SLC27A) gene family encodes fatty acid transport proteins (FATPs) and includes 6 members. During fetal and postnatal periods of development, the growing brain requires a reliable supply of fatty acids. Because autism spectrum disorders (ASD) are now recognized as disorders caused by impaired early brain development, it is possible that functional abnormalities of SLC27A genes may contribute to the pathogenesis of ASD. Here, we confirmed the expression of SLC27A3 and SLC27A4 in human neural stem cells derived from human induced pluripotent stem cells, which suggested their involvement in the developmental stage of the central nervous system. Additionally, we resequenced the SLC27A3 and SLC27A4 genes using 267 ASD patient and 1140 control samples and detected 47 (44 novel and 29 nonsynonymous) and 30 (17 novel and 14 nonsynonymous) variants for the SLC27A3 and SLC27A4, respectively, revealing that they are highly polymorphic with multiple rare variants. The SLC27A4 Ser209 allele was more frequently represented in ASD samples. Furthermore, we showed that a SLC27A4 Ser209 mutant resulted in significantly higher fluorescently-labeled fatty acid uptake into bEnd3 cells, a mouse brain capillary-derived endothelial cell line, compared with SLC27A4 Gly209, suggesting that the functional change may contribute to ASD pathophysiology.The solute carrier 27A ( Autism spectrum disorders (ASD) are complex neurodevelopmental disorders characterized by impairments in social orientation and communication and repetitive or restricted patterns of interests or behaviors1245The brain is a lipid-rich organ, with nearly 50% its dry weight consisting of lipids910SLC27A3/Slc27a3  is highly expressed in the adrenal gland, testis, ovary, lung, and the neonatal and adult brain. SLC27A4/Slc27a4  is expressed in the intestine, skin, brain, kidney, liver and heart, as well as trophoblasts of the placenta and endothelial cells. Additionally, only Slc27a3 and Slc27a4 were expressed in mouse brain capillary-derived endothelial cells (bEnd3)121314Fatty acid transport proteins (FATPs) solute carrier 27A (SLC27A) family includes six members in humans and mice (SLC27A1/Slc27a1 to SLC27A6/Slc27a6). Among FATP family members, Slc27a3 mRNA levels are significantly higher in embryonic mouse brain than in newborn or adult micein vivo model of Slc27a3 loss of function has not yet been reported. The expression pattern of Slc27A3 protein suggests that it may play an important role in brain developmentet al.Slc27a4 deficient embryos exhibited normal fetal growth. However, they did not perform any neuronal analyses including behavioral assessment, and it is worthy of examining a potential role of the SLC27A4 gene in functional brain disorders including ASD.Although it is known that SLC27A3 and SLC27A4 in ASD. First, we investigated the role of SLC27A3 and SLC27A4 in neural development by examining the expression of these genes during neuronal differentiation from human induced pluripotent stem cells (hiPSCs) and also in mouse fetal brain. Further, we resequenced SLC27A3 and SLC27A4 in 267 ASD patients and 1140 control samples. We focused on identifying nonsynonymous variants, and assessed the associations of these variants with ASD, along with the functional evaluation of the associated variant.Thus, in this study, we investigated possible roles for SLC27A3) and FATP4 (encoded by SLC27A4), because these proteins may be key molecules for fatty acid uptake into the fetal brain (SLC27A3 and SLC27A4 mRNA expression levels in human induced pluripotent stem cell (hiPSC) (201B7) derived-neurosphere cells and hiPSC derived-differentiated neurons . This mutation is predicted to be benign by “PolyPhen2”1820In the sequencing analysis, we found that the iPSC clone does not have any mutations in the http://www.proteinatlas.org/). These results suggest that fatty acids, including PUFA, may be taken up into endothelial cells and cortical cells through SLC27A3 (Slc27a3) and SLC27A4 (Slc27a4) during brain development.Furthermore, we assessed the Slc27a3 (Fatp3) and Slc27a4 (Fatp4) protein expression patterns in mouse fetal brain (E18.5). Slc27a3 and Slc27a4 proteins were expressed in both endothelial cells (CD31 positive cells) and cortical cells  in the mouse brain . The expSLC27A genes encoding fatty acid transporter proteins may modify the risk for neurodevelopmental disorders, including ASD. We first performed a resequencing analysis for the SLC27A3 and SLC27A4 genes in 267 ASD samples. Polymorphism screening detected a total of 24 and 13 different variants of SLC27A3 and SLC27A4, respectively . Since we identified a large number of variants, especially in the SLC27A3 gene, we suspected the presence of duplications in this gene locus. We performed genomic quantitative PCR for the SLC27A3 genomic interval to determine whether the SLC27A3 gene had any copy number variations. We found no evidence of duplications (or deletions) in the genomic region that spanned the SLC27A3 gene , we performed association analyses separately for males and females  was significantly less frequent among male ASD patients as compared with male control samples. However, this significant result was not maintained after correcting for multiple testing (n = 12 × 2). SLC27A4 c.625G>A (p.Gly209Ser) was significantly less frequent among male ASD patients and was significantly more frequent among female ASD patients as compared with control samples. The significant result found for males was not maintained after correcting for multiple testing (n = 5 × 2). In contrast, the significant result for females was maintained after correcting for multiple testing (n = 5 × 2) (SLC27A4 p.Ser209 carriers had those CNVs. We compared the Autism Diagnostic Interview-Revised (ADI-R) scores among genotypes  of the SLC27A4 variant in female ASD patients. There was no significant genotype-dependency on the phenotypes  . We conf= 5 × 2) . To inveenotypes . When boSLC27A3 c.553G>A (p.Gly185Arg), c.1,477C>T (p.Arg493Cys), SLC27A4 c.250G>A , and c.272C>T (p.Thr91Met) were exclusively found in ASD patients ; however, this significance did not withstand correction for multiple testing (n = 12) ((n = 12) .SLC27A4 p.Ser209 was the only variant that showed an empirical association with ASD in the current genetic study SLC27A genes (SLC27A3 and SLC27A4) that are expressed in the fetal brain with ASD.Abnormal fatty acid metabolism has been suggested to play a crucial role in ASD pathophysiologySLC27A4 p.Gly209Ser was significantly more frequent in ASD patients and that this substitution caused increased fatty acid uptake into bEnd3 cells in vitro. SLC27A4 Ser209 is a known variant that is associated with insulin resistance syndromeSLC27A4 Ser209 allele and ASD may link metabolic syndrome to ASD at the molecular level.One of the limitations of the current genetic study is that the sample size is rather small. Bearing this in mind, our results showed that SLC27A4 p.Gly209Ser substitution increases the intrinsic transporter activity of a protein. One possibility is that this substitution affects CoA synthase activity that is coupled to transporter activity. Indeed, the Gly209 residue is located in the AMP binding domain . Estrogen produced by the actions of aromatase may promote SLC27A4 expression in males. Therefore, the differences in balance of sex hormone levels between males and females during a critical period of brain development may underlie the different effects of the SLC27A4 polymorphism for the risk of ASD. In addition to the p.Gly209Ser mutation, a excess of potentially functional variants (missense and ins/del) of SLC27A4 was observed in the female ASD group  SLC27A4 p.Gly209Ser was associated with ASD although a caveat the small sample size of this study; and (3) this variant had functional consequences with regard to controlling endothelial long-chain fatty acid transport. Further studies with larger sample sizes and more extensive functional assessments including a comparison of fatty acid uptake activity between SLC27A4 G209 and Ser209-harboring hiPSCs and acyl-CoA ligase activity for long-chain fatty acids will be needed to determine the precise roles of SLC27A3 and SLC27A4 in ASD pathogenesis.To conclude, in the present study we found that: (1) the TM Select (Life Technologies) and dissociated into single cells by pipetting. Cells were plated into a T75 flask and cultured in medium hormone mix (MHM) supplemented with B27 (Life Technologies), 20 ng/mL of bFGF, 10 μM Y-27632 (Wako), and 10 ng/mL of human leukemia inhibitory factor (hLIF)  in 4% oxygen for 14 daysHuman-induced pluripotent stem cells (hiPSCs) GAPDH: Hs02758991_g1, SLC27A3: Hs00950760_g1, SLC27A4: Hs00192700_m1), and an ABI 7900 sequence detection system, according to the manufacturer’s instructions. The GAPDH gene was used as an internal control (Applied Biosystems). A PCR assay was performed with test and standard samples simultaneously and with no template controls on the same plate. A standard curve was generated by plotting the cycle of threshold values against input quantity  for both the GAPDH gene and the target genes for each PCR assay. All real-time quantitative PCR data were acquired using ABI PRISM 7900 Sequence Detection System (SDS) v2.4 (Applied Biosystems). The expression level of a target gene relative to that of the GAPDH gene (target gene/GAPDH gene) was determined.Total RNA was extracted from hiPSC derived neurosphere cells and hiPSC derived-differentiated neurons using a miRNeasy Mini Kit . Single stranded cDNA was synthesized using SuperScript VILO Master Mix . mRNA expression levels were determined by real-time quantitative PCR by using TaqMan Gene Expression Master Mix, transcript-specific minor groove binding (MGB) probes  (C57BL/6NCrlCrlj (B6) inbred mouse strains were obtained from Japan’s Charles River Laboratories. Mice were housed in groups of four in standard cages in a temperature and humidity-controlled room with a 12-h light/dark cycle (lights on at 08:00) and provided free access to standard lab chow and tap water. All experiments were performed between 10:00 and 14:00. Our experimental procedures were approved by the RIKEN Animal Ethics Committee. The animal experiments were carried out in "accordance" with the approved guidelines. For immunohistochemical studies, at least five mice at E18.5 were examined. Mice were deeply anesthetized with sodium pentobarbital and then transcardially perfused with 4% paraformaldehyde in 0.01 M phosphate-buffered saline (PBS). Brains were removed and further immersion-fixed in the same fixative at 4 °C for 16 h. Coronal sections (14 μm thick) were prepared using a cryostat . These sections were washed with Tris-buffered saline that contained Tween 20 . For immunostaining, cryostat sections were incubated with primary antibodies at 4 °C for 18 h. To detect antigen localization, sections were incubated with appropriate secondary antibodies at 4 °C for 2 h. For immunocytochemical studies, hiPS cells, hiPS-derived neurospheres and hiPS-derived neurons were used. They were fixed with 4% paraformaldehyde in 0.01 M PBS at room temperature (RT) for 20 min, and washed with TBST followed by incubated with primary antibodies at RT for 1 h. After that, they were incubated with appropriate secondary antibodies at RT for 1 h. Information on the primary and secondary antibodies and other reagents are given in SLC27A3 and SLC27A4 genes, we evaluated 267 Japanese ASD patients  and 1140 Japanese control subjects . In the analysis of SLC27A4 p. Gly209Ser, we examined an expanded control sample set . For the transmission disequilibrium test and de novo mutation analysis, 201 patient-parents trio samples (603 samples) and 267 pedigree samples that included 66 incomplete trios (only single parents were available) were examined, respectively. ASD patients from pedigrees were the same as those used for resequencing and case-control association analyses. All of our study subjects resided in central Japan. A diagnosis of ASD was made using DSM-IV and Interview-Revised (ADI-R) criteriahttp://www.wma.net).For resequencing and association analyses of all the protein-coding exons of the SLC27A3 or SLC27A4 genes were screened for polymorphisms by direct sequencing of polymerase chain reaction (PCR) products. The primers used for amplification are listed in SLC27A3 (RefSeq: NM_024330.1) and SLC27A4 (RefSeq: NM_005094.3) were based on the UCSC February 2009 draft assembly of the human genome database (http://www.genome.ucsc.edu), and the NCBI database (http://www.ncbi.nlm.nih.gov/) was searched for known single nucleotide polymorphisms (SNPs). Custom TaqMan SNP Genotyping Assays (Applied Biosystems) were used to score the identified missense SNPs using the TaqMan assay methodGenomic DNA was isolated from blood samples obtained from our human subjects using standard methods. All the coding exons and exon/intron boundaries of the SLC27A3 were analyzed by real-time genomic quantitative PCR for exons 1, 4, and 10 using the TaqMan method (Applied Biosystems). MLC1 at chromosome 22q13.33 was used as a normal copy number control gene. For quality control, PFKFB1 on chromosome Xp11.21 was used to determine whether our genomic quantitative PCR accurately detected differential dosages of the X chromosome between male and female control samples. For the analysis of CNVs in the 16p11.2 and 15q11.2 regions, the RNaseP gene was used as a normal copy number control gene. No copy number polymorphisms have been documented in the MLC1, PFKFB1 and RNaseP genes within the Japanese population. For genomic quantitative PCR, DNA solutions were first quantified using an ultraviolet spectrophotometer and further quantified using a TaqMan RNase P Detection Reagent kit (Applied Biosystems). Primers sequences used for these analyses are listed in Insertions/deletions within the genomic length of SLC27A3 and SLC27A4 mRNA expression using Human Placenta Marathon-Ready cDNA . The primers used for amplification are listed in GAPDH was used for quality control. The PCR products were separated on an agarose gel.We performed semi-quantitative analysis of SLC27A4 cDNA that covered the open reading frame was generated by PCR using cDNA derived from established lymphoid cell lines of ASD children who were heterozygous for wild-type (Gly209) and mutant (Ser209) SLC27A4 alleles and the following primer set; SLC27A4 forward: 5′-ATGCTGCTTGGAGCCTCTCTGG-3′, SLC27A4 reverse: 5′-CAGCTTCTCCTCGCCTGCCT-3′. Each amplified cDNA was cloned into the mammalian expression vector pcDNA3.1/V5-His-TOPO (Life Technologies), which is driven by the CMV promoter and enables TA-cloning of the PCR products and adding V5 and His tags (epitopes) to the C-termini of expressed proteins. Because the vector is supplied as a linearized TA cloning vector, it is very difficult to obtain an empty vector. Therefore, we used the inverted SLC27A4 cDNA-containing construct as a control, which was obtained in a single transformation along with the construct containing the SLC27A4 cDNA in the normal direction. The inverted cDNA is predicted to encode a very short peptide due to the early termination codon, suggesting that no functional or biologically active protein can be produced. Plasmid structures were verified by sequencing. Cells were seeded in 8-well chamber slides  and cultured as described above using growth medium that contained 1% fatty acid-free bovine serum albumin (FF-BSA) and no fetal calf serum. bEnd3 cells were transfected with human SLC27A4-Gly209-pcDNA3.1/V5-His-TOPO and human SLC27A4-Ser209-pcDNA3.1/V5-His-TOPO simultaneously or with the control construct using a Lipofectamine LTX transfection system together with Plus-reagent (both from Life Technologies), according to the manufacturer’s instructions. At 24 h after transfection, cells were washed with PBS-1% FF-BSA and incubated at 37 °C for 3 min with PBS that contained 1% FF-BSA and 20 μM C1-BODIPY-C12 (Sigma-Aldrich). Cells were washed vigorously, and fixed with 4% paraformaldehyde at room temperature (RT) for 10 min. After fixation, cells were first incubated with 0.2% Triton X-100 in PBS (PBST) at RT for 5 min and then incubated with 1% blocking reagent  in PBST at RT for 30 minThe mouse endothelial cell line bEnd3  was cultured in high glucose DMEM  supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 100 U/ml of penicillin, and 100 U/ml of streptomycin. Human http://imagej.nih.gov/ij/). These experiments were repeated at least three timesFor immunocytochemistry, cells were incubated with primary antibodies  at RT foP values for gene expression levels in hiPSC-derived neurospheres and neurons were determined using a Mann Whitney test. P values for our genetic association analysis were determined by Fisher’s exact test. A transmission disequilibrium test (TDT) was performed using PLINK v1.07 software (http://pngu.mgh.harvard.edu/~purcell/plink/). P values for fatty acid uptake analysis were determined using Bonferroni’s multiple comparisons test. P < 0.05 was considered significant.How to cite this article: Maekawa, M. et al. Investigation of the fatty acid transporter-encoding genes SLC27A3 and SLC27A4 in autism. Sci. Rep.5, 16239; doi: 10.1038/srep16239 (2015).


Human antibody 10E8 targets the conserved membrane proximal external region (MPER) of envelope glycoprotein (Env) subunit gp41 and neutralizes HIV-1 with exceptional potency. Remarkably, HIV-1 containing mutations that reportedly knockout 10E8 binding to linear MPER peptides are partially neutralized by 10E8, producing a local plateau in the dose response curve. Here, we found that virus partially neutralized by 10E8 becomes significantly less neutralization sensitive to various MPER antibodies and to soluble CD4 while becoming significantly more sensitive to antibodies and fusion inhibitors against the heptad repeats of gp41. Thus, 10E8 modulates sensitivity of Env to ligands both pre- and post-receptor engagement without complete neutralization. Partial neutralization by 10E8 was influenced at least in part by perturbing Env glycosylation. With unliganded Env, 10E8 bound with lower apparent affinity and lower subunit occupancy to MPER mutant compared to wild type trimers. However, 10E8 decreased functional stability of wild type Env while it had an opposite, stabilizing effect on MPER mutant Envs. Clade C isolates with natural MPER polymorphisms also showed partial neutralization by 10E8 with altered sensitivity to various gp41-targeted ligands. Our findings suggest a novel mechanism of virus neutralization by demonstrating how antibody binding to the base of a trimeric spike cross talks with adjacent subunits to modulate Env structure and function. The ability of an antibody to stabilize, destabilize, partially neutralize as well as alter neutralization sensitivity of a virion spike pre- and post-receptor engagement may have implications for immunotherapy and vaccine design. As vaccination, immunoprophylaxis and immunotherapies are becoming increasingly feasible approaches to combat HIV/AIDS, understanding the activity of relevant anti-HIV antibodies is crucial. Antibody 10E8 defines a key vulnerability on the envelope spikes of a vast majority of HIV isolates but mechanisms of resistance to this neutralizing antibody are incompletely understood. Our findings show how partial neutralization of HIV can occur through apparent partial occupancy by 10E8 of HIV spikes that is accompanied by specific, antibody mediated effects on spike stability, infectivity and sensitivity to various inhibitors of HIV. We reveal a previously unappreciated mechanism of spike-antibody recognition where consequences on viral infectivity by 10E8 binding are dependent on interactions between subunits of the virion spike that modulate its stability and recognition properties. HIV vaccine development and immunoprophylaxis involving 10E8-like antibodies and their target, the gp41 MPER, may have to consider functional relationships involving the MPER and antibody occupancy at the base of trimeric spikes. The membrane proximal external region (MPER) of HIV-1 is an important target on the transmembrane subunit gp41 as it is linked to a highly conserved sequence motif and epitopes of several broadly neutralizing antibodies Advances in both vaccine development and immunoprophylaxis are needed to combat HIV/AIDS Models of the MPER typically focus on peptide monomers, either on micelles, lipid bilayers or in solution Although antibodies can reach an occupancy level of three per Env spike e.g. W672A with 4E10), others diminish peptide binding but enhance neutralization (e.g. I675A with Z13e1) Models have depicted the MPER at the base of unliganded spikes where it might interact with other elements of gp41 in vivo, and so has relevance to immunotherapy and vaccine approaches.We wished to gain new insight into MPER mediated neutralization using 10E8. We discovered an unexpected mechanism in which HIV-1 becomes partially neutralized by 10E8 wherein potency is high against a ‘neutralizable’ fraction of virus infectivity. Whereas 10E8 readily occupies all three protomers of wild type unliganded spikes it partially and inefficiently occupies MPER mutant unliganded spikes, indicative of a hindrance to further occupancy by 10E8 on adjacent subunit(s). Here, 10E8 seems to bind but remarkably not fully inhibit HIV-1 spikes from mediating infection of target cells. Moreover, with the ‘non-neutralizable’ fraction of virus infectivity we find that 10E8 can kinetically alter stability and ligand-binding properties of Env spikes pre-receptor engagement as well as post-receptor engagement in distinct ways. These features define a novel mechanism of HIV-1 neutralization involving the MPER, accessibility by antibody to the MPER, and interactions between MPER and adjacent elements of Env. The novel mechanism described for 10E8 conceivably might also influence HIV-1 facing 10E8-like antibodies Table S1). Of 21 Ala mutants covering positions 660–680 of the MPER, only mutants W672A, F673A, W680A and K683A were partially neutralized by 10E8, i.e. neutralization curves plateaued with less than full neutralization . One mutant, N671A, was less sensitive to 10E8 but became fully neutralized by 10E8 at high concentration . Importantly, in all cases partial neutralization was consistently reproducible. The nature of amino acid substitution also had an effect on 10E8 neutralization. Mutants containing conservative substitutions of aromatics for aromatics (F673W/Y and W672Y/F) were fully neutralized whereas substitutions from hydrophobics to hydrophilics (F673R/Q) were partially neutralized with maximum plateaus at 50–60% . Antibody 2F5 to an MPER epitope upstream of 10E8 neutralized all of the mutants completely and more potently than wild type virus  were very similar to that of wild type HIV-1 for these inhibitors ; this was also true in the SF162 Env background (data not shown). In fact, the F673L mutants were hypersensitive to MPER antibodies 2F5 and Z13e1 . FcγRI improves on-rates of antibodies against receptor-activated gp41, particularly MPER antibodies Partial neutralization using dose-saturating concentrations of 10E8 conceivably might be due to an MPER mutant having enhanced fusion kinetics that limits the time in which 10E8 can act post-receptor engagement. We therefore tested the sensitivity of mutant F673L to fusion inhibitors C34 and 5-Helix, which act post-receptor engagement on NHR and CHR regions of the gp41 pre-fusion intermediate, respectively. We found that ICZ13e1 and2. HencFigure S3B, C, and D). We also used 10E8 IgG produced both transiently in 293 cells and using a stable CHO-K1 cell line as well as Fab 10E8 prepared by enzymatic digestion. Both in-house 10E8 IgG preparations, a sample from the NIH ARRRP and the Fab 10E8 molecule all showed partial neutralization against the F673L mutant, however the potency with the Fab was found to be lower . Hence, partial neutralization by 10E8 appears to occur independently of target cell type, antibody format or solubility state of the antibodyWe considered that characteristics specific to TZM-bl target cells, or heterogeneity or molecular size of 10E8 IgG might be causing partial neutralization with 10E8. However, incomplete neutralization of HIV-1 mutant F673L was also observed using U87.CD4.CCR5 cells and HOS.CD4.CCR5 cells with plateaus similar to that of TZM-bls (-/- (293S) cells and in 293T cells treated with kifunensine (Kif), which results in relatively homogeneous Man5-Man9 and Man9 glycan residues, respectively -/- or Kif-treated cells were tested in a neutralization assay and were still partially neutralized by 10E8, but plateaus were shifted from 42%  to 82% and 88% neutralization, respectively . Kif treatment also slightly reduced viral infectivity and cleavage efficiency , while it caused gp120 and gp41 to run slightly faster on SDS-PAGE . However, these effects of Kif on processing and function of Env were equal against wild-type and mutant so the changes in maximum neutralization by 10E8 are not a simple function of diminished infectivity or cleavage. To see if glycans on gp41 were responsible for limiting neutralization by 10E8, we individually ablated the four N-linked glycosylation sequons (NGS) in gp41 on an F673L mutant background and tested these double mutants in a neutralization assay. None of the NGS mutations affected the sensitivity of the F673L mutant to control antibodies 2F5 or 4E10; however, N625Q increased the maximum level of 10E8 neutralization from 41% to 65% . The other three NGS knockouts were no more or less sensitive to 10E8 neutralization. Whereas contributions from other glycans or factors besides glycosylation cannot be ruled out, the results above suggest that complex glycan on Env and the glycan at N625 of gp41, can significantly affect the maximum neutralization achieved by 10E8.Partial neutralization could indicate differences in glycosylation that result in sensitive and resistant viral subpopulations. The crystallographic defined epitope of 10E8 has only protein elements, but glycans conceivably could affect MPER accessibility. We therefore produced virions in GnTIe.g. 5-Helix and C34) as well as antibodies to gp41  or gp120 . For comparison, we similarly fixed a somewhat lower sub-neutralizing concentration of 10E8 IgG to use against wild type JR2 and wild type SF162 viruses. Indeed, the results confirmed our speculation. Remarkable differences were observed in the potency of a number of different inhibitors and antibodies against Env due to the presence of 10E8, seen most significantly against the F673L mutants . This effect was highly pronounced for weakly neutralizing antibodies 17b and b6  that might be particularly sensitive to increases in Env rigidity. Finally, the presence of 10E8 can even affect the IC50 of antibodies against wild type Tier 1A isolate SF162 in the absence of the F673L mutation, and the directionality of the shift in IC50s with wild type SF162 caused by 10E8 is the same as with the F673L mutant for cognate antibody, though the magnitude in the shift is less (and data not shown). Importantly, the presence of human serum  did not affect the level of partial neutralization of the F673L mutant by 10E8 suggesting that the effects we observed may also occur in vivo (data not shown).There are several notable observations to make on the neutralization experiments performed in the presence and absence of 10E8. First, the effect of 10E8 on the ICh 2F5 and4, whic(i) neither makes Env globally neutralization sensitive nor globally resistant but has more specific effects on both gp41 and gp120, as well as (ii) modulates both receptor-naïve and receptor-activated spikes since 10E8 antagonizes ligands like sCD4 but potentiates ligands like C34, which exclusively target unliganded and receptor-activated spikes, respectively. Moreover, 10E8 modifies neutralization sensitivity of wild type HIV-1 (e.g. SF162). Notably implicit with the strongest of the observed effects of 10E8 on neutralization profiles of HIV-1 is the perhaps contraintuitive notion that 10E8 may be binding to most, if not all, Env spikes with at least partial subunit occupancy and without fully inhibiting their function.The results above show that the presence of 10E8 50 but not an IC80 against F673A mutant HIV-1 Figure S5). Compared to control Fabs PG9 and b12, which bind to one and three gp120 subunit(s) on Env We speculated that partial neutralization of MPER mutants by 10E8 might relate to stoichiometry of 10E8 binding to Env trimers. Addressing antibody occupancy post-receptor engagement is not straightforward. However, blue native (BN) PAGE can be useful for addressing stoichiometry with unliganded Env Because 10E8 can bind to Env before or after detergent-solubilization, or both, we performed a washout step prior to adding detergent so that only Fab already bound to native trimers would remain. This procedure had a modest effect on 10E8 binding to wild type JR-FL spikes, verifying that 10E8 can bind to unliganded Env on virions, although it likely binds more efficiently post-solubilization Figure 7i.e. 0.1 µg/ml) decreased the half-life of wild type JR2 from 13.6 h to 8.7 h, indicating that it too destabilizes functional Env spikes over time (90) at which an Env variant of HIV-1 loses 90% of its infectivity in one hour 90 of wild type JR2 from 49°C to 43°C in a dose dependent manner, indicating that 10E8 decreases wild type Env stability at both physiologic and elevated temperatures  of F673L from 45°C to 50°C in a dose dependent manner  of mutants W672A, W680A and K683A by 5–6°C  infectivity decay of F673L at physiologic temperature remained relatively constant but at high concentrations (∼500 µg/ml) 10E8 reproducibly increased its half-life slightly from 3.6 h to 4.4 h . Hence, when 10E8-Env complexes bound by 10E8 are exposed to elevated temperatures they are stabilized, but may become altered to be less prominent on BN-PAGE analysis, perhaps through aggregation, while unbound spikes dissociate into individual subunits.We next assessed whether effects of 10E8 on Env function directly relate to effects on the oligomeric state of Env. First, wild type JR-FL and F673A mutant virions were subjected to a heat gradient in the presence or absence of 10E8 Fab and Env was analyzed using BN-PAGE. Unexpectedly, we found that binding of 10E8 stabilized a fraction of both wild type and MPER mutant Env trimers against temperatures that caused dissociation of Env spikes in the absence of 10E8  roughly 10-fold with SF162 and JR2 wild type HIV-1 when virus is pre-incubated with 10E8 up to 20 hours rather than the standard 1 hour prior to adding to target cells , which is consistent with our findings above that mobility on BN-PAGE of 10E8-bound F673A spikes did not change with incubation times of up to 22 hours prior to running the gel . However, neutralization of F673L virus by 10E8 could be completely washed away prior to adding virions to target cells . This agrees with our BN-PAGE data that showed that the apparent affinity of 10E8 on the MPER mutant spike is relatively low and is readily washed off  90) of TM20.13 or M20490 BMR 211 Env spikes (90) of the clade C Envs, as both sCD4 binding and thermostability are properties of Env in its unliganded (pre-attachment) state.MPER polymorphisms such as L673 occur naturally in different individuals infected with clade C isolates (i.e. effects of 10E8 on thermostability and sensitivity to sCD4) can vary and be distinct from effects of 10E8 on receptor-activated Env (i.e. effects of 10E8 on sensitivity to fusion inhibitors). Taken in sum, our analysis shows that 10E8 can alter ligand recognition properties of functional clade C Env spikes with both naturally occurring and introduced MPER mutations.To further determine whether 10E8 would alter stability or ligand recognition of clade C viruses, we tested F673 mutants of two other clade C Envs. Thus, an F673A mutant of an otherwise 4E10 sensitive clade C isolate, COT6 HIV-1 Env has evolved to sequester its most conserved surfaces from recognition by neutralizing antibodies of the host. With the MPER this likely involves steric limitations imposed by Env and viral membrane both pre- and post-engagement. However, molecular details are lacking on how MPER antibodies overcome these limitations. Here, we reveal novel mechanisms of 10E8 activity not accounted for by prior models. First, we confirmed that antibody 10E8 causes unusual partial neutralization with certain Envs. We showed that 10E8 occupied an apparent maximum of two gp41 subunits of an MPER mutant trimer instead of three subunits observed with its wild type counterpart that was fully neutralized. Clade C isolates with natural MPER polymorphisms were also partially neutralized by 10E8 suggesting that this phenotype could evolve during natural infection under antibody pressure. Second, we found that 10E8 functionally destabilizes unliganded Envs while functionally stabilizing mutant Env counterparts, the latter activity of which is unprecedented for a virus-neutralizing antibody. Third, we found that the presence of 10E8 can significantly alter the sensitivity of Env to neutralization by antibodies and inhibitors to gp120 and gp41. A quaternary model incorporating behavior of each MPER on trimeric Env we think provides for a superior account of these observed effects of MPER recognition by 10E8-like antibodies.There has been uncertainty as to whether MPER antibodies act on a pre-hairpin intermediate In the unliganded state, 10E8 can functionally stabilize mutant Envs to heat and physiological decay, and can also inhibit neutralization by sCD4 and CD4bs antibodies. However, these pre-attachment effects were limited to certain unstable Envs. In contrast, the clade C isolates in which L673 occurred naturally were not stabilized to heat in the presence of 10E8 and only ligands that bind post-attachment had activities affected by 10E8 binding. MPER antibodies bind to unliganded Env better with variants that adopt a more open conformation, but with many primary isolates can only bind post-attachment e.g. 102-106-fold drop in IC50) Figure S2) such as W672F and F673W destabilize Env JR2 but do not lead to partial neutralization presumably because high affinity of 10E8 for the MPER is maintained Since Ala mutations to residues W672, F673, W680 and K683 in JR2 all caused similar partial neutralization as well as altered sensitivity to heat and ligands in presence of 10E8, it seems that a more general disruption of the MPER is sufficient for these effects. These mutations would affect recognition of the CDR H3 and adjacent residues of 10E8 based on existing structural data One antibody is typically sufficient to neutralize one HIV-1 spike Figure S9). This would also explain why saturation of the mutant spike by 10E8 does not fully block 2F5 neutralization and why the effect also occurs with ligands that bind distal to 10E8. Flexibility of the MPER How 10E8 can diminish neutralization by other MPER antibodies cannot be completely clear without detailed structural information. However, recent structures of disulfide-stabilized soluble gp140 trimers omit the MPER 50s or maximum neutralization percentages of 10E8 to 10E8-peptide affinity data by Huang et al produced no obvious relationships (unpublished observations). Second, the epitopes of 2F5 and 4E10 are not well exposed on resting primary spikes suggesting an unmasking of elements of Env upon CD4 engagement that allows antibody recognition Our results are most consistent with a model in which antibody and MPER interact and function in the specific context of trimeric Env. First, there is a known lack of correlation between neutralization and antibody binding to monomeric MPER peptides Examination of the literature turned up no equivalent mechanism to 10E8 partial neutralization. Although several different neutralization mechanisms have been described for MPER antibodies, including antibody-induced shedding of gp120 10E8 has a reported weak affinity for membranes For vaccine design, partial occupancy of Envs by MPER antibodies or B cell receptors (BCRs) elicited early in a primary response could alter the structure and immunogenicity of Env. Trimeric immunogens could be identified that promote or discourage specific quaternary features of the native MPER. Neutralizing antibodies that saturate all three MPERs of the HIV-1 spike in a clash free manner may be the most potent and therefore most desirable to elicit. Approaches to enhance immunogenicity of the MPER on native spikes are also desired, including prime-boost strategies using MPER specific immunogens equipped with compatible T cell epitopes  and Vaccination, immunotherapy and immunoprophylaxis are becoming increasingly attractive approaches to combat HIV/AIDS In conclusion, our work shows that in order to gain a full picture of neutralization at the base of the trimeric spike, that consideration be given not only to the interaction antibody makes with a single MPER but also to the stability and recognition properties of adjacent, unoccupied MPERs and subunits of trimeric Env both pre- and post- receptor engagement.-.E- were obtained through the NIH AIDS Research and Reagent Program (ARRRP), contributed by J. Kappes and X. Wu and by N. Landau, respectively. Env complementation plasmid pSVIIIexE7pA−YU2 was kindly provided by J. Sodroski (Harvard) and the envelope genes JR2 − using the KpnI and XhoI sites as described previously HIV-1 backbone plasmids pSG3ΔEnv and pNL4-3.Luc.RHIV-1 antibodies were obtained from the following sources (target epitope and subunit in parentheses): 10E8  IgG heavy and light chain DNA expression vectors were kindly provided by M. Connors  and IgG was produced in house, Fab 10E8 was prepared using Endoproteinase Lys-C (Promega) digestion according to the manufacturer's protocol. IgGs 2F5 and 4E10  were purchased from Polymun (Vienna). IgGs Z13e1 , 8K8 , and DN9  - cells were a gift from H.G. Khorana (MIT) +CXCR4+CCR5+), TZM-bl FcγRI cells +CCR5+), and HOS cells (CD4+CCR5+) were obtained through the ARRRP, contributed by J. Kappes and X. Wu, by D. Montefiori and G. Perez, by H. Deng and D. Littman, and by N. Landau, respectively. TZM-bl cell lines were maintained in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U of penicillin/ml, and 100 µ g/ml of streptomycin. U87.CCR5 cells lines were maintained in DMEM supplemented with 15% FBS, 2 mM L-glutamine, 100 U/ml of penicillin, and 100 µg/ml of streptomycin, 1 µg/ml puromycin, and 1 µg/ml G418. HOS cells were maintained in the same medium as TZM-bl but with the addition of 1 µg/ml puromycin.HEK-293 cells were from the ATCC, and 293 GnTI-.E- at a mass ratio of 1 3.5 was mixed with transfection reagent polyethylene imine . Kifunensine  was added to HEK-293 cells 30 min prior to transfection Pseudotyped viruses were produced by transfection of HEK-293 cells. DNA comprising Env plasmid and pSG3ΔEnv or pNL4-3.Luc.R5 per well) were seeded in 96 well plates 24 hours prior to assay. The virus and inhibitor mixture was incubated at 37°C, and then added to TZM-bl cells. Infectivity was measured 48 h post infection using a luciferase assay system (Promega) and a Synergy HT Microplate Reader (Bio-Tek). Data was processed using Prism 5.0 software (Graphpad). Washout neutralization assays were performed as described previously Single cycle viral entry neutralization assays were performed using TZM-bl cells, unless otherwise indicated. TZM-bl FcγRI cells, U87.CCR5 cells, and HOS cells were also used, as indicated. Cells  in PBS. Aggregate was pelleted by centrifugation at 22,000×90) was determined using Prism 5.0 software . In half-life infectivity decay experiments, virus and antibody were co-incubated at 37°C for various time intervals and infectivity of virus was determined using TZM-bl indicator cells. Data was plotted using a non-linear, one phase exponential decay equation (plateau constraint  = 0) and t1/2 was determined using Prism 5.0 software.Temperature gradient infectivity assays were determined using a gradient PCR block  as previously described g at 4°C) and resuspended 100-fold concentrated in PBS. For gel mobility shift assays, virions were pre-incubated with antibodies for 30 min before preparation for BN-PAGE. In some cases virus was pelleted in a microcentrifuge for 45 min at 4°C and the buffer was exchanged to remove unbound antibody prior to detergent treatment. For heat gradient BN-PAGE, virions were exposed to a temperature gradient for 1 hour, as detailed above, prior to detergent solubilization. BN-PAGE was performed as previously described Virions were produced by transfection using molecular clone plasmid pLAI, pelleted in an Optima ultracentrifuge , and divided by the distance shifted by Fabs b12 and PG9 that are assumed to bind three and one Fab(s) per trimer, respectively. To estimate antibody affinity for the detergent-solubilized Env trimer, BN-PAGE blots were again analyzed using ImageJ software and the percentage of trimer that remained unshifted at each concentration was calculated by comparing the band intensity to that of samples with no antibody added.Figure S110E8 IgG partially neutralizes JR2 mutants W672A and F673L with a maximum plateau that is consistent between replicate experiments #1 and #2 despite presence or absence of a downward slope at very high concentrations of 10E8 and despite a tendency of 10E8 to aggregate. (A) In experiment #1 the neutralization plateau remains stable whereas in experiment #2 the neutralization curve shows a plateau that is followed by a downward slope at high concentrations of 10E8. Each datum point in both experiments is an average of a duplicate with error bars shown, although error within experiment was extremely small. The experimental artifact or element responsible for the difference in curve shape between replicate experiments is currently undetermined. (B) 10E8 IgG was deliberately aggregated by concentration (see tion see  and as a(TIFF)Click here for additional data file.Figure S2Effect of conservative or non-conservative substitutions at positions 672, 673 and 683 in the MPER on neutralization of HIV-1 by 10E8. Selected conservative or non-conservative mutations to (A) F673, (B) W672, and (C) K683 were introduced into JR2 and the corresponding viruses tested for neutralization sensitivity to 10E8.(TIFF)Click here for additional data file.Figure S3Partial neutralization of HIV-1 F673L by 10E8 is not restricted to TZM-bl cells. Neutralization sensitivity of HIV-1 JR2 mutant F673L to MPER antibodies on (A) U87.CD4.CCR5 and (B) HOS.CD4.CCR5 as target cells. (C) Neutralization sensitivity of HIV-1 JR2 and mutant F673L with 10E8 IgG on TZM-bl FcγRI reporter cells. (D) Partial neutralization plateau percentages of 10E8 IgG on various target cells.(TIFF)Click here for additional data file.Figure S4Glycosylation state of MPER mutant Env influences extent of maximum neutralization by 10E8. (A) The four conserved N-glycosylation sites (NGS) in gp41 were individually mutated on a JR2 F673L Env background and corresponding viruses were tested in neutralization assays against 10E8 (left), 4E10 (middle) and 2F5 (right). (B) JR2 F673L virions, engineered with an E168K mutation to generate the PG9 epitope, were produced in the presence or absence of the glycosidase inhibitor kifunensine (Kif), which prevents the formation of complex glycans so glycosylation remains high mannose (i.e. Man9 residues). Viruses were tested against 10E8 (left), 2F5 (middle), and the Kif-sensitive antibody, PG9 (right) C) Neutralization of JR2 (right panels) and cognate F673L mutant (left panels) by 2F5, 10E8 and sCD4 using virus produced in either 293S (GnTI-/-) cells, a cell line that is unable to generate complex glycans so glycosylation comprises Man5 up to all Man9 residues (top panels), or 293T cells (bottom panels).(TIFF)Click here for additional data file.Figure S5Kifunensine (Kif) treatment moderately impairs Env processing and function. Replication competent JR2 wild-type (wt) and F673A virions were produced by transfection of 293T cells in the presence or absence of 25 µM Kif. (A) Relative infectivity of virions was analyzed by infection of TZM-bl target cells for 48 hours. (B) Apparent incorporation of gp120 was analyzed using a lectin-capture ELISA detected using gp120 antibodies (b12 and F425-B4e8). Results were normalized for p24 content as determined by p24 ELISA. (C) Cleavage efficiency of Env was determined using SDS-PAGE followed by Western blot with a cocktail of gp120 antibodies. Similar results were observed using gp41 antibodies.(TIFF)Click here for additional data file.Figure S6Env trimers have greater thermal resistance in the presence of 10E8 but grow fainter on BN-PAGE Western blots without accumulation of decay products. HIV-1 JR-FL wild type virions were heated at 37°C or 57°C for 1 hour in the presence or absence of 100 µg/ml Fab 10E8. Env was subsequently detergent-solubilized and analyzed using BN-PAGE. Western blots were stained using antibody cocktails specific for the gp41 MPER , gp41 cluster I and II epitopes , or gp120 . T = gp120/gp41 trimer.(TIFF)Click here for additional data file.Figure S710E8 maximum neutralization plateaus with HIV-1 mutant virus incubated at physiological conditions for an extended time period. SF162 and JR2 F673 mutants were either used fresh or incubated at 37°C for 20 hours prior to the addition of 10E8. Incubated virus and antibody mixture was incubated for 1 hour at 37°C and then added to TZM-bl cells.(TIFF)Click here for additional data file.Figure S8Neutralization of HIV-1 JR2 wild type and F673L mutant with and without ligand washout. Wild type (left panel) and mutant F673L (right panel) virus were treated with inhibitors (A) 10E8, (B) C34, and (C) b12, and incubated 1 hour at 37°C. Virus was pelleted and washed to remove unbound inhibitor before adding to TZM-bl cells.(TIFF)Click here for additional data file.Figure S9Cartoon models portraying possible mechanism of effects of 10E8 on HIV-1 Env trimer structure-function. (A) The unliganded Env spike of SF162 F673 mutant in the absence or presence of 10E8 showing exposure of receptor binding site. Left panel: Wild type SF162 (not shown) and cognate F673L virus are fully sensitive to soluble CD4, b6 and 17b in the absence of 10E8, indicating spontaneous exposure of CD4bs (purple circle) and elements of the inner domain of gp120 (dark shading). Right panel: Binding by 10E8 (green) stabilizes conformations of the SF162 F673 spike in which inner domains of gp120 are less exposed (inward arrows) causing interference with the ability of b6 and 17b to neutralize the virus and reduced sensitivity to sCD4. (B) Prefusion intermediate of gp41 showing stabilizing effect of 10E8 on exposure of the heptad repeats of receptor-activated gp41. Left panel: The NHR coiled coil (yellow cylinders), CHR regions (blue strands), and MPER (red strands) of the metastable (blurred lines) pre-fusion intermediate of gp41, are sensitive to DN9/8K8/C34 (pink), 5-Helix (not shown), and MPER antibodies , respectively. Right panel: Binding by 10E8 (rightmost green) stabilizes conformations of the pre-fusion intermediate NHR and CHR regions of gp41 that are favorable for ligand binding, but less favorable for binding by other MPER antibodies (leftmost green). (C) Presence of 10E8 stabilizes function of MPER mutant Envs of SF162 and JR2. Left panel: Env spikes containing MPER mutations that destabilize unliganded Env (see Right panel: Presence of Fab 10E8 (green) increases thermostability of the MPER mutant spikes in their receptor-naïve state. (D) Occupancy of wild type JR-FL and MPER mutant Env trimers by 10E8. Left panel: Fab 10E8 (green) readily occupies all three gp41 subunits (yellow) of wild type, unliganded Env, which also destabilizes the functional trimer over time. Right panel: Fab 10E8 (green) binds with less apparent affinity (not depicted) and with lower subunit occupancy (up to two Fabs per spike) for spikes containing the F673A mutation, which stabilizes the functional trimer. Partial subunit occupancy by 10E8 also limits exposure of the CD4 binding site and the 10E8-unbound MPER (depicted by an X), as in (TIFF)Click here for additional data file.Table S1HIV-1 isolates and variants used in this study with cognate amino acid sequences in the membrane proximal external region (MPER).(DOCX)Click here for additional data file.Table S2Effect of presence of 4E10 on the sensitivity of F673L mutants to MPER and CD4bs antibodies and inhibitors.(DOCX)Click here for additional data file.


MCHR2 shares its promoter region with MCHR2-AS1, a long antisense non-coding RNA. The aim of this study was to determine whether tagging single nucleotide polymorphisms (tSNPs) of MCHR2 and MCHR2-AS1 are associated with the body mass index (BMI) in the psychiatric and in the general population. The influence of MCHR2 and MCHR2-AS1 tSNPs on BMI was firstly investigated in a discovery psychiatric sample (n1 = 474). Positive results were tested for replication in two other psychiatric samples  and in two population-based samples . In the discovery sample, TT carriers of rs7754794C>T had 1.08 kg/m2 (p = 0.04) lower BMI as compared to C-allele carriers. This observation was replicated in an independent psychiatric sample . The association of rs7754794C>T and BMI seemed stronger in subjects younger than 45 years (median of age). In the population-based sample, a moderate association was observed  among younger individuals (<45y). Interestingly, this association was totally driven by patients meeting lifetime criteria for atypical depression, i.e. major depressive episodes characterized by symptoms such as an increased appetite. Indeed, patients with atypical depression carrying rs7754794-TT had 1.17 kg/m2 (p = 0.04) lower BMI values as compared to C-allele carriers, the effect being stronger in younger individuals . This study provides new insights on the possible influence of MCHR2 and/or MCHR2-AS1 on obesity in psychiatric patients and on the pathophysiology of atypical depression.Obesity development during psychotropic treatments represents a major health issue in psychiatry. Melanin-concentrating hormone receptor 2 (MCHR2) is a central receptor involved in energy homeostasis.  Compared to the general population, patients with chronic severe mental disorders have an estimated shorter life expectancy of 15 to 25 years due to the psychiatric disorder and/or physical comorbidities (i.e obesity or other metabolic disorders) but also in the use of psychotropic treatments, togetheMCH gene have been observed to be lean, having decreased feeding behavior and increased energy expenditureMCHR2 and MCHR2-AS1 where then selected using HapMap Genome Browser . Ten tagging polymorphisms within MCHR2 and twelve within MHCR2-AS1 were obtained by limiting the search to SNPs with a minor allele frequency >5% in the Caucasian population and r2 cutoff of 0.8. MCHR2 tagging SNPs were customized and added to the Illumina 200K cardiometabochip[MCHR2-AS1 tagging SNPs were added to the cardiometabochip. Due to technical issues, proxies of SNPs were chosen in some cases  was fitted, adjusting for age, gender, smoking status, current psychotropic drug and comedications potentially inducing weight gain (MCHR2 or MCHR2-AS1 SNPs (D’ and r2). The haplo.stat package of R was used for haplotype analysis. P-values were defined using asymptotic chi-squared tests of haplo scores.Associations between tagging SNPs of ght gain , allowinS1 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.


The aim of present study was to investigate which set of proteins is involved in H2S-regulated metabolism or signaling pathways. Spinacia oleracea seedlings were treated with 100 µM NaHS, a donor of H2S. Changes in protein expression profiles were analyzed by 2-D gel electrophoresis coupled with MALDI-TOF MS. Over 1000 protein spots were reproducibly resolved, of which the abundance of 92 spots was changed by at least 2-fold . These proteins were functionally divided into 9 groups, including energy production and photosynthesis, cell rescue, development and cell defense, substance metabolism, protein synthesis and folding, cellular signal transduction. Further, we found that these proteins were mainly localized in cell wall, plasma membrane, chloroplast, mitochondria, nucleus, peroxisome and cytosol. Our results demonstrate that H2S is involved in various cellular and physiological activities and has a distinct influence on photosynthesis, cell defense and cellular signal transduction in S. oleracea leaves. These findings provide new insights into proteomic responses in plants under physiological levels of H2S.Hydrogen sulfide (H At low concentration, H2S has an obvious signaling regulatory function in plants. For instance, H2S could promote seed germination of wheat and ameliorate copper-induced damage of plasma membrane integrity in root tips 2S has also been reported to counteract chlorophyll loss and reduce oxidative damage due to osmotic stress in sweet potato seedling leaves 2S through enhancing the activities of antioxidant enzymes and decreasing the accumulation of toxic ions 2S concentration has been shown to promote the embryonic root length of Pisum sativum2S could enhance photosynthesis through promoting chloroplast biogenesis, photosynthetic enzyme expression and thiol redox modification in Spinacia oleracea seedlings 2S plays an ameliorative role in protecting barley seedlings against aluminum toxicity by inducing the activities of antioxidant enzymes, increasing citrate secretion and the gene expression of citrate transporter, and enhancing the protein expression of PM H+-ATPase 2S may delay senescence of cut flowers and prolong flower vase life in a wide spectrum of botanical species, including herbaceous and woody plants. In addition, H2S also could prolong the postharvest shelf life of strawberries and play an antioxidative role in fruits 2S may interfere with plant's normal growth and metabolism. For instance, high concentrations of H2S may impair photosynthetic electron transport and depress plant growth Hydrogen sulfide  mixture. Two-week-old seedlings were transferred to 1/2 Hoagland's solution (pH 6.0) in a controlled growth chamber with a light/dark regime of 15/9 h, relative humidity of 80%, temperature of 21/27°C and a photosynthetically active radiation (PAR) of 190 µmol m−2 s−1. NaHS was purchased from Sigma and used as an exogenous H2S donor as described by Hosoki et al., S. oleracea leaves were collected and immediately frozen in liquid N2 and stored at −80°C for subsequent experiments. Each experiment was repeated at least three times.Seeds of et al.Twenty leaves with the same leaf position were collected from control and NaHS treated seedlings, respectively, and then flatted on clean coordinate paper. Leaves were photographed with a digital camera at the same image resolution. Leaf area was calculated with pixels as described previously by Xiao Leaves were removed from the same leaf position and immediately weighed to obtain the leaf fresh weight (FW). Leaves were subsequently placed into vials filled with distilled water for 24 h, then blotted to remove excess water and re-weighed to determine the leaf turgid weight (TW). Leaves were dried to a constant weight at 65°C and re-weighed to obtain the leaf dry weight (DW). Leaf relative water content (RWC) was calculated as (FW–DW)/(TW–DW)×100 Chlorophyll content was measured according to Lichtenthaler Pn) was measured using a portable photosynthesis system  on the third fully developed leaf of each seedling. Air temperature, light intensity, CO2 concentration and air relative humidity were maintained at 25°C, 800 µmol m−2 s−1, 380 µl l−1, and 90%, respectively. Pn was expressed on a leaf area basis.The net photosynthetic rate  and the mixture was further vortexed for 30 min. Proteins were precipitated by adding five volumes of ammonium sulfate-saturated methanol and incubated at −20°C for at least 4 h. After centrifugation as described above, the protein pellets were re-suspended and rinsed with ice-cold methanol, followed by washing with ice-cold acetone twice, and spun down at 15,000 g, 4°C for 10 min after each washing. The final washed pellets were air-dried and dissolved in lysis buffer containing 8 M urea, 2 M thiourea, 4% (w/v) 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 1% (w/v) DL-dithiothreitol (DTT) and 1% (v/v) IPG buffer (pH 4–7). Protein concentrations were determined by the Bradford assay et al., Total proteins were extracted by the phenol procedure t test (P<0.05) were selected and identified by MALDI-TOF MS. To compensate for subtle differences in sample loading or gel staining/destaining during individual repeat experiments, We normalized spot volumes based on total intensity of valid spots were calculated for each 2-DE gel and used for statistical calculations of protein abundance. For some missing values, we manually checked, and a consistent reproducibility between normalized spot volumes was found in the three replicates (SDS-PAGE gels were stained with Coomassie Brilliant Blue (CBB) R–250 and then scanned at 600 dots per inch (dpi) resolution using a scanner . Gels were analyzed using PDQuest software . For each gel, a set of three images was generated, corresponding to the original 2-D scan, the filtered image and the Gaussian image. The Gaussian image, containing three-dimensional Gaussian spots, was used for the quantification analysis. After normalization and background subtraction, a matchset was created by comparing the control gels. The intensity of each spot was determined using the spot quantification tool and expressed using the ratio of pixel intensity of a single spot to the pixel intensity of all spots on the gels. The intensity of each spot was normalized by the local regression model of the software to compensate for gel-to-gel variation plicates . Each ex4HCO3 for at least three times until the color of CBB was removed, then washed several times with water and completely dried in a vacuum centrifuge. Depending on protein amount, 2–3 µl of 0.1 mg µl−1 modified trypsin  in 25 mM NH4HCO3 was added to the dehydrated gel spots. After 30 min incubation, 7 µl of 25 mM NH4HCO3 was added to submerge the gel spots and left at 37°C overnight. After digestion, the gel slices were washed with 0.1% trifluoroacetic acid (TFA) in 50% v/v acetonitrile (ACN) three times to acquire the peptides. Matrix was prepared by dissolving α-cyano-4-hydroxycinnamic acid (CHCA) in 50% ACN and 0.1% TFA. Ten microliters of matrix solution was added into the dry peptides, and vortexed for 30 min. 0.5 µl of peptide was mixed with 0.5 µl of matrix solution. A total of 1 µl of reconstituted in-gel digest sample was spotted onto an Anchorchip target plate. The dried sample on the target plate was washed twice with 1 µl of 0.1% TFA, and left for 30 s before solvent removal. MALDI-TOF MS analysis  was used to acquire the peptide mass fingerprint (PMF). A standard peptide mixture was spotted adjacent to all samples for external calibration, followed by internal mass correction using peptide ions generated by trypsin autoprotolysis (m/z 842.5 and m/z 2211.10). Spectra were analyzed using the flexAnalysis software . Then, the measured tryptic peptide masses were transferred through the MS BioTool program  as inputs to search against the taxonomy of green plants in the NCBI  database. The parameters of PMF were as follows: 100 ppm tolerance as the maximum mass error, MH+ monoisotopic mass values, allowance of oxidation (M) modification, allowance for one missed cleavage, and fixed modification of cysteine by carboxymethyl . Potential matches were identified by considering the Mascot score, the putative functions and differential expression patterns on 2-DE gels. Several criteria were used to assign a positive match with a known protein. These were as follows: (i) Protein identifications were validated manually, ensuring that at least 4 peptides matched. (ii) The coverage of protein sequences by the matching peptides had to reach a minimum of 10%. (iii) The score that was obtained from the analysis with the Mascot software indicated the probability of a true positive identification and had to be at least 70. Positive matches were BLAST searched against the UniPort (http://www.uniprot.org) and/or NCBI protein (http://www.ncbi.nlm.nih.gov) databases for updated annotation and identification of homologous proteins. The identified proteins were searched with the UniPort and TAIR databases to find out if their functions were known, then they were further classified using Functional Catalogue software (http://mips.gsf.de/projects/funcat).Protein spots showing significant changes in abundance during the treatments were excised manually from colloidal CBB stained 2-DE gels and protein digestion with trypsin was performed as follows. Briefly, gel slices were first destained with a 1∶1 (v/v) solution of methanol and 50 mM NHP<0.05) . For proteomic experiment, protein samples for 2-DE gel image analysis were extracted from three independent seedlings grown in three different pots in the same growth chamber. Thus, for Control and H2S treatment, three independent biological replicates were performed in 2-DE gel image analysis. The ratio of H2S and CK in the P<0.05) provided by PDQuest software as mentioned earlier.Values in figures were expressed as means ± SE. The statistical significance of the data was analyzed using an univariate analysis of variance (2S could significantly affect growth and photosynthesis in S. oleracea seedlings. As shown in S. oleracea seedlings treated with NaHS was profoundly increased (P<0.01) by 40% compared to control plants. Similarly, dry weight of S. oleracea seedlings under NaHS treatment was significantly increased (P<0.01) from 0.32 g per seedling to 0.47 g per seedling (P<0.05) (P<0.01) in seedlings treated with NaHS (S. oleracea seedlings (P<0.01) (S. oleracea seedlings treated with NaHS (Hseedling . The rel(P<0.05) . In addiith NaHS . Consist(P<0.01) . Howeverith NaHS .2S affected amino acid metabolism, we measured the contents of 17 kinds of amino acid in S. oleracea seedlings after NaHS treatment. As shown in S. oleracea seedlings under NaHS treatment.To investigate whether a low concentration of H2S-induced increased in plant growth, we performed 2-DE to identify differences in the whole protein profiles of S. oleracea seedlings under 100 µM NaHS treatment compared with control plants. Representative images are presented in pI) ranging from 4 to 7 and molecular weight (MW) ranging from 12 to 110 KDa . The reproducibility decreased to still significant levels with decreasing protein abundance , which plays a key role in photosynthetic CO2 assimilation and plant growth, was up-regulated after H2S treatment. This result is consistent with protein expression data obtained using the western blotting method (data not shown). Similarly, these results were supported by physiological measurements , whose expression was significantly increased under NaHS treatment. The LHCB proteins are the apoproteins of the light-harvesting complex of photosystem II (PSII), which are normally complexed with chlorophyll and xanthophylls and serve as the antenna complex Fv/Fm) is reached in the presence of H2S 2S appears to have a positive effect on the process of light capture in photosynthesis.It's well known that large amounts of ATP are needed by plants to provide sufficient energy for growth, development and photosynthesis S-adenosylmethionine (SAM) via the following pathway: SAM→1-aminocyclopropane-1-carboxylic acid (ACC)→ethylene 2S treatment, indicating that ethylene biosynthesis may also be associated with H2S signaling pathway. Interestingly, Liu et al.2S content in Arabidopsis seedlings. Meanwhile, they also found that the generation of H2S induced by NO might mediate ethylene-induced stomatal closure in Arabidopsis2S treatment, suggesting that H2S could enhance plant defense by increasing the level of JA. Interestingly, Hou et al.2S may function downstream of H2O2 in JA induced-stomatal closure of Vicia faba. In addition, JA could enhance the generation of endogenous H2S and L-cysteine desulfhydrase activity in guard cells of Vicia faba leaves. Therefore, we hypothesized that the signaling pathway of JA may also be associated with H2S signaling networks. Lipoxygenases (LOXs) are non-heme iron-containing dioxygenases widely distributed in plants and animals 2S treatment. Therefore, we speculated that H2S signaling function may be related with JA pathway to some extent. Glutaredoxins (Grxs) are small oxidoreductases of the thioredoxin family of proteins regulating the thiol redox state of several proteins 2S treatment obviously up-regulated this protein expression, indicating that H2S may be involved in thiol redox modification and cell redox homeostasis, in line with our previously published paper The gaseous hormone ethylene plays multiple roles in regulating plant growth and development 2S treatment. For instance, cc-nbs-lrr resistance protein (spot 19), putative NBS domain resistance protein (spot 59), Rp1-like protein (spot 26) and RPM1-interacting protein 4 (spot 30). These proteins are essential regulators of plant defenses and play central roles in resistance against infection by pathogens 2S could protect plants from pathogen infection by increasing these protein abundances. Further, glycine-rich proteins (GRPs), containing >60% glycine, have been found in the cell walls of many higher plants and form a group of structural protein components of the wall in addition to extensins and proline-rich proteins 2S treatment.In this study, several proteins involved in plant response to biotic and abiotic stresses were identified under H2S. Therefore, we hypothesized that H2S might be involved in the pollination process and elongation of the pollen tube and this physiological function of H2S is the same as that of NO signaling molecule in flower plants Pollination and elongation of the pollen tube are important processes for the normal growth and development of flower plants S. oleracea seedlings treated with 100 µM NaHS, a donor of H2S. These results showed that H2S affected plant growth and the accumulation of organic compounds. In addition, it's well known that aminotransferase and methyltransferase are key links between carbon and nitrogen metabolism Substance metabolism is the basic life activity and is vulnerable to environmental stresses in plants. As shown in 2S may play a crucial role in plant defense by increasing the biosynthesis of cell wall related compounds.Carbon compounds in the cell wall serve as an important physical barrier, having a significant role in cell defense against various external stresses 2S may function as a signaling molecule in lipid, fatty acid and isoprenoid metabolic pathways. As we known, in plants, acyl-CoA dehydrogenase is involved in the β-oxidation process of fatty acid 2S may be involved in regulating the β-oxidation process of fatty acid by changing the protein expression of acyl-CoA dehydrogenase.We also identified several lipid, fatty acid and isoprenoid metabolism related proteins, including three up-regulated proteins, i.e., serine palmitoyltransferase 2 (spot 24), adenylate isopentenyltransferase (spot 33) and putative acyl-CoA dehydrogenase (spot 65), and three down-regulated proteins, i.e., putative plastid 1-deoxy-D-xylulose 5-phosphate reductoisomerase precursor (spot 68), lipase class 3 family protein (spot 69) and cyclopropane-fatty-acyl-phospholipid synthase (spot 84). These results indicate that H2S treatment, indicated that H2S signaling molecule could accelerate the biosynthesis process of protein in plants. PRP-like protein, which is involved in mRNA processing and regulation of the timing of the transition from a vegetative to reproductive phase in plants, was also found to be obviously increased under H2S treatment. Besides, Skp1 is a core component of the Skp1-Cullin-F-box (SCF) family of E3 ubiquitin ligases and serves to tether the rest of the complex to an F-box protein, which provides specificity for binding ubiquitin ligase substrate proteins and plays a role during embryogenesis and early postembryonic development, especially during cell elongation and division 2S treatment, indicating that H2S could inhibit protein degradation and delay embryogenesis in plant. In addition, HIR proteins are a group of proteins involved in hypersensitive reaction (HR). They belong to the PID  superfamily, whose members function in cell proliferation, ion channel regulation and cell death 2S treatment, indicating that H2S may act as a signaling molecule that regulates the plant immunity by changing the expression of HIR protein. Finally, the two-component response regulator ARR 15 is transcriptional activator that binds specifically to the DNA sequence 5′-[AG]GATT-3′ and functions as a response regulator involved in the His-to-Asp phosphorelay signal transduction system for cytokinin and meristem stem cell maintenance 2S treatment, suggesting that H2S may be involved in His-to-Asp phosphorelay signal transduction system.Regulation of gene expression is achieved at several levels, i.e., transcriptional, post-transcriptional, translational, and post-translational. Several proteins implicated in transcription, protein synthesis and modification were identified in the present study, including three ribosomal proteins , PRP-like protein (spot 63), putative Skp1 (spot 70), hypersensitive-induced response (HIR) protein (spot 76), two-component response regulator ARR 15 (spot 92). Among them, three ribosomal proteins, whose abundances were increased following H2S treatment, indicating that H2S-enhanced plant immunity may be associated with the regulation of WRKY TFs. Besides, a previous study in animal research has shown that H2S, as an endogenous gaseous signal molecule, could induce the opening of KATP channels 2S could affect potassium channels in plants. Interestingly, in our experiment, we found that H2S increased the protein expression of potassium channels (spot 31) in S. oleracea seedlings. Moreover, our unpublished data also has shown that H2S could increase K content and promote K+ influx in barley seedlings roots under high salt stress by changing the expression of K channel related genes. In addition, García-Mata and Lamattina have reported that H2S could induce stomatal closure and participate in the ABA-dependent signaling pathway, possibly through the regulation of ABC transporter proteins in guard cells 2S treatment. It's well known that phytochromes are a family of photoreceptors that modulate the expression of a large number of light-responsive genes and control plant growth and development 2S signaling pathway. In our study, we identified phytochrome C (spot 37) and found that its expression was up-regulated under H2S treatment, suggesting that H2S could promote plant growth and development and these processes were associated with the signal pathway of phytochrome. Further research need to widen our understanding of the relationship between phytochrome and H2S signal in plants.It's well known that WRKY transcription factors (WRKY TFs) are a large family of regulatory proteins involved in various plant processes but most notably in coping with diverse biotic and abiotic stresses 2S treatment affected actin and tubulin expressions, suggesting that H2S might be involved in cellular homeostasis. 14-3-3 proteins are known to bind certain phosphorylated proteins to complete phosphoregulation events. In plants, 14-3-3 protein binding activity includes the regulation of key metabolic enzymes, such as nitrate reductase and sucrose synthase +-ATPase 2S treatment, indicating that H2S may function in the signal transduction regulatory events involving 14-3-3 protein. However, the detailed mechanism of cross-talk between H2S and 14-3-3 protein in signal transduction regulatory events is still unclear.In the present study, we identified several proteins with binding function or cofactor requirement and cellular components, including beta tubulin (spot 15), lipid binding protein (spot 38), protein binding protein (spot 41), 14-3-3e protein (spot 60), 14-3-3 protein Lil 1433-3 (spot 62), cytosolic phosphoglucose isomerase (spot 85) and actin (spot 89). Among them, actin and tubulin dynamics have important functions in cellular homeostasis. HS. oleracea seedlings respond to the H2S signaling molecule. Quantitative analysis of more than 1000 highly reproducible proteins on 2-DE profiles identified 92 proteins which expressions were significantly changed in response to H2S. These proteins were classified into 9 functional groups, the main one being energy production and photosynthesis associated proteins, followed by cell rescue, development and defense related proteins. In addition, transcription, protein synthesis, folding, modification related proteins and proteins with binding function or cofactor requirement and cellular components formed a large part of the identified proteins. Cellular signal transduction and metabolism related proteins were also found. Taken together, the above-mentioned results indicated that H2S played important roles in a set of proteins associated with energy production, photosynthesis, metabolism, cell rescue, cell defense and protein synthesis, folding and signal transduction, etc. On the basis of above results, we also studied another important question regarding H2S signaling in plant cells, the localization of its sub-cellular target, and proposed a schematic model of systematic response mechanism of S. oleracea seedlings to H2S  and Log10(CK-R2) ratio (A), Log10(CK-R2) and Log10(CK-R3) ratio (B), Log10(CK-R1) and Log10(CK-R3) ratio (C), Log10(H2S-R1) and Log10(H2S -R2) ratio (D), Log10(H2S –R2) and Log10(H2S –R3) ratio (E), Log10(H2S -R1) and Log10(H2S –R3) ratio (F), with correlation coefficients of 0.984, 0.985, 0.981, 0.982, 0.979 and 0.977, respectively.(DOC)Click here for additional data file.Table S1Spot volumes of differentially expressed protein (92) of Spinacia oleracea with NaHS treatment for 30 d. Labeled R1, R2 and R3 stand for Replicate 1, Replicate 2 and Replicate 3 for Control or NaHS treatment, respectively.(DOC)Click here for additional data file.Table S2Details of identified proteins (92) and peptides list of each protein in Spinacia oleracea leaves after treatment with 100 µM NaHS.(DOC)Click here for additional data file.


IL7 receptor gene. If Gfi1 levels fall below a threshold, Id1 expression increases and renders E2A unable to function, which prevents hematopoietic progenitors from engaging along the B lymphoid lineage.A regulatory circuit that controls myeloid versus B lymphoid cell fate in hematopoietic progenitors has been proposed, in which a network of the transcription factors Egr1/2, Nab, Gfi1 and PU.1 forms the core element. Here we show that a direct link between Gfi1, the transcription factor E2A and its inhibitor Id1 is a critical element of this regulatory circuit. Our data suggest that a certain threshold of Gfi1 is required to gauge E2A activity by adjusting levels of Id1 in multipotent progenitors, which are the first bipotential myeloid/lymphoid-restricted progeny of hematopoietic stem cells. If Gfi1 levels are high, Id1 is repressed enabling E2A to activate a specific set of B lineage genes by binding to regulatory elements for example the  B-lymphocytes are the principal antibody producing cells and are indispensable for an efficient humoral immune response. B cell differentiation begins in the bone marrow (BM) by the generation of multipotent progenitors (MPPs) from hematopoietic stem cells (HSCs) , 2. A suA very complex network involving many transcription factors and cytokines such as Flt3L , 12 and The transcription repressor Gfi1 is a key element of this network orchestrating progenitor cell fate between myeloid and lymphoid lineages . Indeed,To further understand how Gfi1 is functioning in early B lymphoid commitment and differentiation, we decided to study its participation in transcription factor regulatory circuits in early lymphoid and multipotential progenitors. We report here that a threshold level of Gfi1 protein expression is required to support B cell differentiation. We identify LMPPs as critical hematopoietic progenitors that require high levels of Gfi1 expression to repress Id1 to specifically sustain B-cell commitment. We propose a model in which Gfi1 is required to maintain Id1 at low levels, which leave E2A active to ensure the expression of those E2A target genes that are important for B cell differentiation.2 and all efforts were made to minimize suffering. GFI1 KO, GFI1 KI, GFI1-GFP and GFI1-P2A mice used in this study have been described previously , including the affy [Expression profiling was carried out using Affymetrix arrays according to published procedures . Raw datthe affy  and Limmthe affy . Data waLSK cells were sorted and cultured in the presence of Flt3L and IL-7 for 1 hr under stroma-free conditions. Morpholinos were added to the culture in the presence of EndoPorter followed the manufacturer's instructions . After 4 hours, progenitor cells were transferred on OP9 cells. After 4 days, the cells were harvested from the co-culture and cultured in the presence of new cytokines and morpholinos with Endoporter for 4 hours. Then the cells were transferred on a new fresh OP9 cell layer. Every 4 days, cells were transferred on new OP9 cells and new cytokines were added.CGGCACTGCCACTGGCGACCTTCAT; Anti-Id1 #2: AGAACAGAGTGTGGGAAGAGAACAA; Control: CCTCTTACCTCAGTTACAATTTATA were used as previously described in kosan et al. [The morpholino sequences: Anti-Id1 #1: n et al. .Two tailed student’s t-test was used to calculate p-values where indicated. A p-value ≤0.05 was considered as statistically significant.E2A, Foxo1 and EBF1, GSE21978; Brg1 and Ikaros, GSE66978; PU.1, GSE21512.Supplemental method about FACS stainings and RNA isolation are provided in Gfi1 gene is replaced by the human Gfi1 cDNA (termed “KI” for “knockin”) [+CD19+, B220+IgM-, B220lowIgM+ and B220highIgM+ B cells , in which Gfi1 is functionally disabled by a mutation in its N-terminal SNAG repressor impairing binding to LSD1 [+ cells in the blood of the transplanted recipient CD45.1 mice, the percentage of cells expressing B220 and CD19 was also lower in mice transplanted with KD BM cells compared to mice transplanted with WT and KI cells while the percentage of myeloid cells was not affected  . During nockin”) . This prnockin”)  and comp B cells  in the Bjections , excludi to LSD1 , we obse to LSD1  were unaaffected  suggesti+CD43+BP-1-HSA-) and B  were observed in KD and KO mice (-CD19-B220+CD43+) were similar between all the mice suggesting that the few cells present in fraction A of KO or KD mice underwent regular differentiation. Another gating strategy [A significant reduction in the frequencies of B cell progenitors contained in both Hardy fractions A  compartment, which includes HSCs and the early lymphoid progenitors. Higher frequencies of LSK cells were found in KO and P2A mice confirming earlier studies [-IL7R+Flt3+Sca1medc-Kitmed) were severely reduced in KO, P2A and KD mice , which were severely decreased in KD, KO and P2A mice  and ST-HSC progenitors (Lin-c-Kit+Sca1+Flt3-CD48highCD150+) were equally increased in KD and KO mice compared to KI or WT mice . The Gfi1 expression levels in heterozygous mice did not cause an increase in the LSK population or a decrease in the LMPP subset showing that a 50–60% expression level of Gfi1 is sufficient to support B cell differentiation. The decrease of LMPPs and CLPs in KD and KO mice is not due to an increase of the cell death compared to KI and WT mice Click here for additional data file.S1 FigGfi1 flox/flox mouse (WT) treated with pIpC and Mx-cre Tg, Gfi1 flox/flox mice (MxcreTg) treated with pIpC to delete Gfi1. Lamin B was used as a loading control. (B) and (C) FACS analysis of bone marrow B cells from Mx-cre Wt, Gfi1 flox/flox mouse (WT) treated with pIpC and Mx-cre Tg, Gfi1 flox/flox mice (MxcreTg) treated with pIpC to delete Gfi1. Bone marrow cells were stained for B220 and CD19 (B) or B220 and IgM (C). (D) FACS analysis of B220 and CD19 in the bone marrow of WT and P2A mice. (E) FACS analysis of B220 and IgM in the bone marrow of WT and P2A mice. (F) Cell numbers of B220+ CD19+ cells in the WT and P2A bone marrow. At least three mice were used to determine absolute numbers of B cell subsets in bone marrow.(A) Western blot Gfi1 from thymocytes isolated from Mx-cre Wt, (PDF)Click here for additional data file.S2 Fig+CD43+ cells were electronically gated and analyzed for HSA and BP-1 expression to identify Fraction A , Fraction B , Fraction C  and Fraction C’ . (B) CD19 expression on pro B cells from WT and P2A mice. (C) Absolute numbers of Fraction A-C’ from WT and P2A mice. (D) Expression of Lin-, CD19, B220, and CD43 on bone marrow cells from WT and P2A mice. Lin−CD19− cells were analyzed for the expression of B220 and CD43. (E) Absolute numbers of pre pro B cells in WT and P2A bone marrow. At least three mice were used to determine absolute numbers of B cell subsets in bone marrow.(A) Flow cytometry analysis of B220, CD43, HSA, and BP-1 surface expression on bone marrow cells from WT or P2A mice. B220(PDF)Click here for additional data file.S3 Fig(A) LSK cell numbers in WT and P2A bone marrow. (B) CLP numbers in WT and P2A bone marrow. (C) Cell numbers of HSC, MPP and LMPP cells in the WT and P2A bone marrow. (D) BM cells from WT, KI, Het, KD and KO mice were stained with control isotypes to determine the CLP population gate. At least three mice were used to determine absolute numbers of B cell subsets in bone marrow.(PDF)Click here for additional data file.S4 Fig-Sca1+c-Kit+Flt3-), (B) MPP (Lin-Sca1+c-Kit+Flt3low) or (C) CLP (Lin-Il7R+Sca1+c-Kit+) were sorted and cultured on OP9 stromal cells in the presence of IL-7 (10 ng/mL) and Flt33L (5 ng/mL) for 10 or 12 days. Then cells were analyzed for B220 and CD19 expression by flow cytometry. The cell number on the FACS plots for each mouse is indicated in bold. (D) Gfi1 flox/flox (Gfi1fl), MB1-cre (MB1/WT) mice were analyzed for B cells in the bone marrow and spleen. (E) Sorted CLPs were cultured on OP9-DL1 cells in the presence of SCF (10 ng/mL), FLT3L (5 ng/mL) and IL-7 (1 ng/mL) for at least 15 days. The cells were analyzed for CD44, CD25, CD4 and CD8 expression. All FACS plots are representative of at least two independent experiments and at least three mice were used to determine absolute numbers of B cell subsets in bone marrow and spleen.(A) HSC (Lin(PDF)Click here for additional data file.S5 FigIl7r, Flt3, Ebf1, Pu.1, E2a, Id1, Id2 and Id3 expression in sorted LSK cells from WT, KI, KD and KO mice was measured by real time qPCR. Expression of the genes was normalized to Gapdh and is presented relative to cDNA from WT cells. (C) PU.1 expression in Lin- cells from WT, KI, KD and KO BM was measured by western blot. β-actin was used as a loading control. (D) Id2 and Id3 expression in lineage negative cells from WT, KI, KD and KO mice measured by western blot. β-actin was used as a loading control. (E) Ccr9, Notch1, Rag1 and Dntt expression in sorted LSK cells from WT, KI, KD and KO mice was measured by real time qPCR. Expression of the genes was normalized to Gapdh and is presented relative to cDNA from WT cells. Real time PCR are representative of at least 3 independent experiments and the western blot scans are representative of two different experiments.(A) Table showing the expression log2 fold change of selected up- and down-regulated genes between sorted WT and KO LSKs. (B) (PDF)Click here for additional data file.S6 FigIl7r, Flt3 and Id1 expression in sorted pre-pro B cells from WT, KI, KD and KO was measured by real time qPCR. Expression of Il7r, Flt3 and Id1 was normalized to Gapdh and presented as the fold increase relative to cDNA from WT cells. (B-H) E2A, PU.1, Brg1, EBF1, Foxo1 and Ikaros targeted regulatory regions from RAG1 null pro-B cells by ChIP-seq across the Il7r, (C) Flt3, (D) Ccr9, (E) Ebf1, (F) Notch1, (G) Dntt, (H) Rag1 loci.(A) (PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


During May 19–September 28, 2013,The U.S. influenza surveillance system is a collaborative effort between CDC and federal, state, local, and territorial partners. CDC uses eight data sources to collect influenza information , six of §During May 19–September 28, WHO and NREVSS collaborating laboratories in the United States tested 52,150 respiratory specimens for influenza; 2,013 (3.9%) tested positive for influenza . During Of the 2,013 specimens positive for influenza in the summer months of 2013, 1,403 (70%) were influenza A viruses, and 610 (30%) were influenza B viruses. Influenza B viruses were reported more frequently than influenza A viruses from late May until early June, and influenza A (pH1N1) viruses were more commonly reported than influenza A (H3N2) viruses from mid-June to September. Of the 1,403 influenza A viruses, 621 (44%) were subtyped: 402 (65%) were influenza A (pH1N1) viruses, 201 (32%) were influenza A (H3N2) viruses, and 18 (3%) were influenza A (H3N2)v viruses. Influenza viruses were reported from the District of Columbia, Puerto Rico, and 45 states in all 10 U.S. Department of Health and Human Services regions.During May 19–September 28 data from ILINet indicated that the weekly percentage of outpatient visits to ILINet providers for influenza-like illness (ILI)Between May 19 and September 28, a total of 20 cases of influenza A variant viruses (18 [H3N2]v and two [H1N1]v) were reported from five states . The 20 cases reported resulted in one influenza A (H3N2)v–associated hospitalization and no deaths. Although cases have been identified from five states, Indiana reported 14 (70%) of the 20 cases. In all 20 cases, contact with swine in the week before illness onset was reported. No ongoing community transmission of these viruses has been detected. The median age of patients was 6.5 years (range: 2–69 years); 65% were female .During May 19–September 28, typical seasonal patterns of influenza activity occurred in the temperate climate Southern Hemisphere countries. In Australia and New Zealand, influenza activity began in early August and decreased in mid-September. Influenza A viruses predominated in both countries with influenza A (H3N2) viruses identified more frequently than influenza A (pH1N1) viruses. Influenza B viruses were also identified in both countries. In South Africa, after a peak in influenza activity caused by influenza A (pH1N1) in June, a second, smaller peak was observed in early August because of increased influenza A (H3N2) and influenza B virus circulation. In temperate areas of South America, influenza activity peaked in June and declined through September. Influenza A viruses were reported more frequently than influenza B viruses, and influenza A (pH1N1) was the predominant virus reported by Argentina, Chile, and Uruguay. Influenza A (H3N2) viruses predominated in Paraguay  and influenza A H3N2) viruses. In tropical South America, influenza A (pH1N1) viruses predominated, with two peaks of activity: the first in June, primarily the result of activity in Brazil and Columbia, and a second peak in late July, the result of increased activity in Ecuador and Peru. South Asia and Southeast Asia saw a decrease in influenza activity during September. Different combinations of types and subtypes of influenza cocirculated in several countries, including Cambodia, India, China, Vietnam, and Thailand. In temperate climate countries in North America and Europe, influenza activity remained low, with small numbers of influenza A (H3N2), influenza A (pH1N1), and influenza B viruses identified. During February 19–September 28, a total of 135 cases of human infection with avian influenza A (H7N9) virus were reported -like virus, an influenza A (H3N2) virus antigenically like the cell-propagated prototype virus A/Victoria/361/2011 , and a B/Massachusetts/2/2012-like (B/Yamagata lineage) virus  viruses, 85 influenza A (H3N2) viruses, and 30 influenza B viruses. A subset of these viruses was genetically characterized. Most viruses tested were propagated in mammalian cell cultures; isolation rates of current human influenza viruses are higher in mammalian cell cultures than in eggs. However, egg-propagated (EP) vaccine viruses are used widely for production of influenza vaccines because most influenza virus vaccines are egg-based. Propagation of influenza viruses in eggs might lead to isolation of viruses that differ antigenically and genetically from viruses from corresponding clinical samples isolated in mammalian cell cultures. In addition, mammalian cell-propagated (CP) viruses are genetically more representative of viruses present in original clinical specimens . TherefoAll but two (99%) influenza A (pH1N1) viruses analyzed  were antigenically similar to EP A/California/7/2009, the influenza A (pH1N1) vaccine component. Most EP and CP influenza A (pH1N1) viruses analyzed during this time had similar antigenic properties. All 85 influenza A (H3N2) viruses  were antigenically similar to many current CP reference viruses, including A/Victoria/361/2011 and A/Texas/50/2012. The majority (60%) of CP influenza A (H3N2) viruses collected and characterized since February 2013 were antigenically similar to the EP A/Texas/50/2012 vaccine virus, while less than 10% of these viruses were antigenically similar to EP A/Victoria/361/2011. For this reason, WHO recommended that the EP A/Texas/50/2012 vaccine virus replace EP A/Victoria/361/2011 as the influenza A (H3N2) vaccine component for the 2013–14 Northern Hemisphere and the 2014 Southern Hemisphere influenza seasons  belonged to the B/Yamagata lineage , and were antigenically similar to the B/Massachusetts/2/2012 virus, the influenza B component for the 2013–14 Northern Hemisphere trivalent vaccine. The remaining 17 viruses tested  belonged to the B/Victoria lineage and were antigenically similar to the B/Brisbane/60/2008 virus, which is the B/Victoria-lineage component of the 2013–14 Northern Hemisphere quadrivalent influenza vaccine. Globally, B/Yamagata lineage viruses predominated during February 1–September 28 , and 140 were from U.S. specimens . Two of the 93 influenza A (pH1N1) viruses from the United States were found to be resistant to oseltamivir and contained the H275Y substitution in the neuraminidase gene; all the viruses were sensitive to zanamivir. High levels of resistance to the adamantanes (amantadine and rimantadine) persist among influenza A (pH1N1) viruses and influenza A (H3N2) viruses currently circulating globally  pdm09 (pH1N1), influenza A (H3N2), and influenza B viruses were identified sporadically. Twenty cases of influenza A variant viruses (influenza A [H3N2]v and influenza A [H1N1]v) were detected in five states, all of which were associated with swine contact. The majority of recent seasonal influenza viruses are antigenically similar to the influenza vaccine for the 2013–14 season.What are the implications for public health practice?To prevent influenza and its associated complications, influenza vaccination is recommended in all persons aged ≥6 months. Year-round influenza surveillance provides critical information for planning interventions to prevent and control influenza, developing vaccine recommendations and antiviral treatment guidance, and presenting information to the media and public regarding the progress and severity of the influenza season.During May 19–September 28, 2013, influenza A (pH1N1), influenza A (H3N2), and influenza B viruses cocirculated worldwide. In the United States, similar levels of seasonal influenza viruses were detected compared with summer months of previous years (excluding the 2009 pandemic), and influenza A viruses were predominant. Although neither the influenza viruses that will predominate nor the severity of influenza-related disease during the 2013–14 season in the United States can be predicted, antigenic characterization of viral isolates submitted during the summer demonstrated that the majority of influenza viruses were antigenically similar to the influenza vaccine strains contained in the 2013–14 Northern Hemisphere vaccine.Compared with the summer of 2012, fewer human infections with novel influenza A viruses were identified in the United States in the summer of 2013. Since the first identification of H3N2v viruses in humans, direct contact with swine has been documented in most cases, but limited person-to-person spread is suspected in a small number. Consistent with the age distribution of patients, serologic studies suggest there is little or no existing cross-reactive antibody to H3N2v in young children, but a substantial proportion of adolescents and younger adults have cross-reactive antibodies ,9. WhereAnnual influenza vaccination remains the best method for preventing influenza and its associated complications . For optTreatment with influenza antiviral medications is recommended as early as possible for patients with confirmed or suspected influenza  who have severe, complicated, or progressive illness; who require hospitalization; or who are at higher risk for influenza-related complications .¶¶¶¶http://www.cdc.gov/flu/weekly. Additional information regarding influenza viruses, influenza surveillance, influenza vaccines, influenza antiviral medications, and novel influenza A virus infections in humans is available at http://www.cdc.gov/flu.Influenza surveillance reports for the United States are normally posted online weekly and are available at