bulking materials  were added into digester 1 along with the feedstock to prevent substrate compaction and floatation. Digester 2 was operated under agitated condition without adding the bulking agent. The digester content was continuously mixed at 180 rpm by using a 50.8 mm × 9.5 mm PTFE coated polygon bar and a large volume magnetic stirrer (Bel-Art ScienceWare Cool Stirrer). Two experimental trials were carried out. In trial 1, both digesters were inoculated with 3 L inoculum taken from an anaerobic digester that has been digesting with sugar beet tailings for months. Trial 1 ended at day 18 when the gas production from both digesters was less than 0.05 L@STP LTotal Solids (TS) and Volatile Solids (VS) contents were determined for the feedstock sugar beet tailings 4 and CO2) was analyzed using Fisher Model 1200 Gas Partitioner. sCOD concentration was determined using Hach method 8000. VOA analysis was conducted using Shimadzu gas chromatograph (GC-9AM equipped with a flame ionization detector) for acetic, propionic, isobutyric, butyric, isovaleric and valeric acid concentrations Daily biogas production from the digesters was measured by a positive displacement gas meter. Gas composition , digester 1 liquor and digester 2 liquor. Digester liquors were sampled at day 3, 5 and 18 for trial 1 and day 1, 4, 8, 11, 12 and 14 for trial 2 . A total 17 samples were analyzed.Total DNA was extracted and purified by using FastDNA Kit  and PowerClean DNA Clean-up Kit  respectively, according to the manufacturer's instruction. The quality of DNA was verified by agarose gel electrophoresis. Extracted DNA was stored at −20°C until further use.2+, and 200 µM of dNTP ] and 1 µL of barcoded primers (100 pmoles each). The amplification protocol was as follows: initial denaturation at 94°C for 3 minutes, followed by 30 denaturation cycles at 94°C for 45 seconds, annealing at 50°C for 30 seconds, and extension at 65°C for 90 seconds, with a final extension for 10 minutes at 65°C. The tree replicated PCR products were combined for each sample, purified using the QIAquick PCR Purification Kit  and quantified using on-chip gel electrophoresis with Agilent 2100 Bioanalyzer and DNA Lab Chip Kit 7500.For each sample, the V4 hypervariable region of16S rRNA gene was PCR-amplified using the F515/806R primer set that was designed for accurate phylogenetic placement of a broad range of archaeal and bacterial taxa with few biases Because each sample was amplified with a known tagged primer, an equimassic mixture ampicon from different samples could be sequenced simultaneously. Among the total 17 samples, 9 samples were sequenced simultaneously in the first batch, and 8 samples in the second batch. The quantity of each PCR product was made equal within a batch: 432 ng of each PCR product in batch 1 and 480 ng in batch 2.www.mothur.org/wiki). First, sequences were de-noised and filtered, and chimeric sequences were removed (chimera.unchime) to improve data quality. Second, qualified sequences were clustered to operational taxonomic units (OTUs) defined by a 97% similarity level. Third, diversity analyses and diversity index calculations were performed for Chao1 richness estimation, Good's coverage and rarefaction curves. The variability of community composition between samples was evaluated with Principal Component Analysis (PCA), which is a multivariate ordination method that visually represents distance between samples. More similar communities would be placed closer in the ordination. A similarity matrix of Yue and Clayton (ThetaYC) distances that take into account of both membership and relative abundance was calculated to determine each sample's position in the PCA ordination. Statistical analyses were conducted using the mothur package Batch 1 and 2 were sent to Interdisciplinary Center for Biotechnology Research (ICBR) at University of Florida for pyrosequencing using a 454 GS-FLX sequencer . The raw sequence data were sorted based on the sample specific barcode for batch 1 and 2, respectively, and primer and barcode sequences were then trimmed from the sorted sequences. The trimmed sequences from batch 1 and 2 were then combined and processed through mothur (The sequence data are available from the NCBI Sequence Read Archive (Run SRR1283194).TS and VS contents of sugar beet tailings, loading quantities and packing density were determined for 2 trials and presented in 4 production rate for digester 1 peaked at 0.70 m3 d−1 (kg VS)−1 on day 5, and 0.34 m3 d−1 (kg VS)−1 on day 11 for digester 2. Trial 2 was started by flooding digester 1 and 2 with digester liquor left in trial 1. Digester 1 reached higher CH4 production rate during trial 2 than during trial 1 (0.94 m3d−1(kg VS)−1 on day 4), whereas digester 2 exhibited similar production rate (0.35 m3d−1(kg VS)−1 on day 7). Both digesters reached their maximal production rate earlier than during trial 1.During trial 1, CH4 yield for digester 1and 2 in both trials were shown in 4 yield of 0.37 m3 CH4 at Standard Temperature and Pressure (STP) (kg VS)−1 and digester 2 achieved CH4 yield of 0.24 m3 CH4 at STP (kg VS)−1 at the end of trial 1. At the end of trial 2, the CH4 yield was 0.35 m3 CH4 at STP kg VS−1 for digester 1 and 0.21 m3 CH4 at STP kg VS−1 for digester 2.Cumulative CHProfiles of soluble COD (sCOD) and VOA concentration of digester 1 and 2 were shown in The main VOAs detected in both digesters are acetic, butyric and propionic acids. In digester 1, acetic acid was detected with the highest concentrations among VOAs at the beginning of each trial, reaching a maximum around day 3 to 5. Its concentration decreased rapidly to a negligible amount as methane was produced. Similarly, but to a less extent, butyric acid slightly accumulated at the beginning of both trials and then rapidly disappeared from day 4 to 7. Propionic acid was present at constantly low concentration.In digester 2, the concentration of acetic and butyric acid peaked between day 8 to day 11, and the degradation was delayed compared to that in digester 1. Profiles of propionic acid showed no significant degradation, resulting in an evident accumulation that reached 1000 mg/L in trial 2.A total number of 6,993 sequences with average length of 204 bps was obtained from 14,773 raw sequence reads after the quality improving process. The number of sequences for different samples ranged from 126 to 994. Of the total sequences obtained, 149  represented lineage from archaea domain. Across 17 samples, 1,137 OTUs (defined at 97% sequence similarity level) were identified. All samples had Good's coverage above 70% and the rarefaction curves were provided in The variability of community composition was evaluated by a PCA plot . In the Thermotogales and Petromonas were found dominant in the inoculums, digester 1 and digester 2 in general. At beginning of trial 1, the community composition in digester 1 and digester 2 diversified and shifted greatly from that of the inoculums. Phylotype Bacillaes gained dominance at day 3 but decreased quickly with progression of the digestion. In trial 2, changes in the community composition were less dynamic.1,137 OTUs were taxonomically classified to 117 different phylotypes at similarity threshold of 80%. Top 23 phylotypes with highest relative abundance were selected and analyzed for each sample . The dynAcetanaerobacterium, Ruminococcus and Ruminococcaceae were detected in digester 2 and their relative abundance followed an increasing pattern with development of the digestion. These phylotypes were either not detected or detected at a very low abundance in digester 1. In addition, phylotype Anaerobaculum exhibited a different distribution between digester 1 and 2. While Anaerobaculum gradually developed and reached relative abundance of 15% in digester 1 (after 15 days), it was identified with constantly low abundance in digester 2.Despite the aforementioned similarities, digester 1 and 2 differed from each other in many ways with regard to the community compositions. Phylotype Methanoculleus and Methanosarcina were found abundant among methanogens. In digester 1, the relative abundance of methanogens increased over time, peaking at 5% for trial 1 (day 5) and 8% for trial 2 (day 22), respectively. In contrast, the development of methanogens in digester 2 was slow and the relative abundance never exceeded 1.4% (day 18) in trial 1. It continuously reduced in trial 2 until no significant detection was obtained near the trial end. Selected phyloptyes  suggested a lower microbial diversity as verified by the Chao1 richness estimation (results not shown) in comparison with digester 1. Interestingly, the community evolving between digester 1 and 2 seemed to follow a similar path from day 0 to day 5 but subsequently developed in separate ways to disparate compositions. This could imply the effect of agitation was not instantaneous but rather cumulative.Anaerobaculum has been known to degrade peptide and a limited number of carbohydrates Anaerobaculum-related species being dominant Anaerobaculum seemed to play a role in propionate degradation, even though it has not been recognized as a syntrophic propionate utilizing bacterium. It can be postulated that the low abundance of Anaerobaculum in digester 2 probably resulted in accumulation of propionic acid.16s rDNA sequences reads were compared to the entries of RDP database, and assigned to phylogeneic groups. However, a large portion of sequences were classified to an unclassified phylotype, suggesting the complexity of microbial communities in anaerobic digestion is yet to be characterized. Among identified phylotypes, Methanoculleus and Methanosarcina species. Members of Methanoculleus are hydrogenotrophic methanogens Methanosarcina species are mostly acetoclasic but also able to use H2Methanosarcina were generally more abundant than from Methanoculleus. The relative abundance of Methanosarcina was seen to follow a dynamic that coincided with the digestion performance. The marked increase from day 3 to day 5 in trial 1 and day 1 to day 4 in trial 2 corresponded to the high CH4 production rate and the significant reduction of acetic acid during a similar time frame. Methanosarcina spp. has been reported to have higher growth rates and tolerance to pH changes and could potentially lead to stable methenogensis in anaerobic digestion 4 production and the accumulation of propionic acid in digester 2 particular for trial 2.Archaeal (mostly methanogens) versus bacterial community ratio generally agreed with the reported methanogen proportions ranging from 0.1% to 15% of the total microbial population Acetanaerobacterium, Ruminococcus and Ruminococcaceae were found with high relative abundance in digester 2. Those species were closely related and all belonged to family Ruminococcaceae, which are known to degrade cellulose and produce hydrogen (H2) as one of the fermentation products Ruminococcus species has been widely used for H2 production from a variety of feedstockRuminococcus were reported to produce propionate other than ethanol as fermentation products, which could also lead to propionic acid accumulation Phylotypes 2) production is energetically unfavorable due to proton being a poor electron acceptor. The development of syntrophic communities allows H2 production to become energetically favorable and sustain degradation of organic compounds and production of CH4. Due to that syntrophic metabolism, methanogenic activity has to be suppressed in order to produce free H2 or it would have been readily converted to CH4 in anaerobic digestion. pH control is desirable for H2 production because methanogenic activity drops sharply in an acidic environment 2 at low partial pressure has been reported in anaerobic processes operated at neural or near neutral pH Ruminococcus related bacteria produced free H2 in digester 2 even though it was typically considered energetically unfavorable. Some studies seemed to support this speculation. Rychlik and May investigated the effect of Methanobrevibacter smithii on growth rate, organic acid production and specific ATP activity of Ruminococcus albus in the co-ulture Ruminococcus albus did not receive energetic advantage from co-culturing with the methanogen and the syntrophic metabolism was not preferred. Zhou et al investigated the effect of methanogenic inhibitors on methaneogens and three rumen bacteria Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciensRuminococcus may not always rely on the syntrophic relationship with methanogens to grow and produced free H2 in digester 2. Its concentration was expected to be very low that was not analyzed in biogas composition, but may have exerted enough inhibition on methanogensis as discussed below.In anaerobic digestion, hydrogen could be generated through fermentation of intermediate products as sugars or VOAs Desulfotomaculum, Pelotomaculum and Syntrophomonas were detected in both digester 1 and 2 at low proportion Click here for additional data file.Table S1Substrate characteristic and loading quantities for digester 1 and 2.(DOCX)Click here for additional data file.Table S2Taxonomic composition of digester 1 and digester 2 at the rank of phylum, order and genus.(DOCX)Click here for additional data file.


Cross talk between Fgf and Hedgehog (Hh) signaling was critical for the specification of GABAergic interneurons and oligodendrocytes. The expression of fgf16 in the forebrain was down-regulated by the inhibition of Hh and Fgf19 signaling, but not by that of Fgf3/Fgf8 signaling. The fgf16 morphant phenotype was similar to that of the fgf19 morphant and embryos blocked Hh signaling. The results of the present study indicate that Fgf16 signaling, which is regulated by the downstream pathways of Hh-Fgf19 in the forebrain, is involved in forebrain development.Fibroblast growth factor (Fgf) signaling plays crucial roles in various developmental processes including those in the brain. We examined the role of Fgf16 in the formation of the zebrafish brain. The knockdown of  The forebrain becomes regionally subdivided into the telencephalon and diencephalon during early embryonic brain development in vertebrates. The telencephalon is further subdivided into the rostrally positioned subpallial  telencephalon and more caudally located pallial  telencephalon. The diencephalon is comprised of the hypothalamus, zona limitans intrathalamica (ZLI), ventral thalamus, dorsal thalamus, and pretectum Fgf8 specifies rostral telencephalic fate and represses caudal telencephalic fate in mice and zebrafish fgf3 in zebrafish affects the expression of genes that have been implicated in the development of the forebrain fgf3 and fgf8 functions revealed that fgf3 and fgf8 possessed a unique and combinatorial function in regional patterning of the forebrain and hindbrain Fgf15 knockout mice demonstrated that Fgf15 repressed rostral telencephalic fate fgf19, which is the Fgf15 orthologue in zebrafish, is known to be essential for development of the ventral region of the telencephalon and diencephalon in zebrafish Fgfs comprise a large family of at least 22 members in vertebrates Fgf16, which was originally identified in the rat heart, is predominantly expressed in the heart at adult stages Fgf16 is expressed in the heart, inner ear and brown adipose tissue during embryonic development in mammals Fgf16 knockout mice have been reported and their phenotypes may potentially be affected by genetic backgrounds. Fgf16 knockout mice on a C57BL/6 background exhibited a decrease in the proliferation of embryonic cardiomyocytes and pathophysiological roles in the postnatal heart, whereas the cardiac phenotype of Fgf16 knockout mice on a 129/B6 background has not yet been examined Fgf16 knockout mice on a Black Swiss background died at approximately E11.5 fgf16 is expressed in zebrafish in the pectoral fin bud and forebrain in addition to the otic vesicle fgf16 knockdown zebrafish embryos indicated that fgf16 is an apical ectodermal ridge (AER) factor that is crucial for pectoral fin bud outgrowth fgf16 morphants display morphological abnormalities in the brain. However, these abnormalities have not yet been elucidated in detail.fgf16 during brain development in zebrafish. Our results demonstrated that fgf16 was critical for cell proliferation in the forebrain and midbrain. fgf16 was also critical for development of the ventral region of the telencephalon and diencephalon, and was implicated in the specification of γ–aminobutyric acid (GABA)ergic interneurons and oligodendrocytes in the telencephalon and diencephalon. On the other hand, fgf16 was not implicated in the specification of tectal and tegmental fates. fgf3, fgf8 and fgf19 have also been shown to be involved in the specification of GABAergic interneurons and oligodendrocytes in the ventral region of the forebrain fgf16 and fgf3, fgf8, and fgf19 in the forebrain.In the present study, we examined the roles of Danio rerio) were maintained, according to The Zebrafish BookZebrafish  or universal control MO (5 ng) was injected into the two-cell embryos of zebrafish. fgf3 MO (10 µg/µl) and fgf8 MO (20 µg/µl) were injected at a volume of 0.15–0.25 nl into the two-cell embryos. fgf19 MO was injected at 10 µg/µl into the four central blastomeres of 16-cell embryos.Morpholino oligonucleotides (MOs) were synthesized by Gene-Tools, LLC . MOs were diluted in Danieau buffer fgf16, full-length fgf16 cDNA was amplified by PCR and inserted into the vector pCS2+ fgf16 mRNA was made by invitro transcription using SP6 polymerase . mRNA was diluted to 0.5 ng/µl with distilled water and injected at a volume of 0.5 nl into 2-cell embryos.To construct Proliferating and apoptotic cells were detected using a rabbit polyclonal anti-phosphorylated histone H3 (H3P) (Upstate Biotechnology) antibody and the DeadEndTM colorimetric detection kit (Promega), respectively Whole mount immunostaining was performed as described previously Cyclopamine  fgf16 was expressed in the pectoral fin bud and also that the knockdown of fgf16 function resulted in the absence of fin bud outgrowth at 5 days post-fertilization (dpf) fgf16 morphants exhibited abnormalities at 5 dpf fgf16 morphants were morphologically distinguishable from the wild type at 24 hours post-fertilization (hpf). fgf16 morphants showed morphological abnormalities in the forebrain at 24 hpf  constriction and exhibited a failure to evaginate laterally in the midbrain at 24 hpf . On the other hand, control MO-injected embryos developed normally during embryogenesis fgf16 RNA with fgf16 MO1 rescued the brain defects caused by fgf16 MO1 (n = 10/13)  . These rfgf16 is expressed in the brains of zebrafish embryos during 18–36 hpf fgf16 has not yet been examined in detail in the brain during neural development. We here examined the spatiotemporal expression pattern of fgf16 in the zebrafish embryonic brain in detail using whole mount insitu hybridization. The expression of fgf16 was first detected in the most ventral part of the anterior telencephalon primordium at 14 hpf  was specifically detected in the mitotic cells in mitotic phase (M-phase) fgf16 morphants was significantly lower than that in wild-type embryos at 24 hpf , B.fgf3, fgf8, and fgf19 have been implicated in patterning events in the zebrafish forebrain fgf16 morphants at 24 hpf. The expression of emx1, which is normally detected in the pallial domain of the telencephalon, was observed in the entire region of the telencephalon in fgf16 morphants (n = 28/32)      . Further= 15/16) . In cont= 27/31) . On the = 21/21) . The ect= 13/13) . In contfgf16 affected diencephalic specification. In addition to the ventral telencephalon, dlx2 is normally expressed in the ventral thalamus. The expression of dlx2 in the ventral thalamus was reduced in fgf16 morphants at 24 hpf (n = 27/31)     . On the ctively) . We also= 14/14) . On the = 14/14) . Thus, tfgf16 morphants showed morphological abnormalities in the MHB constriction and midbrain. Therefore, to investigate whether fgf16 was involved in MHB development, we examined the expression of fgf8 in fgf16 morphants at 24 hpf. The expression of fgf8 was detected in the MHB of fgf16 morphants (n = 27/27)  , which ifgf16 was involved in specification of the midbrain. Otx2 is an important player in the regulation of midbrain patterning otx2 was unaffected in the midbrains of fgf16 morphants at 24 hpf (n = 13/13) , respectively  . Furtherectively . These rfgf16 MO affected neuronal differentiation in the forebrain, the expression of the basic helix-loop helix (bHLH) proneural gene, ngn1, was analyzed in fgf16 morphants at 24 hpf. The expression of ngn1 was unaffected in the dorsal telencephalon of fgf16 morphants, whereas it was reduced in the diencephalon (n = 11/11)   . We then= 15/20) . These rgad1 encoding glutamic acid decarboxylase was found to be expressed specifically in GABAergic interneurons fgf16 had any effects on GABAergic interneuron differentiation in the forebrain, gad1 expression was analyzed in fgf16 morphants at 28 hpf. gad1 was expressed in the subpallial telencephalon and nucleus of the tract of the postoptic commissure (nTPOC) in the forebrain fgf16 morphants, the expression of gad1 was severely reduced in both the ventral telencephalon and the nTPOC (n = 27/28) (fgf16 morphants. GABA-immunoreactive cells were not detected in the forebrains of fgf16 morphants at 3 dpf (n = 20/20)  (fgf16 was involved in the formation of myelinating oligodendrocytes. PLP (proteolipid protein)/DM20 is a marker of oligodendrocyte differentiation and is expressed in newly formed oligodendrocyte progenitor cells, well before myelination plp was not detected in the forebrains of fgf16 morphants at 4.5 dpf (n = 12/12) (plp in the hindbrain disappeared in fgf16 morphants at 4.5 dpf (n = 10/12) (fgf16 morphants at 4.5 dpf (n = 11/11)  2.2, the postmitotic marker of glutamatergic neurons, was analyzed in fgf16 morphants at 28 hpf. In fgf16 morphants, the expression of slc17a6a was unaffected in both the pallial telencephalon and diencephalon (n = 14/14)  . We also= 20/20) . Oligode= 14/20) . Further= 12/12) . In addi= 10/12) . The imm= 11/11) , B. Thes= 14/14) . This refgf16 led to abnormalities in the regionalization and generation of specific cell types such as GABAergic interneurons and oligodendrocytes in the forebrain. Hh signaling is critical for regulating the expression of fgf3, fgf8, and fgf19 in the forebrain and that of fgf19 and fgf22 in the midbrain Fgf16 was responsive to Hh signaling. Since the alkaloid cyclopamine completely blocked Hh signaling at the level of Smoothened, which transduces Hh signals, in zebrafish Fgf16 in embryos treated with cyclopamine. In embryos treated with cyclopamine, fgf16 expression was lost in the forebrain at 16 and 25 hpf  (fgf16 expression in the midbrain was lost in embryos treated with cyclopamine (n = 10/10)  forebrain patterning and promotes the GABAergic neuronal/oligodendrocyte lineage restriction of forebrain stem cells ctively) . Further= 10/10) . All con= 10/10) . These rfgf16 led to abnormalities in the regionalization and generation of specific cell types such as GABAergic interneurons and oligodendrocytes in the forebrain. fgf3 and fgf8 are also involved in the regional patterning and generation of GABAergic interneurons and oligodendrocytes in the forebrain. The inhibition of both fgf3 and fgf8 was shown to result in defects in the expression of genes associated with early patterning functions and the specification of GABAergic interneurons and oligodendrocytes in the forebrain fgf16 was detected later than that of fgf3 or fgf8. Therefore, to examine whether the expression of fgf16 was affected by the inhibition of both fgf3 and fgf8 during forebrain development, we examined its expression in fgf3/8 double morphant embryos at 24 hpf. The expression of fgf16 was unaffected in the forebrains of fgf3/8 double morphant embryos (n = 22/24) (Fgf3 MO and Fgf8 MO led to a reduction in the expression of fgf16 in the midbrain at 24 hpf (n = 22/24)   . In cont= 22/24) . This re= 12/14) . Thus, fFgf8 is required for MHB development, and the MHB is crucial for proliferation and patterning in the midbrain fgf8 has not been implicated in growth of the forebrain fgf16 knockdown significantly inhibited cell proliferation and led to a reduction in the size and morphological abnormalities in the forebrain and midbrain. fgf16 morphants showed normal expression patterns of fgf8 in the MHB and had normal MHB-specific characteristics. This result indicated that a decrease in cell proliferation in the midbrains of fgf16 morphants was not due to a defect in the MHB. Thus, fgf16 functions are required to promote cell proliferation in the forebrain and midbrain.Fgf signaling regulates the proliferation and differentiation of specific neuronal cell types in the forebrain and midbrain fgf16 was first detected in the most anterior part of the ventral telencephalon at 14 hpf. fgf16 morphants exhibited the expanded expression of markers for the pallial telencephalon, emx1 and tbr1, and decreased expression of markers for the subpallial telencephalon, dlx2, at 24 hpf. These results suggested the loss of subpallial fate in the telencephalon of fgf16 morphants. Reduced cell proliferation in the telencephalon was observed in fgf16 morphants. Therefore, subpallial cells may be formed in smaller numbers due to reduced cell proliferation caused by the inhibition of fgf16. However, the expanded expression of ngn1 and slc17a6a was not detected in the ventral telencephalon of fgf16 morphants, which suggested that ventral cells in the telencephalon of fgf16 morphants were not formed in smaller numbers. Furthermore, fgf16 knockdown did not appear to transform cell fate specification from subpallial to pallial cells, and did not induce differentiation into dorsal neuronal cell types in the subpallial telencephalon. The ectopic expression of otx2 was detected in the ventral telencephalon of fgf16 morphants. Thus, Fgf16 is involved in patterning of the ventral forebrain, whereas the ventral telencephalon does not develop into the pallium following the inhibition of fgf16.The expression of dlx2 was decreased in the ventral thalamus by the inhibition of fgf16 at 24 hpf, whereas that of shh was unaffected in the ventral region. Furthermore, the expression of pax6a was normally detected in the diencepharon of fgf16 morphants at 24 hpf. These results demonstrated that the ventral thalamus was initially induced in fgf16 morphants. Therefore, fgf16 is necessary for maintaining of the characteristics of the ventral thalamus. In contrast, tectum- and tegmentum-specific characteristics were unaffected in the midbrains of the fgf16 morphants. This result indicated that fgf16 may be involved in regulating cell proliferation, but not dorsoventral patterning during midbrain development. In contrast, fgf16 may be involved in both the establishment of the subpallial telencephalon and ventral thalamus as well as the regulation of cell growth during forebrain development.In the diencepharon, the expression of ngn1 was unaffected in the dorsal telencephalon of fgf16 morphants. Furthermore, slc17a6a expression was also detected normally in the dorsal telencephalon of fgf16 morphants. On the other hand, the expression of isl1 was reduced in the ventral telencephalon, anterior ventral thalamus, and epiphysis, which suggested that fgf16 may be involved in neuronal differentiation in the ventral region, but not the dorsal region in the forebrain. However, slc17a6a expression was detected normally in the ventral thalamus of fgf16 morphants. These results indicated that fgf16 was not required for the specification of glutamatergic neurons in the forebrain.Ngn1 is known to be sufficient for conferring neuronal identity on uncommitted precursors and plays an important role in neurogenesis dlx2 was reduced in the forebrains of fgf16 morphants. Dlx2 was shown to be involved in the specification of GABAergic interneurons and oligodendrocytes in the telencephalon GAD1, when ectopically expressed in cortical explants olig2, expressed in oligodendrocyte precursors, is necessary and sufficient for the generation of oligodendrocytes throughout the neuraxis fgf16 knockdown resulted in a severe reduction of the expression of gad1 and olig2 in the ventral telencephalon and diencephalon. GABA-immunoreactive cells were also lost in the forebrains of fgf16 morphants, which indicated that GABAergic neurons did not fully differentiate in fgf16 morphants. plp expression and CC1 immunoreactivity also disappeared in fgf16 morphants, which suggested that the oligodendrocytes did not terminally differentiate into myelinating cells in fgf16 morphants. These results demonstrated that fgf16 was involved in the specification of GABAergic interneurons and oligodendrocytes in the ventral telencephalon and diencephalon. On the other hand, the knockdown of fgf16 did not strongly stimulate apoptosis in the forebrain. This result suggested that the survival of GABAergic interneurons and oligodendrocytes was unaffected by fgf16. Accordingly, Fgf16 appears to be crucial for the differentiation of GABAergic interneurons and oligodendrocytes, but not for that of glutamatergic neurons in the forebrain.The expression of Shh plays a mitogenic role in the brain and the ectopic expression of Hh target genes causes human cancers such as Basal Cell Carcinoma or medulloblastoma, a granule cell tumor fgf16 morphants as well as Shh mutant mice. Furthermore, the expression of fgf16 in the forebrain and midbrain was markedly reduced by the inhibition of Hh signaling at 16 and 25 hpf. These results indicated that Fgf16 may function downstream of Hh activity in cell proliferation in the forebrain and midbrain. On the other hand, Fgf8 participates in the growth of the midbrain, whereas Fgf3 and Fgf8 are not required for growth of the forebrain fgf3 and fgf8 led to a reduction in the expression of fgf16 in the midbrain, whereas it was unaffected in the forebrains of fgf3/8 double morphant embryos. Thus, fgf3 and fgf8 expressed in the MHB may regulate cell proliferation in the midbrain by activating the expression of fgf16 in the midbrain.olig2fgf16 morphants as well as smu/smo mutants exhibited the suppressed specification of GABAergic interneurons and oligodendrocytes in the forebrain. Hh signaling specifies GABAergic interneurons and oligodendrocytes via fgf3, fgf8, and fgf19 in the ventral forebrain, and this ensures the expression of pan-ventral transcription factors, such as dlx2 and olig2, whereas Fgf19 has distinct functions independent from those of Fgf3 and Fgf8 fgf19 led to a reduction in the expression of fgf16 in the forebrain, whereas the expression of fgf16 was unaffected in fgf3/8 double morphant embryos. This result indicates that fgf16 expression in the forebrain is regulated by Fgf19, but not by Fgf3/Fgf8. Thus, the effects of Hh activity on the differentiation of GABAergic interneurons and oligodendrocytes may be mediated through Fgf19-Fgf16 pathways in the forebrain.In addition to cell proliferation, Hh signaling is required for patterning in the telencephalon and the generation of GABAergic neuronal/oligodendrocyte progenitors from ventral forebrain stem cells via the activation of fgf16 expressed in the developing brain plays crucial roles in brain development. fgf16 is involved in cell proliferation in the forebrain and midbrain. fgf16 is also involved in the development of the ventral region and specification and differentiation of GABAergic interneurons and oligodendrocytes in the forebrain. On the other hand, fgf16 was not required for the specification of tectal and tegmental fates. Furthermore, the expression of fgf16 was dependent on Hh and fgf19. The present results suggest that crosstalk between Fgf16 signaling and Fgf19 and Hh signaling may be crucial for cell proliferation, regionalization, and cell type specification during forebrain development.In conclusion, the present results indicated that Figure S1fgf16 morphants. At 24 hpf, apoptotic cells in the brain of the wild-type (A) and fgf16 MO1-injected (B) embryos were marked via TUNEL. Lateral views with anterior to the left and dorsal to the top.Apoptosis in the brain of (TIF)Click here for additional data file.Figure S2fgf16 morphants.  Dorsal views of wild-type embryos (A) and fgf16 morphants (B), labeled to show CC1/APC immunoreactivity at 4.5 dpf.Oligodendrocyte differentiation in the hindbrain of (TIF)Click here for additional data file.


Hairy and Enhancer of Split genes (HES) are expressed in the inner ear, their full array of functions still not being disclosed. We have previously shown that zebrafish her9 acts as a patterning gene to restrict otic neurogenesis to an anterior domain. Here, we disclose the role of another her gene, her4, a zebrafish ortholog of Hes5 that is expressed in the neurogenic and sensory domains of the inner ear. The expression of her4 is highly dynamic and spatiotemporally regulated. We demonstrate by loss of function experiments that in the neurogenic domain her4 expression is under the regulation of neurogenin1 (neurog1) and the Notch pathway. Moreover, her4 participates in lateral inhibition during otic neurogenesis since her4 knockdown results in overproduction of the number of neurog1 and deltaB-positive otic neurons. In contrast, during sensorigenesis her4 is initially Notch-independent and induced by atoh1b in a broad prosensory domain. At later stages her4 expression becomes Notch-dependent in the future sensory domains but loss of her4 does not result in hair cell overproduction, suggesting that there other her genes can compensate its function.The generation of sensory neurons and hair cells of the inner ear is under tight control. Different members of the  Drosophila atonal gene was first identified in 1993 atonal orthologs, atoh1, is initially expressed in the sensory epithelium to later restrict in nascent hair cells atoh1 results in complete absence or ectopic differentiation of hair cells atoh1 genes are found in the inner ear, which act in two distinct phases of sensory development atoh1b appears in a broad prosensory territory to subsequently get restricted to smaller sensory domains. There, atoh1a is induced by atoh1b to direct the differentiation of hair cells Distinct proneural genes from the atonal basic helix-loop-helix (bHLH) superfamily control cell fate specification in the inner ear. atonal proneural family, neurog1, is used to define the otic neurogenic domain where otic progenitors will acquire a neuronal lineage Hes5 is induced and suppresses hair cell fate while promoting supporting cell fate Hes5 is expressed complementary to Delta1 expressing cells in the neurogenic domain Her4 is one of the zebrafish orthologs of mammalian Hes5 and it has been implicated in the control of primary neurogenesis, brain regeneration and neuronal target innervation her4 expression depends on Notch, while does not in the trigeminal sensory ganglia her4 in the inner ear, thus we aimed to study its function and regulation during neural and sensory specification. Here we show that zebrafish her4 is expressed in the neurogenic and sensory domains and requires neurog1 and atoh1b for its expression, respectively. Moreover, her4 in the neurogenic domain is dependent on Notch, while initial broad induction of her4 in the presumptive sensory domain does not require Notch but atoh1b. However, later on Notch restricts her4 expression to the future sensory maculae. Furthermore, we show that loss of her4 mediates lateral inhibition during neurogenesis, but not during sensorigenesis.Another member of ta52bhineurog11059y83her4 DNA containing 22 bp of 5′ UTR sequence of her4 cDNA is controlling EGFP expression. Homozygous mutant mib and neurog1 embryos were obtained by pairwise mating of heterozygous adult carriers. mib descendant embryos were genotyped after in situ hybridization. Embryos were developed in an incubator at 28.5°C in system water containing methylene blue and staged after counting somite number. Embryonic stages are given as hours post-fertilization (hpf) at 28.5°C Zebrafish were maintained at the PRBB Animal Facility under standard conditions. The zebrafish protocols followed the guidelines and were approved by the IACUC, Comité Ético de Experimentación Animal-Parc de Recerca Biomèdica de Barcelona (CEEA-PRBB); approved number JIB-08-1098P2. The zebrafish strains used were the following: wild-type (AB), mibin situ hybridization were performed as previously described atoh1b and atoh1aneurog1her4deltaBSynthesis of antisense RNA and whole-mount her4-MO her4 mRNA transcript with sequence: 5′-ATT GCT GTG TGT CTT GTG TTC AGT T-3′. Her4-MO was injected at concentration 0.025 mM and its efficiency was assessed by the specific loss of GFP signal from the Tg(her4:EGFP)y83 transgenic line atoh1b-MO MOs were obtained from Gene Tools. Embryos were injected at 1-cell stage. The Dechorionated zebrafish embryos were incubated with 50 µM SU5402 , a potent pharmacological inhibitor of Fgf signalling. Incubations were done at 28.5°C, starting at 10 hpf until the sacrifice of the animals at 16 hpf. The final solution was done in E3 medium from the 5 mM SU5402 stock solution (kept at −20°C in DMSO). For control treatments, embryos were incubated in an equivalent concentration of dimethyl sulfoxide .Pictures were acquired in a Leica DRM microscope or in Leica MZFLIII stereomicroscope using a Leica DFC300 FX camera and the Leica IM50 software. Adobe Photoshop 7.0.1 software was used for photograph editing.her genes in zebrafish, orthologous to mammalian Hes genes, consists of 21 genes based on data published on Ensembl (Zv9). We analysed the expression of 10 her genes during otic development and only her6, her4 and her9 were found to be expressed in the otic vesicle from 12–14 hpf to 24 hpf (data not shown). We have previously demonstrated a role for her9 during otic patterning in restricting neurogenesis to the anterior domain, downstream of retinoic acid (RA) signalling her4 gene. Previous studies have analysed the development of the neurogenic and sensory territories separately, however since both domains develop simultaneously, a complete picture of the development of both territories should be taken into account. Moreover, the sequential phases of Notch activation is still not well defined. Consequently, we have generated a precise map of the expression of her4 at early stages of neuro- and sensorigenesis together with the expression of atoh1b, atoh1a and neurog1. her4 expression is first observed at 12 hpf throughout a broad band of cells just adjacent to the hindbrain. The pattern at this stage is strikingly similar to the one from atoh1b (compare her4 is only detected at the future anterior (utricular) and posterior (saccular) sensory maculae with higher levels of expression than atoh1b .The family of  compare . By 13 h compare . At 16 h compare . atoh1a  onwards . At 20 hr macula  . Note that neurog1 expression begins in the neurogenic domain at 15 hpf but her4 expression is not initiated there until two hours later  compared to heterozygous embryos that retain her4 expression  as well as atoh1b (n = 5/5) confirming that both have similar regulatory requirements  to assess the possible regulation of her4 by atoh1b. her4 expression precedes the onset of atoh1a in the otic placode, thus we reasoned that atoh1b instead of atoh1a was the best candidate for testing the upstream regulation of her4. Indeed, in atoh1b-MO injected embryos, expression of her4 is abrogated in the anterior and posterior sensory epithelia at 15 hpf .In order to distinguish between these possibilities, we have blocked t 15 hpf ; n = 5/5 maculae ; n = 5/5 compare '. The exher4 is a target of atoh1b in the sensory domain and depletion of atoh1b function has no effect on her4 neither on neurog1 expression in the neurogenic domain.In summary, her4 with the proneural gene neurog1 by using the hi1059neurog1 mutant line neurog1-/- mutants obtained from heterozygotic crosses of hi1059neurog1 adults, her4 is not expressed in the neurogenic domain at 24 hpf  line Her4-MO sequence binds specifically to the 5′UTR affecting EGFP translation. In control embryos, strong EGFP is visible in the neural tube, while in 0.025 mM her4-MO injected embryos EGFP expression is completely abolished . In cross-sections, an expansion of neurog1 expression is also observed . As we have previously shown that atoh1b is upstream of her4, this result indicates that, as observed for neurog1, her4 does not feedback on atoh1b.We could expect a similar role of her4 becomes dependent of Notch, most probably participating in Notch-mediated lateral inhibition and repressing the differentiation of sensory progenitors to hair cells. If this hypothesis is true, we expected to find increased numbers of hair cells in her4-MO embryos. Intriguingly, the number of cells expressing atoh1a at 24 hpf and 28 hpf, as a readout of committed hair cells did not increase in her4-MO (atoh1a (n = 5/13).We have seen above that at later stages  her4-MO  and was her4 in inner ear development and its relationship with proneural genes and Notch signalling. In the neurogenic domain her4 and neurog1 expressions are correlated spatially, with a temporal delay between neurog1 induction and her4, suggesting an intermediate step. The fact that in the neurogenic domain her4 expression depends on Notch, positions Notch as the intermediary pathway that activates her4 downstream of neurog1. Moreover, depletion of her4 leads to an increase in the population of neurons. This is similar to what was previously reported for her4 role in primary neurogenesis her4 nor Hes5 has already been analysed directly in inner ear neurogenesis . The data demonstrate for the first time that in the inner ear, as in the CNS, her4 participates in Notch-mediated lateral inhibition to control the final number of neuronal cells.Here we have explored the role of her4 is regulated differently. In the presumptive sensory territory her4 expression is highly dynamic and identical to atoh1b. It initially encompasses a broad medial territory of the otic placode to progressively restrict to the future anterior and posterior maculae. Initial her4 expression requires atoh1b and Fgf signalling but not Notch, indicating that it cannot be assumed that her4 is always regulated by Notch. Nonetheless, in an intermediate developmental period, in the CMD Notch regulates negatively but not positively her4 in the CMD. Our work thus shows that her4 is not the downstream target of Notch to repress atoh1b expression. her6, another member of her repressors, cannot perform this role since is not expressed in the CMD at 12.5–13 hpf y83 embryos and also it resulted in expansion of deltaB expression. One of the most plausible explanations is that contrary to what happens in the neurogenic domain, other her genes compensate for her4 loss in the sensory domain. Her6, a Hes1 ortholog, is expressed exclusively in the sensory domain already from 12.5 hpf Click here for additional data file.


With gold nanoparticles (AuNPs) is possible to develop nanoscale devices that can interact with chemical and biological systems. The phenomenon explored in these nanosystems is called Localized Surface Plasmon Resonance (LSPR), which promotes electromagnetic wave oscillation electronics on these small metallic structures. It is interesting to note that this resonance is directly linked to the size of the nanoparticles, the nature of the dielectric material and support environment where the device is being studied ,2.3C6H5O7) and Sodium Borohydride (NaBH4). Was expected to demonstrate the viability of these two reducing agents and highlight the potential differences obtained in each of the mechanisms. Since knowledge is the ability of stabilizing citrate ions and the strong reducing action of NaBH4 [This work makes a comparison between results obtained in the synthesis of AuNP's reduction method using a Sodium Citrate NaC6H5O7 an4) 2.5 × 10-4 M, 5 mL of 1% NaBH4 and 5 mL of Na3C6H5O7 also 1%.In these experiments consisted initially in the preparation of the following aqueous solutions: 100 mL Tetrachloroauric Acid  .4 did not generate nanoparticles with a reasonable size for the LSPR occur. Phenomenon responsible for the peak at 530 nm in the spectrum of sodium citrate (Na3C6H5O7).The UV-Vis spectrophotometry shows the absorption of the samples obtained in experiments, it is noted that the easy synthesis with NaBH4 lot nuclei initiators nanoparticles were generated, but reduced in size. Na3C6H5O7 already has a good ability to stabilize, thus required temperature rise in the synthesis process to increase capacity reduction. Nuclei being formed ions favor the stabilization phase of growth, thus generating nanostructures favorable process RPSL to 530 nm.Due to capacity reduction of NaBH