Constitutively activated oncogenic signaling via genetic mutations such as in the EGFR/PI3K/Akt and Ras/RAF/MEK pathways has been recognized as a major driver for tumorigenesis in most cancers. Recent insights into tumor metabolism have further revealed that oncogenic signaling pathways directly promote metabolic reprogramming to upregulate biosynthesis of lipids, carbohydrates, protein, DNA and RNA, leading to enhanced growth of human tumors. Therefore, targeting cell metabolism has become a novel direction for drug development in oncology. In malignant gliomas, metabolism pathways of glucose, glutamine and lipid are significantly reprogrammed. Moreover, molecular mechanisms causing these metabolic changes are just starting to be unraveled. In this review, we will summarize recent studies revealing critical gene alterations that lead to metabolic changes in malignant gliomas, and also discuss promising therapeutic strategies via targeting the key players in metabolic regulation.  Malignant glioblastomas (GBM) are aggressive brain tumors, which are resistant to radiation and chemotherapy ,2,3,4. IIn recent years, the understanding of the regulation of tumor metabolism has significantly improved. Accumulating evidence show that tumor cells reprogram their metabolism to meet high energy demands, coordinate markedly elevated biosynthetic processes and energy production, which in turn promote rapid growth and division of tumor cells ,18,19,20In this review, we will focus on the basic metabolism pathways in GBM including glucose, glutamine, and lipid metabolism, and we will discuss the reprogramming of metabolic pathways and the molecular link between oncogenic signaling pathways and key metabolic regulators.In normal tissues, ATP is primarily generated in mitochondria via complete oxidative phosphorylation (OXPHOS) of glucose. Conversely, only 10% of ATP is generated from glycolysis in which glucose is converted to lactate . IntriguHexokinase (HK) functions in the first step of glycolysis and irreversibly catalyzes the phosphorylation of glucose to glucose-6 phosphate (G6P)  Figure . HexokinPKLR and PKM in mammals. R and L isoforms are derived from PKLR, expressed in erythrocytes and the liver [PKM gene, PKM1 contains exon 9 and PKM2 contains exon 10, respectively [PKM show different properties, which results in the different activity of PKM1 and PKM2. PKM1 is constitutively active, but PKM2 can be regulated by fructose-1,6-bisphosphate. Recently, splicing repressors of hnRNP A1, hnRNP A2  and PTB (polypyrimidine tract binding protein) were reported to directly bind to the flanking intron of exon 9 and repress the use of exon 9. Downregulation of these splicing repressors increased PKM1 expression levels in cancer cells [PKM2 is the M2 isoform of pyruvate kinase (PK) . PK funche liver . M1 and ectively . Exon 9 er cells .CCND1 and MYC. C-Myc further upregulates glucose transporter (GLUT) and HK2 to promote glycolysis  is an enzyme that catalyzes the oxidative decarboxylation of isocitrate to produce α-ketoglutarate, and also generate NADH in mitochondria or NADPH in cytoplasm . In the In 2008, IDH1 and IDH2 mutations were first reported in low grade of gliomas and secondary GBM ,61,62. IRecently, CpG island methylation profiling was performed to identify the difference between IDH1-R132 mutant and IDH1-R132 wild type GBM. The profiling analysis showed a similar methylation pattern in IDH1-R132 mutant GBM and a newly-described CpG island methylator phenotype (CIMP) . The phep = 0.01) [In addition to glucose, glutamine is another major energy source for cells ,77 Figu. Glutami = 0.01) . Interes = 0.01) . CompareLipids consist of phospholipids, fatty acid, cholesterol, triglycerides, cholesterol esters, sphingolipids and others, which are critical components to form cellular membranes ,91,92,93Lipid metabolism has been found to be largely altered in cancers ,97,98,99N-terminus of SREBPs will be released from the membrane after sequential cleavage by the membrane-bound serine proteases, S1P and S2P [N-terminus enters into the nucleus to promote its downstream gene expression [Sterol regulatory element-binding proteins (SREBPs) are important transcription factors that regulate lipid metabolism thereby playing a crucial role in the regulation of lipogenesis and cholesterol uptake . There a and S2P ,107. Thepression ,108,109.de novo fatty acid synthesis [Interestingly, SREBP-1 has been found to be highly activated in GBM and other cancers ,112,113.ynthesis  /PI3K/Akt signaling in GBM  Figure . More inCholesterol is also an important component of cell membranes ,115. In Recently, low density lipoprotein receptor (LDLR) has been found to be upregulated in GBM patient tissues, xenograft tumors and cell lines, and this upregulation is correlated with high levels of cholesterol esters in GBM cells ,121. Thede novo synthesis, and efflux (Cholesterol homeostasis is regulated by uptake, d efflux . When ced efflux . Interesd efflux . TherefoMetabolic reprogramming is a key feature driving oncogenesis in cancers. Recent studies have revealed that glucose, glutamine and lipid metabolism are largely altered in GBM and facilitate its malignant growth. Oncogenic signaling pathways are found to regulate these metabolic alterations. Targeting metabolic reprogramming is a novel and promising therapeutic strategy to treat cancers. Further understanding of the metabolic alterations in GBM will definitely shed light on developing effective approaches to abrogate GBM malignant growth, and also provide insights to overcome GBM resistance with current therapies.


In order to increase the transcriptome data and accelerate genetic improvement for this breed, we performed Transcriptome sequencing of Liaoning cashmere goat on a Roche 454 platform yielded 804,601 high-quality reads. Clustering and assembly of these reads produced a non-redundant set of 117,854 unigenes, comprising 13,194 isotigs and 104,660 singletons. Based on similarity searches with known proteins, 17,356 unigenes were assigned to 6,700 GO categories, and the terms were summarized into three main GO categories and 59 sub-categories. 3,548 and 46,778 unigenes had significant similarity to existing sequences in the KEGG and COG databases, respectively. Comparative analysis revealed that 42,254 unigenes were aligned to 17,532 different sequences in NCBI non-redundant nucleotide databases. 97,236 (82.51%) unigenes were mapped to the 30 goat chromosomes. 35,551 (30.17%) unigenes were matched to 11,438 reported goat protein-coding genes. The remaining non-matched unigenes were further compared with cattle and human reference genes, 67 putative new goat genes were discovered. Additionally, 2,781 potential simple sequence repeats were initially identified from all unigenes.denovo assembled, and annotated, providing abundant data to better understand the Liaoning cashmere goat transcriptome. The potential simple sequence repeats provide a material basis for future genetic linkage and quantitative trait loci analyses.The transcriptome of Liaoning cashmere goat was deep sequenced,  Capra hircus) is famous for its fine cashmere quality, high cashmere yield, and stable genetic character. Although this population was bred in the 1980s, a little is known regarding the molecular mechanism and regulation of cashmere growth in this breed. Genetic studies of Liaoning cashmere goat had been performed in the past decade, which mainly focused on characterization and expression of functional genes [Cashmere is an important commodity in China and Liaoning cashmere goat  or transcriptome sequencing is an efficient way to generate functional genomic-level data for an organism, especially non-model organism. ESTs provide comprehensive information about the transcriptome  and have animals -20, plan animals ,21-24 an animals ,25-27.de novo transcriptome sequencing of the female Liaoning cashmere goat using Roche 454 GS FLX platform. 804,601 high-quality reads were assembled into a non-redundant set of 117,854 unigenes, comprising 13,194 isotigs and 104,660 singletons. Functional annotation and comparative analysis were performed on those 117,854 unigenes, providing an invaluable new resource for functional genomics and biological research in Liaoning cashmere goat. Finally, potential simple sequence repeats we identified could be useful for genetic linkage mapping and other genetic studies.In this article, we performed de novo assembly of Liaoning cashmere goat genes [Although the Roche 454 GS FLX platform incurs a high cost for sequencing, the read length of the output sequences are more than adequate for the at genes ,29. To aMelitaea cinxia (197 bp) [Acropora millepora (440 bp) [P. contorta (500 bp) [Pristina leidyi (707 bp) [Megalobrama amblycephala (730 bp) [Junco hyemalis  [197 bp) , Acropor(440 bp) , P. cont(500 bp) , Pristin(707 bp) , and Meg7 bp , Ac,248 bp) . About 1All unigenes (isotigs and singletons) were analyzed by EMBOSS software  for gene- 5. 24,614 unigenes had significant BLASTp matches, including 5,340 isotigs (40.47%) and 19,274 singletons (18.42%). Similarly low matching percentages of isotigs and singletons have been reported in non-model species, such as in Macrobrachium nipponense (40.76% contigs and 17.71% singletons) [Junco hyemalis (49% isotigs and 11% singletons) [Scophthalmus maximus (44.84% contigs and 7.55% singletons) [All putative protein sequences were subjected to a BLASTp similarity search against the non-redundant protein database of NCBI (www.ncbi.nlm.nih.gov), with a cutoff E value of <1egletons) . Bos taurus (60.37%) sequences. The top 20 species distribution of BLAST matches is shown in Capra hircus (goat) or Ovis aries (sheep) sequences. In principle, the unigenes of the Liaoning cashmere goat should be more similar to these two species, because they all belong to the Caprinae. We speculated that this might have resulted from an underrepresentation of goat and sheep protein sequences in the NCBI Nr database. To confirm this speculation, we collected the total number of protein sequences in NCBI Nr database for the top 20 species, then calculated the percent of matched unigenes in the total sequences of NCBI Nr database for each species in the top 20  against the Swiss-Prot and TrEMBL database, and then using the GoPipe software[Gene ontology (GO) is an international classification system for standardized gene functions, offering a comprehensive description of the gene properties and their products in any organism. All putative protein sequences  of Liaoning cashmere goat were assigned for GO terms based on sequence similarity, using BLASTp  is a database where orthologous gene products may be classified. All unigenes were subjected to a search against the COG database for functional prediction and classification. Some unigenes had multiple COG functions. 46,778 unigenes showing significant homology to entries in the COG database at NCBI were classified into 25 COG categories . Among t- 5 was performed. 3,548 unigenes, comprising 1,728 isotigs and 1,820 singletons, had KEGG Orthology (KO) numbers , a bidirectional BLAST search against Kyoto Encyclopedia of Genes and Genomes (KEGG) protein database with a cutoff E value of <1e numbers S2. Acco numbers S2. The In addition, the 3,548 KEGG annotated unigenes could be categorized into six different functional groups . The lar-20. Of the 117,854 assembled unigenes, 66,351 (56.30%) showed one alignment to an existing sequence. After processing and filtering, 42,254 unigenes were aligned to 17,532 different specific genes. The coverage of every gene was also calculated . To orient the Liaoning cashmere goat unigenes derived from RNA-seq, we performed BLASTn alignment  against the sequences of Capra hircus genomes. Among a total 117,854 unigenes, 97,236 (82.51%) were mapped to 30 chromosomes of Capra hircus . 35,551 (30.17%) unigenes, comprising 6,935 isotigs and 28,616 singletons, were mapped to 11,438 (51.58%) Capra hircus protein-coding genes  and Homo sapiens (human) reference genes, using the BLASTn algorithm with a cutoff E value of <1e-20. Of 82,303 non-matched unigenes, 15,779 were aligned to 6,060 cattle reference genes, and 6,837 were aligned to 2,880 human reference genes  unigenes were annotated, including 9,289 (70.40%) isotigs and 44,864 (42.87%) singletons. This result will greatly increase the number of annotated sequences for the Liaoning cashmere goat in the NCBI database. In addition, all Liaoning cashmere goat unigenes were searched against 22,175  reported and wereng genes S5. To fce genes S6. By mCapra hircus protein-coding genes but matched to 8,940 cattle and human reference genes. After statistical investigation, among the 8,940 cattle and human reference genes, 272 genes whose full-length were covered more than 50% by our Liaoning cashmere goat unigenes. Moreover, we collected coding sequences (CDS) of the 272 genes, and through comparative analysis, 67 cattle genes whose coding sequence were completely covered by 67 unigenes , followed by AACTG/AGTTC , AT/AT , AGC/CTG  and AAAT/ATTT   are polymorphic loci present in genomic DNA, and are distributed in both coding and non-coding regions . They corcus skin, whose t, 3.80%) S9. The rtebrates, but difrtebrates. SSRs can be used for subsequent marker development, genetic linkage and quantitative trait loci analysis. To make these SSR markers be useful for Liaoning cashmere goat breeding or other studies, we employed primer 3 to design primer pairs for each SSR. After removing the microsatellites without sufficient flanking sequence for primer design, 958 primer pairs from 2,781 (34.45%) SSRs were designed S10. Howde novo transcriptome sequencing of the female Liaoning cashmere goat using the 454 GS FLX system was performed for the first time. 1,044,032 raw reads were de novo assembled into 117,854 unigenes. All unigenes were then evaluated and functionally annotated by comparing with the existing protein databases, such as NCBI NR database, Swiss-Prot and TrEMBL database, COG database, and KEGG protein database. Further comparative analysis revealed that 54,153 (45.95%) unigenes got annotated and 67 putative new goat genes were discovered. Moreover, 2,781 SSRs were initially identified among the unigenes, which can be used for subsequent marker development, genetic linkage and quantitative trait loci analysis. Overall, our study on the Liaoning cashmere goat transcriptome provides a valuable resource that will lead to a better understanding of the Liaoning cashmere goat transcriptome.In this study, All animal procedures and study design were conducted in accordance with the Guide for the Care and Use of Laboratory Animals , and were approved by the animal ethics committee of Northwest A & F University.The most important issue for transcriptome sequencing was the preparation of fresh and healthy tissue samples. Therefore, three adult female Liaoning cashmere goats were selected from Liaoning Province of China in 2012. The 13 tissue samples including heart, liver, spleen, lung, kidney, pancreas, skin, muscle, ovary, stomach, intestines, brain and arteries were freshly collected. All samples were immediately frozen in liquid nitrogen and stored at -80°C until use.260:OD280 ratio of 1.9- 2.1 and an OD260:OD230 ratio of 2.0 -2.5 were used for subsequent analysis. Integrity and size distribution were checked with an Agilent 2100 Bioanalyzer  RNA 6000 Pico LabChip . Total RNA was extracted from the 13 tissues using the TRIzol Reagent  according to the manufacturer’s instructions. DNase I  was used to remove potential genomic DNA contamination from the RNA samples. The quality of the total RNA was determined using a Nanodrop spectrophotometer . Only RNA samples with an ODTM mRNA purification kit (Ambion). RNA quality and quantity was determined again at the end of this process. First-strand cDNA was synthesized using GsuI-oligo dT as reverse transcription primers, 10 mg of the mRNA as template, and 1000 units of Superscript II reverse transcriptase (Invitrogen), at 42°C for 1 h. The mRNA 5' cap structure was then oxidized using NaIO4  and connected to biotin. The biotin-coupled mRNA/cDNA was screened by Dynal M280 beads (Invitrogen), and the first-strand cDNA was released by alkaline dissociation. Subsequently, adapters were added to the first-strand cDNA using DNA ligase , and the second-strand cDNA was synthesized using Ex Taq polymerase (TaKaRa). Finally, the polyA and 5' end adapters were removed by Gsu I digestion.We pooled equivalent amounts of total RNA from each sample  and delivered it to Shanghai Hanyu Bio-Tech Co., Ltd.  for cDNA synthesis and 454 sequencing. Pure mRNA was subtracted using the Micropoly(A) PuristFragmentation of synthesized cDNA was performed by nebulization, which shears double-stranded cDNA into fragments ranging from approximately 300 to 800 base pairs. A single-strand template DNA (sstDNA) library was constructed using a GS DNA Library Preparation kit  following the manufacturer’s protocol. The sstDNA was attached to beads using a GS emPCR kit (Roche Applied Science), and an emulsion-based clonal amplification was performed. After amplification, Roche GS-FLX 454 pyrosequencing was conducted, according to the manufacturer’s recommendations.1,044,032 raw sequences were obtained from Roche 454 pyrosequencing, with an average length of 416 bp. Newbler Software v. 2.5 (provided with the Roche GS FLX sequencer) was used for sequence assembly, using default parameters. Prior to assembly, we removed the nonsense sequences, including adapters added for reverse transcription and 454 sequencing, primers and low quality sequences, using Newbler. Raw data were deposited in NCBI Sequence Read Archive (SRA) under the accession number SRA064946. All unigene sequences (>200 bp) were deposited to the Transcriptome Shotgun Assembly (TSA) database. This Transcriptome Shotgun Assembly project has been deposited at DDBJ/EMBL/GenBank under the accession GAFC00000000. The version described in this paper is the first version, GAFC01000000.-5. Blast hits annotated with uninformative terms such as "unknown", "uncharacterized" and "hypothetical" were omitted from analyses. Ultimately, 24,614 unigenes had significant BLASTp matches. All unigenes were analyzed by the EMBOSS software package  for gene-5. The results were then sorted by GO categories using gene2go in the GoPipe software [http://www.ncbi.nlm.nih.gov/COG/) to predict possible functional classifications. KEGG Orthology (KO) numbers were assigned to unigenes using a bidirectional BLAST search against the KEGG protein database with a cutoff E value of <1e-5. The sequences were mapped to the KEGG pathways according to the KO numbers in the pathway database. To annotate unigenes with gene ontology (GO) terms, sequences were compared against the Swiss-Prot and TrEMBL database using the BLASTp algorithm, with a cutoff E value of <1esoftware . BLASTp -20. 42,254 unigenes aligned to 17,532 different specific genes, because multiple unigenes sometimes matched to one specific gene. We collected the full-length versions of the 17,532 different specific genes, and then calculated the length of every specific gene covered by the 42,254 unigenes. The results of the coverage analysis are shown in The assembled sequences were compared with the sequences in the NCBI non-redundant nucleotide databases using the BLASTn algorithm with a cutoff E value of <1eCapra hircus genome and compared with the reported 22,175 Capra hircus protein-coding genes, using BLASTn with a cutoff E value of <1e-20. The remaining non-matched unigenes were also aligned to Bos taurus (cattle) and Homo sapiens (human) reference genes, using BLASTn with a cutoff E value of <1e-20. 18,602 unigenes were matched to 8,940 cattle and human reference genes. Among the 8,940 cattle and human reference genes, 272 genes whose full-length were covered more than 50% by unigenes. To discover the putative new goat genes, the coding sequences (CDS) of 272 cattle and human reference genes collected from NCBI. Through analysis, 67 genes whose coding sequence were completely covered by 67 Liaoning cashmere goat unigenes.To further annotate and identify the assembled sequences, all the sequences  were mapped to http://pgrc.ipk-gatersleben.de/misa/), which accepts FASTA-formatted sequence files. The parameters were adjusted to identify perfect mono-, di-, tri-, tetra-, penta-, and hexanucleotide motifs with a minimum of 20, 10, 7, 5, 4 and 4 repeats, respectively. The maximum number of bases interrupting two SSRs in a compound microsatellite was set at 100. Primer 3 software [SSRs were detected among the 117,854 unigenes using MIcroSAtellite Click here for additional data file.Table S2KEGG summary of Liaoning cashmere goat unigenes.(XLSX)Click here for additional data file.Table S3The statistics of gene coverage rate.(XLSX)Click here for additional data file.Table S4The result of Liaoning cashmere goat unigenes mapped to goat genome sequence.(XLSX)Click here for additional data file.Table S5The result of Liaoning cashmere goat unigenes aligned to 22,175 goat protein-coding genes.(XLSX)Click here for additional data file.Table S6The result of non-matched unigenes compared with cattle and human reference genes.(XLSX)Click here for additional data file.Table S7The analysis result of 67 Liaoning cashmere goat unigenes compared with 67 cattle reference genes.(XLSX)Click here for additional data file.Table S8Summary of the 67 putative novel goat genes.(DOCX)Click here for additional data file.Table S9Frequency statistics of classified repeat types (considering sequence complementary).(XLSX)Click here for additional data file.Table S10Summary of 958 SSRs and their primers.(XLSX)Click here for additional data file.


PubChem provides a 3-D neighboring relationship, which involves finding the maximal shape overlap between two static compound 3-D conformations, a computationally intensive step. It is highly desirable to avoid this overlap computation, especially if it can be determined with certainty that a conformer pair cannot meet the criteria to be a 3-D neighbor. As such, PubChem employs a series of pre-filters, based on the concept of volume, to remove approximately 65% of all conformer neighbor pairs prior to shape overlap optimization. Given that molecular volume, a somewhat vague concept, is rather effective, it leads one to wonder: can the existing PubChem 3-D neighboring relationship, which consists of billions of shape similar conformer pairs from tens of millions of unique small molecules, be used to identify additional shape descriptor relationships? Or, put more specifically, can one place an upper bound on shape similarity using other "fuzzy" shape-like concepts like length, width, and height?x, Qy, and Qz) were binned and their frequency of occurrence was examined. Per molecular volume type, this effectively produced three different maps, one per quadrupole component , of allowed values for the similarity metric, shape Tanimoto (ST) ≥ 0.8.Using a basis set of 4.18 billion 3-D neighbor pairs identified from single conformer per compound neighboring of 17.1 million molecules, shape descriptors were computed for all conformers. These steric shape descriptors included several forms of molecular volume and shape quadrupoles, which essentially embody the length, width, and height of a conformer. For a given 3-D neighbor conformer pair, the volume and each quadrupole component . This efficiency increased linearly as a function of ST threshold in the range 0.8-0.99. The Qx filter was consistently the most efficient followed by Qy and then by Qz. Use of a monopole volume showed the best overall performance, followed by the self-overlap volume and then by the analytic volume.The efficiency of these relationships  as a function of ST threshold was determined in a test run of 13.2 billion conformer pairs not previously considered by the 3-D neighbor set. At an ST ≥ 0.8, a filtering efficiency of 40.4% of true negatives was achieved with only 32 false negatives out of 24 million true positives, when applying the separate Qxyz filter in a "real world" test of 3-D neighboring of 4,218 chemicals of biomedical interest against 26.1 million molecules in PubChem reduced the total CPU cost of neighboring by between 24-38% and, if used as the initial filter, removed from consideration 48.3% of all conformer pairs at almost negligible computational overhead.Application of the monopole-based QBasic shape descriptors, such as those embodied by size, length, width, and height, can be highly effective in identifying shape incompatible compound conformer pairs. When performing a 3-D search using a shape similarity cut-off, computation can be avoided by identifying conformer pairs that cannot meet the result criteria. Applying this methodology as a filter for PubChem 3-D neighboring computation, an improvement of 31% was realized, increasing the average conformer pair throughput from 154,000 to 202,000 per second per CPU core. PubChem is an open and free resource of the biological activities of small molecules -4. PubChThe PubChem3D neighboring uses as a measure of molecular shape similarity the shape Tanimoto (ST) ,10, giveAA Vand BB Vare the self-overlap volumes of conformers A and B, respectively, and AB Vis the common overlap volume between A and B. The 3-D neighboring requires finding the maximum shape similarity between static compound 3-D conformations, as dictated by AB Vin Equation 1, to calculate ST, a computationally intensive step. It is highly desirable to avoid this overlap computation, especially if it can be determined with certainty that a conformer pair cannot meet the criteria to be a 3-D neighbor. As such, PubChem employs a series of filters, based on the concept of volume, to effectively ignore approximately 65% of all conformer neighbor pairs during 3-D neighboring, thus dramatically accelerating processing  The false negative count of 30 removed by Of the three volume types utilized, the monopole-based quadrupole filters, Fmp filters are used in a series , of 4.18 billion 3-D neighbor pairs resulting from PubChem 3-D neighboring of 17.1 million single conformer molecules were analyzed. The maximum quadrupole differences between neighbor conformers were determined. This examination demonstrated a distinct dependency of shape similarity upon quadrupole variation. With some slight modification of fringe regions, the results of this analysis were turned into computationally inexpensive, yet highly effective set of filters capable of removing 3-D conformer pairs that cannot meet a required shape similarity, using only knowledge of the volume and steric quadrupole moments of the conformer pair. When applied in the context of shape similarity searching, these filters can significantly improve throughput performance by avoiding expensive superposition optimization computation of conformer pairs that cannot possibly meet a pre-defined shape similarity search threshold.x Qfilters eliminated a larger portion of the compound pairs than the y Qand z Qfilters. When the filters were used in a series simultaneously, they could eliminate 30~40% of non-neighbor pairs, with the removal of a negligible amount of potential neighbors. For example, the xyz Qfilter based on the monopole volumes , monopole volume (mpV), self-overlap volume (soV), and steric shape quadrupole moments  were computed for the theoretical conformer of all 17.1 million molecules. See Figures At the time of project initiation, PubChem 3-D neighboring of 17,143,181 unique molecules  had been completed using a single conformer per compound, yielding 4,182,412,802 3-D neighbors. Using the Shape Toolkit from the OpenEye Software , the anaThe quadrupole filters developed for pre-screening conformer-pairs based on quadrupole differences as a function of shape similarity ST threshold were generated using the following steps:1) The 4.18 billion 3-D neighbor pairs and their associated data were obtained from PubChem.mp, VsoV, and anV) and quadrupole components  of the compound pair for each 3-D neighbor were converted into integers using Equations 4 and 5 to yield BinSize was 5.0 Å3 for all three volume types and 2.5 Å5, 0.5 Å5, and 0.1 Å5, for x, QyQ, and zQ, respectively.2) The volumes (3) For each of the three binned volume types, the following was performed using the 3-D neighbor pairs (in this case using a) Of the two conformers in a 3-D neighbor, the one with the smaller b) For each of the three binned quadrupole components, and using x Qdifference between the two molecules.i) 3-D neighbors were binned according to three indices, ii) The neighbor count for all (x Qfilter based on monopole volumes (iii) The 4) To obtain filters effective at an ST threshold other than ≥ 0.80, first restrict the original 4.18 billion 3-D neighbor pairs to those at or above the desired ST threshold and repeat step 3.bin Qvalue. In an original map [panel (a) of Figure bin Qvalue. Similarly, any empty bins within the range of bin Qvalue.Figure bin Qvalues starting with the maximum. As lesser Δbin Qvalues are considered in this correction, greater Δbin Qvalues are considered at the Δbin Qvalue being considered. A pseudo-code implementation of this procedure is shown in Figure This procedure is performed for all unique ΔTo test the efficiency of the quadrupole filters devised, two sets of molecules were chosen. One set contains molecules in the PubChem CID range of 1 ~ 25,000,000, and the other contains those in the CID range of 25,000,001~25,001,000. Because a theoretical conformer was not generated for all CIDs or because compound records were not "live", the two datasets had 17,488,897 and 753 molecules, respectively. All-by-all comparison between the two sets gives 13,169,139,441 CID pairs. Using the first diverse conformer for each compound, the ST values for these 13.2 billion pairs were computed using ROCS  from OpeQuery set) were neighbored against 26,157,365 compound records (Search set), representing the entire "live" PubChem3D contents as of Oct. 2010, using up to 1, 3, 5, 7, and 10 diverse conformers per compound for both compound sets. Timing and efficiency differences with our earlier work are given in Tables One aspect of this effort is to examine the change in real-world efficiency of PubChem3D neighboring processing when using quadrupole filters while computing the 3-D "Similar Conformers" relationship. To achieve this, the set of 4,218 biologically relevant chemical structures with known pharmacological actions from our earlier efforts  was usedThe authors declare that they have no competing interests.SK analyzed the quadrupole differences of the 3-D neighbors, generated the quadrupole filters, and wrote the first draft. EEB supervised the project and revised manuscript. SHB reviewed the final manuscript. All authors read and approved the final manuscript.Quadrupole filters. A zip archive of text files containing information on the maximum quadrupole differences as a function of molecular volumes.Click here for file


Intracerebral hemorrhage (ICH) is a common type of fatal stroke, accounting for about 15% to 20% of all strokes. Hemorrhagic strokes are associated with high mortality and morbidity, and increasing evidence shows that innate immune responses and inflammatory injury play a critical role in ICH-induced neurological deficits. However, the signaling pathways involved in ICH-induced inflammatory responses remain elusive. Toll-like receptor 4 (TLR4) belongs to a large family of pattern recognition receptors that play a key role in innate immunity and inflammatory responses. In this review, we summarize recent findings concerning the involvement of TLR4 signaling in ICH-induced inflammation and brain injury. We discuss the key mechanisms associated with TLR4 signaling in ICH and explore the potential for therapeutic intervention by targeting TLR4 signaling. Intracerebral hemorrhage (ICH) is the least treatable type of stroke and has devastating consequences. HemorrhSecondary damage following ICH is triggered by the presence of intraparenchymal blood, which subsequently activates cytotoxic, oxidative and inflammatory pathways,10. The Toll-like receptors (TLRs) belong to a large family of pattern recognition receptors that play a key role in innate immunity and inflammatory responses,22. Thirin vivo study shows that activation of TLR4 by heme causes ICH-induced inflammatory injury via the MyD88/TRIF signaling pathway and that effective blockade of TLR4 by its antibody suppresses ICH-induced inflammation[It has been reported that TLR4 is upregulated in a rat model of ICH,30 and tammation. Thus, tTLR4 is expressed in microglia, the resident macrophages of the brain. Microglia are activated within minutes after ICH,35 and sMicroglial cells are activated within minutes after the onset of ICH,35. ActiBesides microglia, other blood-derived inflammatory cells, such as leukocytes and macrophages, are also activated after ICH and contribute to ICH-induced brain injury. Neutropin vivo and in vitro[Cytokines are well-known to be associated with inflammation and immune activation. Althoug in vitro,28,34,49 in vitro. Consist in vitro. Single- in vitro. Similar in vitro,54.NF-κB, a transcription factor involved in inflammatory responses, is also activated after ICH, leading to upregulation of gene expression that contributes to brain injury,55. ActiSeveral lines of evidence have shown that NF-κB is activated by RBCs and plasma via signaling pathways involving free radicals, cytokines and glutamate receptors. It is wTLRs, especially TLR4, are involved in the inflammatory responses and neuronal damage associated with cerebral ischemia,65. Expret al.[The roles of TLR4 in inflammation and neurological impairment following ICH have been studied recently, using TLR4-knockout mice, by our research group and others,31. CompTLR4 is expressed in various cell types in the central nervous system, including microglia, neurons and astrocytes, as well as in peripheral blood cells, such as leukocytes, macrophages and platelets,33,70,71et al.[Microglial activation in response to ICH contributes to ICH-induced brain injury by releasing cytokines, and inhibition of microglial activation has been shown to improve neurological function in animal models of ICH-41. Receet al. reportedet al..et al.[Using blood transfer experiments in which TLR4-deficient blood was injected into the brains of WT mice and WT blood into brains of TLR4-deficient mice, Sansing et al. found thTherefore, TLR4 signaling on resident microglia and on blood cells within the hemorrhage is critical for ICH-induced inflammatory injury. Because TLR4 is expressed on resident microglia which are activated within minutes after ICH,35, the TLRs are a group of Class I transmembrane proteins that consist of ectodomains, transmembrane domains, and intracellular TIR domains. TLRs recognize and bind various ligands via leucin-rich repeats in the extracellular ectodomains. The receptor-ligand binding results in conformational changes of the receptor, subsequently leading to recruitment of intracellular adaptor proteins, including MyD88, TRIF, TIRAP, or TRAM. Once reTLR4 interacts with two distinct adaptor proteins (MyD88 and TRIF) and therefore activates two parallel signaling pathways to initiate the activation of transcription factors that regulate expression of proinflammatory cytokine genes,26,73. TThe MyD88 pathway of TLR4 signaling activates not only the NF-κB pathway but also the activating protein1 (AP-1) pathway. AP-1 is a dimeric protein composed of members of the Jun, Fos, and α-fetoprotein families of proteins. AP-1 acInterestingly, lively and Schlichter showed that the expression of inflammatory mediators after ICH is age-dependent. They ex, in vivo animal studies show that heme triggers a TLR4 signaling pathway involving both MyD88 and TRIF[Because the brain is a sterile organ without any pathogens derived from bacteria or viruses, the endogenous molecules released after ICH become TLR stimulators. Several endogenous molecules have been reported as TLR4 ligands, including heme, fibrinogen, high-mobility group protein B1 (HMGB1), heat shock proteins, hyaluronan, oxidized low-density lipoprotein and amyloid β,69,83-87and TRIF, whereasand TRIF. The ligand TRIF,90. UndeMany endogenous TLR4 ligands are known to be released during ICH. Some TLR4 ligands are crucial for activating TLR4 to trigger ICH-induced inflammation and inflammatory cytokine expression,31,91,92A deeper understanding of TLR4 signaling pathways should enable development of potential therapeutic targets for prevention and treatment of ICH. There are two ways to block TLR signaling: direct blockade by removal of TLR ligands  and inhibition of TLRs and their downstream signaling pathways. Effective removal of deposited blood and disintegrated cells by promoting hematoma clearance has been demonstrated to reduce ICH-induced neurological deficits in a mouse model of ICH,37. HemaAs TLR4 signaling plays an important role in ICH-induced inflammatory injury, TLR4 inhibition should be beneficial. TLR antagonists have been developed for a number of inflammatory and autoimmune diseases-95 and iet al. reported that this antibody blocked only LPS-induced, but not hemin-induced, TLR4 activation in macrophages, suggesting that different TLR4 receptor conformations exist in response to different ligands[We have found that a specific antibody (Mts50) blocked TLR4 signaling in a mouse model of ICH. TLR4 an ligands. The disMany TLR4 antagonists have been reported to produce anti-inflammatory effects-95. SomeIn addition to these antagonists, many agents with multiple pharmacological mechanisms have been found to have neuroprotective effects in animal models of ICH via inhibition of TLR4 signaling. For example, oxymatrine, which has anti-inflammatory, antioxidative, and antiapoptotic activities, suppresses TLR4 and NF-κB gene expression and decreases production of proinflammatory cytokines, such as IL-6, TNF-α, and IL-1β. GinkgolParadoxically, TLR4 activation with low doses of LPS prior to ischemic brain injury protects against subsequent severe ischemic injury-109. TheRBC lysis occurs at approximately 24 hours after the onset of ICH and continues for the next several days, leading to release of cytotoxic hemoglobin. HemogloS-transferase, in the brain after ICH[Haptoglobin, an abundant protein in blood plasma, has the ability to bind hemoglobin. In the brain, it is produced and released by oligodendrocytes, thereby protecting the brain against extravascular hemoglobin toxicity. Expressfter ICH. The antfter ICH.in vitro and in vivo models of ICH[Peroxisome proliferator-activated receptorγ (PPARγ) plays an important role in augmenting phagocytosis and promoting hematoma absorption,37. PPARs of ICH,37, and s of ICH,37.in vitro and in vivo after ICH, enhance phagocytosis in vitro and increase hematoma absorption in vivo[ROS are produced after ICH and contribute to ICH pathogenesis,5,13,34.n in vivo. The PPAn in vivo. Phagocyn in vivo.in vitro and in vivo experiments, PPARγ activators reduced expression of proinflammatory genes, including TNF-α, IL-1β,MMP9, and inducible nitric oxide synthase[PPARγ can also induce neuroprotection after ICH via anti-inflammatory effects. In both synthase. PPARγ isynthase,55, whicsynthase,117.Microglia/macrophages express the scavenger receptor CD36, which has been reported to assist in phagocytosis-mediated removal of RBCs after ICH. PPARγ aIncreasing evidence has shown that TLR4 signaling plays important roles in ICH-induced inflammatory brain injury by stimulating activation of microglial cells, infiltration of leukocytes, and production of cytokines and chemokines,31. The There are many challenges to be overcome before inhibition of TLR4 signaling can be used in the prevention and treatment of ICH. Though inhibition of TLR4 signaling by anti-TLR4 antibodies or deletion of TLR4 genes can effectively reduce ICH-induced neurological deficits in mice, specifiBrain injury following ICH is triggered by the presence of intraparenchymal blood. The mechanisms of ICH-induced brain injury are numerous, including cytotoxic, oxidative, and inflammatory pathways. It would be beneficial to develop a medical treatment that promotes hematoma resolution and inhibits cytotoxic, oxidative, and inflammatory insults following ICH. For example, PPARγ activators reduce ICH-induced brain injury by improving hematoma resolution and reducing oxidative injury,37. AnotHb: Hemoglobin; HMGB1: High-mobility group box 1 protein; HO: Heme oxygenase; ICH: Intracerebral hemorrhage; IFN-β: Interferon β; IL-1β: Interleukin-1β; LPS: Lipopolysaccharide; MyD88: Myeloid differentiation factor 88; NF-κB: Nuclear factorκB; Nrf2: NF-E2-related factor2; PPARγ: Peroxisome proliferator-activated receptorγ; RBC: Red blood cell; TIR: Intracellular Tollinterleukin 1 receptor; TIRAP: TIR domain-containing adaptor protein; TLR: Toll-like receptor; TNF-α: Tumor necrosis factorα; TRAM: TRIF-related adaptor molecule; TRIF: TIR domain-containing adaptor-inducing interferon; WT: Wild type.The authors declare that they have no competing interests.All authors read and approved the final manuscript. FH collected literatures and reviewed the literatures. WPF reviewed the literatures. WYC proofreaded and corrected the manuscript. ZY revised the manuscript. YQW wrote the manuscript and approved the final version of the manuscript.


Growing knowledge about the cytokine network response has led to a better comprehension of mechanisms of pathologies and to the development of new treatments with biological drugs, able to block specific molecules of the immune response. Indeed, when the cytokine production is deregulated, diseases often occur. The understanding of the physiological mechanism of the cytokine network would be useful to better comprehend pathological conditions. Moreover, since the immune system and response change their properties with development, differences in patients' age should be taken into account, both in physiological and in pathological conditions. In this study, we analyzed the profile of 48 cytokines and chemokines in the serum of healthy subjects, comparing adults (≥18 years) with young children and children (1–6 and 7–17 years). We found that a certain number of cytokines were not being produced in healthy subjects; others showed a constant serum level amongst the groups. Certain cytokines exhibited a downward or an upward trend with increasing age. The remaining cytokines were up- or downregulated in the group of the children with respect to the other groups. In conclusion, we drew some kinds of guidelines about the physiological production of cytokines and chemokines, underling the difference caused by aging.  Inflammatory response is driven by a complex network of mediators and signaling pathways. For example, cytokines regulating inflammatory response include interleukins that are responsible for communication between white blood cells, chemokines that promote chemotaxis, and interferons that have antiviral effects. Moreover, these molecules are involved in both innate and adaptive immunity, playing a significant physiological role in lymphoid tissue ontogenesis, organogenesis, vasculogenesis, and tissue repair , 2. WhenThe investigation of a large panel of systemic chemokines and cytokines is important to understand the complete mechanism of immunological changes observed in patients suffering, for example, from inflammatory and autoimmune pathologies. In order to evaluate the significance of cytokines and chemokines modulation in pathological conditions, however, it is necessary to establish the physiological range of these molecules in healthy subjects.Moreover, even if accumulating evidence suggests that pediatric and adult onset of pathologies often shares similar clinical presentations and basic therapeutic algorithms, we must recognize that children are not miniature adults. Even in the physiological parameter, there are issues specific to children that must be considered in case of disease to ensure prompt diagnosis and an appropriate medical management. Since cytokines and chemokines drive the immune response and the inflammatory processes, it is clear that their expression, at least for some of these molecules, is expected to vary with the growth of the individual, with the immune system achieving gradually a complete maturation .As information on immune cell responses in infancy and early childhood is scarce, we conducted an observational study in which a large cytokine and chemokine panel is analyzed in young children, children, and adults.The aim of the study is to create guidelines to refer to, especially evaluating if differences in the age of the subjects correspond to differences in the production of cytokines and chemokines.The study was approved by the medical ethics review board of the Institute for Maternal and Child Health, IRCCS “Burlo Garofolo,” Trieste . For a child to be eligible, informed consent had to be obtained from parents or caregivers. For ethical reasons we restricted our study population of infants and young children to those who had to undergo a medically indicated peripheral venous blood sampling before elective surgical interventions or within the scope of elective diagnostic procedures. Furthermore, subjects of any age were excluded from the study if they had an acute or chronic infectious disease, any clinically significant disorder, and if they were on any medication with known influence on immunological factors . Patients' histories of breast feeding, vaccinations, previous infectious diseases, and allergy were documented but not evaluated as covariates in the study.Subjects aged more than 18 years were included in one group (≥18). Subjects under the age of 18 years were divided into two groups: young children (1–6 years) and children (7–17 years), considering that in pediatric clinic, diseases that occur before the sixth year of life are defined as early onset pathologies.Whole blood has been collected in a covered test tube without anticoagulants and allowed to clot by leaving it undisturbed at room temperature for 20 minutes. Clot has been then removed by centrifuging at 1500 ×g for 10 minutes in a refrigerated centrifuge. The resulting supernatant was designated serum. Following centrifugation, the liquid component (serum) has been immediately transferred into a clean polypropylene tube using a Pasteur pipette. The samples were maintained at 2–8°C while handling and immediately analyzed, avoiding freeze-thaw cycles because this is detrimental to many serum components.β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12(p70), IL-13, IL-15, IL-17, Eotaxin, Basic FGF, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, PDGF-BB, MIP-1β, RANTES, TNF-β, VEGF, IL-1α, IL-2Rα, IL-3, IL-12(p40), IL-16, IL-18, CTACK, GRO-α, HGF, IFN-α2, LIF, MCP-3, M-CSF, MIF, MIG, β-NGF, SCF, SCGF-β, SDF-1α, TNF-α, TRAIL) was performed on serum samples using a magnetic bead-based multiplex immunoassays (Bio-Plex)  following manufactures' instructions. Data from the reactions were acquired using the Bio-Plex 200 reader, while a digital processor managed data output and the Bio-Plex Manager software returned data as Median Fluorescence Intensity (MFI) and concentration (pg/mL).The analysis of a 48 cytokines and chemokines panel (including IL-1P) values were calculated on the basis of two-tailed tests. Analysis was performed using GraphPad Prism software version 5.0 . A P value of less than 0.05 was considered for statistical significance, unless established by the adjustment for multiple comparisons.For each set of experiments, values were analyzed by calculating medians and interquartile ranges (IQRs). Box plots were used to show median, IQR, and Tukey whiskers values. The nonparametric Kruskal-Wallis test followed by the Dunn's multiple comparison test was used when appropriate. Probability , IFN-α2, LIF, MCP-3, β-NGF, and TNF-β) were under the lower limit of detection (LLOD) in all subjects, either because levels were very low or because these molecules are not produced by healthy subjects , Basic FGF, G-CSF, GM-CSF, IP-10, MCP-1, MIP-1elopment .β, IL-18, GRO-α, MIF, SCF, and SCGF-β in the adult group were significantly lower if compared to both groups of children. Moreover, IL-2Rα, SDF-1α, and TRAIL showed a similar trend, even if the difference was significant between the adult and only one of the other groups , that is overexpressed in several autoimmune diseases, such as ankylosing spondylitis, psoriatic arthritis, ulcerative colitis, Crohn's disease, and rheumatoid arthritis [Other cytokines and chemokines, involved in several diseases, are already targets of biological drugs . For exarthritis –12. Anakrthritis . Mepolizrthritis .α and IL-1β, for example, are known to be molecules with a strong proinflammatory role [Several cytokines were not found in the sera of healthy subjects or were below levels of detection. Some of these molecules are distinctive of a pathological situation or of an inflammatory response. Both IL-1ory role , IL-15 hory role , and IL-ory role .β, IL-18, GRO-α, MIF, SCF, SCGF-β, IL-2Rα, SDF-1α, and TRAIL, showing that these molecules are mainly produced during childhood and youth with respect to adulthood.A decreasing trend has been found for MIP-1On the contrary, IL-17 and Eotaxin showed an increasing trend, correlated with the age.γ, PDGF-BB, and TNF-α) showed more complex profiles, with a marked difference in the production in the children group (7–18 years), probably caused by the physiological changes that occur during adolescence. Similar findings were described in other studies [Other cytokines (IL-4, IL-6, IL-13, IFN-It must be taken into account that this work was conducted on subjects' serum, and thus the levels we found refer to mature and circulating cytokines and chemokines. Moreover, no clear explanation exists to understand how sex, sex hormones, and/or chromosomes affect the immune system and why the female/male ratio is high in several autoimmune and autoinflammatory diseases , 22. TheIn conclusion, even though the findings must be confirmed by further studies with larger cohorts of patients, guidelines were drawn to assist in evaluating the levels of cytokines and chemokines in the serum of healthy subjects of different age, in order to better comprehend the production of these molecules in physiological conditions.


H17 cell, B cell, and neutrophil responses. Clinical manifestations include recurrent skin and lung infections, serum IgE elevation, connective tissue repair and development alterations, and the propensity for vascular abnormalities and tumor development. Signal transducer and activator of transcription 3 (STAT3) signaling, dedicator of cytokinesis 8 (DOCK8) signaling, and tyrosine kinase 2 (TYK2) signaling alterations have been implicated in 3 forms of hyper-IgE syndrome.Hyper-IgE syndrome is a primary immunodeficiency marked by abnormalities in the coordination of cell-cell signaling with the potential to affect T STAT3 gene, located on chromosome 17q21, encodes a transcription factor that dimerizes with itself, signal transducer and activator of transcription 1 (STAT1), or STAT4 after cognate receptor activation and translocates to the nucleus to affect transcription in response to multiple cytokines  and in the DHR2 domain (important for binding to Rho family GTPases)[ome 9p24 . Loss ofome 9p24 . Both RAome 9p24 . Rho GTPome 9p24 -39. Dock GTPases),41 .+ T-cell stimulation and clonal proliferation [+CD45RA+CCR7+ circulating T cells; to a lesser degree, there is a decline in CD8+CD45RA-CCR7-/+ memory T cells and an increase in a senescent population of effector memory CD45RA+ (TEMRA) cells with the phenotype CD8+CD57+CX3CR1+CD27-CD28-CD127- [H17 cell functional responses; however, these findings do not appear as prominent as in AD-HIES [+ and CD8+ long-lived memory T-cell survival [B and T lymphocytes express the most DOCK8 . Hence, feration . Further8-CD127- . Some pa AD-HIES . A mousesurvival . Engelhasurvival  postulatTYK2 is located on chromosome 19p13.2 [Tyrosine kinase 2 (TYK2) is in the family of Janus kinase molecules, of which there are 4 known in humans.  19p13.2 -49. TYK2 19p13.2 -58. The  19p13.2 ,60 an SH 19p13.2 -64 and a 19p13.2   manifestations of AD-HIES are rare. One case report described a 26-year-old man with sigmoid diverticulitis that progressed to a pelvic abscess. Resolution required drainage, intravenous antibacterial and antifungal agents, and sigmoid colectomy for successful treatment . There hAD-HIES patients also have an increased risk of developing lymphomas. This risk may be as high as 259-fold over the general population . A revieS. aureus are still prominent, but affected subjects have a higher rate of viral infections, which include molluscum contagiosum, herpes simplex virus, and varicella . Food and environmental allergies are common [S. pneumoniae, Haemophilus influenza, Pneumocystis jiroveci, Proteus mirabilis, P. aeruginosa, Cryptococcus, respiratory adenovirus, and respiratory syncytial virus [Patients with DOCK8 immunodeficiency syndrome (DIDS), the most common form of AR-HIES, have a slightly different presentation from that described above Table . The seve common ,43. Asthe common ,43. Recual virus . MortaliThe typical facial presentation of a broad nose, prominent forehead, and facial asymmetry, as well as retention of primary teeth, is absent. Scoliosis is rare in this subset. Mucocutaneous candidiasis is common in DIDS.Haemophilus influenza[There is an increase in neurologic manifestations in DIDS, including facial paralysis, hemiplegia, and CNS vasculitis . Reporteinfluenza,43. Cereinfluenza. In the influenza. The disinfluenza.In the GI tract, salmonella enteritis and giardiasis have been reported. Mortality from malignancy is far higher in DIDS than in AD-HIES. Skin-originating cancers are more common and include fatal metastatic squamous cell carcinoma and fatal cutaneous T-cell lymphoma-leukemia . Burkitt+ and low CD8+ counts but normal CD4+ to CD8+ ratio [DIDS laboratory findings can reveal, in addition to elevated IgE and eosinophilia seen in AD-HIES, low absolute lymphocyte counts, low total T-cell counts, with low CD48+ ratio ,43. Neut8+ ratio ,43. IgG 8+ ratio ,43.TYK2, but in different locations of the gene. Furthermore, the patients were of different ethnic backgrounds and had varied clinical symptoms. Both patients lacked features typical to AD-HIES, such as skeletal or dental alterations and both patients had susceptibility to intracellular Mycobacterium bovis after Bacille Calmette-Geurin (BCG) vaccination, but with variable severity. Both had episodes of cutaneous herpes infection but with variable severity.Two patients with TYK2 deficiency have been reported ,86. The Brucella spp after ingestion of unpasteurized cheese. The patient was treated with a 6-week antibiotic course and was found to have neurobrucellosis and pneumonia 1 month later. The patient was severely debilitated after this episode with left temporal, occipital, and bilateral parietal brain infarcts accompanied by sensorineural hearing loss and residual cognitive impairment. At age 11, he had an episode of herpes zoster involving the right maxillary branch of the trigeminal nerve [The patient with more severe infections was of Turkish descent, had an early termination codon in TYK2 at amino acid position 767 caused by a 9-base pair DNA deletion in exon 16, had mildly elevated IgE (to a maximum of 218 IU/mL), no atopy, asthma, skin candidiasis, boils, folliculitis, or cold abscesses. This patient developed 2 disseminated infections with intracellular organisms. Specifically, 6 months after vaccination with BCG, he was noted to have axillary lymphadenopathy at age 8 months. This infection relapsed after treatment with isoniazid and became generalized at age 21 months, affecting the cervical, axillary, and inguinal lymph nodes bilaterally. Laboratory assessment during chronic infection demonstrated normal nitroblue tetrazolium test, normal serum IgG, IgM, IgA, and mildly elevated IgE. He had no relapse after 18 months of treatment. The patient was subsequently infected at age 8, with the intracellular organism al nerve .+ T cells were stimulated with IL-2 and anti-CD3 [The patient with more mild infections was of Japanese descent, had an early termination codon at amino acid position 90 caused by a frame shift mutation at DNA coding regions 70 to 89, had elevated IgE (2100 IU/mL), and susceptibility to viruses, fungi, mycobacteria, and intracellular and extracellular bacteria. He was noted to have atopic dermatitis-like rash in the first month of life, skin abscesses, oral candidiasis, recurrent otitis media, sinusitis, pneumonias, molluscum contagiosum, and herpes simplex infection of the skin and mucosa. He was also noted to develop 2 infections with intracellular infections. The first infection was to BCG at age 22 months. The second infection was to non-typhi salmonella gastroenteritis at age 15. Peripheral blood laboratory assessment revealed normal quantitative T, B, and natural killer cells and normal neutrophil function. In vitro peripheral blood mononuclear cell studies from the patient demonstrated higher basal class I HLA expression, complete defects in type I interferon responses, and failure to make interferon γ after IL-12 stimulation. In vitro studies also revealed blunted IL-6 and IL-10 responses with either absent or attenuated feedback inhibition through suppressor of cytokine signaling (SOCS)3 expression after cytokine stimulation. In vitro studies also revealed increased IL-5 and IL-13 when naive CD4anti-CD3 .Diagnosis of HIES is based on clinical and laboratory findings originally described in a NIH HIES scoring criteria consisting of 21 features . AlthougSTAT3 mutation include: abscesses of internal organs, other severe infections, pneumatoceles, nail/mucocutaneous candidiasis, bone fractures with minimal trauma, scoliosis, and a family history of HIES [The features identified in the NIH scale that are most suggestive of a  of HIES . A subje of HIES . Attempt of HIES .H17 cells, and a definitive diagnosis would require a known STAT3 mutation [STAT3 mutations, the features unique to the AR-HIES secondary to DOCK8 were not addressed.Revised clinical guidelines for the diagnosis of STAT3 mutant HIES have been proposed by Woellner et al. A possibSTAT3 mutation analysis is currently available commercially and used in the research setting, given the large size of the gene and expense of this endeavor. The use of high resolution polymerase chain reaction-based DNA-melting assays to identify and screen patients for STAT3 mutations are under investigation [STAT3 mutations in AD-HIES patients and is expected to identify such mutations in 90% of AD-HIES patients [STAT3 mutations identified [tigation . This astigation . This tepatients . To dateentified .T-cell receptor excision circle (TREC) analysis in newborn screening to detect severe combined immune deficiency identified 3 consanguineous siblings with DIDS to have low TRECs in one sibling and undetectable TRECs in the older siblings aged 4 and 6 . These fTYK2 sequencing is available at specialized research laboratories.Commercially available tests for diagnosis of DOCK8 and TYK2 deficiency are limited. DIDS diagnostic considerations include assessment for low serum IgM, low T-cell numbers, and low T-cell proliferative responses. DOCK8 and S. aureus and fungal infections is essential as these patients often display fewer of the traditional findings and symptoms associated with these infections [The importance of early recognition and treatment of fections . Althougfections ,94. Vira+ and CD15+ cells being identified as donor 21 days after HCT. After discharge, the subject did not show evidence of any skin infections. Unfortunately, long-term follow-up was unable to be performed as the patient was hospitalized and died 58 days after transplantation due to Klebsiella pneumoniae bacteremia. The authors speculate that the transplant recipient's congenital asplenia contributed to morbidity and mortality [Hematopoietic cell transplantation (HCT) has been used in treating both the autosomal recessive and autosomal dominant forms of the disease. Limited information exists regarding long-term outcome; however, many experts recommend transplantation for DIDS, as long-term outcome without transplantation is poor owing to the risk for fatal infections, malignancy risk and CNS infarction, and bleeding . Successortality . Anotherortality .+ cell analysis. After transplantation, both subjects demonstrated normalized IgE levels, which remained normal for 14 and 10 years after transplantation, respectively. In addition, neither subject has had recurrent infections requiring hospitalization, and both have been free of skin infections [H17 counts were normal [Some experts recommend using HCT in AD-HIES patients with malignancy because other disease-related effects can be managed without HCT. The role of HCT in the treatment of AD-HIES is less consistent than reports on patients with DIDS. One case series reported 2 subjects with AD-HIES and non-Hodgkin lymphoma who underwent allogeneic HCT with successful engraftment, and complete donor chimerism was confirmed by CD3fections . Osteopoe normal .The authors declare that they have no competing interests.


Lipocalin-2 is a novel adipokine with connection to insulin resistance. In this study, we aimed to investigate the association of serum lipocalin-2 with glucose metabolism and other metabolic phenotype in a large-scale Chinese population.We evaluated serum lipocalin-2 in a cross-sectional sample of 2519 Chinese aged from 50 to 82 year in a Shanghai downtown district by ELISA. Glucose, insulin, lipid profile, inflammatory markers, and adipokines were also measured.p = 0.009) after adjustment of age, gender, smoking, alcohol drinking, family history of diabetes, serum CRP, serum adiponectin, serum CXCL5, HOMA-IR, BMI, and waist/hip ratio. The OR for participants with impaired glucose regulation and type 2 diabetes was 1.31 .Serum lipocalin-2 was significantly higher in subjects with isolated impaired fasting glucose, isolated impaired glucose tolerance, combined impaired fasting glucose/impaired glucose tolerance and newly-diagnosed type 2 diabetes than in those with normal glucose regulation. Lipocalin-2 elevation was clearly associated with a higher risk for impaired glucose regulation (OR 1.30 for each 10 ng/ml increase in serum lipocalin-2, 95% CI 1.23-1.62, Our findings suggest that elevated serum lipocalin-2 is closely and independently associated with impaired glucose regulation and type 2 diabetes. Obesity is a major risk factor for insulin resistance. It is well known that adipocytes could secrete a variety of biologically adipokines, which are thought to contribute to the development of insulin resistance and cardiovascular disease -3. LipocLipocalin-2, also known as neutrophil gelatinase-associated lipocalin, neu-related lipocalin, siderocalin, uterocalin, and 24p3, belongs to the lipocalin super family. It shows high affinity to several small hydrophobic ligands such as retinoids, fatty acids, pheromones, steroids and iron . LipocalRecent studies in animal models showed that lipocalin-2 also plays an important role in systemic insulin sensitivity and glucose homeostasis ,11. LipoTo better evaluate the role of lipocalin-2 in glucose metabolism, insulin resistance and chronic Low-grade systemic inflammation, we explored the association of serum lipocalin-2 levels with chronic inflammatory marker, insulin resistance and glucose metabolism states in a large-scale Chinese population. We examined lipocalin-2 distribution in 2519 middle-aged and older Chinese subjects in a cross-sectional study and explored the association of serum lipocalin-2 levels with glucose metabolism as well as other metabolic parameters.The design and recruitment of the population-based cross-sectional study has been described in detail elsewhere . In 2007A standardized questionnaire was used by trained physicians to collect information such as age, sex, smoking (yes/no), alcohol drinking (yes/no), and self-reported diabetes, hypertension, dyslipidemia. According to participants' responses to the corresponding questions, family history of diabetes was classified as yes or no.2), or overweight (24-28 kg/m2), or obesity (≥ 28.0 kg/m2), according to the criteria for Chinese individuals [All subjects were assessed after overnight fasting for at least 10 h. The details of anthropometric measurements including height, weight, waist circumference, hip circumference and blood pressure were carried by trained physicians. BMI was calculated as weight in kilograms divided by the square of height in meters and categorized as normal weight . Fasting insulin was determined by radioimmunoassay . Insulin resistance was estimated using homeostasis model assessment index-insulin resistance (HOMA-IR).Type 2 diabetes and prediabetes were defined by 2005 ADA criteria . A fastiThe serum CRP, adiponectin, CXCL5 and lipocalin-2 were determined in duplicate by ELISA with Duoset kit  as recommended by the manufacturer. The ELISA system had an intraassay coefficient of variation of 3.1-9.5% and an interassay coefficient of variation of 4.3-10.7%, respectively.Normally distributed data were expressed as means ± SD, whereas variables with a skewed distribution were reported as median (interquartile range) and log transformed to approximate normality before analysis. Categorical variables were represented by frequency and percentage. Univariate and multivariable stepwise regression analysis were used to investigate the association of serum lipocalin-2 with clinical and biochemical characteristics. The stepwise regression procedure was used to obtain determinants of serum lipocalin-2, potential confounding variables including age, gender, smoking, alcohol drinking, family history of diabetes, serum CRP, serum adiponectin, serum CXCL5, HOMA-IR, BMI, waist circumference, and waist/hip ratio were controlled in the regression models. To study the association of impaired glucose regulation (IGR) with lipocalin-2, we defined subjects with normal glucose regulation as 0 (n = 1143), and impaired glucose regulation as 1 (n = 984), and excluded type 2 diabetic patients from logistic regression analysis. For the association of combined impaired glucose regulation and type 2 diabetes with lipocalin-2, we defined subjects with normal glucose regulation as 0 (n = 1143) and combined impaired glucose regulation and type 2 diabetes as 1 (n = 1376) in the logistic regression analyses. Homogeneity of groups was determined when the means showed significant differences. Means of these groups were compared by the Student-Newman-Keuls method. All statistical analysis was performed with the SPSS Statistical Package . A p value of less than 0.05 was considered to be statistically significant.Our study involved 1143 subjects with NGR and 392 with newly diagnosed type 2 diabetes . However, serum C-reactive protein was not an independent determinant for serum lipocalin-2 (p > 0.05).Body mass index, waist circumference, logp < 0.001) and 2 h OGTT plasma glucose  in total population. Serum lipocalin-2 concentrations were significantly higher in subjects with isolated IFG, isolated IGT, IFG + IGT, and newly diagnosed type 2 diabetes compared with those of subjects with NGR  after adjustment for age, gender, BMI, and family history of diabetes. There was no statistical difference between the four groups with glucose metabolism dysregulation  after adjustment for age, gender, smoking, alcohol drinking, family history of diabetes, serum CRP, serum adiponectin, serum CXCL5, HOMA-IR, BMI, waist/hip ratio, serum triacylglycerol, total cholesterol, HDL-cholesterol and LDL-cholesterol. The OR for subjects with impaired glucose regulation and type 2 diabetes was 1.31 .Remarkably, as presented in Table In this study, we found a strong correlation between serum lipocalin-2 levels and impaired glucose regulation as well as type 2 diabetes in a middle-aged and elderly Chinese population. Moreover, this association is independent of potential confounders.Lipocalin-2 has been reported to be elevated in certain metabolic disorders, including obesity, polycystic ovary syndrome and type 2 diabetes ,13,17. IPancreatic β-cell dysfunction and insulin resistance are hallmarks of type 2 diabetes. We found that fasting serum insulin and HOMA-IR were clearly associated with lipocalin-2 even in stepwise regression analysis, suggesting that this protein might be an indicator for β cell function and insulin resistance in humans. Lipocalin-2 has been proposed to be an iron delivery protein . LipocalIncreasing evidences suggest a critical role for PPARγ in the regulation of insulin sensitivity. However, the exact role of lipocalin-2 in obesity-induced insulin resistance remains controversial. Jin et al. observed developed insulin resistance and decreased adipose PPARγexpression in the absence of lipoclain-2 mice, suggesting that lipocalin-2 is a critical selective modulator of PPARγactivation . Jun et Visceral fat accumulation has been well-associated with adipose tissue inflammation and insulin resistance . In presType 2 diabetes was associated with wide pathophysiological pathways, including inflammation . CRP is One of the interesting findings of the present study is that the concentration of lipocalin-2 is also associated with CXC ligand 5 (CXCL5). Elevated CXCL5 levels were strongly and independently associated with insulin resistance . CXCL5 iTo our knowledge, this is the first study to investigate the association of serum lipocalin-2 and the development of type 2 diabetes as well as impaired glucose regulation in Chinese. The main strength of our study is that all diabetic participants were newly diagnosed. None had used glucose-lowering treatment or insulin. Hence, the confounding effect of glucose-lowering treatment could be excluded. Anyway, there are still some limitations in this work. First, HbA1c levels were not measured in this study, some patients with diabetes might have been missed. Second, No causal relationship between lipocalin-2 levels and the risk of impaired glucose regulation or diabetes can be drawn, since the study was cross-sectional designed.In conclusion, our study indicates that serum lipocalin-2 is independently correlated with impaired glucose regulation and type 2 diabetes. Although the underlying pathophysiological mechanisms remain unclear, it seems that insulin resistance and chronic low-grade systematic inflammation may be involved in this association.The authors declare that they have no competing interests.Conceived and designed the experiments: ZY, RH Performed the experiments: YH, ZY, QL Analyzed the data: ZY Contributed reagents/materials/analysis tools: JW, XT, LC, MH, XW, BL, ZZ, WZ, SQ. Wrote the paper: YH All authors read and approved the final manuscript.


Rice black-streaked dwarf virus (RBSDV) causes great losses in rice, maize and wheat production in Asian countries. The use of serological methods for RBSDV detection depends on the availability of antibodies. In this study, three highly sensitive and specific murine monoclonal antibodies (MAbs) against RBSDV antigens were produced using crude extracts from tumors of RBSDV-infected maize as the immunogen, and two serological assays, antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA) and dot enzyme-linked immunosorbent assay (dot-ELISA) were developed for RBSDV detection.-1) with the crude extract of infected maize, 1:10,240, 1:20,480, 1:20,480  with the crude extract of infected rice, 1:5,120, 1:10,240, 1:10,240  with the crude extract of infected wheat, 1:9,600, 1:9,600, 19,200  with the crude extract of infected planthopper. The newly developed ACP-ELISA could detect the virus in the infected maize, wheat, rice tissue crude extracts diluted at 1:81,920, 1:20,480, 1:10,240 , respectively, and in individual viruliferous planthopper extract diluted at 1:19200 . The dot-ELISA was proved to detect the virus in the infected maize, wheat and rice tissue crude extracts diluted at 1:320 , and in individual viruliferous planthopper extract diluted at 1:1,600 , respectively. Field plants (915) and planthopper samples (594) from five provinces of China were screened for the presence of RBSDV using the two developed serological assays. The results indicated that 338 of the 915 plant samples and 19 of the 594 planthopper samples were infected by RBSDV.All three MAbs reacted strongly and specifically with the crude extracts from RBSDV-infected plant and planthopper tissues. The detection endpoints of three MAbs  in ACP-ELISA were respectively 1:40,960, 1:40,960, 1:81,920 (w/v, g mLThe newly developed ACP-ELISA and dot-ELISA were highly sensitive and specific to detect RBSDV in field plant and planthopper samples. The field survey demonstrated that RBSDV is widespread in rice, maize and wheat crops in Jiangsu, Zhejiang, Shandong provinces of China. Fijivirus in the family Reoviridae, is mainly transmitted by the small brown planthopper (Laodelphax striatellus) in a persistent and propagative manner, but not transmitted via eggs [Rice black-streaked dwarf virus (RBSDV), a member of the genus via eggs -3. RBSDVvia eggs ,3-5. Thevia eggs . The disvia eggs . RBSDV ovia eggs ,8. The ovia eggs ,3. In Chvia eggs ,8-10. Invia eggs . Virionsvia eggs ,13.RBSDV virions are non-enveloped, icosahedral, double-shelled particles with 75 to 80 nm in diameter and short surface spikes and contain 10 segments ranging from 1.8 to 4.5 kb of linear double-stranded genomic RNA (designated S1–S10) ,14. MostCurrently, some approaches have been used for detection of RBSDV: reverse transcription (RT)-polymerase chain reaction assay (PCR) , RT-loopth immunization, the spleen cells of the immunized mice were used for hybridoma preparation. Via cell fusion, cell culture, antibody detection and cell cloning, three hybridoma lines  secreting MAbs against RBSDV antigens were obtained and injected intraperitoneally into BALB/c mice to produce ascitic fluids, respectively. The IgG yields of MAbs from ascitic fluids ranged from 2.32 to 7.78 mg mL-1  reacted strongly with the crude extracts from RBSDV-infected rice, maize and wheat plant tissues, but not with RDV-, SRBSDV-, RRSV-, RSV-infected rice and healthy rice, maize and wheat plant tissues  with the crude extract of infected maize, 1:10,240, 1:20,480, 1:20,480  with the crude extract of infected rice, 1:5,120, 1:10,240, 1:10,240  with the crude extract of infected wheat, 1:9,600, 1:9,600, 19,200  with the crude extract of infected planthopper  of the three MAbs  in ACP-ELISA were respectively 1:40,960, 1:40,960, 1:81,920  or with horseradish peroxidase (HRP) in ACP-ELISA were determined by phalanx tests. The results of the three independent ACP-ELISA assays revealed that RBSDV was readily detected in infected plant tissues when the MAb and the goat anti-mouse IgG conjugated with AP were used at the dilutions of 1:6,000 and 1:7,000, respectively. Meanwhile, RBSDV was readily detected in the planthopper samples when the MAb and the goat anti-mouse IgG conjugated with HRP were used at the dilutions of 1:5,000 and 1:7,000, respectively.-1, pH9.6) of RBSDV-infected field plant and planthopper samples were used to determine the sensitivities of the developed ACP-ELISA. The results showed that the ACP-ELISA could detect the virus in infected maize, wheat, rice crude extracts diluted at 1:81,920, 1:20,480, 1:10,240 , respectively, and in individual planthopper crude extract diluted at 1:19200   Figure A, B. Spe) Figure A, 3B. ThThe optimal working dilutions of the MAb 5G5 and the goat anti-mouse IgG conjugated with -AP or HRP in dot-ELISA were also determined by phalanx tests and the result demonstrated that the optimal dilutions for the detection of the virus in plant samples were 1:5,000 for MAb 5G5 and 1:7,000 for the goat anti-mouse IgG conjugated with AP. To detect the virus in planthopper samples, the optimal dilutions of the MAb and the goat anti-mouse IgG conjugated with HRP were 1:4,000 and 1:6,000, respectively.-1), and in individual viruliferous planthopper crude extract diluted at 1:1,600   Figure C, D.CP gene sequences available in GenBank, and the results showed that the sequences of the amplified products shared 94-99% identity with the RBSDV CP gene sequences in GenBank. Those detection results indicate that RBSDV is widespread in Jiangsu, Zhejiang and Shandong provinces of China  were then developed. Three MAbs reacted with the crude extracts from the RBSDV-infected plant tissues and viruliferous planthopper, but not with RDV-, SRBSDV-, RRSV- or RSV-infected rice plants, healthy plant tissues or non-viruliferous vectors.Fijivirus in the family Reoviridae, is transmitted to rice and maize by the white backed planthopper (Sogatella furcifera) in a persistent manner [SRBSDV that is a new species in the genus t manner ,28. Recet manner ,27-29. Bt manner ,28. HencIn order to forecast and control the disease, we have developed two reliable, rapid and efficient serological methods for specific and sensitive detection of RBSDV. Because RBSDV infection is an important problem in cereal crop production in Southeast Asian countries, the serological detection methods developed in this work will support further investigations on the epidemiology of RBSDV and detect the virus more efficiently and economically than the previously available assays.-1). In this study, the ACP-ELISA could detect RBSDV in infected plant tissue extracts at the dilution of above 1: 10,240  and in individual viruliferous vector extracts at the dilution of 1:19200 , while dot-ELISA could detect RBSDV in infected plant tissue extracts at the dilution of 1: 320  and in individual viruliferous vector extracts at dilution of 1:1,600   (1 g tumor tissues in 3 mL buffer). The ground homogenate was centrifuged at 5000 × g for 5 min at 4°C. The supernatant was used to immunize five eight-week-old BALB/c mice as described previously [The white tumors of RBSDV-infected maize plants from Jiangsu Province were ground to fine powder with a mortar and pestle in liquid nitrogen and homogenized further in PBS in 0.02 mol Leviously .Animal experiments were carried out using female BALB/c mice provided by the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences  at the Research Center of the Laboratory of Animal Science, Zhejiang College of Traditional Chinese Medicine, Hangzhou, China. The animal experiments were performed according to the Principles of the Helsinki accords. The experimental protocols were approved by the Animal Ethics Committee of Zhejiang University, Hangzhou, China.Preparation of hybridomas secreting anti-RBSDV MAbs were performed as described by Wu et al. . Hybrido′, 5, 5′-Tetramethylbenzidine (TMB) substrate . The OD405 or OD450 were measured with a Microplate Reader Model 680 . Samples were considered to be positive when absorbance values were at least three times greater than the negative controls.The optimal working concentrations of the MAb and the goat anti-mouse IgG conjugated with AP or with HRP (Sigma-Aldrich) for ACP-ELISA were determined by phalanx tests as described previously 30). Detection of RBSDV in plant tissues or in planthopper vectors was performed following the procedure as described by Shang et al. [. Detecti-1 PBS (1 g plant tissues in 10 mL PBS). Individual planthopper was placed in a 0.5 mL centrifuge tube containing 50 μL PBS, and mashed with a toothpick. The plant and planthopper homogenates were centrifuged at 5000 × g for 3 min at 4°C. Negative and positive controls were the healthy and RBSDV-infected plant tissues or non-viruliferous and viruliferous planthoppers. The supernatants (2 μL) were spotted onto nitrocellulose membranes  and air-dried at room temperature. The nitrocellulose membrane was soaked in PBST containing 5% dried skimmed milk powder  for 30 min to block protein-free areas. Then, the membrane was incubated in suitably diluted or serial two-fold diluted MAb for 1 h at 37°C. After washing four times with PBST and the membrane was incubated in goat anti-mouse IgG conjugated with AP or HRP for another 1 h. Finally, after washing five times with PBST, the membrane was color-developed in NBT/BCIP or TMB substrate solution . Positive samples developed either purple or blue color within 10–25 min.The dot-ELISA Procedures were performed as described previously with slight modifications . BrieflyCP gene  sequences available in GenBank, the RBSDV S10-specific forward primer (5′- ATGGCTGACATAAGACTCGA-3′) and reverse primer (5′- TCATCTTGTCACTTTGTTTAG -3′) (nucleotides 22–1698 of S10) were designed to amplify the RBSDV CP gene using a standard RT-PCR protocol described previously [Based on RBSDV The authors declare that they have no competing interests.JW, YN and HL did most experiments and drafted the manuscript. LR did the RT-PCR for RBSDV detection. XZ and YZ conceived of the study, and participated in its design and coordination. XZ, YZ and JW proof-read and finalized the manuscript. All authors read and approved the final manuscript.


Lawsonia intracellularis is the etiological agent of proliferative enteropathy (PE), causing mild or acute hemorrhagic diarrhea in infected animals. Here we report the genome sequence of strain N343, isolated from a sow that died of hemorrhagic PE. N343 contains 24 single nucleotide polymorphisms and 90 indels compared to the reference strain PHE/MN1-00. Lawsonia intracellularis is a Gram-negative, obligate intracellular bacterium which has been identified as the causative agent of proliferative enteropathy (PE). PE is endemic in pigs, causing gross pathological changes to the lower intestine through hyperplasic lesions and a thickening of the mucosal tissue   using Mira . There are 3 plasmids that are 27,133 bp, 39,878 bp, and 194,613 bp in size, with G+C contents of 29.0, 29.2, and 32.9% and containing 29, 24, and 104 predicted CDSs, respectively. There are two rRNA gene operons and 44 tRNA genes representing all standard amino acids except pyrrolysyl. Comparative analysis between N343 and PHE/MN1-00 identified 8 single-nucleotide polymorphisms (SNPs) and 70 insertion/deletions (indels) in intragenic regions, and 16 SNPs  and 20 indels in intergenic regions.L. intracellularis N343 include the genes required for glycolysis, the pentose phosphate pathway, and the biosynthesis of peptidoglycans and lipopolysaccharides. However, components of the tricarboxylic acid (TCA) cycle and those required for the synthesis of specific amino acids are absent. Strain N343 contains 57 genes involved in ATP binding cassette (ABC)-type transport systems. Two-component signal transduction systems involved in sensing nitrogen availability and chemotaxis are present, as are the genes required for flagellum assembly. A wide range of genes related to virulence, adhesion, and invasion were identified, including genes predicted to be involved in type III secretion and genes belonging to the type V autotransporter (AT) protein family. Of these, two classical ATs were located on the chromosome, with the remaining located on plasmids 2 and 3.The genomic features of CP004029 (chromosome) and CP004030, CP004031 and CP004032 (plasmids 1 to 3).Genome and plasmid sequences are available at DDBJ/EMBL/GenBank under accession numbers


Helicobacter pylori (HP) infection. Subsequent eradication of the bacteria resulted in improvement of his thrombocytopenia. The clinical course remained uneventful at 15-month follow-up, consistent with monoclonal B-cell lymphocytosis. The observed clonal expansion with plasmacytic differentiation may have occurred under the influence of HCV with HP infection.A 71-year-old Japanese male patient infected with HCV was diagnosed with thrombocytopenia. Histological examination of the bone marrow aspirate showed numerous lymphoid aggregates with Russell bodies. Immunohistochemistry and flow cytometric analysis demonstrated clonal expansion of CD5+ CD23+ B cells. Russell bodies were positive for IgM and lambda immunoglobulin light chain. The patient also underwent gastric biopsy, which revealed  During histological examination of the bone marrow, lymphoid aggregates are occasionally found. Those lymphoid aggregates may be either neoplastic or reactive, yet the histological distinction can be difficult , as it oHelicobacter pylori (HP), are well known to drive B-cell proliferation, causing different monoclonal or polyclonal lymphoplasmacytic diseases, and this is reviewed by Chiba and Marusawa [Some infective agents, such as hepatitis C virus (HCV) and Marusawa . In patiMarusawa , 5. ReceMarusawa . RBs areMarusawa . AccordiHere, we describe a clonal B-cell population with a CLL-like phenotype aggregated in the bone marrow and show plasmacytic differentiation without clinical progression in a case infected with both HCV and HP.6/L; absolute number of lymphocytes, 1, 170 × 106/L; red blood cell count, 495 × 1010/L; platelet count, 35, 000 × 106/L; aspartate aminotransferase, 23 IU/L; alanine aminotransferase, 11 IU/L. Total protein was 7.3 g/dL. Antigen of hepatitis B virus was negative, and HCV antibody was positive. No abnormality of serum or urine protein was noted. The differentiated nuclear cells from bone marrow aspirate showed 4% myeloid precursors, 1% myelocytes, 7% metamyelocytes, 34% neutrophils, 3% eosinophils, 0.3% monocytes, 17% lymphocytes, 0.2% plasma cells, and 33% erythroblasts. Although clot section from the aspirated bone marrow showed numerous lymphoid aggregates suggesting lymphomatous involvement, the patient was observed at our clinic without therapy for 15 months. Meanwhile, he also underwent gastric biopsy, which revealed chronic gastritis with HP, but no RB  (Vysis), which revealed no fusion signal of cyclin D1 and the Ig heavy chain genes.Clot section from the aspirated bone marrow showed numerous lymphoid aggregates composed of small lymphocytes with numerous Russell bodies at the periphery Figures  and 2 inIn the present case, clonal expansion of B-cells with a CLL-like phenotype remained in the bone marrow for 15 months without clinical manifestation, consistent with MBL. Of note, each lymphoid aggregate was surrounded by numerous IgM-positive RB, indicating plasmacytic differentiation. Although histological differential diagnosis of the present case would include lymphoplasmacytic lymphoma, characterized by admixture of small lymphocytes and plasma cells with IgM expression, CD5 expression and clinical indolence seen in the present case are not typical of lymphoplasmacytic lymphoma. Mantle cell lymphoma, another CD5-positive B-cell tumor, can be also negated due to the lack of a cytogenetic hallmark or chromosomal translocation involving the CyclinD1 gene.6/L, and the majority of such clonal populations show a CLL (CD5+/CD23+)-like immunophenotype, yet they will not progress to overt CLL. Such a condition has been recognized as MBL, for which the diagnostic criterion is simply based on the detection of a clonal expansion of peripheral B-cells. However, it has been suggested that almost all MBL have some degree of bone marrow involvement, yet detailed understanding of the bone marrow in MBL remains to be clarified [In western countries, more than 4% of the general population over the age of 40 harbor clonal B-cell populations with an absolute number of peripheral B-cells of less than 5, 000 × 10 CD5+/CD2+-like imIn the pathway of B-cell differentiation, activation-induced cytidine deaminase (AID) is a physiologic genome mutator essential for somatic hypermutation and class switch recombination of Ig genes. AID is induced by nuclear factor kappa B activation via both HCV and HP, causing gene mutations in Ig and non-Ig genes, consistent with the notion that HCV and HP drive B-cell proliferation and directly modulate cellular function, thus giving cell growth advantages and resistance to apoptosis . In factHP infection is also associated with B-cell proliferation such as some gastrointestinal lymphomas. Recently, RB gastritis, a variant of chronic gastritis with numerous RB, has been reported to be associated with HP infection, and the disappearance of RB upon follow-up biopsy after the eradication of HP has been described, thus supporting the causative role of HP in the development of RB . In the


Arthrobacter sp. strain PAO19 is a polyphosphate-accumulating organism isolated from maize rhizosphere soil. Here we report its genome sequence, which may shed light on its role in phosphate removal from water bodies. To our knowledge, this is the first genome announcement of a polyphosphate-accumulating strain of the genus Arthrobacter. Arthrobater, with maximum identity to that of A. nicotianae strain DSM 20123. Arthrobacter spp. strains are among the most frequently isolated, indigenous, aerobic bacterial genera found in soils. Members of the genus are metabolically and ecologically diverse and have the ability to survive in environmentally harsh conditions for extended periods of time  and phosphorus (P) nutrients, is one of the most serious water pollution problems . N limit of time , 8. To d strains , 10. In  strains , 12. In Arthrobacter sp. strain PAO19 was performed using an Illumina Hiseq2000 with 101-bp paired-end reads. The reads were trimmed and assembled de novo using SOAPdenovo2 (de novo assembly yielded 499-fold coverage of a 3,637,746-bp draft genome contained in 47 contigs with an average GC content of 59.52%. The contig N50 was approximately 246 kb, and the largest contig assembled was approximately 448 kb. A total of 3,451 ORFs, 41 tRNAs, and 3 rRNAs were predicted from the draft genome.The genome sequencing of Pdenovo2 . The prePdenovo2 –16, respPdenovo2 . The de In functional annotation of the genome, we identified as being involved in the metabolisms of phosphorus and inorganic polyphosphate the genes for polyphosphate kinase domain-containing protein at contig 1 and contig 8, polyphosphate kinase and exopolyphosphatase at contig 1, polyphosphate-glucose phosphotransferase at contig 9, low-affinity inorganic phosphate transporter at contig 11, phosphate ABC transporter substrate-binding protein PstS at contig 7 and contig 23, phosphate ABC transporter inner-membrane subunits PstC and PstA and phosphate ABC transporter ATP-binding subunit PstB at contig 19, phosphate transport system regulatory protein PhoU at contig 24, and alkaline phosphatase at contig 6.ATKN00000000. The version described in this paper is the first version, ATKN01000000.This whole-genome shotgun project has been deposited in DDBJ/EMBL/GenBank under the accession no.