HAC1 mRNA to generate the bZIP transcription factor Hac1, which subsequently activates its target genes to increase the protein-folding capacity of the ER. This cellular machinery, called the unfolded protein response (UPR), is believed to be an evolutionarily conserved mechanism in eukaryotes. In this study, we comprehensively characterized mutant phenotypes of IRE1 and other related genes in the human fungal pathogen Candida glabrata. Unexpectedly, Ire1 was required for the ER stress response independently of Hac1 in this fungus. C. glabrata Ire1 did not cleave mRNAs encoding Hac1 and other bZIP transcription factors identified in the C. glabrata genome. Microarray analysis revealed that the transcriptional response to ER stress is not mediated by Ire1, but instead is dependent largely on calcineurin signaling and partially on the Slt2 MAPK pathway. The loss of Ire1 alone did not confer increased antifungal susceptibility in C. glabrata contrary to UPR-defective mutants in other fungi. Taken together, our results suggest that the canonical Ire1-Hac1 UPR is not conserved in C. glabrata. It is known in metazoans that active Ire1 nonspecifically cleaves and degrades a subset of ER-localized mRNAs to reduce the ER load. Intriguingly, this cellular response could occur in an Ire1 nuclease-dependent fashion in C. glabrata. We also uncovered the attenuated virulence of the C. glabrata Δire1 mutant in a mouse model of disseminated candidiasis. This study has unveiled the unique evolution of ER stress response mechanisms in C. glabrata.Proper protein folding in the endoplasmic reticulum (ER) is vital in all eukaryotes. When misfolded proteins accumulate in the ER lumen, the transmembrane kinase/endoribonuclease Ire1 initiates splicing of  Candida glabrata has lost the canonical UPR, but instead possesses the RIDD pathway and is relatively tolerant to ER stress. The transcriptional response to ER stress was dependent mainly on calcium signaling mediated by the protein phosphatase calcineurin in C. glabrata. Our results provide novel insights into ER quality control mechanisms and are useful for understanding evolutionary biology and the development of antifungal agents targeting the UPR.The majority of secretory and transmembrane proteins are structurally matured in the endoplasmic reticulum (ER). The accumulation of misfolded proteins in the ER (ER stress) activates the ER-resident stress transducer Ire1, which has two distinct outputs: the unfolded protein response (UPR) and regulated Ire1-dependent decay (RIDD). The UPR is a transcriptional response to increase the protein folding capacity of the ER. RIDD induces degradation of ER-localized mRNAs to reduce the ER load. To date, the UPR has been believed to be an evolutionarily conserved pathway in almost all eukaryotic species, while RIDD has been found only in metazoans. Recent studies in several pathogenic fungi revealed that the UPR is implicated in antifungal resistance and virulence, and thus it has attracted attention as a therapeutic target. Here, we demonstrate that the important fungal pathogen  In eukaryotic cells, the majority of secretory and transmembrane proteins are folded and modified in the lumen of the endoplasmic reticulum (ER). Contingent on proper folding, they are either transported to the Golgi apparatus or degraded Saccharomyces cerevisiae, the ER-resident transmembrane stress transducer Ire1, which contains an ER luminal sensing domain and cytosolic protein kinase/endoribonuclease domains, is responsible for detecting unfolded proteins and triggering the UPR HAC1 mRNA to excise the intron, allowing translation of the basic-leucine zipper (bZIP) transcription factor Hac1 that subsequently induces transcription of the UPR target genes In response to ER stress, eukaryotic cells activate a signaling pathway, termed the unfolded protein response (UPR), to induce a comprehensive gene expression program that adjusts the protein-folding capacity of the ER , an N-linked glycosylation inhibitor, and dithiothreitol (DTT), an inhibitor of disulfide bond formation. Compared to bilities . Other rC. glabrata IRE1 and HAC1 orthologs were identified by BLASTp searches using the NCBI (http://www.ncbi.nlm.nih.gov/BLAST/) and Genolevures (http://cbi.labri.fr/Genolevures/elt/CAGL) databases. The respective amino acid sequences of S. cerevisiae IRE1 (YHR079c) and HAC1 (YFL031w) were used as queries. The deduced amino acid sequence of C. glabrata IRE1  displayed 53.6% similarity and 35.8% identity with that of S. cerevisiae IRE1. As presented schematically in C. glabrata Ire1 consists of typical Ire1-domain structures C. glabrata IRE1 is syntenic with S. cerevisiae IRE1 situated on chromosome VIII, but the 3′ region of C. glabrata IRE1 is orthologous to S. cerevisiae chromosome IV  in the C. glabrata genome, exhibiting 36.9% similarity and 24.2% identity with the deduced amino acid sequence of the unspliced form of S. cerevisiae HAC1. It has been known that functional orthologs of transcription factors often display limited sequence similarity outside their DNA-binding domains C. glabrata HAC1 is not highly conserved at the overall sequence level, but contains a bZIP domain at the N-terminal region. The putative DNA binding domain of C. glabrata Hac1 displays strong sequence similarity to those of other Hac1 homologs  contains the 252 nucleotides intron, which is excised by Ire1 to generate the induced form of HAC1 (iHAC1)  . The welno acids . Howeverice site .C. glabrata, we constructed a mutant of each in which the entire open reading frame (ORF) of IRE1 or HAC1 was deleted, and examined growth on plates containing TM and DTT , suggesting that Hac1 is not a downstream target of Ire1 in C. glabrata.Ire1-mediated splicing of  and DTT , they weHAC1 ORF in S. cerevisiae and C. glabrata. In S. cerevisiae, HAC1 mRNA was efficiently spliced upon ER stress induced by 1.5 µg/ml TM or 5 mM DTT  using only the bZIP domain sequences of S. cerevisiae Hac1, C. neoformans Hxl1, and Homo sapiens Xbp1 as described previously HAC1, the following eight putative genes were identified in the C. glabrata genome (YAP1), CAGL0J06182g (SKO1), CAGL0K02585g (YAP3), CAGL0M10087g (YAP3), CAGL0L02475g (GCN4), CAGL0F03069g (CAD1/YAP2), CAGL0F01265g (YAP7), and CAGL0M08800g (YAP6). We sequenced the RT-PCR products of these mRNAs, which were obtained from C. glabrata wild-type cells treated with TM at concentrations of 1.5 and 5 µg/ml for 3 h, but none of them displayed evidence of a splicing event (data not shown). The results suggest that another Hac1 homolog is unlikely to exist in C. glabrata.To address the possibility that a a genome : CAGL0H0C. glabrata Ire1 could complement S. cerevisiae Ire1 function, C. glabrata IRE1 was expressed under the S. cerevisiae ADH1 promoter in the S. cerevisiae Δire1 mutant. After treatment with 1.5 µg/ml TM for 3 h, a spliced form of S. cerevisiae HAC1 was generated in the wild-type strain, but not in the Δire1 strain regardless of the presence of C. glabrata IRE1 . As expected, C. glabrata Ire1 did not rescue the growth of the S. cerevisiae Δire1 mutant in the presence of TM , PDI1 (protein disulfide isomerase), DER1 (ER-associated degradation), and FPR2 (ER protein trafficking), was examined. Compared to the wild-type strain, the expression levels of these genes were decreased in the Δhac1 mutant, but reversed by the heterologous expression of C. glabrata HAC1 in the mutant . Since sustained activation of the UPR is toxic to cells C. glabrata HAC1 and the induced form of S. cerevisiae HAC1 (iScHAC1) similarly impaired cell growth even under normal growth conditions . These results indicate that C. glabrata Hac1 was matured without splicing in S. cerevisiae. In agreement with this, C. glabrata Hac1 also rescued growth of the S. cerevisiae Δire1 mutant in the presence of TM  pathway are also required for the ER stress response in S. cerevisiaeC. glabrata using four C. glabrata mutant strains lacking a key component of these signaling pathways, including Ire1, the regulatory B subunit of calcineurin Cnb1, the calcineurin-regulated transcription factor Crz1, and the last member of the PKC1-MAPK cascade Slt2. While all mutants displayed wild-type growth on a drug-free control plate, the Δire1, Δcnb1, and Δslt2 mutants exhibited decreased tolerance to both TM and DTT  with accession number GSE29855. It has been reported that at least 381 genes, corresponding to more than 5% of the ORFs in the genome, were induced in response to TM and DTT under the control of the UPR in S. cerevisiaeC. glabrata wild-type strain after treatment with TM for 3 h , which are well-known UPR targets in S. cerevisiaeKAR2 in the presence of high concentrations of TM and DTT were similar to those observed in the presence of 1.5 µg/ml TM -3′5′-GNGGCTCA-3′C. glabrata KAR2. These results support our KAR2 expression data, collectively suggesting that transcriptional induction of KAR2 in response to ER stress is mediated by the calcineurin-Crz1 pathway, but not by Ire1 signaling, in C. glabrata.Transcriptional induction of the ER-resident chaperone  mutants . These rire1 mutant at a relatively early phase (0.5 h) of ER stress  in the nucleotide-binding pocket of Ire1 kinase to asparagines abolish phosphorylation, but preserve RNase activity C. glabrata (C. glabrata kinase-dead IRE1 (IRE1-KD) harboring the corresponding mutations  in the Ire1 kinase domain. The growth defects of the Δire1 null mutant in the presence of TM and DTT were complemented by wild-type IRE1, but not by IRE1-KD  containing a 10 residue (D973-Y982) internal deletion within the nuclease domain  inhibited the transcriptional upregulation of CAGL0K11946g and CAGL0L06776g by DTT but had no or little effect on the mRNA dacay of nd ECM33 , indicatexposure . AlthougC. glabrata, we examined the growth of the Δire1 mutants under various stress conditions. Pathogenic fungi commonly encounter a variety of adverse environmental conditions, such as low oxygen availability and nutrient depletion, in the infected host. It has been reported in A. fumigatus that IreA is required for growth in hypoxia and under iron-depleted conditions, which is induced by adding the Fe2+ chelator bathophenantroline disulphonate (BPS) to the culture medium C. glabrata, the loss of Ire1 had no apparent effect on cell growth under low oxygen tension , suggesting that neither Ire1 nor Hac1 is primarily involved in cell wall stress response in C. glabrata.It is also reported in several pathogenic fungi, including C. neoformans and A. fumigatusIRE1 alone did not affect azole susceptibility in C. glabrata , was more susceptible to azole antifungals than either single mutant . These results suggest that calcineurin and Ire1 serve redundant roles in cell growth under certain stress conditions in C. glabrata. Collectively, these phenotypic analyses revealed that loss of Ire1 alone does not induce diverse phenotypes in C. glabrata unlike in other fungi.Importantly, disruption of Ire1/IreA confers a drastic increase in azole susceptibility in glabrata . Howevere mutant . The res defects . None ofC. glabrata that calcineurin and Slt2 are required for virulence in murine models of disseminated candidiasis C. glabrata virulence has not been examined. The C. neoformans Δire1 and A. fumigatus ΔireA mutants are avirulent in murine models of systemic cryptococcosis and invasive aspergillosis, respectively, which can be explained primarily by their growth defects at 37°C C. glabrata, the loss of Ire1 did not affect cell growth at 37°C (generation time of the wild-type and Δire1 strains in Yeast-peptone-dextrose (YPD) broth was 75 and 78 min, respectively). Therefore, we compared the virulence of the C. glabrata wild-type, Δire1, and IRE1-complemented strains in a mouse model of disseminated candidiasis. Immunocompetent mice infected with the Δire1 mutant displayed significantly reduced fungal burden in both kidney and spleen relative to those infected with the wild-type and Ire1-complemented strains  is much longer than those in other species and has mutations in the Ire1 recognition motifs at both splice sites, suggesting that the Ire1-dependent unconventional splicing mechanism may not be present in C. glabrataC. glabrata HAC1 complemented S. cerevisiae Hac1 functions without splicing, and C. glabrata HAC1 mRNA was not spliced by Ire1 even in ER-stressed C. glabrata cells. Furthermore, Hooks and Griffiths-Jones Candida species such as Candida parapsilosis, Candida lusitaniae, and Candida guilliermondii, despite the fact that the HAC1 ORFs are intact in these Candida species. These findings suggest that these species may have lost the unconventional splicing mechanism, but instead acquired an alternative mechanism to regulate the UPR. Future studies are warranted to determine whether Hac1 is involved in the ER stress response in these species.According to the current evidence, the UPR regulated by Ire1-Hac1 signaling is highly conserved and is a key pathway to cope with ER stress in eukaryotes ire1 strains with respect to growth defects in the presence of ER stress-inducing agents (e.g. TM and DTT) in all eukaryotic species tested so far, and the UPR mediated by Ire1-Hac1 signaling has been believed to be a phylogenetically conserved pathway in eukaryotes C. glabrata Ire1 plays a role in the ER stress response in a Hac1-independent manner. Exploration of potential Ire1 targets in this fungus by a genome-wide analysis will be of interest in future investigations . The results of our gene expression assays suggest that Ire1 does not mediate transcriptional response to ER stress in C. glabrata, raising the question of how Ire1 is involved in the ER stress response in this fungus.Although some Hac1-independent functions of Ire1 have been reported in some eukaryotic species XBP1 splicing and RIDD D. melanogasterS. cerevisiae, RIDD does not occur C. glabrata genes and the previously identified S. cerevisiae gene that were transcriptionally downregulated by the UPR in response to TM HAC1/XBP1 mRNA at the highly conserved splice junctions C. glabrata Ire1 may not need to switch between specific and nonspecific modes of cleavage. We are currently investigating how the RIDD activity is regulated in this fungus.In metazoans, ER stress triggers two distinct outputs of Ire1's nuclease activity, S. cerevisiaeC. glabrata. In S. cerevisiae, calcineurin and Slt2 are also involved in the ER stress response through different mechanisms 2+ homeostasis. TM increases Ca2+ uptake by stimulating the Cch1-Mid1 high affinity Ca2+ channel, while calcineurin dephosphorylates the Cch1 subunit of the channel to inhibit Ca2+ influx and prevents nonapoptotic cell death in S. cerevisiaeC. glabrata, loss of either calcineurin or Slt2 resulted in decreased tolerance to ER stress, and the additional deletion of IRE1 in the Δcnb1 and the Δslt2 backgrounds had synthetic effects on viability loss in the presence of TM. The results suggest that Ire1 functions in parallel with the calcineurin and Slt2 MAPK pathways to cope with ER stress in C. glabrata, consistent with previous findings in S. cerevisiaeAlthough the only known substrate of the Ire1 kinase is Ire1 itself in S. cerevisiaeC. glabrata, phenotypes of the Δire1 mutant were investigated in various stress conditions in view of clinical significance. The UPR is critical for azole tolerance in C. neoformans and A. fumigatus, and thus loss of either Ire1/IreA or Hxl1/HacA leads to increased susceptibility to azole antifungals in these species C. glabrata , but it is now known that the signaling pathway is involved in various stress responses from yeast to humans S. cerevisiae and C. albicans cells C. glabrata  and all of the institutional regulations and guidelines for animal experimentation after pertinent review and approval by the Institutional Animal Care and Use Committee of Nagasaki University under protocol number 0904130747.C. glabrata and S. cerevisiae strains used in this study are listed in 2+ chelator BPS  or the bacterial siderophore DFO  were added to SC media at final concentrations of 20 and 50 µM, respectively.The C. glabrata and S. cerevisiae was performed using a lithium acetate protocol as described previously C. glabrata deletion mutants were constructed using a one-step PCR-based technique as described previously C. glabrata parent strains were transformed with the deletion construct, and the resulting transformants were selected by tryptophan or histidine prototrophy. PCR and Southern blotting were performed to verify that the desired homologous recombination occurred at the target locus.The primers and plasmids used in this study are listed in C. glabrata and S. cerevisiae genes were amplified from the genomic DNA of CBS138 S. cerevisiae HAC1 mRNA was obtained from BY4742 cells treated with 1.5 µg/ml TM for 3 h. The extracted RNA was then reverse-transcribed using a QuantiTect Reverse Transcription kit , and the resulting cDNA was used as a template to amplify the mature S. cerevisiae HAC1, iScHAC1 (“i” for induced).To generate complementation plasmids, C. glabrata Ire1 to asparagines using the KOD-Plus-Mutagenesis Kit , and mutagenic primers CgIRE1-mut-F2171 and CgIRE1-mut-R2170. Similarly, pCgACT-PIRE-ND was created by introducing a 10 residue (D973-Y982) internal deletion within the nuclease domain of C. glabrata Ire1 using mutagenic primers, CgIRE1-F2947 and CgIRE1-R2916. All of the plasmids constructed using PCR products were verified by sequencing before use.The plasmid pCgACT-PIRE was used as the template to generate pCgACT-PIRE-KD and pCgACT-PIRE-ND. pCgACT-PIRE-KD was generated by mutating two residues, D723 and K725, in the kinase domain of Spot dilution tests and time-kill assays were performed as described previously HAC1 mRNA splicing was analyzed using a modification of a reported protocol S. cerevisiae cells grown in SC medium lacking leucine were treated with 1.5 µg/ml TM or 5 mM DTT for 3 h. Logarithmic-phase C. glabrata cells grown in SC medium were treated with either TM (5 and 10 µg/ml) or DTT (5 and 10 mM) for 1 and 3 h. These cells were harvested, and total RNA was extracted using a FastRNA Red Kit  according to the manufacturer's instructions. cDNA was synthesized from 1 µg of total RNA using a QuantiTect Reverse Transcription kit (Qiagen) in a final volume of 20 µl, and 5 µl of resulting cDNA were then used as the template for individual PCR. The amplified PCR products were analyzed by both electrophoresis on a 1% agarose gel and DNA sequencing. For Northern blot analysis, twenty micrograms total RNA was separated on a 2% agarose gel and transferred to a Hybond-N+ membrane , which was incubated in PerfectHyb Hybridization Solution (Toyobo) with probes directed against the 5′ region of the HAC1 ORF. All probes were generated by PCR with primers listed in 32P]dCTP using a Random Primer DNA Labeling Kit Ver.2.0 . Images were obtained using Image Reader FLA-5000 .Total RNA extraction and cDNA synthesis were performed as described above, and 3 µl of resulting cDNA were used as the template for individual PCR with a QuantiTect SYBR Green PCR kit (Qiagen). qRT-PCR was carried out in triplicate in a 96-well plate format, using a 7500 Real-Time PCR System . The mRNA abundance of the target genes was normalized to 18S rRNA (CAGL0L13398r). The qRT-PCR assays were repeated at least twice on independent occasions.C. glabrata cells were grown in SC medium to exponential phase (OD600 = 0.8) and exposed to 1.5 µg/ml TM at 30°C. Total RNAs were extracted using the FastRNA Red Kit (Qbiogene). The quality of RNA was checked with a RNA 6000 Nano Kit and Agilent 2100 Bioanalyzer. Double-stranded cDNA was synthesized using the Invitrogen SuperScript Double-Stranded cDNA Synthesis Kit and oligo (dT) primers. The resulting cDNA samples were labeled with Cy3 using the NimbleGen One-Color DNA Labeling Kit and subsequently hybridized to a custom-made 4×72 K C. glabrata array  wherein each chip measures the expression levels of 5,217 genes from C. glabrata CBS138 with six 60-mer-probe pairs per gene, with two-fold technical redundancy. The arrays were washed using the NimbleGen Wash Buffer Kit and scanned with a NimbleGen MS 200 Microarray Scanner. Data were quantified using the NimbleScan v2.6 software and normalized as described previously http://www.candidagenome.org/cgi-bin/GO/goTermFinder) with default parameters C. glabrata GAS2 gene was amplified from the genomic DNA of CBS138 and fused to the lacZ reporter in pEM14 C. glabrata wild-type and Δire1 strains containing pEM14-GAS2 were grown in SC-ura broth, adjusted to 1×107 cells/ml, and then incubated in the presence and absence of 3 mM DTT for 2 h. β-galactosidase assay was performed as described previously g for 30 min at 4°C. Protein concentrations were determined by the Bio-Rad Protein Assay  using bovine serum albumin as a standard. β-galactosidase activities were measured using the β-Galactosidase Enzyme Assay System (Promega) according to the manufacturer's instructions and calculated in Miller units (nmoles/min/mg of protein) at 37°C A 699-bp DNA fragment containing the 5′ untranslated region (UTR) and the first 17 codons of the C. glabrata cells were harvested, washed, resuspended in sterile saline, and adjusted to 4×108 cells/ml after counting the number of cells using a hemocytometer. The actual CFU in the inocula were confirmed by plating serial dilutions of cell suspension on YPD plates. Groups of 8 female, 8-week-old, BALB/c mice  were injected with 0.2 ml of the C. glabrata cell suspension via the lateral tail vein. The mice were euthanized 7 days after injection and the spleen and bilateral kidneys were then excised. No mice died before euthanasia in this experiment. Appropriate dilutions of organ homogenates were plated on YPD plates. Colonies were counted after 2 days of incubation at 30°C and the CFUs per organ were calculated. Statistical analyses were performed using the Kruskal-Wallis test with Dunn's multiple comparison post-test .Animal experiments were performed as described previously C. glabrata, groups of 7 female BALB/c mice  were immunosuppressed by intraperitoneal administration of cyclophosphamide at a concentration of 200 mg/kg/day on days −3, −2, and −1 of infection, and housed under sterile conditions. The mice were inoculated intravenously with 0.2 ml of C. glabrata cell suspensions (1×108 and 1×107 cells/ml) on day 0. Checks were made for dead or moribund mice in a blinded fashion twice daily over a period of 16 days. A group of 4 immunosuppressed mice was injected with sterile saline instead of C. glabrata cell suspension, and no mice died due to immunosuppression only. Survival was plotted on a Kaplan-Meier curve for each C. glabrata strain, and the log rank (Mantel-Cox) test was used for pairwise comparison of percent survival (GraphPad Prism 5). A P value of <0.05 was considered statistically significant. These animal experiments were conducted on two separate occasions to ensure reproducibility.To examine mortality of mice with disseminated candidiasis due to C. glabrata: 18S rRNA (9488051); ACT1 (2890423); CAD1/YAP2 (2887750); CAGL0A04081g (2886450); CAGL0F04829g (2887774); CAGL0I07249g (2889193); CAGL0I08591g (2888926); CAGL0K11946g (2889962); CAGL0L06776g (2890816); CAGL0M04191g (2891538); CAGL0M12320g (2891428); CNB1 (2890566); CRZ1 (2891693); DER1 (2891627); ECM33 (2891194); FPR2 (2888506); GAS2 (2891237); GAS4 (2887679); GCN4 (2890908); HAC1 (2890416); HIS3 (2890838); IRE1 (2887705); KAR2 (2887030); PDI1 (2886594); SKO1 (2889579); SLT2 (2889880); TRP1 (2886909); YAP1 (2888604); YAP3/CAGL0K02585g (2890264); YAP3/CAGL0M10087g (2891349); YAP6 (2891289); YAP7 (2887954).S. cerevisiae: ACT1 (850504); ADH1 (854068); CCH1 (853131); DER1 (852500); FPR2 (852131); HAC1 (850513); IRE1 (856478); KAR2 (853418); MID1 (855425); PDI1 (850314); PGK1 (850370); SLT2 (856425).A. fumigatus HacA/Hac1 (3506096); A. fumigatus IreA/Ire1 (AEQ59230); A. nidurans HacA (CBF87535); C. albicans Hac1 (3639758); C. albicans Ire1 (3640823); C. elegans Xbp1 (175541); C. neoformans Hxl1 (3255200); C. neoformans Ire1 (3255994); D. melanogaster Xbp1 (44226); H. sapiens Xbp1 (7494); P. pastoris HAC1 (8199414); T. reesei HAC1 (AJ413272); Y. lipolytica HAC1 (2906724).Others: Figure S1Comparison of cell growth in the presence of ER stress between various fungal species. Logarithmic-phase cells of each fungal strain were harvested after appropriate incubation in YPD broth. Cell concentration was adjusted with optical density at 600 nm, and then 5 µl of serial 10-fold dilutions were spotted onto YPD or synthetic complete (SC) plates containing either tunicamycin (TM) or dithiothreitol (DTT) at the indicated concentrations. Plates were incubated at 30°C for 48 h.(TIF)Click here for additional data file.Figure S2IRE1 and HAC1 loci between S. cerevisiae and C. glabrata. (A) Schematic representation of the C. glabrata IRE1 (CAGL0F03245g) locus compared to syntenic regions of the S. cerevisiae genome. Colored arrows indicate orthologs in the two species. White arrows indicate S. cerevisiae genes of which orthologs are not located around IRE1 in the C. glabrata genome. (B) Synteny analysis of the C. glabrata HAC1 (CAGL0K12540g) and S. cerevisiae HAC1 loci.Synteny relationships of the (TIF)Click here for additional data file.Figure S3HAC1 in C. glabrata. The entire open reading frame (ORF) of HAC1 was replaced with the deletion construct containing a TRP1 marker. Southern blot analysis of ClaI-digested genomic DNA using a HAC1 probe, which was designed within the HAC1 ORF, identified the predicted 4.3 kb band in the parent strain 2001HT (Δhis3 Δtrp1) but no band in the Δhac1 mutant TG141 (Δhac1::TRP1 Δhis3). A second Southern blot analysis using a TRP1 probe confirmed that the desired homologous recombination had occurred at the HAC1 locus without ectopic integration of the deletion construct in the mutant.Disruption of (TIF)Click here for additional data file.Figure S4C. glabrata wild-type (CBS138) cells were treated with TM or DTT at the indicated concentrations for 1 and 3 h. Expression levels of KAR2 (A) and other known UPR targets (B) were examined by qRT-PCR as described in qRT-PCR analysis to validate expression profiles of representative UPR target genes in the presence of tunicamycin (TM) and dithiothreitol (DTT). (TIF)Click here for additional data file.Figure S5C. glabrata strains: WT, 2001T; Δcnb1, TG161; Δcrz1, TG171; Δslt2, TG151; and Δire1, TG121.Genome-wide gene expression profiles in response to tunicamycin (TM) exposure for 0.5 h. Hierarchical clustering of genes whose expression levels were changed more than 1.5-fold after treatment with 1.5 µg/ml TM for 0.5 h. Genes were clustered with average linkage. (TIF)Click here for additional data file.Figure S6C. glabrata. (A) Logarithmic-phase cells of each C. glabrata strain were adjusted to 2×107 cells/ml, and then 5 µl of serial 10-fold dilutions were spotted onto YPD plates. One plate was incubated under normal (aerobic) condition for 44 h, and the other plate was incubated under conditions of low oxygen tension (hypoxic) for 68 h. (B) Serial dilutions of C. glabrata cells were prepared as described above and spotted onto synthetic complete (SC) plates containing either 20 µM bathophenantroline disulphonate (BPS) or 50 µM desferrioxamine (DFO) in the presence and absence of 1 mM ferric chloride (FeCl3). Plates were incubated at 30°C for 24 h.Ire1 is dispensable for growth under conditions of hypoxia and iron depletion in (TIF)Click here for additional data file.Table S1C. glabrata bZIP transcription factors identified by a BLASTp search using the bZIP domain sequences of S. cerevisiae Hac1, H. sapiens Xbp1, and C. neoformans Hxl1 as queries.Putative (DOC)Click here for additional data file.Table S2List of genes whose expression levels were altered by tunicamycin treatment for 3 h.(XLSX)Click here for additional data file.Table S3Classification of the putative Slt2-repressed genes.(XLSX)Click here for additional data file.Table S4List of genes whose expression levels were altered by tunicamycin treatment for 0.5 h.(XLSX)Click here for additional data file.Table S5Classification of the putative Ire1-repressed genes.(XLSX)Click here for additional data file.Table S6Strains used in this study.(DOC)Click here for additional data file.Table S7Primers used in this study.(DOC)Click here for additional data file.Table S8Plasmids used in this study.(DOC)Click here for additional data file.


Recent studies of antibodies in human plasma from HIV-1-infected and immunized individuals have revealed an important role for the V1/V2 region of gp120 in an antiviral response, and recent evidence that protection in the RV144 vaccine trial correlated with the presence of V1/V2-specific antibodies suggests that the V1V2 region is an important target for candidate HIV-1 vaccines. However, the function of V1V2-specific antibodies is not well understood. In this study we present data about the development, subtype specificity, and neutralization activity of such antibodies present in plasma from several hundred infected North American and African subjects.Human plasma from infected individuals were screened for neutralization activity versus a panel of subtype B, subtype C, and other Tier 2 pseudoviruses, and were titrated for binding activity against the consensus subtype B, C and A/E V1/V2 fusion glycoproteins, expressed via fusion to a fragment of the MuLV gp70 sequence. This system expresses V1/V2 domains in their native glycosylated and conformational forms. V1V2-specific antibodies were isolated from selected plasma by immunoaffinity chromatography on gp70-V1/V2 antigen columns, and characterized for neutralizing activity against various HIV-1 pseudoviruses.50s generally in the 1-20 μg/ml range.Most (>80%) of the HIV-1 infected subjects possessed robust levels of V1/V2 binding activity versus the three antigens. Interestingly, the development of V1/V2-reactive antibodies tracked with the development of autologous neutralizing antibodies in several subjects infected with subtype C viruses. Immunoaffinity-purified V1/V2-specific antibodies from selected broadly neutralizing plasma samples also possessed broad neutralization activities, with ICHighly cross-reactive V1V2-specific antibodies were present in almost all broadly neutralizing human plasmas at large concentrations, and frequently possessed modest neutralizing activities against a range of isolates, including tier 2 viruses. Additional information about the nature of these antibodies and their target epitopes would help elucidate their potential roles in protection against infection.


Drosophila melanogaster. This region includes 57 predicted protein-coding genes. Seventeen of these genes are in six clusters that appear to have arisen by tandem duplication. Within this region we found 23 complementation groups that are essential for zygotic viability, and we have identified the transcription units for 18 of the 23. We also found a 55 kb region in 72D that is nonessential. Flies deficient for this region are viable and fertile. Within this nonessential region are 48 DNA sequences of 12 to 33 base pairs that are completely conserved among 12 distantly related Drosophila species. These sequences do not have the evolutionary signature of conserved protein-coding DNA sequences, nor do they appear to encode microRNAs, however, the strong selection suggests functions in wild populations that are not apparent in laboratory cultures. This region resembles dispensable gene deserts previously characterized in the mouse genome.We have investigated a region of ∼310 kb of genomic DNA within polytene chromosome subdivisions 72A to 72D of  Drosophila melanogaster genome has been intensely studied for over 100 years. Recently, the sequencing of the majority of the genomic DNA has revealed much about the structure and organization of the genome Drosophila Gene Disruption Project to mutagenize the genome with transposable element insertions have generated a collection of transposon insertions that tag about two-thirds of all annotated protein-coding genes Drosophila Gene Disruption Project to determine the level of saturation for gene function disruption among the tagged genes. In addition, we identified a large dispensable region reminiscent of gene deserts found in the mouse genome The Df(3L)th102. These mutations define 22 complementation groups. One mutant chromosome failed to complement mutations in two adjacent genes (11 Arf72A3DNApol-delta), and is probably a small deletion. The essential complementation groups and the number of alleles that we recovered for each are shown in l(3)72Ds] represented by a single allele.After EMS mutagenesis, we recovered 188 mutations that failed to complement 01318kst mutant chromosome was reported to carry a second-site lethal mutation, 01318l(3)72Dq(http://flybase.org/reports/FBgn0028257.html), which failed to complement Df(3)st-f13. We could not confirm the existence of 01318l(3)72Dq, as the 01318kst mutant strain from the Bloomington Stock Center complemented both Df(3L)st-f13 and Df(3L)th102 for viability. Another complementation group that was mapped to this region is E(smoDN)B-leftE(smoDN)B-left is allelic to l(3)72Dh. Finally, Daniel Kalderon and co-workers screened for mutations that failed to complement Df(3L)brm11, and identified six complementation groups [l(3)72CDa through l(3)72CDf] that failed to complement both Df(3L)brm and Df(3L)st-f13l(3)72CDa corresponds to l(3)72Db, l(3)72CDe corresponds to l(3)72Dc, and l(3)72CDf corresponds to l(3)72Da. In addition, we confirmed the location of their complementation group l(3)72CDc, which is the 23rd essential gene within the region deleted by Df(3L)th102. We were unable to confirm their other two complementation groups. We found that M3l(3)72CDb complemented Df(3L)th102. We also found that the l(3)72CDd complementation group is an artifact. It is represented by a single mutant chromosome that failed to complement two deletions, Df(3L)brm11 and Df(3L)st-f13. The L2l(3)72CDd mutant chromosome was assumed to carry a single lethal mutation in the region of overlap missing in both deletions Df(3L)brm11 is caused by an Arf72A mutation, which we have named L2Arf72A. This is the only lethal mutation on this chromosome within Df(3L)th102, since the lethality over Df(3L)th102 was rescued by the Arf72A transgene, +t10.8Arf72ADf(3L)st-f13, is caused by a second mutation, L2l(3)72-73a, which also failed to complement Df(3L)st-g24.We also tested mutations from other groups that were previously mapped to this region of the genome. The Df(3L)th102 th102 for viability, indicating that the lethality of the insertion chromosome is not due to disruption of the associated gene. Eleven of the transposon insertion mutants failed to complement one of our complementation groups. The complementation groups that failed to complement each transposon insertion mutant are shown in Nine of our complementation groups in CG32155 gene from two alleles of the l(3)72Dp complementation group. Both alleles were isolated on the iso-1 third chromosome. The 1l(3)72Dp mutation has seven base pairs deleted and three base pairs inserted (a net loss of four base pairs). This should frame shift the CG32155 protein after residue T32, causing the addition of six amino acid residues (VFTSMV) before a stop codon truncates the protein. The 3l(3)72Dp mutation is a GC to AT transition that changes amino acid residue W128 to a stop codon, prematurely truncating the CG32155 protein. We sequenced the CG32154 gene from two alleles of the l(3)72Dr complementation group. Both alleles were isolated on the 1 e4red chromosome, which differs from the iso-1 sequence at amino acid 206 (S in iso-1 and C in the 1 e4red marked chromosome). Each allele has one additional amino acid change from the parental chromosome, both caused by TA to CG transitions. The 1l(3)72Dr mutation changes amino acid residue C323 to R and the 2l(3)72Dr mutation changes amino acid residue N258 to S. We sequenced the Taspase1 gene from seven l(3)72Dl alleles. The Taspase1 alleles 1, 3, 5, 6, 7, 8, and 9 change amino acid residues D75 to V, P98 to L, C74 to Y, E253 to K, G197 to E, P98 to L, and G252 to S, respectively. All of the Taspase1 alleles died at the pharate adult stage when heterozygous to Df(3L)th102. Although Taspase1 cleaves the homeotic transcriptional regulator Trithorax Taspase1 mutants is probably due to the perdurance of maternally encoded gene products. We tried to eliminate the maternally encoded gene products by making germ-line clones 6Taspase1 and 8Taspase1, but the Taspase1 mutant clones failed to produce mature eggs.We assigned three of the remaining complementation groups to transcription units by sequencing candidate genes (suggested by the deficiency mapping) from homozygous mutants. We sequenced the EP3292P{EP}DNApol-delta to recover 14DNApol-delta, which failed to complement l(3)72Ac alleles. Using all of the information above, we have been able to assign 18 of the 23 complementation groups to transcription units.Finally, we used imprecise excision of the P element insertion CG17026, CG17029, CG17028, and CG17027 in the top panel of brahma gene, and encodes putative inositol monophosphatases that are 41%–77% identical to each other. There are four genes in all Drosophila species except D. willistoni (three genes) and D. yakuba (eight genes). In the more distantly related dipteran, the mosquito Anopheles gambiae, there is only a single ortholog in the intron of the brahma gene. The next most distal cluster of three related genes . We were unable to identify an ortholog for these proteins in A. gambiae. The most proximal cluster of tandemly related genes is a pair of genes  and four predicted genes in the proximal cluster . In addition, the two clusters are distantly related to each other. In the D. melanogaster iso-1 strain, there is an X element non-LTR transposon insertion between the two clusters. This X element insertion is not present in the Canton S strain (data not shown). No cDNAs have been isolated for any of the genes in either cluster, nor did any of them show expression in the high-throughput RNA sequencing (RNA-seq) data from the modENCODE project Df(3L)Exel6128/Df(3L)BSC559 transheterozygotes and Df(3L)Exel6128/Df(3L)BSC560 transheterozygotes] that includes both clusters and another predicted gene, CG13073 (bobbed mutations (mutants in the rDNA clusters on the X and Y chromosomes) and Minute mutations . These phenotypes only appeared after several generations of inbreeding and could not be rescued by two large paternally inherited duplications ST1 and DpL131-D3). We believe that the phenotype is not caused by deleting this region, but by a maternal-effect mutation somewhere else in the genome. Another overlapping pair of deletions, Df(3L)Exel6128 and Df(3L)st4, delete about two-thirds of the same dispensable region (Df(3L)Exel6128/Df(3L)st4 transheterozygous flies for several generations did not reveal the same Minute-like phenotype as observed when inbreeding the Df(3L)Exel6128/Df(3L)BSC559 or Df(3L)Exel6128/Df(3L)BSC560 transheterozygous flies.Two additional clusters of related genes in 72D are shown in  CG13073  and 3. We region . InbreedGyc76C duplication in the iso-1 strain) Six clusters of genes in the 72A–D region of the genome appear to have arisen by tandem gene duplication. These clusters include 17 of the 57 predicted protein-coding genes. In a previous study of the 76B–D region, we identified four clusters of related genes, which included 17 of the 80 protein-coding genes  in these 37 essential genes. Thus, each mutagenized third chromosome that we screened with both deletions was 9.4×104 base pairs of open reading frame tested. We did not screen all of our mutagenized chromosomes with both deletions. Therefore, we have confined our analysis to the 3009 EMS-treated third chromosome lines that we screened with both chromosomal deletions. We thus tested a total of 2.8×108 base pairs (9.4×104 base pairs on each of the 3009 chromosomes) for lethal mutations in our 37 identified essential genes. We recovered 130 mutations, which corresponds to one mutation in every 2.2×106 base pairs , or one mutation per 2200 kb. This is the estimate for mutations that cause a lethal mutant phenotype. We can compare this to the estimates for the frequency of mutations that alter DNA sequence (but may not necessarily cause a mutant phenotypes). The latter frequencies range from 1 mutation per 273 kb to 1 mutation per 476 kb What proportion of mutations decrease gene function enough to cause a mutant phenotype? We can estimate this proportion from the data reported here and in our previous work CG5151 and CG5018 in 72D9–10 (a region of ∼78 kb) may be similar to gene deserts that have been described in mammalian genomes CG13073). At least 55 kb are dispensable. Two gene deserts in the mouse genome were deleted and were also dispensable Drosophila using the flanking CG5151 and CG5018 genes, which are evolutionarily conserved. The region varies in size from slightly less than 60 kb in D. yakuba to almost 78 kb in D. simulans and D. melanogaster. No protein-coding genes are conserved among all species. We identified the ortholog of CG13073 in this region in all species except D. persimilis. In the subgenus Drosophila, CG11196 is present in this region, while in the subgenus Sophophora, CG11196 is located between Nup44A and Hey on Muller element C (44A2 in D. melanogaster). We do not know which location for CG11196 is the ancestral, as the A. gambiae CG11196, Nup44A, and Hey orthologs are in three different locations. While there are few, if any, genes conserved between CG5151 and CG5018, there are many DNA sequences conserved. We identified 64 DNA sequences between 12 and 43 base pairs in length that are completely conserved among 12 Drosophila species. Forty-eight of the conserved sequences (7 through 54) are within the region deleted by both Df(3L)BSC559 and Df(3L)Exel6128. The conserved sequences are in D. melanogaster, but are clustered. For example, several pairs of sequences are separated by only a single variant nucleotide. Sequences 13 and 14 have 55/56 base pairs conserved, sequences 20 and 21 have 46/47 base pairs conserved, sequences 30 and 31 have 43/44 base pairs conserved, sequences 32 and 33 have 56/57 base pairs conserved, and sequences 45 and 46 have 51/52 base pairs conserved. These sequences do not have the evolutionary signatures of conserved protein-coding DNA sequences or of microRNAs D. melanogaster, their strong evolutionary conservation suggests functions critical in nature.The region between http://flybase.org/) unless otherwise noted. For the Taspase1 germ-line clones, three independent recombinants with hs)}2AP{FRT}2AP{ovoD1-18}3L P{FRT, and were then tested for lethality when heterozygous to Df(3L)th102.Flies were raised on a yeast/cornmeal/molasses/Tegosept medium at 25°. All mutations and chromosome aberrations are described in Lindsley and Zimm iso-1 strain 1red and 4e. Both third chromosomes were made isogenic prior to mutagenesis. We recovered mutations that failed to complement Df(3L)th102 from two different experiments. Following the nomenclature suggested by Lindsley and Zimm l(3)72Aa through l(3)72Ae for those complementation groups distal to Df(3L)st-f13, and l(3)72Da through l(3)72Ds (excluding l(3)72Dq) for those complementation groups within Df(3L)st-g24. We did not use the name l(3)72Dq for any of our complementation groups, since that name was already in use (http://flybase.org/reports/FBgn0028257.html). The first experiment to recover mutations that failed to complement Df(3L)th102 was previously described Df(3L)th102; 26 of those mutations that failed to complement Df(3L)brm11 (but complemented Df(3L)st-f13) were previously reported Df(3L)th102 are alleles of l(3)72Dml(3)72Dm or its alleles in our description of the genes within the 72A–D region, because the l(3)72Dm mutations complemented deletions that overlap Df(3)th102, and are probably allelic to a second-site mutation on the Df(3L)th102 chromosome. In the second experiment, we generated balanced lines 1red and 4e. Only those lines in which few or no flies homozygous for the mutagenized third chromosome survived were subsequently tested by crossing to Df(3L)th102/TM6B virgins. We tested 1938 lethal-bearing third chromosome lines and recovered 88 mutations that failed to complement Df(3L)th102.Males were fed ethyl methanesulfonate (EMS) as described Df(3L)st4 was determined by PCR amplification of DNA fragments from Df(3L)Exel6128/Df(3L)st4 transheterozygous flies. The distal breakpoint of Df(3L)st4 is proximal to the DNA sequence CCGTTACACGTTGTACACC  and distal to the sequence CGAGGAGTTAAGGTCTCAG .We used inverse PCR http://genome.ucsc.edu/) http://blast.ncbi.nlm.nih.gov/Blast.cgi). For the determination of evolutionarily conserved amino acids, we used EvoPrinter Version 1.1 (http://evoprinter.ninds.nih.gov/index11.html) D. erecta, D. yakuba, D. ananassae, D. pseudoobscura, D. persimilis, D. virilis, D. mojavensis, and D. grimshawi) with available BLAT files. Only those amino acid residues that were identical in all nine Drosophila species were counted as evolutionarily conserved. For the determination of evolutionarily conserved base pairs, we used EvoPrinterHD (http://evoprinter.ninds.nih.gov/evoprintprogramHD/evphd.html) For the evolutionary comparisons, we used both BLAT on the UCSC Genome Browser website (Table S1Sequences (12 base pairs or longer) from the 72D region that are conserved among Drosophila species.(DOC)Click here for additional data file.


Interestingly, among those immune parameters, the frequency of circulating Th17 cells was higher in stage III HCC patients than in stage I HCC patients (P = 0.015) and healthy donors (P<0.001). Moreover, an increased frequency of circulating Th17 cells was observed 30 days after TACE (Th17D30) compared with the baseline level (P = 0.036). Kaplan-Meier analysis demonstrated that Th17D30 was positively associated with overall survival  and time to progression . Multivariate Cox analysis revealed that Th17D30 was an independent prognostic factor for OS  and TTP . These results provide a potential prognostic marker for stage III HCC patients undergoing TACE and may be useful for identifying patients who can benefit from adjuvant immunotherapies.Transarterial chemoembolization (TACE) has therapeutic effects in patients with unresectable hepatocellular carcinoma (HCC), but its impact on the cellular immune response during disease progression is largely unknown. Here we conducted a prospective study to evaluate the effect of TACE on immune status and to identify prognostic immune markers governing treatment success. In this study, 51 stage III HCC patients, 28 stage I HCC patients (TNM classification) and 20 healthy donors were enrolled. Flow cytometry and cytometric bead array were used to evaluate the circulating immune cell subsets, including CD4 Hepatocellular carcinoma (HCC) is among the most prevalent tumor types, with an increasing incidence and mortality worldwide + T helper cell subset, termed Th17 cells, has been described. Th17 cells have potent pro-inflammatory properties and play an active role in inflammation and autoimmune diseases HCC is usually derived from inflamed fibrotic and/or cirrhotic liver with intensive immune cell infiltration. Thus, the immune status may largely influence the biologic behavior of HCC + T cells , CD8+ T cells, NK cells and NKT cells, and plasma cytokines in unresectable HCC patients who underwent TACE.Immune markers predicting patient prognosis or response to therapy may advance the potential of personalized therapy in cancer treatment This study protocol was approved by the Ethics Committee of the Sun Yat-sen University Cancer Center as stipulated by the Declaration of Helsinki, and written informed consent was obtained from all subjects. All of the samples were anonymously coded in accordance with local ethical guidelines.From June 2009 to May 2010, a total of 1,196 patients were newly diagnosed with HCC in our institution, 329 underwent TACE and 161 met the inclusion criteria but 104 patients refused to take part in the study. In total, 57 patients were enrolled into this study. Six patients were excluded from this study because they were lost to follow-up. Finally, 51 patients with stage III HCC were included. The diagnosis of HCC was based on the diagnostic criteria for HCC used by the European Association for the Study of the Liver All 51 patients with stage III HCC received TACE treatment (Group 1) and 28 patients with stage I HCC who underwent surgical resection or radiofrequency ablation were included as the control group (Group 2). Patients did not receive any other treatment within 30 days after TACE. Control blood samples were obtained from 20 healthy donors attending the Guangzhou Blood Center (Group 3) and all of the controls were negative for hepatitis B, hepatitis C, HIV and syphilis. The clinicopathologic characteristics of all patients were summarized in Transarterial chemoembolization treatment was performed following the techniques described previously All of the stage III HCC patients were closely followed until June 15, 2012. The median duration of follow-up for the stage III patients was 12.7 months (range 1.0–34.8 months). The routine follow-up program included a serum alpha-fetoprotein (AFP) assay, abdominal ultrasonography, and liver function tests at intervals of one month for the first year and every three months thereafter 0) and 30 days after treatment (D30). Blood samples from 20 healthy donors and 28 stage I HCC patients before treatment were analyzed as controls. All the 51 stage III HCC patients were assessed before treatment and 30 patients returned for the immunological follow-up 30 days after treatment. For phenotype analysis, peripheral blood mononuclear cells (PBMC) were isolated from fresh heparinized blood by Ficoll density gradient centrifugation Blood samples from stage III HCC patients were withdrawn immediately before treatment -conjugated anti-CD3, phycoerythrin-Texas Red (ECD)-conjugated anti-CD3, fluorescein isothiocyanate (FITC)-conjugated anti-CD4, phycoerythrin-cyanin 7 (PE-Cy7)-conjugated anti-CD4, FITC-conjugated anti-CD8, phycoerythrin (PE)-conjugated anti-CD8, PE-conjugated anti-IFN-γ , FITC-conjugated anti-CD3-FITC, PE-Cy7- conjugated anti-CD56 , Alexa Fluor 647 (AF647)-conjugated anti-IL-17A, APC-conjugated anti-FoxP3 and PE-conjugated anti-Perforin . For intracellular cytokine detection, cells were stimulated for 5 h with Leukocyte Activation Cocktail (BD Biosciences), stained with surface markers, fixed and permeabilized with IntraPre Reagent (Beckman Coulter), and then stained with anti-IL-17A and anti-IFN-γ. For FoxP3 analysis, the APC anti-FoxP3 Staining Set (eBioscience) was used according to the manufacturer's instructions. Data were acquired using a Cytomics™ FC500 (Beckman Coulter) and analyzed with CXP analysis software (Beckman Coulter).Plasma cytokine levels of IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, and TNF-α were evaluated using the human Th1/Th2/Th17 cytokine Cytometric Bead Array (CBA) Kit (BD Biosciences) according to the manufacturer's instructions. Fluorescence was detected using a Cytomics™ FC500 (Beckman Coulter), and the data were analyzed using CBA analysis software (BD Biosciences). The assay sensitivities for IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, and TNF-α were 2.6, 4.9, 2.4, 4.5, 18.9, 3.7 and 3.8 pg/mL, respectively.U-test. The Wilcoxon signed-rank test was used for paired samples. Correlations between variables were determined by Spearman's rho tests. The Kaplan-Meier method was applied to assess actuarial survival using median value as a cut-off to separate patients into a high and low group. The log-rank test was applied to compare the groups. Univariate analysis was performed to assess significant clinicopathological and immunological parameters that influence overall survival and time to progression after treatment. A multivariate analysis was performed by Cox regression for significant variables in the univariate analysis. P value of less than 0.05 was considered significant.Data analysis was performed using SPSS 13.0 software , and the data were expressed as the mean ± SD for continuous variables. The statistical significance of the difference between two groups was determined using the non-parametric Mann-Whitney + T cells , CD8+ T cells, NK cells, and NKT cells, and cytokine levels  in peripheral blood from 51 stage III HCC patients, 28 stage I HCC patients before treatment and 20 healthy donors. In general, the frequencies of total T cells, CD4+ T cells, CD8+ T cells, NK cells and NKT cells in HCC patients did not differ significantly from healthy donors (+ T cell subsets and found that the frequencies of IL-17-producing CD4+ T (Th17) cells, IFN-γ-producing CD4+ T (Th1) cells and CD4+CD25+FoxP3+ cells (Treg) were all increased in stage III HCC patients compared with healthy donors , whereas the other cytokines were below the level of sensitivity for detection of the CBA kit in most samples  and 30 days after TACE (D30) (30 compared with D0 in these patients (30 (data not shown). Taken together, our data indicate that TACE may have a long-term effect on the Th17 cell population and augment its frequency in peripheral blood.Previous studies have shown that TACE induces necrosis and causes inflammation with cytokine production, which may favor immune activation and presentation of tumor-specific antigens CE (D30) . A signipatients , whereaspatients . In addipatients . Further0 (Th17D0) and D30 (Th17D30). Kaplan-Meier analysis revealed that Th17D30 rather than Th17D0 (P = 0.007) and TTP (P = 0.009). Patients with higher Th17D30 had significantly longer OS (median: 22.9 months) or TTP (median: 7.2 months) than patients with lower Th17D30 . Moreover, five of the six patients who did not experience disease progression within one year after TACE were in the high Th17D30 group.Since circulating Th17 cells were significantly increased after TACE in stage III HCC patients, we next investigated the association of Th17 cells with the survival in those patients. We analyzed relevant clinical information and correlated the data with the frequencies of circulating Th17 cells at Dn Th17D0  was posi0 to D30 only occurred in a part of the patients . Then, we evaluated the prognostic value of the change of Th17 levels represented by Th17D30/Th17D0 in univariate Cox regression for OS and TTP. The results showed that it could not yet be considered as a prognostic variable probably due to the limited number of patients (D30/Th17D0) and TTP . In addition, the ten patients with extreme good prognosis also had higher ThD30 than the other patients  and TTP  in stage III HCC patients.In univariate analysis , Th17D30+CD4+ T cells  cells  and time to progression (B) by the frequency of Th17 cells before TACE (n = 51).(TIF)Click here for additional data file.Table S1Comparison of the frequencies of various lymphocyte subsets in healthy donors and HCC patients.(DOC)Click here for additional data file.Table S2Plasma levels of Th1, Th2 and Th17 associated cytokines in healthy donors and HCC patients.(DOC)Click here for additional data file.Table S3Effects of TACE on circulating lymphocyte subsets in stage III HCC patients who underwent TACE.(DOC)Click here for additional data file.


During this process, cells can develop immortality with genomic instability, although the mechanisms by which this is regulated are unclear. Here, we show that a growth-arrested cellular status is produced by the down-regulation of histone H2AX in normal cells. Normal mouse embryonic fibroblast cells preserve an H2AX diminished quiescent status through p53 regulation and stable-diploidy maintenance. However, such quiescence is abrogated under continuous growth stimulation, inducing DNA replication stress. Because DNA replication stress-associated lesions are cryptogenic and capable of mediating chromosome-bridge formation and cytokinesis failure, this results in tetraploidization. Arf/p53 module-mutation is induced during tetraploidization with the resulting H2AX recovery and immortality acquisition. Thus, although cellular homeostasis is preserved under quiescence with stable diploidy, tetraploidization induced under growth stimulation disrupts the homeostasis and triggers immortality acquisition.Normal cells, both  Cancer is a disease associated with genomic instability and the accumulation of mutations A recent genomic analysis of various cancers revealed that massive genomic rearrangements, including loss of heterozygosity (LOH) and chromosomal translocation, amplification and deletion, do not gradually accumulate over time, as conventionally thought, but appear to be acquired in a single catastrophic event in vitro by the activation of oncogenes In the initial stages of carcinogenesis, cells are subjected to oncogenic stress, resulting in the accumulation of DNA replication stress-associated lesions and the onset of barrier responses such as senescence and apoptosis in vitro, is tightly associated with mutations in the Arf/p53 module Arf and p53 under functional regulation mediate longevity and cancer prevention Arf and p53 in MEFs has a protective effect from immortalization The development of cancer, as well as the onset of immortality in cells This study focused on the mechanism by which normal cells under serial proliferation regulate homeostasis preservation and abrogation and sought to identify the regulatory target of p53. Our results illustrated two distinct conditions that could result in growth-arrested cells: (i) cells that maintain continuous quiescence by down-regulating H2AX (a variant of core histone H2A) under p53 regulation and stable-diploidy maintenance; and (ii) cells that develop tetraploidy and immortality under continuous growth stimulation, characterized by the accumulation of γH2AX foci. Thus, oncogenic stress under growth stimulation triggers catastrophic tetraploidization that leads to immortalization in association with the accompanying mutation of the Arf/p53 module and recovery of H2AX expression and growth activity.MEFs cultured under the standard 3T3 protocol (Std-3T3) senesce in association with oxygen sensitivity To determine the DNA lesion status induced by accelerated growth stimulation, γH2AX foci were compared in growth-arrested MEFs (P9) under both conditions . As expeTo address why γH2AX foci do not form under tSD-3T3 conditions, the expression level of H2AX at P9 was determined. As shown in in vitro and in vivo.To address whether H2AX diminution is a general occurrence, H2AX expression was compared in normal human fibroblasts (NHFs) and MEFs. Decreased H2AX was observed in both cell types at growth-arrested stage after serial proliferation , suggestH2AX is also diminished during premature senescence induced by DNA damage. Using early passage MEFs (P2), H2AX diminution was observed when senescence was induced by treatment with hydroxyurea (HU) to induce DNA replication stress  and withTo directly address the impact of H2AX reduction, H2AX was knocked down in early passage NHFs, which induced cellular quiescence with senescent cell characteristics; cells adopted a flattened and enlarged morphology and showed an increase in senescence-associated β-galactosidase activity . Since tThe above results illustrate that cellular quiescence is produced when cells maintain stable diploidy and diminished H2AX expression. In these cells, the H2AX level is less than 100-fold compared to that in actively growing cells. To study the effect of growth stimulation in cells with an H2AX-diminished quiescent status, complete medium (DMEM with 10% FBS) was added to quiescent MEFs prepared under tSD-3T3 conditions . In thesTo explore the effects of the change in H2AX status, the response of H2AX to DNA replication stress was compared between primary and immortalized MEFs. While H2AX in primary MEFs was down-regulated after HU treatment, this did not occur in immortalized MEFs , which ip53 knockout (KO) MEFs were cultured. Unlike normal primary MEFs, but similar to immortalized MEFs  growth arrested normal cells with senescent morphology can be defined as either (a) those in a continuous quiescent status with largely diminished H2AX level or (b) those in a transient status with inducing genomic instability and the resulting onset of immortality, under which cells accumulate γH2AX foci; (iii) to protect cells from immortality, one of the critical roles of p53 is the induction of growth-arrest via the down-regulation of H2AX with cellular quiescence. Cells in H2AX diminution-associated quiescence are shown in the cause of mature and premature senescence, during which cells show senescent morphology  normal cells generally achieve quiescent status with diminished H2AX level both rphology , probablrphology . HoweverSince growth-arrested cellular status with senescent morphology is directly induced by H2AX-knockdown , H2AX doin vivo and in vitro but not growing cells in early passages and in organs from young mice  there is no signal of the H2AX gene with ChIP-on-CHIP analyses against p53 Except for tumors associated with specific chromosomal translocation, development of most cancers as well as Together, our results provide a rationale for the regulation of cellular homeostasis preservation. By prohibiting immortality development and preserving quiescent cell status, p53 induces an H2AX diminution-mediated quiescent status. However, this status is abrogated by continuous growth stimulation, which results in the induction of genomic instability with mutation of the Arf/p53 module, which leads into H2AX recovery, the restoration of growth activity, and immortality acquisition .Mice were treated in accordance with the Japanese Laws and the Guidelines for Animal Experimentation of National Cancer Center. All experiments were approved by The Committee for Ethics in Animal Experimentation of National Cancer Center .p53-KO MEFs were prepared from day 13.5 embryos of wild type and p53(+/−) mice Cells were cultured as described previously DSB damage was induced by neocarzinostatin  treatment. For induction of DNA replication stress, MEFs were treated with hydroxyurea (HU).Antibodies against γH2AX  and H2AX (Bethyl) were used for immunostaining and Western blot analysis. Antibodies against β-actin , PCNA (Santa Cruz) and histone H3  were used for Western blot analysis. Prior to immunostaining with primary and secondary antibodies, cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100/PBS for 10 min. Western blot analysis and confocal microscopy were performed as described previously 5′-TTGCTTCAGCTTGGTGCTTAG-3′; H2axr, AACTGGTATGAGGCCAGCAAC; β-actinf, CATCCAGGCTGTGCTGTCCCTGTATGC; and β-actinr, GATCTTCATGGTGCTAGGAGCCAGAGC; Trp53-F, CGGATAGTATTTCACCCTCAAGATCCG; Trp53-R, AGCCCTGCTGTCTCCAGACTC; Sidt2-F, CGGAAGGCTGGTTTCTGAGTTTCCG; Sidt2-R, CTGTAAACGCCAAGGACCAGAA; Phlda3-F, CGGTCCATCTACTTCACGCTAGTGACCG; Phlda3-R, TGGATGGCCTGTTGATTCTTGA; Gapdh-F, AACTTTGGCATTGTGGAAGG; Gapdh-R, ATGCAGGGATGATGTTCTGG. The amplified products by AmpliTaq Gold (Applied Biosystems) were separated on a 2% agarose gel and visualized with ethidium bromide. Otherwise, real-time PCR assay was carried out using Power SYBER green PCR Master kit (ABI).Total RNA was extracted from MEFs with the RNeasy system (Sigma). RNA (0.8 µg) was reverse-transcribed using a cDNA Archive kit (Applied Biosystems) and subjected to PCR. The following PCR primers were used: H2axf, Mitotic cells were prepared by treatment with 20 ng/ml nocodazole for 6 h and then collected. The collected cells were swollen hypotonically with 75 mM KCl for 15 min, and then fixed with Carnoy's solution (75% methanol/25% acetic acid) for 20 min. After changing the fixative once, the cells were dropped in Carnoy's solution onto glass slides and air-dried. The slides were stained with 4% Giemsa (Merck) solution for 10 min, washed briefly in tap water, and air-dried.Figure S1Representative images of MEFs during the lifespan. MEFs cultivated as in (TIF)Click here for additional data file.Figure S2H2AX diminution is also observed in adult mice organs. Samples were prepared from five week (5W), five month (5M) and seven- or nine-month-old mice (7M or 9M). Compared to five months old organs, H2AX protein level is diminished in Testis (9M), Brain (7M), and Colon (7M), in which the diminution levels are lower than those in Liver, Spleen, and Pancreas. In Heart and Thymus, H2AX levels did not altered the alteration in through 5 weeks old to 7 or 9 months old.(TIF)Click here for additional data file.Figure S3H2AX diminution is also shown in damage induced premature senescence. Premature senescence was induced with NCS treatment as shown schematically in the top, in which each red arrowhead represents 100 ng/µl NCS treatment. Premature senescence by damage was induced with H2AX diminution, in which cells showed typical senescent morphology of flattened and enlarged.(TIF)Click here for additional data file.Figure S4H2AX transcript is decreased in quiescent MEFs. Decrease in H2AX mRNA level in senescing MEFs was observed by RT-PCR (right panel) and is compared with protein diminution (left panel).(TIF)Click here for additional data file.Figure S5H2AX over-expression accelerates immortality development in MEFs with tetraploidy.A. Experimental scheme of H2AX over expression. After transfection of H2AX-over expressing (H2AX-OE) or empty control vectors into early passage MEFs (P3), the transformed MEFs were selected, re-plated, and maintained in complete medium until immortalized cells appeared. B. Growth curves of MEFs during the experiments in A. MEFs before transfection and re-plating, MEFs transfected with H2AX-over-expressing vector, and MEFs transfected with empty control vector are indicated by black closed squares, red open circles, and black open diamonds, respectively. MEFs over-expressing H2AX showed accelerated development of immortality. C. H2AX status was determined as indicated in the figure. Although senescence was induced in the transfected and selected MEFs, H2AX over-expressing MEFs show higher levels of H2AX after the selection resulting in the development of immortality with H2AX recovery. D. Representative MEF images during accelerated immortality development with H2AX over-expression and controls. MEFs transfected with the H2AX over-expressing vector showed an efficient escape from senescence, while MEFs carrying the negative control vectors remained senescent with a flattened and enlarged morphology. E,F. Genomic instability status in immortalized MEFs (IP3) that were developed with H2AX over-expression was assessed by flow-cytometry (E) and Giemsa staining of M-phase chromosome (F).(TIF)Click here for additional data file.Figure S6p53 expression in senescing MEFs. To determine p53 expression in the cause of senescence, the expression levels of p53 and the targets (Sidt2 and Phlda3) that are likely associated with tumor suppression were compared between early passage (P2) and senescent MEFs (P7) under tSD-3T3 conditions. Along with H2AX diminution under p53 proficient background after serial cultivation, the expressions of Sidt2 and Phlda3 were observed in senescent MEFs (P7), in which the change in the expressed p53 transcript is limited.(TIF)Click here for additional data file.Figure S7p53 activation shown by miR34a expression in primary wt-MEFs after damage is not directly associated with H2AX expression levels at least for transcript regulation.A. To confirm p53 dependent DNA damage response, wt- and p53−/−-MEFs in primary and immortal were treated with 200 ng/ml neocarzinostatin (NCS) for 6 hours and the expression of p53-target miR34a was assessed. As expected, miR34a expression was shown after NCS treatment in primary wt-MEFs (wild type) but neither in immortalized wt-MEFs nor in p53−/−-MEFs. B. To determine the p53-activation associated change in the expression levels of H2AX transcript, mRNA levels of H2AX in MEFs treated as in A were analyzed. Whereas p53 is activated after NCS treatment in primary wt-MEFs, H2AX transcript levels were stable, suggesting no direct regulation by p53 transcription factor for H2AX expression. The PCR primers for miR34a were used from miRNA-specific primers (ABI) with snoRNA202 (ABI) for the control. Real-time PCR assay was carried out TaqMan microRNA assay kit (ABI).(TIF)Click here for additional data file.


Among all mammals, fetal growth and organ maturation must be precisely synchronized with gestational length to optimize survival at birth. Lack of pulmonary maturation is the major cause of infant mortality in preterm birth. Whether fetal or maternal genotypes influence the close relationship between the length of gestation and lung function at birth is unknown. Structural and biochemical indicators of pulmonary maturity were measured in two mouse strains whose gestational length differed by one day. Shorter gestation in C57BL/6J mice was associated with advanced morphological and biochemical pulmonary development and better perinatal survival when compared to A/J pups born prematurely. After ovarian transplantation, A/J pups were born early in C57BL/6J dams and survived after birth, consistent with maternal control gestational length. Expression of genes critical for perinatal lung function was assessed in A/J pups born after ovarian transfer. A subset of mRNAs important for perinatal respiratory adaptation was selectively induced in the A/J pups born after ovarian transfer. mRNAs precociously induced after ovarian transfer indicated an important role for the transcription factors C/EBPα and CREB in maternally induced lung maturation. We conclude that fetal lung maturation is determined by both fetal and maternal genotypes. Ovarian transfer experiments demonstrated that maternal genotype determines the timing of birth and can influence fetal lung growth and maturation to ensure perinatal survival. Premature birth is associated with impaired maturation and function of various organs, limiting perinatal adaptation. Pulmonary immaturity and associated deficiency of pulmonary surfactant are the major factors affecting survival of preterm infants worldwide RDS in preterm infants is caused by the lack of pulmonary surfactant, which is required to reduce surface tension at the air-liquid interface in the alveolus after birth. In preterm infants, lack of maturation of lung structure also contributes to respiratory dysfunction. Differentiation of respiratory epithelial cells lining the peripheral lung increases in later stages of lung morphogenesis. Lung maturation is associated with 1) expansion of peripheral lung saccules and the formation of septae to produce alveoli, 2) production and secretion of pulmonary surfactant required to reduce surface tension at the air-liquid interface after birth, 3) clearance of lung fluids after birth, and 4) thinning of the alveolar walls and the expansion of the pulmonary vascular bed to facilitate gas exchange across the alveolar capillary barrier Relationships between gestational length and fetal lung maturation were assessed in C57BL/6J and A/J mice. While C57BL/6J dams gave birth on embryonic (E) day 19.5±0.9 hr, A/J dams delivered pups on E20.5±1 hr as recently reported During the perinatal period, the lung undergoes dramatic structural changes required for respiration at birth. Peripheral lung saccules dilate, the mesenchyme thins, pulmonary vascularity increases, and the epithelium differentiates to produce cuboidal alveolar type II and squamous alveolar type I cells. Dilation of the peripheral lung parenchyma at E16.5 and E17.5 was delayed in A/J compared to C57BL/6J mice . During Cebpa, Lpcat1, Sftpb, and Sftpc increased in both strains from E15.5 to E18.5  and surfactant associated proteins. Fetal lung SatPC, a critical component of surfactant, increased with advancing gestation in both strains of mice, increasing earlier in C57BL/6J fetuses . Likewisto E18.5 . In relaA/J mice . SimilarA/J mice . The incA/J mice .Sftpa1, Sftpb, Sftpc, Sftpd, Abca3, and Slc34a2, increased markedly with advancing gestation in both strains, increasing earlier in C57BL/6J fetuses. In contrast, expression of those mRNAs in “Cluster 2,” including Nkx2.1, Pdgfa, and Lpcat1, increased moderately across gestational ages in both strains. mRNAs in “Cluster 3,” including Foxa2, Nfatc3, Nr3c1, and Etv5, transcription factors known to be important transcriptional regulators of lung maturation, did not change substantially with advancing gestation, supporting the concept that their activity is not mediated primarily by the level of their transcripts but perhaps by post-transcriptional mechanisms. Consistent with these findings, immunohistochemical staining of FOXA2 was similar in respiratory epithelial cells in both strains . When A/J fetuses from A/J dams (parturition 20.5 days) were delivered two days prematurely at E18.5 by hysterectomy, all A/J pups (n = 27) failed to expand their lungs, became cyanotic and died of respiratory failure soon after birth. However, A/J fetuses delivered one day prematurely at E19.5 from A/J dams maintained oxygenation and survived , indicating that only one additional gestational day was required for functional maturation of the lungs of A/J fetuses. Likewise, when C57BL/6J mice were born one day prematurely at E18.5 from C57BL/6J dams, most of the pups initiated respiration, became oxygenated and survived . Taken together, these data support the concept that maternal-fetal interactions, determined by the maternal genome, influence the length of the normal gestational period and that the maternal environment influences lung maturation in these mouse strains.Napsa, Eln, Fabp5, Scnn1g, Sftpc, and Lpcat1 mRNAs, encoding proteins known to play important roles in surfactant lipid synthesis or perinatal lung maturation, were significantly induced in the AJ-OT mice, indicating that the maternal environment regulated these genes. Other subsets of mRNAs did not change and/or were reduced when comparing the AJ-OT with the A/J mice at E17.5 , known to be transcriptional effectors in the Notch pathway and to regulate cell differentiation and post-embryonic development Creb-/- mice at E17.5  and CebpaΔ/Δ gene targeted mice at E18 CebpaΔ/Δ and Creb-/- mice, including Sftpa, Sftpb, Sftpd, Abca3, Scd1/2, Fabp5, Scnn1g, and Mia1. Likewise, differentially expressed mRNAs in Creb-/- and CebpaΔ/Δ mice were closely correlated (Creb1 or Creb3 mRNAs between the two strains (data not shown). The potential role of CREB1 was further assessed in transfection assays using promoters of several maturation-associated target genes in vitro, selecting genes that were induced in the AJ-OT pups at E17.5 and/or PN1 , occurred precociously in AJ-OT pups born early in C57BL/6J SCID dams. mRNAs induced precociously in the AJ-OT pups were associated with strain dependent differences in lung SatPC and lung weight observed during normal fetal lung development. While a number of genes regulating surfactant homeostasis were induced when AJ-OT pups were carried to term in the C57BL/6J SCID dams, many others were regulated in a strain-dependent manner and were not influenced by the maternal environment. Our studies demonstrate a remarkable, coordinate development of lung structure, as well as conducting and peripheral airway epithelial cell maturation, that coincides with the induction of biochemical and genetic markers of lung maturation that were regulated in a strain dependent manner. mRNAs regulating surfactant proteins and lipids, fluid homeostasis, and innate defense were induced with advancing gestation in a strain dependent manner.Lung maturation proceeded more rapidly in the C57BL/6J mice, as indicated by the earlier sacculation of peripheral airspaces and the expression of mRNAs regulating surfactant lipid and proteins. Likewise, the developmental increase in mRNAs associated with surfactant homeostasis, including those encoding proteins required for normal surfactant production and/or lipid homeostasis , transcription factors known to regulate cell differentiation and post-embryonic development nt study .Nr3c1) increased modestly with advancing gestation in both strains of mice. Nr3c1 is required for normal lung maturation and perinatal survival, influencing the expression of a number of genes important for surfactant production Nr3c1 mRNA increased with advancing gestation, its levels were not clearly influenced by maternal strain.Expression of the glucocorticoid receptor  was calculated at the midpoint of the dark cycle, prior to the appearance of the plug. Females were monitored continuously from E14.5 using infrared lighting and closed circuit, infrared-video cameras . Video recordings identified the birth of the first pup to assign gestational length C57BL/6J and A/J mice between 6 and 8 weeks of age were housed in humidity- and temperature-controlled rooms on a 12-hour light:dark cycle at The Jackson Laboratory . Mice were maintained in a pathogen-free environment in accordance with protocols approved by the Jackson Laboratory Animal Care and Use Committee. Mice were allowed food and water Transfer of A/J ovaries  to C57BL/6J-SCID (black) mice was performed as described previously Pregnant C57BL/6J and A/J dams were euthanized at E18.5 or E19.5 (A/J only) and fetuses were removed from the uterus and amniotic membranes. Resuscitation of the pups was performed by clearing nostrils and mouth and gently massaging the pups until they moved and breathed on their own. Pups were considered dead if attempts to resuscitate pups failed after 20 minutes. After resuscitation, pups were kept on a warming pad and observed for at least 2 hrs.Lung tissue preparation, immunohistochemistry, morphometry, RNA and lipid extraction were performed as described 5 per well in 6-well plate and transfected 24 hours later with Fugene 6  according to manufacturer's instructions. Effects of CREB1 and Notch1 were assessed in promoter assays tested on the mouse Sftpc (−4.8 kb), mouse Abca3 (−2.6 kb), mouse Lyz1 (−1.0 kb), mouse Lyz2 (−1.0 kb) and mouse Lpcat1 (−2.5 kb) genes with either pGL2 or pGL3 as the backbone vector . Methods are provided in Mouse lung epithelial (MLE-15) cells were maintained as previously described Figure S1Delayed expression of C/EBPα in the A/J fetuses during the perinatal period. Immunostaining for C/EBPα was performed on C57BL/6J and A/J lungs from fetal mice harvested at E15.5, 16.5, E17.5, E18.5, and E19.5 (A/J only). At E15.5, C/EBPα was detected in both epithelial (arrows) and mesenchymal cells (arrowheads). At E16.5, C/EBPα was increasingly restricted to the epithelium in C57BL/6J mice, while still present in both tissue compartments in the A/J mice. During the saccular stage, C/EBPα increased in both strains. At E18.5, C/EBPα was increased in alveolar regions and was detectable in the conducting airway epithelium (Br). While C/EBPα expression was present in most epithelial cells lining the distal conducting airways (asterisk) of C57BL/6J mice on E18.5 (G), fewer epithelial cells expressed C/EBPα in A/J lungs (H). By E19.5, C/EBPα expression in A/J fetal lungs was similar to that in C57BL/6J fetuses at E18.5 (I). Scale bar: 100 µm.(TIFF)Click here for additional data file.Figure S2FOXA2 is similar in C57BL/6J and A/J. Immunostaining for FOXA2, a transcription factor expressed in lung epithelial cells, was performed on C57BL/6J and A/J lungs from fetal mice harvested at E15.5, 16.5, E17.5, E18.5, and E19.5 (A/J only). Ontogenic changes in FOXA2 staining were similar in respiratory epithelial cells of both mouse strains. Scale bar: 100 µm.(TIFF)Click here for additional data file.Figure S3Earlier expression of CCSP in conducting C57BL/6J mice. Staining for CCSP (a Clara cell marker), was performed on C57BL/6J and A/J fetal mouse lungs harvested at E15.5, 16.5, E17.5, E18.5, and E19.5 (A/J only). At E17.5 and E18.5, CCSP immunostaining was increased in bronchiolar epithelial cells in the C57BL/6J compared to A/J fetuses. By E19.5, CCSP staining in A/J was similar to that seen at E18.5 C57BL/6J fetuses. Scale bar: 30 µm.(TIFF)Click here for additional data file.Figure S4Earlier expression of acetylated tubulin in conducting airways of lungs from C57BL/6J than in A/J mice. Immunohistochemical staining for acetylated tubulin (a ciliated cell marker), was performed on C57BL/6J and A/J lungs from fetal mice harvested at E15.5, 16.5, E17.5, E18.5, and E19.5 (A/J only). α-Tubulin was detected as early as E16.5 in the bronchiolar epithelium of C57BL/6J fetuses. α-Tubulin was first detected at E18.5 in A/J mice. α-Tubulin was similar in E19.5 A/J and E18.5 C57BL/6J mice. Scale bar: 20 µm.(TIFF)Click here for additional data file.Figure S5mRNA expression profiles during lung development. Dynamic mRNA expression profiles of 53 genes at different gestation ages for the A/J mice were correlated with body weight (BW), lung weight (LW), SatPC , and morphometric measurements  at corresponding gestational ages using multivariate correction function from JMP 9 . The heat map was generated based on data from A/J mice using Ward's minimum variance method to estimate cluster similarity. Gradients in the red and green color range indicate positive and negative correlation, respectively. The levels of mRNAs in red clusters were highly correlated with ontogenic changes in lung SatPC and fractional area of airspace; mRNAs in blue clusters were moderately correlated with SatPC, but closely correlated with fractional area of airspace; mRNAs in green clusters correlated well with the fractional area of the tissue compartment.(TIFF)Click here for additional data file.Figure S6Prenatal and postnatal growth after ovarian transfer. Body weight (A), lung weight (B), and lung-to-body weight ratios (C) of OT-B6 and OT-AJ mice were measured at E17.5 and at PN1. Values are means ± SEM of 12 animals per strain derived from n = 6 dams; *p< 0.05, for OT-AJ vs. OT-B6. Lung weights and LW/BW ratios were increased when A/J fetuses were born to C57BL/6J dams. SatPC was measured in lungs from B6-OT and AJ-OT mice at E17.5 and PN1 (D-E). Results are expressed as the means ± SEM of 12 animals per strain.(TIFF)Click here for additional data file.Figure S7Hey1 and Hes1 mRNAs were compared in whole lung mRNA from C57BL/6J and A/J pups at each age. Statistical differences were assessed by ANOVA, * indicate p<0.05.(TIFF)Click here for additional data file.Table S1Common cis-elements in the promoter regions of genes induced in AJ-OT mice at E17.5 and at PN1.(DOCX)Click here for additional data file.Table S2Differentially expressed mRNAs in Creb-/- and Cebpa/ΔΔmice.(DOCX)Click here for additional data file.Data S1Online Data Supplement – Materials and Methods.(DOCX)Click here for additional data file.


Increasing evidence supports the contribution of local inflammation to the development of Alzheimer's disease (AD) pathology, although the precise mechanisms are not clear. In this study, we demonstrate that the pro-inflammatory protein S100A9 interacts with the A Knockdown of S100A9 expression improves cognition function in Alzheimer's disease model mice (Tg2576) and these animals also exhibit reduced amyloid plaque burden It has been speculated that inflammation is directly related to Alzheimer's disease. Studies performed in transgenic animals suggest that cerebral amyloid deposition is increased in inflammatory conditions To investigate the relationship between S100A9 and amyloid plaques, we have studied the interaction between the main component of the amyloid plaque , minor distinctions were observed in the 200 to 230 nm wavelength range . The twoThe effect of S100A9, Ad to 3 d . During To explore the interaction between S100A9 and Aobserved . AccordiThe quantities of S100A9 monomer and homodimer were estimated by integrating the intensities of all bands using Image J software. Based on the SDS PAGE data, the freshly dissolved S100A9 contained 74% monomers and 26% homodimers. After incubation with cells for 3 d, the fraction of homodimer dropped to 8.4%, which suggests that the reduction in the quantity of S100A9 dimers is caused by its interaction with cells. A similar decrease in the S100A9 dimer content was observed when S100A9 was mixed with various concentrations of A1–40 . AccompaThe induction of the S100A9 gene was found in the brains of AD patients and AD animal models, Tg2576 and CT-Tg mice The consequences of S100A9/Ae formed . The hei fibrils . Conformormation . In cont spectra . The pos spectra . The foriability . The redon cells . Sharingnd A1–42 . Similargregates . It is wWith a constant amount of S100A9 monomers, we observed a great decrease in S100A9 dimer content from 26% to 8% . Such vaatically . AdditioS100A9 homodimer is shown to be expressed independently during inflammation bic core . With noOverall, the results of this study, combined with those in previous reports, suggest that AThe proteins used in these experiments were recombinant S100A9 (MRP14) and AA ThT stock solution was made by dissolving 2.5 mM Thioflavin-T (Merck-Schuchardt) in phosphate buffer  and filtered before use. This stock solution was diluted 50-fold in the phosphate buffer to generate the working solution. Protein samples of 10 CD spectra were recorded with a JASCO J-720 spectropolarimeter equipped with a PTC-343 temperature controller. For each sample, 3 spectra were acquired and averaged in the range of 190 nm to 250 nm, using a 5 nm/min scan speed, and 1 nm resolution. The quartz cells had a 1 mm optical path. The contribution from the buffer was subtracted, and the results are presented in relative ellipticity. The difference ellipticity in SDS and native polyacrylamide gel electrophoresis (PAGE) was carried out on 15% Tris gels. 5 volume of protein sample was mixed with 1 volume of loading buffer (1.6 ml Tris-HCl (1 M), 4 ml SDS (10%), 2 ml glycerol (100%), 1 ml The operation procedure of GEMMA instrument was as published previously AFM measurements were obtained with a PicoPlus AFM (Agilent) in tapping mode using a 100 nm scanner in ambient conditions. Acoustically driven cantilevers with etched silicon probes of the TESP model (diameters of 10nm and less)  were used. We applied a resonance frequency in the 170 kHz to 190 kHz range, a scan rate of 1 Hz or less, and a resolution of 512 pixels×512 pixels. Height, amplitude and phase data were collected simultaneously in trace and retrace modes to avoid scan artifacts. Amyloid samples were deposited on the surface of freshly cleaved mica (GoodFellow) for 5 min, washed 3 times with 200 SH-SY5Y (ATCC CRL-2266) were cultured in Dulbecco's modified Eagle medium supplemented with 10%(v/v) FBS. Mixed cortical neuron cultures were prepared from embryonic day 16.5 rat embryos. Animals used in this study were maintained in strict accordance with the approval granted by UmeTo evaluate cell viability, 10 Figure S1A for (a) 1 d; (b) 3 d. The scale bars denote 600 nm and 2000 nm, respectively. The insertions show the cross-section profile of the fibrils corresponding to the marked positions. After 1 d incubation, proto-fibrils with a height of 0.5 nm were observed. After 3 d, the surface was dominated by fibrils with a height of 2 nm, which is typical for amyloid mature fibrils. As the fibrils observed upon the addition of S100A9 (f S100A9  carried (TIF)Click here for additional data file.Figure S2CD septra (a) The transition in the secondary structures of (a) S100A9 (0.2 mM); (b) A(TIF)Click here for additional data file.Figure S3AFM height images of S100A9 (0.2 mM) (a) incubated at pH 3.0 and 57(TIF)Click here for additional data file.Figure S4Measurements of NHSY5Y cell line viability by WST1 assay in the presence of (a): freshly dissolved A(TIF)Click here for additional data file.Figure S5(a) Luminescent cytotoxicity assay of neuron cells in presence of 0.02 mM S100A9 and S100A9/A(TIF)Click here for additional data file.Figure S6AFM images of (a) A(TIF)Click here for additional data file.


Pre-existing immunity is an important factor countering the pandemic potential of an emerging influenza virus strain. Thus, studying of pre-existing immunity to the 2009 pandemic H1N1 virus (2009 H1N1) will advance our understanding of the pathogenesis and epidemiology of this emerging pathogen. In the present study, sera were collected from 486 individuals in a hospital in Shanghai, China, before the 2009 H1N1 influenza pandemic. The serum anti-hemagglutinins (HA) antibody, hemagglutination inhibition (HI) antibody and neutralizing antibody against the 2009 H1N1 were assayed. Among this population, 84.2%, 14.61% and 26.5% subjects possessed anti-HA antibody, HI antibody and neutralizing antibody, respectively. Although neutralizing antibody only existed in those sera with detectable anti-HA antibody, there was no obvious correlation between the titers of anti-HA and neutralizing antibody. However, the titers of anti-HA and neutralizing antibody against seasonal H1N1 virus were highly correlated. In the same population, there was no correlation between titers of neutralizing antibody against 2009 H1N1 and seasonal H1N1. DNA immunization performed on mice demonstrated that antibodies to the HA of 2009 pandemic and seasonal H1N1 influenza viruses were strain-specific and had no cross-neutralizing activity. In addition, the predicted conserved epitope in the HA of 2009 H1N1 and recently circulating seasonal H1N1 virus, GLFGAIAGFIE, was not an immunologically valid B-cell epitope. The data in this report are valuable for advancing our understanding of 2009 H1N1 influenza virus infection. Many infectious diseases display epidemic wave patterns due to interaction between pathogen antigens and the pre-existing host immunity barrier The current strategy for influenza virus control and prevention is primarily dependent on vaccination along with therapeutic and prophylactic use of neuraminidase inhibitors In this study, to evaluate the presence of antibodies against 2009 H1N1 in the general population in Shanghai, China, IgG levels and neutralizing activity against both 2009 pandemic and a seasonal H1N1 virus were assessed in 486 serum samples collected prior to 2009 pandemic influenza outbreak. The mechanism(s) of cross-protection were studied by immunization with homologous and heterologous HA-encoding plasmids in mice model.In total, 486 serum samples (age ranging from 16 to 88) were obtained from in-and out-patient subjects without influenza virus vaccination history, in Changhai Hospital, Shanghai, China, from December, 2008 to February, 2009. Subjects were considered to have not been exposed to 2009 H1N1 influenza virus because the final sample was taken 3 months prior to the onset of the pandemic. Another 27 samples were taken from 20–22-year-old healthy volunteers inoculated with the 2009 H1N1 inactivated vaccine in December, 2009. All study subjects were selected on the basis of a questionnaire designed to exclude any person with symptoms possibly associated with influenza-like illness in the previous 6 months. All donors gave written informed consent for research use of blood samples. The study design was approved by the Ethical Committee of Second Military Medical University.Madin-Darby canine kidney (MDCK) cells, and human embryonic kidney (HEK) 293T cells were obtained from American Type Culture Collection  and grown in Dulbecco's modified essential medium  supplemented with or without 10% fetal bovine serum.The cDNA fragments encoding the full-length HA of A/California/05/2009 (GenBank Accession No. FJ966952) strain and a seasonal H1N1 isolate  were synthesized and inserted into the vector pVRC to produce 2009 HA and seasonal HA expression constructs as we reported previously Galanthus nivalis lectin  in phosphate-buffered saline (PBS) and incubated overnight at 4°C. Plates were washed with washing buffer (0.05% Tween 20 in PBS), and non-specific binding sites were saturated with BSA buffer (3% BSA and 0.05% Tween 20 in PBS). Crude cell lysates from 2009 HA, seasonal HA expression plasmids, or mock vector transfected HEK 293T cells were added to the plates at 100 µL/well and incubated for 2 h at room temperature (RT). After extensive washing, serial two-fold dilutions of serum in BSA buffer, stating with a 1∶100 dilution, were added (100 µL/well) and incubated for 40 min at RT. The plates were washed, incubated with horseradish peroxidase-conjugated anti-mouse or anti-human IgG , diluted 1∶10,000 in BSA buffer, and incubated for 40 min at RT. Plates were again washed, color was developed using 3,3′,5,5′-tetramethylbenzidine (TMB) substrate, and the absorbance at 450 and 630 nm measured. A two-fold increase in mean signal over that in mock-coated wells was considered to be HA antibody-positive. The highest positive dilution was determined to be the antibody titer.Anti-HA antibody was assayed as described previously 2009 pandemic H1N1 and seasonal H1N1 viruses were isolated and confirmed by regular influenza virus survey in Fujian Province. The viruses were grown in chorioallantoic fluid of 10-day-old embryonated chicken eggs and purified by sucrose gradient centrifugation. Virus particles were prepared in PBS, aliquoted, and stored at −80°C until use. The viral titer was determined by titration in MDCK cells and the tissue culture infectious dose affecting 50% of the cells (TCID50) was calculated using the Reed-Muench formula HI assays were performed according to standard methods as described recently Escherichia coli XL1-blue and purified using Qiagen Giga columns, according to the manufacturer' instructions. Female, 7-week-old BALB/c mice were purchased from SIPPR-BK Experimental Animal Co. Ltd, Shanghai, China. Mice were injected with 2009 HA or seasonal HA plasmids (100 µg in 100 µL) in both tibialis anterior muscles. Control mice were injected with 100 µg mock vector. Each mouse received a boost immunization 2 weeks later. Sera were collected at 2-week intervals, inactivated at 56°C for 30 min and stored at −80°C until required.The 2009 HA and seasonal HA expression plasmids were propagated in All animal experiments were conducted in accordance to the recommendations in the Guide for the Care and Use of Laboratory Animals of the Second Military Medical University and the Rules for the Medical Laboratory Animal (1998) from Ministry of Health, China. The protocol was approved by the Animal Care and Use Committee of Second Military Medical University.3, and incubated with mixed sera of the vaccinated subjects (diluted 1∶100 in the same buffer) for 40 min at RT. Cells were then washed twice with PBS and incubated with FITC-conjugated rabbit anti-human IgG . Fluorescent-positive cells were quantified by flow cytometry using a Cell Lab Quanta SC instrument (Beckman Coulter). The percent neutralization was determined by comparing viral infectivity (denoted as percentage of fluorescent-positive cells) in the presence of serum with the mean infectivity in the absence of serum Wild-type 2009 H1N1 or seasonal H1N1 virus (100 TCID50/well) was incubated with twofold serial dilutions of RDE-treated sera. The mixtures were transferred to MDCK target cells in a 96-well plate, incubated at 37°C in the presence of TPCK-trypsin for 2 h. After washing twice, the cells were cultured in medium without TPCK-trypsin to prevent re-entry of viruses. After 24 hours incubation, the cells were detached using 2 mM EDTA in PBS, washed in PBS buffer containing 2% FBS and 0.05% NaNThree linear B-cell epitope in HA of 2009 H1N1 and an epitope located in hepatitis C virus envelope protein 2, were synthesized by Biovisualab Inc . Peptides were coated onto microplates (1 µg/well) overnight at 4°C. After blocking with BSA buffer, epitope-specific antibody levels in human and mouse sera were assayed as described above.To estimate the value of the MN titer corresponding to the ELISA titer of anti-2009 HA antibodies, we performed a correlation analysis using linear regression models. Data were analyzed using linear regression and multivariable models, Student' t-test, and estimation of geometric mean titers (GMTs) with confidence intervals and corresponding P values, using the SAS software (ver. 9.1). Data were expressed as mean±standard deviations (SD).To investigate pre-existing immunity in the general population in Shanghai, 486 serum samples, obtained pre-pandemic, were assessed for an antibody response to 2009 pandemic H1N1 by in-house-developed ELISA. Of the total number of subjects, 84.2% (409/486) possessed serum antibody against the HA of 2009 H1N1 virus. Positivity rates increased from 56.5% of those born after 1990 to 95.0% born before 1930 . The GMTSerum antibodies that inhibit HA function of 2009 H1N1 virus were determined by HI and MN assay. Sera showed no reactivity with the HA of 2009 pandemic H1N1 virus in ELISA did not display HI and neutralizing activity against 2009 H1N1 virus, and all HI antibody-positive serum samples showed neutralizing activity (data not shown). Similar to HA antibody profile, HI and MN antibody roughly increased with the age. The overall positive rate of HI antibody was 14.61%. HI antibody positive rate increased from 4.3% of those born after 1990 to 30% of those born before 1930 . GMTs of2healthy) was as low as 0.2543, while that of vaccinated subjects (R2vacc) was 0.8435, suggesting that subjects were exposed to 2009 pandemic H1N1-like viruses  were selected for correlation assay. Neutralizing activity did not correspond to the antibody levels of anti-HA. The linear correlation index between IgG and microneutralization antibody titers in general subjects  were further tested for reactivity and MN titer to seasonal human influenza virus H1N1. Sera with high cross-reactive responses to HA of seasonal H1N1 virus also possessed high MN titers to seasonal influenza H1N1 virus  was predicted as the only conserved B cell epitope in HA of 2009 H1N1 and human seasonal H1N1 www.who.int/csr/don/2009_07_06/en). This was not significantly higher than that of seasonal influenza. Pre-existing immunity has been assumed to contribute to the overall low morbidity Despite the initial high mortality in Mexico, 2009 pandemic H1N1 virus caused generally mild symptoms, and overall mortality remained at around 0.45% , MN antibodies rate (26.5%) and HI antibodies rate (14.61%), but all the MN positive sera were also anti-HA positive, and all the HI positive sera were MN positive. Compared with the subjects immunized 2009 H1N1 vaccines, the correlation between anti-HA titers and MN titers of the general population was relatively lower. It may be explained by different epitopes in HA involved in immune response. In this regard, major anti-HA antibodies may target the conserved stalk region of HA, while the neutralizing antibodies were against the hypervariable globular head of HA. On the other hand, as described above, our data may also indicate that the immunity against 2009 H1N1 in general Shanghainese was derived from complicated circulating influenza viruses, this possibility was supported by studies from Taiwan and France A 2009 H1N1 isolate was more pathogenic in mice and ferrets with no previous exposure to influenza, indicating that pre-existing cross-reactive antibodies might offer partial protection against novel influenza strains