Bitter taste receptors (T2Rs) are expressed in the mammalian gastrointestinal mucosa. In the mouse colon, T2R138 is localized to enteroendocrine cells and is upregulated by long-term high fat diet that induces obesity. The aims of this study were to test whether T2R38 expression is altered in overweight/obese (OW/OB) compared to normal weight (NW) subjects and characterize the cell types expressing T2R38, the human counterpart of mouse T2R138, in human colon. Colonic mucosal biopsies were obtained during colonoscopy from 35 healthy subjects (20 OW/OB and 15 NW) and processed for quantitative RT-PCR and immunohistochemistry using antibodies to T2R38, chromogranin A (CgA), glucagon like peptide-1 (GLP-1), cholecystokinin (CCK), or peptide YY (PYY). T2R38 mRNA levels in the colonic mucosa of OW/OB were increased (> 2 fold) compared to NW subjects but did not reach statistical significance (P = 0.06). However, the number of T2R38 immunoreactive (IR) cells was significantly increased in OW/OB vs. NW subjects (P = 0.01) and was significantly correlated with BMI values . In both OW/OB and NW individuals, all T2R38-IR cells contained CgA-IR supporting they are enteroendocrine. In both groups, T2R38-IR colocalized with CCK-, GLP1- or PYY-IR. The overall CgA-IR cell population was comparable in OW/OB and NW individuals. This study shows that T2R38 is expressed in distinct populations of enteroendocrine cells in the human colonic mucosa and supports T2R38 upregulation in OW/OB subjects. T2R38 might mediate host functional responses to increased energy balance and intraluminal changes occurring in obesity, which could involve peptide release from enteroendocrine cells. The gastrointestinal (GI) tract mucosa detects luminal nutrients and non-nutrients through different effectors and sends information to the nervous system to initiate appropriate functional responses ranging from digestion and absorption to defense mechanisms to protect from outside threat –5. The G2+ increase and transmitter release [2+ and induce release of GI peptides from EEC cells in vitro and in vivo, support the concept that TRs play a role in chemosensing in the gut [Taste receptors (TRs) that detect complex tastes such as sweet, savory (umami) and bitter tastes, comprise two major families of G protein coupled receptors, the taste 1 receptors (T1Rs) that function as dimers to detect sweet (T1R2 and T1R3) or umami (T1R1 and T1R3) and the taste 2 receptors, T2Rs that include 25–30 subtypes and detect a large variety of bitter tastants –20. Upon release , 21–25.  release ,17,25–34 the gut ,28,35,36 the gut –39. More the gut ,40.In a previous study, we have shown that in mice, a long-term high fat diet inducing obesity results in a selective upregulation in the distal colon of the T2R subtype, T2R138, which is activated by phenylthiocarbamide (PTC), and the signaling molecule α-gustducin. In contrast, a high fat diet does not affect the T2R108, which is activated by denatonium benzoate . Since lThe aims of this study were to compare the expression of T2R38 in the colonic mucosa of overweight and obese (OW/OB) vs. normal weight (NW) subjects and characterize the types of cells containing T2R38 immunoreactivity (IR) in the human colonic mucosa using samples of colonic biopsies with quantitative RT-PCR, single and double immunofluorescence and confocal microscopy.2 and OW/OB subjects had a BMI ≥ 25.1 kg/m2. Study participants did not have personal or family history of GI conditions such as irritable bowel syndrome or other chronic pain conditions. Additional exclusion criteria for all subjects included: infectious or inflammatory disorders, active psychiatric illness as assessed by structured clinical interview for the DSM-IV , or current tobacco or alcohol abuse. All subjects were compensated for participating in the study, and written informed consent was obtained from all subjects. The study was approved by the University of California Los Angeles (UCLA) Institutional Review Board and was conducted in accordance with the institutional guidelines regulating human subjects research.Male and female healthy individuals ages 18–55 were recruited primarily by community advertisement. A medical history and physical examination and completion of validated questionnaires measuring GI and non-GI symptoms were performed at the screening visit. Lean (NW) subjects were classified as those with a body mass index (BMI) ≤ 25.0 kg/mColonic mucosal biopsies were taken at 30 cm from the anal verge during a flexible sigmoidoscopy, which was performed in the medical procedures unit. Mucosal samples were collected and immediately snap frozen and stored at -80°C for RNA extraction and qRT-PCR expression analysis or fixed for 2 hours in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, and paraffin embedded for immunohistochemistry.Total RNA was isolated from human colonic mucosal biopsies using Qiagen RNeasy Minikit  and DNase I treatment was performed to eliminate genomic DNA contamination . RNA quaSerial (5 μm thick) sections from the colonic mucosa of OW/OB (n = 20) and NW (n = 10) subjects were mounted on poly-L-lysine–coated slides and processed for single and double labeling immunofluorescence  using anThe antibody against T2R38 has been well characterized in human circumvallate papillae . Specifi2, we randomly selected to assess 12 fields per sample in order to determine the average number of cells immunoreactive for T2R38, CgA, CCK, GLP-1 or PYY in an area of 3.4 mm2. Since we counted immunopositive cells on a 5μm section, a 20μm interval between 4 consecutive sections was selected to avoid cell re-counting and therefore related quantitative biases.Quantitative analysis of immunolabeled cells was performed to determine the density of T2R38-IR cells in NW vs. OW/OB subjects, and of T2R38-IR cells coexpressing CgA, CCK, GLP-1 or PYY in both groups. Cell counting was performed in a blind manner by two expert operators using a laser scanning confocal microscope  equipped with an appropriate filter to discriminate different fluorescent fluorophores. Since the 40X objective has a field of view = 0.28 mmt-test, whereas for comparison among different groups of EEC cells in OW/OB and NW subjects we used one-way ANOVA followed by the Bonferroni test . P<0.05 was considered significant. To determine whether T2R38 expression correlated with the BMI of our subjects, we did a correlation analysis between the T2R38 mRNA levels or T2R38-IR cell numbers and the BMI values of the subjects investigated using D’Agostino and Pearson normality test followed by Spearman rank correlation test .qRT-PCR and cell quantification values were expressed as a mean ± standard error of the mean (SEM). Differences in mRNA level and cell number between the two groups of interest (OW/OB vs. NW) were analyzed using the unpaired Student’s 2. Of the 20 OW/OB subjects, there were 11 men and 9 women with a mean age of 36.5 ± 2.8 years (range 20–55) and mean BMI of 32 ± 0.7 kg/m2. Samples from these subjects  were used for qRT-PCR (11 NW and 14 OW/OB) and/or for immunohistochemistry (10 NW and 20 OW/OB).A total of 35 healthy volunteers participated in the study and included 20 OW/OB subjects and 15 lean subjects. The 15 NW subjects were comprised of 7 men and 8 women with a mean age of 34.8 ± 3.0 years (range 22–55) and had a mean BMI of 20 ± 0.5 kg/m= 0.06).Since the levels of T2R38 mRNA in OW and OB subjects were comparable, they were grouped together. There was a marked increase (>2 fold) in the level of expression of T2R38 mRNA in OW/OB compared to NW subjects  , which dT2R38-IR was localized to cells located in the epithelial lining and throughout the glandular epithelium of the descending colon in NW and OW/OB subjects . T2R38-IThere was a highly significant positive correlation between the density of T2R38-IR cells and BMI values (P = 0.001), but not between T2R38 mRNA levels and BMI .All the T2R38-IR cells co-expressed CgA, while not all CgA-IR cells co-expressed T2R38-IR T2R38-IR colocalized with immunoreactivity for CCK, GLP-1 or PYY , peptideT2R38/CgA-IR cells are significantly increased in OW/OB vs. NW subjects, whereas the overall CgA cells population (T2R38 positive and T2R38 negative) was similar in OW/OB and NW subjects This study shows that the bitter taste receptor subtype, T2R38 is expressed by the human colonic mucosa, where it is localized to distinct types of EEC cells, including CCK, GLP-1, and PYY cells. We show for the first time that T2R38 is upregulated in overweight and obese subjects as indicated by the marked and significant increase in number of T2R38-IR cells without changes in the overall density of EEC cells in the colonic mucosa of OW/OB vs. NW subjects. In the OW/OB group, there was also a trend in T2R38 mRNA increase (~2 fold compared to the NW group), but the difference between the two groups did not reach statistical significance. This could be explained by differences between T2R38 transcription and translation or by the smaller number of samples from OW/OB subjects utilized for qRT-PCR analysis compared to those used for the immunohistochemical analysis . T2R38 is confined to EEC cells in the human colonic mucosa, since all epithelial cells identified with T2R38 immunostaining were also immunoreactive for the EEC cell generalized marker, CgA. This differs from the mouse, where the colocalization between T2R138, the mouse counterpart of the human T2R38, and CgA was not complete indicating that other cell types express this T2R138 subtype . This isT2R38 expression was most abundant in EEC cells producing CCK ranging from ~85% of the CCK-IR cells in NW to ~93% in OW/OB individuals. CCK is a gut peptide released by the breakdown of proteins and fats, which modulates gastrointestinal secretion and motility, triggers digestive enzyme release and induces satiation via vagal afferent neurons . T2R38 e11−1012 per gram of contents containing up to a thousand species [Bitter taste has evolved as a central warning signal against harmful substances and elicits defensive responses. Several lines of evidence support that extra-oral T2Rs might serve as defense mechanisms against external or internal threats. In the gut, bitter tastants induce conditioned flavor avoidance, inhibit food intake and gastric emptying, modulate gut efflux membrane transporters and increase intestinal chloride secretion ,35–38,57 species . Since b species ,44,60. I species ,62. Over species ,64. The


In vitro APOBEC3F (A3F) anti-HIV-1 activity is weaker than A3G but is partially resistant to Vif degradation unlike A3G. It is unknown whether A3F protein affects HIV-1 disease in vivo. To assess the effect of A3F gene on host susceptibility to HIV- acquisition and disease progression, we performed a genetic association study in six well-characterized HIV-1 natural cohorts. A common six-Single Nucleotide Polymorphism (SNP) haplotype of A3F tagged by a codon-changing variant  frequency of 48% in European Americans) was associated with significantly lower set-point viral load and slower rate of progression to AIDS (Relative Hazards (RH) = 0.71, 95% CI: 0.56, 0.91) and delayed development of pneumocystis pneumonia (PCP) . A validation study in the International Collaboration for the Genomics of HIV (ICGH) showed a consistent association with lower set-point viral load. An in vitro assay revealed that the A3F I231V variant may influence Vif mediated A3F degradation. Our results provide genetic epidemiological evidence that A3F modulates HIV-1/AIDS disease progression.Human APOBEC3 cytidine deaminases are intrinsic resistance factors to HIV-1. However, HIV-1 encodes a viral infectivity factor (Vif) that degrades APOBEC3 proteins.  A3) gene family serve as intrinsic resistance factors to HIV-1 and other retroviruses. HIV-1 encodes the viral infectivity factor (Vif) protein that degrades APOBEC3 proteins via the ubiquitination-proteosomal pathway. APOBEC3F (A3F), unlike APOBEC3G (A3G), is partially resistant to Vif-mediated degradation. The antiviral activity of the A3 family has largely been demonstrated in in vitro experiments, and there is mounting evidence that A3G genetic variants influence HIV disease progression. It is not resolved if A3F protein affects HIV disease in vivo. To assess the in vivo effect of A3F, we performed a genetic association study of genetic variants in A3F for their influence on HIV- acquisition and HIV disease progression. A common A3F haplotype was associated with a 30% reduced rate of AIDS disease progression, lower set-point viral load and delayed development of pneumocystis pneumonia (PCP) in European Americans. This study provides the first epidemiological evidence that A3F might modify HIV-1/AIDS pathogenesis.Cytidine deaminases of the human APOBEC3 ( A3 genes  tandemly arrayed on chromosome 22. In in vitro experimental systems, several members of the human APOBEC3 family are capable of inhibiting HIV-1 replication to some degree , with A3G and A3F showing evidence of strong inhibitory activity. A3G protein catalyzes deamination of cytosine bases on the DNA minus strand during reverse transcription, inducing guanosine (G)-to-adenosine (A) hypermutation in the HIV-1 provirus . H. H5]. Huprovirus –8. A3G cprovirus –13. It iprovirus , 16–18]–18A3 genprovirus . How A3 in vitro experiments as some reported that A3F activity is as strong as A3G , thedeletion  and A3G  199376C . The effS2 Fig), which allows the addition of seroprevalent patients who had not progressed to AIDS for >10 years (time from seroconversion to AIDS) to the slow progressor category of seroconverters; only seroconverters were included in the fast category (≤10 years) to avoid frailty bias. The prevalence of AIDS protective genotypes 231V/V and 231 I/V was elevated in the slow progressors (Odds ratio (OR) = 0.62, P = 0.004, chi2 test, S2 Fig). The results of both the survival  and defined disease category analyses  support a strong dominant 231V association with delayed progression to clinical AIDS outcome.The protective effect of p. 231V for AIDS progression was also observed in a defined disease category analysis . A Cox model analysis also showed an association of p.231V with delayed progression to PCP . A sensitivity test in the MACS cohort revealed no impact of PCP prophylaxis usage on the association. In a sensitivity test performed in 410 MACS seroconverters, adjustment of PCP prophylaxis (n = 138) did not attenuate the association of p.231V with PCP (RH changed from 0.637 to 0.633). These results demonstrated that p.231V has a protective association with PCP development. In 278 Africa American seroconverters, a nonsignificant protective trend to PCP was seen for p.231V homozygote carriers Multiple different AIDS-defining conditions including opportunistic infections and malignancies are AIDS-defining . In an eP = 0.02). The p.231V carriers were associated with a 0.27 ± 0.65 log10 lower set-point viral load . For comparison, heterozygousity for CCR5 Δ32 may nfection . A3B hasnfection ,49–51, bnfection ,53. The  outcomes. Other s outcomes,56, and  outcomes; given eollow-up . The rolcognized ,58–61.A3F 231V variant as a novel AIDS-modifying genetic factor in European Americans, which shows a similar trend in African Americans with HIV infection. A3G H186R is only found in individuals with African ancestry and is almost absent in European population ), considering SNP spacing and gene coverage. Several other nonsynonymous variants in A3F are reported in the 1000 genome project but were not genotyped here either because they were rare or tagged by other SNPs: rs2020390 (p. A108S) is in near-absolute LD (r2 = 0.94) with rs2076101 (p.I231V) in Europeans. rs34182094 (p. A178T), rs12157816 (p. Y307C), rs13056825 (p.N346S) were absent or infrequent in Utah Europeans (CEU) and Yoruba Africans (YRI).We selected six SNPs that were codon-changing, in a predicted splicing or regulatory site , according to the manufacturer’s protocol. For quality control, water controls were included on each plate and 10% of samples were duplicated. No water contamination or genotype mismatches between duplicates was observed. SNP rs2076101 was also genotyped by a second in-house designed TaqMan assay (sequences available upon request); the resulted genotypes were identical by two assays.A3F rs5750728 T/C with an Infrared EMSA Kit (LI-COR), as previously described [TCCCACCACAT-3’ and 5’-CCCTTTGGCCAGTGCGCCCCACCACAT-3’.EMSA was performed in nuclear extracts from HeLa cell stimulated with PMA using oligonucleotides carrying escribed . The douHEK293T  cells were maintained in Dulbecco's modified Eagle's medium  with 10% fetal bovine serum and penicillin/streptomycin (D-10 medium) and passaged upon confluence. DNA transfection in HEK293T cells was carried out using Lipofectamine 2000 (Invitrogen) as recommended by the manufacturer. For degradation assays, HEK293T cells in 12-well plates were transfected with 1 μg of Vif or 1 μg of empty vector and 0.3 μg of A3F expression vector at a 3:1 ratio. HEK293T cells were collected at 48 h after transfection. Cell lysates were lysed in 1x loading buffer  and boiled for 5 min before separating the proteins by SDS-PAGE. Membranes were probed with various primary antibodies against proteins of interest. Secondary antibodies were alkaline phosphatase-conjugated anti-sheep, anti-rabbit or anti-mouse (Jackson Immunoresearch) antibodies, and staining was carried out with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT) solutions prepared from chemicals obtained from Sigma.pol gene alone  or the pol gene with additional other genes were obtained by the Sanger sequencing from plasma of patients in the Swiss HIV Cohort Study. A3F specific hypermutation was quantified as in the Los Alamos Hypermut2 tool [HIV-1 sequences covering the ut2 tool . Consensut2 tool . Using tut2 tool ,41. To qWe performed analyses using SAS version 9.12 .A3F variants on disease progression to AIDS outcomes by the Cox proportional hazards model (Cox model) and Kaplan-Meier survival curve analyses. We only included seroconverters with known infection dates (midpoint estimation between last seronegative and first seropositive HIV test) in our analysis. The endpoint was clinical AIDS diagnosis using the Centers for Disease Control and Prevention (CDC) 1987 definition of AIDS [CCR5 Δ32, CCR5-59029 , HLA-B*27, HLA-B*57, HLA-B*35Px group , HLA-C [HLA Class I homozygosity for European American , we conservatively include the first two eigenvalues from a PCA analysis (EigenSoft) to correct for population stratification [We assessed the influences of the  of AIDS , i.e. HIiewed in ; HLA-B*5fication .10-transfromed copies of HIV-1 RNA in plasma measured between months 6–33 after seroconversion (2–5 measurements). Viral load measurements exceeding three-fold (0.5 log10) from the average of all remaining points were excluded, as previously suggested [t test for analysis of differences between viral load means of the group carrying 0 or 1 231V allele versus the 231I homozygote group.HIV-1 viral load set-point was defined as the mean loguggested . We usedA3F SNPs from ICGH were obtained from the previous GWAS data [A3F SNPs on HIV-1 viral load in ICGH was assessed using a fixed-effect inverse-variance weighted meta-analysis as previously described [S3 Table. Viral load data from a total of 10395  HIV-1 seropositives was used in the meta-analysis.The International Collaboration for the Genomics of HIV (ICGH) combined the GWAS data from 25 cohorts from Europe and North America, including cohorts used in this study . The genWAS data . The impescribed . A summaA3F variants on HIV-1 infection susceptibility was assessed by comparing allelic frequencies between the HIV-1 infected group comprising seroconverter and seroprevalent persons and the HIV-1 uninfected group composed of persons at risk for HIV. Odds ratios (OR) and P values were obtained by using a conditional logistic regression test. All P values were 2-tailed.The impact of 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.S1 Fig2 level and haplotype block was defined by 95% CI.Data was based on HapMap phase III data in the CEU (Utah Caucasian) population and was plotted with Haploview. The intensity of the box reflects the r(TIFF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S3 FigP values were obtained from the Cox proportional hazards model. P values for survival curves were obtained from the log-rank test.RH and adjusted (TIFF)Click here for additional data file.


Geobacillus stearothermophilus. The E2 protein acts as a scaffold by self-assembling into 60-mer particles, displaying up to 60 copies of the fused target on the surface. Rabbits were immunized with E2 particles displaying MPER and/or the gp41 ectodomain in conjunction with DNA encoding full-length gp160. Only vaccines including E2 particles displaying MPER elicited MPER-specific Ab responses. NAbs were elicited after two immunizations that largely targeted the V3 loop. To overcome V3 immunodominance in the DNA component, E2 particles displaying MPER were used in conjunction with gp160 DNA lacking hypervariable regions V2, V3, or combined V1V2V3. All rabbits had HIV binding Ab responses and NAbs following the second vaccination. Using HIV-2/HIV-1 MPER chimeric viruses as targets, NAbs were detected in 12/16 rabbits after three immunizations. Low levels of NAbs specific for Tier 1 and 2 viruses were observed in all groups. This study provides evidence that co-immunizing E2 particles displaying MPER and gp160 DNA can focus Ab responses toward conserved regions of Env.Developing a vaccine that overcomes the diversity of HIV-1 is likely to require a strategy that directs antibody (Ab) responses toward conserved regions of the viral Envelope (Env). However, the generation of neutralizing Abs (NAbs) targeting these regions through vaccination has proven to be difficult. One conserved region of particular interest is the membrane proximal external region (MPER) of Env located within the gp41 ectodomain. In order to direct the immune response to this region, the MPER and gp41 ectodomain were expressed separately as N-terminal fusions to the E2 protein of  HIV-1 infection continues to be a worldwide epidemic with more than 2.5 million individuals newly infected annually The membrane-proximal external region (MPER), located within the gp41ectodomain, is one of the most conserved regions on Env. It contains epitopes of three well-characterized human bNmAbs: 2F5, 4E10, and 10E8 in vivo in macaques via trancytosis-blocking Abs Although non-NAbs may be associated with vaccine efficacy, as was seen in the most recent Phase III RV144 Thai trial demonstrating transient protection Geobacillus stearothermophilus to display regions of HIV-1 Gag and Env in vitro from denaturing conditions to form the 60-mer scaffold without the need of chaperonins Previous studies in our lab have explored the use of a multimeric scaffold based upon a subunit of the pyruvate dehydrogenase (PDH) complex of Using this system, we showed previously that the E2 multimeric scaffold displaying Gag p17 elicits Gag-specific Abs and T cell responses in mice E2 particles displaying MPER or gp41, or both, in combination with gp160 plasmid DNA, were immunogenic and elicited equivalent gp41-specific Abs; however, only rabbits immunized with E2 particles displaying MPER developed significant binding Abs directed to MPER. All immunized rabbits developed NAbs to HIV-1 SF162 after two immunizations, but the majority of these NAbs were directed to the immunodominant V3 loop. In order to overcome the immunodominance of V3 and other hypervariable regions, gp160 constructs with deletions in the V2, V3, or combined V1, V2, and V3 regions were used in conjunction with the MPER-E2 fusion particles and compared to immunization with full-length (wt) gp160 DNA. The resulting vaccines elicited similar levels of gp140 binding Abs. Epitope mapping of these Abs revealed that the deletion of V3 resulted in increased recognition of MPER peptides compared to the full-length gp160, and increased titers of NAbs after four immunizations. All other groups had equivalent levels of NAbs to HIV-1 SF162. Additionally, 12/16 rabbits immunized with DNA plus MPER-E2 particles developed Abs capable of neutralizing HIV-2/HIV-1 MPER chimeric viruses, but this activity was not correlated with binding to MPER peptides. Sera from all groups demonstrated low-level neutralization of selected Tier 1 and Tier 2 viruses after two or three vaccinations, and the NAbs had greater breadth than those elicited in our prior studies with E2 particles displaying V3 The Oregon Health & Science University West Campus Institutional Animal Care and Use Committee approved this research under protocol number IS000954.Female New Zealand White rabbits  were housed at the Oregon National Primate Research Center (ONPRC) at Oregon Health & Science University in accordance with the standards outlined by the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All studies were performed according to the rules approved by the Institutional Animal Care and Use Committee (IACUC). The ONPRC is an AAALAC-accredited institution. The first study described here was completed with nine rabbits, with three rabbits per group. Two additional rabbits were immunized three times with E2wt particles to generate E2-specific serum. The effect of modifications to the gp160 vector was studied subsequently using 16 rabbits with four rabbits per group. Rabbits were immunized intramuscularly with a total of 200 µg of protein per immunization with Incomplete Freund's adjuvant (IFA). Codon-optimized SF162 gp160 DNA, either full-length or lacking variable loop regions 2, 3, or combined V1, V2, and V3, was delivered intradermally via Gene Gun (Bio-Rad) at a pressure of 400 psi. A total of 36 µg of DNA was delivered in 18 shots of 2 µg DNA each, given in clusters of three non-overlapping positions at six shaven sites . Blood was collected and serum was separated and heat inactivated at 56°C for 1 h before being stored at −20°C.GCGCCCATGGAACAGAACCAACAAGAAAAG and GCGCCCCGGGTACTATCATTATGAATAT, and the oligonucleotide sequence encoding the gp41 extracellular region was amplified using primers GAGAAGAGTGGTGCAGAGAGAAACCATGGCAGTGACGCTAGGAGCTATG and CATTATCCCGGGTTTTATATACCCGGGCCATTTTGATATGTCAAACC; each containing the restriction sites NcoI and XmaI. Cycling conditions for the PCR were as follows: denature at 94°C for 2 min, 10× , 20× , and a final elongation of 72°C for 7 min. The PCR product and the pETE2DISP vector were double digested with NcoI and XmaI (New England Biolabs) and ligated together with T4 DNA ligase (New England Biolabs) before transformation into BL21 (DE3) CodonPlus-RIPL competent cells (Stratagene). In-frame ligation of all constructs was confirmed by sequencing. These constructs encoding N-terminal fusions of MPER or the gp41 extracellular region to the E2 scaffold are annotated in this document as Env(MPER)-E2 and Env(gp41)-E2, respectively.The Env(MPER)-E2 and Env(gp41)-E2 expression vectors were constructed from the previously described pETE2DISP plasmid 600 between 0.8–1.0. Protein expression was induced with the addition of Isopropyl β-D-1 thiogalactopyranoside (IPTG) with a final concentration of 1 mM. Cells were harvested by centrifugation at 5000 g for 5 min at 4°C. Pellets were then resuspended in Lysis buffer (1× BugBuster Protein Extraction Reagent (Novagen), 1xPBS, Benzonase Nuclease (EMD), rLysozyme (EMD), TurboDNase (Ambion), Cyanase (RiboSolutions) and Complete, EDTA-free Protease Inhibitor Cocktail Tablet (Roche)) and incubated at room temperature for 30 min and then at 37°C for 30 min, shaking at 225 rpm.Plasmids encoding E2 wild-type (E2wt)  and Env-E2 fusion proteins were maintained and expressed in BL21 (DE3) CodonPlus-RIPL cells (Stratagene). Cells were grown overnight at 37°C in Luria-Bertani (LB) broth with a final concentration of 100 µg/ml ampicillin and 50 µg/ml of chloramphenicol, shaking at 225 rpm. The following day, the cultures were back diluted 1∶20 and grown to an ODThe soluble fraction containing the E2 monomers was recovered after centrifugation at 10,000 g for 10 min at 4°C and was loaded onto a Sephadex G-25 column  for buffer exchange. Fractions containing E2wt were pooled and loaded onto a Detoxi-gel column (Pierce). E2wt did not bind to the Detoxi-gel column and was recovered in the flow through, which was loaded onto a Q-Sepharose anion exchange column  at 1.0 ml/min. Bound protein was eluted from the column with a 0–60%/400 ml gradient of elution buffer  at 3.0 ml/min., with the E2wt eluting at a salt concentration of 30%. Peak fractions containing E2wt were pooled and concentrated with a 10 kD molecular weight cut off (MWCO) using Amicon Ultra Centrifugal Filter (Millipore). The retentate was loaded onto a Superdex200 gel filtration column  at 1 ml/min using Solubility Buffer 2.2 , 50 mM NaCl, 250 mM L-Arginine (Sigma)). Fractions containing the 1.5 MDa E2wt 60-mer particles were concentrated to 1 mg/ml using the Ultra Centrifugal devices and then stored in Solubility Buffer 2.2 at −80°C.E.coli and were purified from the insoluble fraction following bacterial lysis and centrifugation as described above. Inclusion bodies were washed three times with Inclusion Body Wash Buffer (1 M Guanidine Hydrochloride (GuHCl), 50 mM NaCl, 1 mM DTT, 1× PBS, 10% glycerol, 0.5 M Arginine, pH 7.4) before dissolving in Unfolding Buffer . To produce soluble HIV-1 Env-E2 multimeric particles, E2wt was combined with the Env-E2 inclusion bodies at either a 1∶1 (Env(MPER)-E2:E2wt) or 1∶3 (Env(gp41)-E2:E2wt) molar ratio and allowed to unfold in Unfolding Buffer  rocking at 4°C for a minimum of 3 h. The proteins were transferred to SnakeSkin Dialysis Tubing, 10K MWCO (Pierce) and subjected to step-down dialysis against the following buffers: 4 M GuHCl , 2 M GuHCl , 0 M GuHCl#1 , 0 M GuHCl#2 , and 0 M GuHCl#3 . A final dialysis was performed in Solubility Buffer 2.2. Refolded soluble 60mer particles were confirmed by gel filtration using the Superdex200 gel filtration column , and purity and identity were assessed by SDS-PAGE and Western blot analysis, respectively. Purified multimeric proteins were stored at −80°C.The Env-E2 fusion proteins formed inclusion bodies in Expression, refolding, and identity of recombinant proteins were assessed by SDS-PAGE and Western blot analysis using Invitrogen NuPAGE 4–12% Bis-Tris mini-gels  under reducing conditions. For SDS-PAGE, gels were stained with SimplyBlue SafeStain (Invitrogen). For Western blot analysis, proteins were transferred onto nitrocellulose paper (Invitrogen), blocked with Odyssey blocking buffer (LI-COR Biosciences) overnight at 4°C. The following day, the blot was probed simultaneously with serum from a rabbit immunized with E2wt (1∶8000) and the human mAbs 2F5 or 4E10  for 1 h at room temperature. Primary Abs were prepared in Odyssey Blocking Buffer 1∶1 with 1xPBS, 0.2% Tween-20. Blots were washed 5 times with 0.1% Triton X-100, 1xPBS. Secondary Abs IRDye 680 Goat anti-Rabbit and IRDye 800CW Goat anti-Human (LI-COR Biosciences) were used at 1∶15,000, diluted in Odyssey Blocking Buffer 1∶1 with 1× PBS, 0.2% Tween-20, 0.02% SDS. Membranes were scanned using the LI-COR Odyssey Infrared Imaging System (LI-COR Biosciences) to allow simultaneous two-color detection of E2 and the HIV-1 Envelope 2F5 or 4E10 epitopes. Integrated intensities were used in conjunction with protein concentrations determined by NanoDrop  to calculate protein purity and concentration.Surface plasmon resonance (SPR) biosensor assays were carried out at 25°C using the Biacore T200 instrument . For all experiments, 10 µg/ml of E2-wt, Env(V3)-E2 env sequence using the following primers:The introduction of deletions within the V2 (ΔV2), V3 (ΔV3), and V1V2 (ΔV12) loops of SF162 were previously reported, where identical residues were omitted as described in GGCGCCGGCAAGCTGATCAACTGCAACAACTGCAGCTTCAAGGTG;SK21(V2) GCCGGCGCCCACCTTGAAGCTGCAGTTGTTGCAGTTGATCAGCTT;SK22(V2) GGCGCCGGCGACATCCGCCAGGCGCCCCAACAACAAC;SK25(V3) GCCGGCGCCGTTGTTGTTGGGGCGCCTGGCGGATGTC;SK26(V3) GGCGCCGGCAACTGCAACACCAGTGACCCCCCTGTGCGTG; andSK29(V1V2) GCCGGCGCCCACGCACAGGGGGGTCACTGGTGTTGCAGTT. These primers replaced the encoded variable loop with an encoded Gly-Ala-Gly peptide sequence, highlighted in bold. Plasmids encoding the env lacking the respective variable loops were transformed into XL-10 Gold Ultracompetent Cells, and screened via PCR. Mutations were confirmed by sequencing. The variant loop Env proteins were expressed on the cell surface, as confirmed by immunofluorescence using COS-7 cells (SK30(V1V2) -7 cells . These vPlasmid DNA expressing codon-optimized SF162 gp160 and variable loop deletion variants were precipitated onto 1 µm diameter gold beads, and bullets were prepared according to the manufacturer's instructions (Bio-Rad). Each bullet was loaded with a total of 2 µg of DNA. To verify that the bullets were functional, COS-7 cells were transfected with the DNA carried by the gold beads, as described previously IgG from individual rabbit sera from weeks 6, 14, and 28 was purified by affinity chromatography as previously described 2SO4, and the plates were read at 450 nm (Molecular Devices SpectraMax 190). Optical density (OD) values were calculated for each group by subtracting two times the average background of pre-immune rabbit serum included in triplicate on each plate. Because each region has a different number of peptides due to size differences, these responses were normalized to compare responses between each HIV Env region. Normalized values reported for each region are the sum of responses  divided by the number of peptides per region.Binding Ab responses from individual rabbits to the E2wt protein, HIV-1 Clade B Envelope antigen gp140, gp41 ΔTM , and MPER and V3 linear peptides were measured by ELISA as described previously 50) was calculated according to the method of Reed and Muench Production of pseudoviruses was described previously 2. Supernatants were collected and centrifuged for 5 minutes at 3,000 rpm to remove cellular debris. The collected virus was aliquoted and stored at -80°C until needed. Virus titers were obtained as described above.The HIV-2/HIV-1 chimeric viruses 7312A (wt), 7312A C1 (MPER), 7312A C3 (2F5) and 7312A C4 (4E10) were provided by G. Shaw luc reporter gene expression compared to that obtained in virus control wells containing virus and cells only, such that {[((virus+cells)-background)-((serum+virus+cells)-background)]/(virus+cells)-background} ×100 = % neutralization. Background control wells contained cells only; all samples were tested twice in duplicate. As a negative control, a pool of the pre-immune serum was used and the lowest dilution tested (1∶8) never achieved 50% neutralization. Neutralization dose-response curves were fitted by non-linear regression and a final titer is reported as the reciprocal of the dilution of serum necessary to achieve 50% neutralization. For mAbs and purified IgG, the concentration of Ab required to obtain 50% neutralization (the 50% inhibitory concentration [IC50]) is reported.Serum and purified IgG samples were tested for their ability to neutralize HIV-1 Env-containing pseudoviruses using the single-cycle TZM-bl neutralization assay as described previously 50 of SF162 or 7312A C1 HIV-2/HIV-1 chimera pseuoviruses. Percent change in neutralization (IC50) was calculated as: [(IC50 without peptide)-(IC50 with peptide)/(IC50 without peptide)] ×100.Detection of NAbs specifically directed to V3 and MPER peptides was performed as previously described MAbs 2F5, 4E10, and 10E8 to HIV-1 MPER were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH and purchased from Polymun Scientific . For peptide competition assays and ELISAs, the HIV-1 SF162 V3 peptide (PNNNTRKSITIGPGRAFYATGD), MPER peptide (RRRNEQELLELDKWASLWNWFDITNWLWYIRRRR), MPER N-terminal peptide (NEQELLELDKWASLWN), MPER C-terminal peptide (KFWDKWILSWINIWYLFY) and MPER scrambled peptides (KKKRIYWLWNTIDFWNWLSAWKDLELLEQENKKK) and (WEALNLQLKLNWESDE) were synthesized by Genscript  with a purity >80%. Consensus B gp160 peptides and MPER derivatives: 162 (SQNQQEKNEQELLEL), 163 (QEKNEQELLELDKWA), 164 (EQELLELDKWASLWN) used for epitope mapping were obtained through the NIH AIDS Research and Reference Reagent Program.P-value obtained for all comparisons. Differences were considered statistically significant when P<0.05, as represented by “*”, P<0.01, as represented by “**”, or P<0.001, as represented by “***”. When comparisons are statistically significant, P-values are indicated in the figure legends. For binding and neutralization assays, the results are expressed as the average +/− SEM, unless specified otherwise in the figure legend.Statistical analyses were performed using either the unpaired Student t-test or the repeated measures ANOVA test to compare data sets, with a E2 gene within the plasmid pE2DISP, resulting in an N-terminal fusion of the gp41 ectodomain to the E2 protein (referred to as ‘Env(gp41)-E2’ for the remainder of this manuscript)  were also ligated in frame to the 5′ end of the E2 gene, resulting in an N-terminal fusion of MPER to the E2 protein (referred to as ‘Env(MPER)-E2’ for the remainder of the manuscript)  in step-down dialysis. Env(MPER)-E2 was refolded with equimolar amounts of E2wt, and Env(gp41)-E2 required a 1∶3 ratio of Env(gp41)-E2 fusion protein:E2wt to remain fully soluble.Nucleotides encoding the HIV-1 SF162 extracellular domain of gp41  were ligated in frame to the 5′ end of the linker upstream to the uscript) . To focuuscript) . These rThe final refolded 60-mer particles were purified by gel filtration, which resulted in a single peak in the void volume . While sSurface plasmon resonance (SPR) was used to determine the availability of the MPER on the surface of the Env(MPER)-E2 particles. Binding of the 2F5 NmAb to the Env(MPER)-E2 particles was detected by SPR, indicating that the 2F5 epitope was accessible on the surface of the Env(MPER)-E2 particles . No bindPrevious studies in our lab using the E2 multimeric scaffold displaying the third hypervariable loop (V3) of Env revealed that Ab responses significantly increased when rabbits were co-immunized with the Env(V3)-E2 particles and HIV-1 gp160 plasmid DNA, compared to protein alone or DNA prime, protein boost P<0.001) or Env(MPER)-E2+Env(gp41)-E2 (P<0.01) groups  groups . Minimal) groups . Strong ) groups .Individual rabbit sera were analyzed for neutralizing activity against the autologous HIV-1 subtype B isolate SF162 (Tier 1) in the single round replication TZM-bl neutralization assay and there was very close agreement in all responses in each group. SF162 NAbs were detected after two immunizations in each group, and these titers reached maximum levels following the third vaccination . As an aTwo different approaches were used to determine if MPER-specific NAbs were elicited in this rabbit experiment. In the first approach, purification of MPER-specific Abs from rabbit serum was tested using an MPER-peptide affinity column. Minimal neutralization activity was detected in the elution fractions from each group, however non-specific Ab binding to the column was also observed (data not shown). After multiple attempts, rabbit serum samples were exhausted due to limitation in blood draw volumes. We concluded that the MPER-specific Abs were too low in concentration to obtain reliable results from this experiment. In a second approach, we employed peptide inhibition of neutralization using full-length and N-terminal MPER peptides, and we only observed modest (10%) reduction in NAb titers (data not shown). Therefore, from these sets of experiments we could not delineate if MPER-specific NAbs were elicited using the Env(MPER)-E2 particles co-immunized with DNA.Previous studies have suggested that removal of the V1, V2, or V3 loops may redirect immune responses towards other neutralization determinants We thus designed a comparative immunization study in rabbits to evaluate the effect of removing these variable regions from the DNA component, and the Env(MPER)-E2 immunogen was chosen as the protein co-immunization partner based upon its ability to elicit Abs targeting MPER , 3C. SixP<0.01)  . HoweverP<0.01) .Similar to the first study, pooled sera were further analyzed by peptide ELISAs using overlapping linear peptides spanning the gp160 Env in order to determine how removal of the variable loops from gp160 affected Ab targeting outside of the V3 and MPER epitopes. Furthermore, to investigate the effects of boosting on temporal changes in Ab targeting, pooled rabbit sera were tested at multiple time points post-vaccination: weeks 6, 14, 22, and 28 . As showSince MPER-specific binding Abs were detected at the highest titer after two (wk 6) or three immunizations (wk 14), further epitope mapping was performed on the individual rabbit serum samples from week 14 using various MPER peptides lacking specific residues . In thisP<0.01) (Sera from each rabbit co-immunized with variant gp160 DNA immunogens and Env(MPER)-E2 particles were tested individually for their ability to neutralize the autologous virus, SF162. All vaccine regimens stimulated similar HIV-SF162 NAb titers following a second vaccination . These rP<0.01) . AlthougP<0.01) . As obseP<0.01) , 6B. ComP<0.01) . Neutral50, comparing scrambled with non-scrambled peptides (data not shown). Neutralization by mAbs 4E10 and 2F5 against these viruses , SS1196.1 (Tier1B), JRFL (Tier 2), JRCSF (Tier 2), and YU2 (Tier 2)] and one clade C envelope [ZM109F.PB4 (Tier 1B)] were analyzed for neutralization sensitivity using purified IgG from weeks 6, 14, and 28 in the TZM-bl assay. All groups had NAbs against Tier 1 viruses following the second vaccination wk 6, . These rDespite the challenge of sequence variability inherent in the HIV-1 Env, there are a number of conserved regions that are targets of broad NAbs, including the MPER. The goal of this study was to develop a vaccine strategy that induced Abs and NAbs directed specifically to the MPER. Therefore, we rationally designed, expressed, and purified Env-E2 particles displaying the gp41 ectodomain or MPER as unconstrained polypeptides and tested these particles in rabbits in combination with gp160 full-length DNA or deletion variants to determine comparative effectiveness. Here, we provide evidence for the induction of binding Abs directed to the MPER using Env(MPER)-E2 particles co-immunized with gp160 full-length DNA or gp160 DNA lacking variable loops V2, V3, or V123, as well as the induction of NAbs capable of neutralizing several heterologous HIV-1 pseudovirus isolates at modest levels.Recent vaccine studies have reported advantages to co-immunization with DNA and protein components NAbs were detected in all rabbits immunized with Env gp160 DNA plus Env(MPER)-E2 particles; however, the majority of the NAb response in rabbits immunized with gp160 bearing V3 was directed to the immunodominant V3 region. V3 has structural conservation and can serve as a target for inter-clade NAbs Ab responses directed to the V2 region have been shown to be an important correlate in the RV144 Thai trial 50 values against HIV-SF162. Although these concentrations do not reach the same potency level as the NmAbs 2F5 or 4E10, these concentrations for purified polyclonal IgG are significantly less than doses of passively transferred polyclonal IgG required for protection in macaque studies  Using these new constructs of gp160 full-length or variable loop deleted DNA in combination with Env(MPER)-E2 particles, we found that twelve out of sixteen rabbits developed MPER-directed NAbs as measured by HIV-2/HIV-1 MPER chimeric viruses. These values were similar to the ICInterestingly, the MPER-specific binding Abs elicited by the various regimens required the SLWN residues at position 667–70 to detect MPER-specific binding for the gp160wt and gp160ΔV2 groups. Abs from the gp160ΔV3 and gp160ΔV123 groups bound to slightly different specificities, where weak binding was observed to the DKWA epitope. A prior study by Menendez, et al. found that 2F5 binding to MPER peptides was not dependent on the serine at position 667, but instead binding strength increased as a result of a hydrophobic residue three residues C-terminal to the tryptophan in DKW, in which leucine or tryptophan residues were preferentially selected Because we had some evidence for MPER-specific targeting using these particles, we wanted to determine the breadth of the neutralization. We measured modest neutralization of Tier 1 and 2 viruses in all immunization groups, with most rabbit serum neutralizing 2–3 isolates in addition to SF162. These NAb responses varied following each immunization, with the highest breadth observed early in the vaccination schedule and decreasing over time for the gp160wt, gp160ΔV3 and gp160ΔV2 groups, similar to binding Abs recognizing MPER . Notably50 values dropped following 3, 4 and 5 immunizations showing increased potency of the responses. We consider it promising that the three of the four rabbits with neutralizing activity against more than one of the HIV-2 chimeras  also had NAbs against the 4–6 of the panel of 7 heterologous viruses. The basis of this neutralization may well differ in each of the vaccine groups. We are encouraged by these results when compared to other gp41-based scaffolds, as the potency of the heterologous NAbs  is similar to those found in other published studies Directing responses to the conserved MPER region has been difficult, despite numerous efforts S1 FigureExpression of gp160 Env variants. (A) Western blot analysis of Env variants expressed in 293T cells post-transfection. Total cell lysate was loaded onto the gel and subsequently probed using CHIVIG polyclonal antibodies. (B) Cell surface expression of the Env variants after transfection into COS-7 cells. Immunofluorescence was detected using CHIVIG and FITC-conjugated goat anti-human IgG. The presence of the expressed Env variants was visualized on the cell surface using fluorescence microscopy.(DOCX)Click here for additional data file.S2 FigureLongitudinal linear antibody epitope mapping. Binding of antibodies directed to linear epitopes of Env elicited in rabbits co-immunized with SF162 gp160wt, gp160ΔV3, gp160ΔV2, gp160ΔV123 DNA and Env(MPER)-E2 was determined by linear peptide ELISA. The clade B consensus Env peptides (15-mer with 11 aa overlap) were used to assess responses from pooled sera at weeks 6, 12, 22, and 28 at a dilution of 1∶25. Responses were considered positive if they achieved OD values two-fold over pre-immune sera values. Colors indicate responses of sera from week 6 , week 14 , week 22 , and week 28 .(DOCX)Click here for additional data file.S3 FigureSerological responses following three immunizations. Binding of antibodies directed to MPER peptides in rabbits co-immunized with SF162 gp160wt, gp160ΔV3, gp160ΔV2, gp160ΔV123 DNA and Env(MPER)-E2 was determined by linear peptide ELISA. Neutralization (IC50) against the HIV-2/HIV-1 MPER chimeras C1, C2, and C3 and six HIV-1 pseudoviruses is displayed for individual rabbit serum samples. Colors indicate the potency of the responses as denoted in the key to the right. HIV-1 insert sequences of the C1, C2, and C3 viruses are shown below the table.(DOCX)Click here for additional data file.


Transporter associated with antigen processing 1 (TAP1) I333V gene polymorphism has been suggested to be associated with type 1 diabetes mellitus (T1DM) susceptibility. However, the results from individual studies are inconsistent. To explore the association of TAP1 I333V gene polymorphisms with T1DM, a meta-analysis involving 2246 cases from 13 individual studies was conducted. The pooled odd ratios (ORs) and their corresponding 95% confidence intervals (95% CIs) were evaluated by a fixed-effect model. A significant relationship was observed between TAP1 I333V gene polymorphism and T1DM in allelic , dominant , homozygous , heterozygous  and additive  genetic models. No significant association between TAP1 I333V gene polymorphism and T1DM was detected in a recessive genetic model  in the entire population, especially among Caucasians. No significant association between them was found in an Asian or African population. TAP1 I333V gene polymorphism was significantly associated with increased T1DM risk. V allele carriers might be predisposed to T1DM susceptibility. Diabetes mellitus (DM) is a common dysmetabolic syndrome that causes internal insulin insufficiency or insulin resistance, and contributes to the glucose and lipid metabolism disturbance led by the combined action of genetic and environmental factors. Type 1 DM (T1DM), also known as insulin-dependent DM (IDDM), is an organ-specific autoimmune disease characterized by selective destruction of the insulin-producing islet beta cells of the pancreas [Transporter associated with antigen processing (TAP) gene is located in the class II region between DQB1 and DPB1. The TAP1 gene product is a transporter associated with the endogenous antigen processing and presentation. The TAP1 can transport peptides into the endoplasmic reticulum, where they combine with MHC I molecules. The MHC I molecules and antigen peptide compounds are subsequently expressed in the cytomembrane and recognized by the CD8+ T cell receptor, thereby stimulating a cytotoxic lymphocyte protective immune response [et al. found that MHC I molecules are significantly reduced and depleted of CD8+ T cells in transgenic mice with a disrupted TAP1 gene [TAP1 gene deficiency and mutation might lead to the endogenous antigen processing and transportation barrier, which results in such autoimmune disease as T1DM. The 1207th base adenine (A) of the TAP1 gene is substituted by guanine (G), which results in the wild-type isoleucine  being replaced by valine  at 333rd amino acid.Human leucocyte antigen (HLA) region, also known as major histocompatibility complex (MHC) region, located in 6p21, are the major gene regions for T1DM. Approximately 60% of T1DM susceptibility is attributed to HLA gene, in which class II region is most strongly associated with T1DM. A total of 42% of T1DM susceptibility comes from this region. AP1 gene . TAP1 geTAP1 I333V gene polymorphism and T1DM exist, the results from individual studies remain controversial. In 1994, Cucca et al. reported that no primary association exists between TAP1 I333V gene polymorphism and IDDM in Italy [et al. found that no significant difference existed in the distribution of TAP1 I333V alleles between IDDM patients and normal controls, and they concluded that TAP1 I333V gene polymorphism did not exhibit a primary association with Japanese IDDM [et al. determined the relative risk of TAP1 I333V gene polymorphism and T1DM using single-stranded conformation polymorphism in the United States [et al. reported that TAP1 333V allele was the susceptibility allele for T1DM in a Chinese population [Although some studies on the relationship between ese IDDM . In contd States . In 2004pulation .TAP1 I333V gene polymorphism and T1DM.In this study, a meta-analysis involving 1140 T1DM patients and 1108 controls was conducted to determine the relationship between Transporter associated with antigen processing 1’, ‘I333V’, ‘T1DM’ and ‘polymorphism’ were adopted to search the electronic databases of PubMed, Embase, Web of Science, China National Knowledge Infrastructure and China Biological Medicine Database. The last research was updated on 31 October 2013, and the included publication years ranged from 1992 to 2007.The terms ‘i) evaluation of the TAP1 I333V gene polymorphism and T1DM; (ii) T1DM diagnosis and classification criteria were presented by the American Diabetes Association and revised by the World Health Organization in 1997; (iii) case–control or cohort study published in an official journal; and (iv) the study should follow the Hardy–Weinberg equilibrium (HWE).The chosen studies had to be in accordance with the following criteria: , recessive (VV versus IV+II), dominant (IV+VV versus II), homozygous (VV versus II), heterozygous (IV versus II) and additive (V versus I) genetic models, were adopted. The relationship between TAP1 I333V gene polymorphism and T1DM was compared using odds ratio (OR) and its corresponding 95% confidence interval (CI). Chi-squared-based Q-test was used to calculate the heterogeneity between the individual studies, and significance was set at P < 0.05 [P < 0.05.Six genetic models, namely, the allelic , dominant , homozygous , heterozygous  and additive  genetic models. No significant association between TAP1 I333V gene polymorphism and T1DM was detected in a recessive genetic model  in the entire population, especially among Caucasians. No significant association was found between them among the Asian or African population , dominant (Pheterogeneity = 0.007), heterozygous (Pheterogeneity = 0.004) and additive (Pheterogeneity = 0.043) genetic models. The following meta-regression was performed to explore the source of heterogeneity. In the allelic genetic model, the heterogeneity could only be explained by the publication year (P = 0.026). The entire population was separated into two subgroups based on publication year. The manuscripts published before 1995 belonged to subgroup 1, and the manuscripts published after 1996 were classified into subgroup 2. In subgroup analysis stratified by publication year, significantly increased T1DM risk was detected in subgroup 2 , whereas no significant increase in T1DM risk was found in subgroup 1 . Heterogeneity was observed in subgroup 1 , but not in subgroup 2 . These results indicate that heterogeneity mainly existed in the studies published before 1995, and the conclusions were relatively consistent in the studies published after 1996.Significant heterogeneity was observed in the allelic (PP = 0.039), II genotype number of T1DM group sample size , IV genotype number of T1DM group sample size  and IV genotype number of control group sample size . All the individual studies were classified into two subgroups according to IV1. The studies with IV1 < 30 were grouped into subgroup 1, whereas the studies with IV1 > 30 were grouped into subgroup 2. In the subsequent subgroup analysis stratified by IV1, significantly increased T1DM risk was only found in subgroup 2 . In subgroup 1, no significant increase in T1DM risk was detected . Heterogeneity decreased in two subgroups but remained significant .In the dominant genetic model, heterogeneity could be explained by publication year (P = 1.0 × 10−10), IV0 (P = 0.005), total sample size of control group  and weight (P = 0.002). All the individual studies were divided into two subgroups according to II1. Subgroup 1 was defined as II1 < 40, and subgroup 2 was defined as II1 ≥ 40. In the subsequent subgroup analysis stratified by II1, significantly increased T1DM risk was detected in subgroup 1 . In subgroup 2, no significant increase in T1DM risk was found .In the heterozygous genetic model, heterogeneity could be explained by II1 (P = 0.003), VV genotype number of T1DM group sample size  and VV genotype number of control group sample size . Individual studies were separated into two subgroups according to II1, which was similar to that in the heterozygous genetic model. In the following subgroup analysis stratified by II1, significantly increased T1DM risk was detected in both subgroups . The heterogeneity disappeared in both subgroups .In the additive genetic model, heterogeneity could be explained by II1 (TAP1 I333V gene polymorphism and T1DM (OR = 1).Logistic regression was performed on multivariable-adjusted risks, such as age, sex, region and ethnicity. These risk factors had no effect on the association between TAP1 I333V gene polymorphism and T1DM was found in allelic , dominant , homozygous , heterozygous  and additive genetic models . No significant association between TAP1 I333V gene polymorphism and T1DM was detected in a recessive genetic model .In the subgroup analysis stratified by ethnicities among the Caucasian population, a significant association between TAP1 I333V gene polymorphism and T1DM was found in the allelic , recessive , dominant , homozygous  and heterozygous genetic models . A significant association was found between them in a additive genetic model  gene polymorphisms were chosen, constructed and analysed. The three haplotypes, namely, TAP1A (333I-637A), TAP1B (333V-637G) and TAP1C (333V-637A) haplotypes, were constructed and analysed. In the following meta-analysis, the TAP1A haplotype could decrease T1DM risk . The other two haplotypes, namely, TAP1B and TAP1C, had no association with T1DM risk .The funnel plot and Egger*s test were used to evaluate the publication bias of the individual studies. No publication bias was observed by visual inspection of the Begg*s funnel plot in the additive genetic model Fig. . No signTAP1 I333V gene polymorphism and T1DM in the allelic (OR: 1.35), dominant (OR: 1.462), homozygous (OR: 1.725), heterozygous (OR: 1.430) and additive (OR: 1.348) genetic models, especially among Caucasians. No positive association between TAP1 I333V gene polymorphism and T1DM was observed in a recessive genetic model (OR: 1.384), which could be associated with the less VV genotype number in the general population. Table TAP1 333V allele possibly increased T1DM risk, i.e. V allele possibly conferred T1DM susceptibility to humans. No significant association was found between TAP1 I333V gene polymorphism and T1DM in an Asian or African population. The different results from the different populations were probably associated with the different ethnicities. Moreover, the relatively few papers on Asian and African populations possibly led to the negative results, which implied that large-scale studies should be performed in future.In this meta-analysis, a significant relationship was observed between P < 0.05).Given that heterogeneity existed in the allelic and heterozygote genetic models, meta-regression was performed to determine the source of heterogeneity. In the subsequent heterogeneity source analysis, publication year, IV1 and II1 were suggested to be the central confounding factors to explain the source of heterogeneity in allelic, dominant, heterozygous and additive genetic models  was replaced by guanine (G), which resulted in the corresponding amino acid aspartate  being substituted by glycine  at 637th amino acid, of which 637G allele possibly increased the T1DM risk. Thus, analysis of the TAP1 gene 333IV-637AG haplotypes was performed [TAP1A (333I-637A) haplotype had a protective effect for T1DM, which was in agreement with the aforementioned meta-analysis results. The negative association between the TAP1B (333V-637G) and TAP1C (333V-637A) haplotypes and T1DM was possibly associated with the limited number of studies because only five and three papers were included respectively. Thus, the results need to be further verified by more studies.Transporter associated with antigen processing 1 gene polymorphisms result in different recognition and transport affinity to the same endogenous antigen peptide, and cause different bodies to show various immune responses to the same endogenous antigen, including protective immunity, immune tolerance and autoimmune tendency [TAP1 I333V gene polymorphism and T1DM remains unclear. In 1998, Quadri et al. reported that TAP1 333I allele specifically enhances translocation of model peptides containing basic C-terminal amino acid residue. However, TAP1 333V allele did not show specificity for the peptide with basic amino acid residue C-terminus, which indicated that the TAP1 I333V mutation could lead to the change in specificity of the transported peptides. Some peptides transported by mutated TAP1 gene products might cause the expression deficiency of MHC I molecules, which resulted in the spontaneous termination of immune tolerance, launched the autoimmunity process and caused the destruction of pancreas islet β cells [TAP1 333V allele increased the T1DM risk, which could be associated with the differences in peptide transport. However, this result is still a hypothesis that needs confirmation by future experiments.tendency . The mec β cells . In the et al. found that the IV genotype of TAP1 gene locus 333 was a susceptible gene for T1DM that had a possible function in the production of autoantibodies, such as insulin autoantibody, islet cell antibody and glutamic acid decarboxylase antibody. Thus, the autoimmune response and destruction in the pancreas islet β cells resulted in T1DM. However, the concrete mechanisms by which the TAP1 333 IV genotype promoted autoantibody production remained unclear [In 2004, Sheng  unclear .TAP1 I333V gene polymorphism are still insufficient. The TAP1 expression level was influenced not only by TAP1 I333V gene polymorphism but also by other genetic and environmental factors, such as ethnicity, inflammation state and other immune system diseases. Given that T1DM is a multigenic heredity disease, TAP1 I333V gene polymorphism might be associated with the gene linkage disequilibrium, such as TAP1 Val458Leu, Asp637Gly and Arg648Gln, which increase T1DM susceptibility [The following limitations were noted in the present meta-analysis. Large-scale studies on the association of T1DM with tibility .Transporter associated with antigen processing 1 I333V gene polymorphism was positively associated with increased T1DM risk. Patients with the V allele may be susceptible to T1DM. Our results may help in establishing individual T1DM therapy strategies. In consideration of the limitations, more large-scale studies are needed to elucidate the significance of our conclusions.


E75 in the silkworm, Bombyx mori, processes three mRNA isoforms, BmE75A, BmE75B and BmE75C. From the early 5th larval instar to the prepupal stages, BmE75A mRNA and protein levels in the prothoracic glands display developmental profiles similar to ecdysteroid titer. In the fat body, mRNA levels but not protein levels of all three BmE75 isoforms correlate with ecdysteroid titer; moreover, proteins of all three BmE75 isoforms disappear at the prepupal stages, and a modified BmE75 protein with smaller molecular weight and cytoplasm localization occurs. At the early 5th larval instar stage, treatment of the prothoracic glands and fat body with 20-hydroxyecdysone (20E) and/or cycloheximide (CHX) revealed that BmE75A is 20E primary-responsive at both mRNA and protein levels, while BmE75B and BmE75C exhibit various responses to 20E. At the early wandering stage, RNAi-mediated reduction of gene expression of the 20E nuclear receptor complex, EcR-USP, significantly decreased mRNA and protein levels of all three BmE75 isoforms in both tissues. In conclusion, BmE75 isoforms display stage- and tissue-specific responses to 20E at both mRNA and protein levels; moreover, they are regulated by other unknown factors at the protein level.Resulted from alternative splicing of the 5′ exons, the nuclear receptor gene  Br-C, E74, E75, E93, HR3 and βFTZ-F1) and their downstream genes1The molting hormone  determines major developmental transitions in insects, including molting and metamorphosis. 20E binds to a heterodimer of nuclear receptors, the ecdysone receptor (EcR) and Ultraspiracle (USP), to trigger a transcriptional cascade, including several transcription factor genes , a hinge region, a conserved ligand binding domain (LBD) and a variable C-terminal activation domain AF-234Drosophila, there are four DmE75 mRNA isoforms, DmE75A, DmE75B, DmE75C and DmE75D, which originate from a single copy gene through differential promoter usage and alternative splicing of 5′ exons. The DBDs of DmE75A and DmE75C contain two C4 zinc fingers, the DBD of DmE75B has one zinc finger, while DmE75D lacks a DBD71415DmE75A and DmE75B, whereas its induction of DmE75C expression is slow and weak; moreover, juvenile hormone (JH) also induces DmE75A expression16DmE75-null mutants missing all three isoforms lead to the arrest during mid-oogenesis16DmE75 null mutants exhibit different phenotypes: DmE75A mutants show reduced ecdysteroid titer leading to developmental retardation and molting defects, DmE75B mutants can survive and exhibit normal reproductive performance, whereas DmE75C mutants die within a few days after eclosionDmE75B has no effects on DmHR3-induced DmβFtz-F1 expression and developmental transitionDmβFtz-F1 expression; and the function of DmE75A and DmE75B to inhibit the transcriptional activity of DmHR3 can be reversed by NO8In Bombyx mori, two isoforms of BmE75, BmE75A and BmE75C, were reported a decade ago. Similar to the findings in Drosophila, 20E rapidly and abundantly induces BmE75A expression in the ovary, while its induction of BmE75C expression is slow and weak. Consequently, BmE75A expression proceeds with BmE75C expression during ovary developmentBmE75 isoform, BmE75BIn the silkworm, http://silkworm.genomics.org.cn/silkdb/), the BmE75 gene locus is located on chromosome 10, spanning 130 kb of genomic DNA. Each BmE75 isoform is characterized by a unique N-terminal sequence encoded by one (BmE75A and BmE75B) or two (BmE75C) distinct 5′ exons. These 5′ exons splice to a common set of four 3′ exons for BmE75A and BmE75C, while BmE75B shares only the last three 3′ exons  contains two C4 zinc fingers for binding to DNA in promoter regions of target genes to regulate gene expression, while the DBD of BmE75B (67 aa) contains only one C4 zinc finger and is thus incapable of binding to DNA. The AF-1 of BmE75A, BmE75B and BmE75C varies in sequence and length, implying that they recruit different co-activators or co-repressors onto target gene promoters to regulate gene expression in a ligand-independent manner71415As a result of the arrangement of the BmE75 isoforms at the initiation of the wandering stage, RNAi-mediated reduction of BmE75 expression lowered the protein levels of all there BmE75 isoforms in the fat body 24 hours after RNAi . As determined by quantitative real-time PCR (qPCR), mRNA levels of BmE75A and BmE75B increased approximately 15-fold 1 hour after 20E treatment, no matter whether CHX was present or absent  to day 2 of the prepupal stage (PP2), the developmental expression profiles of all three BmE75 isoforms in the prothoracic glands were examined at both mRNA and protein levels. BmE75A mRNA level is under detectable during the feeding larval stages, and reaches a small peak on L5D7 and a very high peak on PP1 and PP2  and the second peak on PP1 and PP2. BmE75C protein level is much higher than BmE75A and BmE75B protein levels and can be always detected during the fifth instar, but the developmental profiles of protein levels of all three BmE75 isoforms are quite similar to one another . NotablySince the BmE75 isoforms are responsive to 20E treatment in BmN cells, we questioned whether the developmental profiles of the BmE75 isoforms are consistent with that of ecdysteroid titer. As measured by radioimmunoassay (RIA), a small peak of ecdysteroid titer was detected on L5D7, and a big peak was detected on PP1 . In geneBmE75A mRNA level in the fat body is under detectable during the feeding larval stages, and reaches a small peak on L5D7 and a very high peak on PP1 and PP2 , with no apparent change for BmE75A protein level, a slight increase for BmE75B protein level, and a steady decrease for BmE75C protein level . To our BmE75 isoforms, RNAi-mediated reduction of BmE75 expression was performed at the initiation of the wandering stage. Forty-eight hours after RNAi, mRNA levels of all three BmE75 isoforms  provided by the Sericultural Research Institute, Chinese Academy of Agricultural Sciences , were reared with fresh mulberry leaves at 25 °C under 14 h light/10 h dark cyclesth abdominal segment, and hemolymph samples were collected at various developmental stages or after various treatments.The prothoracic glands, peripheral fat body tissues from the 5rp49 is used as a reference gene that has been validated for qPCR analysis in BombyxTotal RNA was isolated using TRIzol (Invitrogen) according to the manufacturer’s instructions. qPCR was performed as previously describedBmE75 isoforms . AB11 USP antibody was a kind gift from Dr. Fotis Kafatos. The primary antibodies, BmE75 (1:5000), AB11 USP (1:2000) and tubulin , were used. The Western blotting images were caught by Tanon-5500 Chemiluminescent Imaging System  / KODAK Medical X-Ray Processor 102 NY14608 (Germany), and quantitative measurements of Western blots were performed using the ImageJ software.A cDNA fragment shared by all three isoforms  was exprBombyx larval were collected, and extracted for ecdysteroids with equal volume of methanol for 3 times. The supernatant after centrifugation was collected and evaporated under 70 °C. RIA was performed to evaluate the supernatants using 20E  as a standard. The rabbit antiserum used was raised against 20E conjugated with human serum albumin. [3H]-ecdysone (approximately 60 Ci/mmol) was obtained from New England Nuclear (USA). Cross-reactions of the antiserum between ecdysone and 20E occurred at a ratio of 1:2.5.Total ecdysteroid titers of the hemolymph samples were determined by radioimmunoassay (RIA) as described previouslyThe fat body tissues were isolated at the indicated times as described previouslyex vivo experiments, newly collected prothoracic gland and fat body tissues from L5D2 larvae were cultured in Grace’s medium  at 25 °C. After pre-incubation for 1 h, the medium was replaced with fresh medium. To determine which BmE75 genes are 20E-primary response genes, 2 μM 20E or/and 10 μg/ml CHX  were added. After incubation for the indicated times, mRNA and protein were isolated for qPCR analysis and Western blotting.BmN cells were maintained in TC-100 medium  supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco). Sf9 cell line was cultured in HyClone SFX medium (Thermo Scientific) supplemented with 5% FBS. Cells were pre-incubated for 1 day before further experiments. For BmE75, a 644 bp common region was amplified by PCR from cDNA using a pair of primers  with T7 RNA polymerase-binding site (5′-GAATTAATACGACTCACTATAGGGAGA-3′) attached to the 5′-end of each primer. The BmE75 double-stranded RNA (dsRNA) was synthesized using T7 RiboMAXTM Express RNAi kit  according to the manufacturer’s instructions. RNAi of BmUSP was conducted as described beforeEGFP dsRNA was used as control. Thirty μg dsRNA per larva was injected at the initiation of wandering stageFor RNAi of BmE75A, BmE75B and BmE75C were cloned into the BamH I - Hind III sites of pFastBac-HTa (Invitrogen) plasmid, and DsRed2 was used as controlΔEGT bacteriaThe full-length cDNAs of t-test and ANOVA. For the t-test: *p < 0.05; **p < 0.01. ANOVA: bars labeled with different lowercase letters are significantly different (p < 0.05). Throughout the study, values are represented as the mean ± standard deviation of 3–8 independent experiments.The experimental data were analyzed using Student’s How to cite this article: Li, K. et al.Bombyx E75 isoforms display stage- and tissue-specific responses to 20-hydroxyecdysone. Sci. Rep.5, 12114; doi: 10.1038/srep12114 (2015).


In vitro treatment of C8161 human melanoma cells with 3D1 mAb shows reductions in anchorage-independent growth and vasculogenic network formation. 3D1 treated cells also show decreases of Nodal and downstream signaling molecules, P-Smad2 and P-ERK and of P-H3 and CyclinB1, with an increase in p27. Similar effects were previously reported in human breast cancer cells where Nodal expression was generally down-regulated; following 3D1 mAb treatment, both Nodal and P-H3 levels are reduced. Noteworthy is the reduced growth of human melanoma xenografts in Nude mice treated with 3D1 mAb, where immunostaining of representative tumor sections show diminished P-Smad2 expression. Similar effects both in vitro and in vivo were observed in 3D1 treated A375SM melanoma cells harboring the active BRAF(V600E) mutation compared to treatments with IgG control or a BRAF inhibitor, dabrafenib. Finally, we describe a 3D1-based ELISA for the detection of Nodal in serum samples from cancer patients. These data suggest the potential of 3D1 mAb for selecting and targeting Nodal expressing cancers.Nodal is highly expressed in various human malignancies, thus supporting the rationale for exploring Nodal as a therapeutic target. Here, we describe the effects of a novel monoclonal antibody (mAb), 3D1, raised against human Nodal.  Melanoma is the most aggressive and deadly form of skin cancer with a median overall survival for advanced stage metastatic disease of less than 6 months . Since tIt appears, therefore, that the selective pressure exerted by the signaling pathway targeting agents can lead to activation or overexpression of alternative signaling events, all too common in melanoma, resulting in resistance and disease progression , 14. EffOur studies have shown how Nodal, an embryonic growth factor of the transforming growth factor-beta (TGFB) superfamily, can play an important role in aggressive human cancer, specifically underlying tumor growth, metastasis and the cancer stem cell phenotype . Typicalin vitro and in vivo using either a polyclonal anti-Nodal antibody or shRNA approach, resulting in significant reduction in tumor cell activity and tumor volume   versus IgG control = 59.3% ± 11.4% (N = 8); P < 0.05]  versus IgG control = 59.4 ± 9.9 (N = 6); P < 0.01]  versus IgG control = 38.8% ± 10.5% (N = 6); P < 0.001], along with a significant increase in 3D1 treated Nude mice of the mean percentage (± SEM) of cells staining for p27  ; BRAFi = 68.7% ± 6.3% (N = 4); IgG = 50.2% ± 7.3 (N = 4); P < 0.05]. Collectively, these findings show the Nodal function-blocking activity and tumor diminishing effect(s) of 3D1 mAb treatment in vivo.Previously, we showed that Nodal expression in melanoma cells harboring the active BRAF mutation is unaffected when these cells are treated with a BRAF inhibitor (BRAFi) . Here, wN = 12) versus median for noninvasive breast cancer = 3509 ]. Thus, these results show that Nodal can be detected in sera from cancer patients with our novel 3D1 mAb based ELISA.Since our previous study showed Nodal expression in tissue samples from a sizable cohort of breast cancer patients , the Nodin vivo resistance mechanisms. Molecular heterogeneity and high rates of somatic base mutations in melanoma can explain, at least in part, the selection of drug resistant clones of those melanoma cells that can ultimately lead to disease progression [Within just a few years the outcomes for the treatment of metastatic melanoma has evolved from little to no difference compared to palliative care, to substantially improved response rates and quality of life of affected patients - thanks to the introduction of novel targeted agents and immune checkpoint inhibitors . Not allgression . The cangression . For insgression , 34. Impgression , and Nodgression . In addigression  is emplogression .in vitro and reduced tumor growth in in vivo xenograft models [Studies have shown that it is possible to target Nodal in metastatic melanoma resulting in inhibition of cell aggressiveness t models -25, 29. t models . Moreovein vitro binding of Nodal to Cripto-1, thus suggesting it can prevent the Cripto-1 co-receptor complex-dependent Nodal downstream signaling. Melanoma cells treated in vitro showed significant reduction of Nodal protein expression levels and reduction of the activated (phosphorylated) forms of SMAD2 and ERK1/2. This treatment was also accompanied by reductions in anchorage independent growth and vasculogenic mimicry, and decreased levels of the cell proliferation-associated molecules Cyclin B1 and P-H3, complemented by an increase of the cell cycle inhibitor p27. Most noteworthy, Nude mice xenograft models, both subcutaneous orthotopic and lung colonization models, treated with 3D1 mAb showed antitumor effects in terms of reduced tumor volume and lung tumor burden, respectively. Equally important, analysis of IHC staining for P-Smad2, p27 and Cyclin B1 in tumor sections confirmed the in vitro results. Also of significance is the effect of 3D1 treatment of breast cancer cells showing the down-regulation of Nodal expression and the accompanying decrease in P-H3. These findings validate key observations of a previous investigation where shRNA knockdown of Nodal expression in MDA-MB-231 and MDA-MB-468 aggressive breast cancer cells resulted in decreased CyclinB1 and upregulation of p27, concomitant with decreased tumorigenicity [In this study, we describe the production and functional validity of 3D1, a novel mAb capable of targeting and functionally blocking human Nodal. We show that 3D1 mAb is capable of robust binding of Nodal and that it binds in proximity to the Cripto-1 co-receptor interacting region encompassing Glu49 and Glu50 , 40. Thegenicity . These dFrom a translational perspective, in addition to types and rates of responses in patients for evaluating clinical outcomes, it is desirable that therapies have specific biomarkers for determining efficacy of treatment or disease relapse. For instance, obtaining circulating tumor cells from patients receiving targeting agents has been proposed for monitoring the emergence of new mutations, which in turn can be targeted with other available agents in a sequential or combinatorial manner . Increasin vitro and in vivo [. Here, we describe a novel Nodal blocking mAb capable of anti-tumor effects demonstrated in melanoma models both in vitro and in vivo. Since Nodal is significantly expressed in melanoma and has been shown to play an important role in its aggressive behavior, this 3D1 mAb holds promise as a novel targeting agent. Nodal expression appears to be independent of the expression of currently targeted molecules and represents a potential alternative or complementary target in non-candidate and candidate melanoma patients for these new therapies. Future pre-clinical studies will determine the optimal dosing schedule with or without sequential or combinatorial associations with other targeting agents to define the best therapeutic approach in aggressive cancers expressing Nodal based on these new scientific findings.Our previous studies have shown that Nodal has the potential as a prognostic and predictive biomarker in several types of human cancers, i.e. patients with high levels of Nodal expression have more aggressive disease, and targeting Nodal in aggressive cancer cells leads to reduced cell proliferation and tumor growth both  in vivo , 21. HerrhNodal) and human Cripto-1 (rhCripto-1) were from R&D Systems . The following antibodies were used for WB: rabbit anti-Nodal ; rabbit anti-P-Smad2 ; rabbit anti-Smad2/3 ; rabbit anti-P-p44/42 MAPK (P-ERK1/2) ; rabbit anti-p44/24 MAPK (ERK1/2) ; mouse anti-actin . The following antibodies were used for immunohistochemistry: goat anti-human Nodal antibody  1:150; rabbit anti-P-Smad2  1:100 - 1:250; anti-p27  1:400; rabbit anti-CyclinB1  1:200.TRIzol and restriction endonuclease enzymes were purchased from Invitrogen . The protein G column was purchased from GE Healthcare. SPR analyses were performed on a Biacore 3000 instrument from GE Healthcare, using CM5 sensor chips and certified HBS buffer . Recombinant human Nodal  in HBS at increasing concentrations (6.0 nM – 100 nM). A 10 mM NaOH solution was used to regenerate the chip surface. For competition assays, rhCripto-1 was covalently immobilized at 5 μg/mL in 10 mM Sodium Acetate buffer pH 4.5 at a flow rate of 5 μL/min onto a CM5 sensor chip, reaching an immobilization level of 600 RU. rhNodal at 5 nM in HBS was incubated with 3D1 at increasing concentrations, so to achieve Nodal:antibody final molar ratios of 1.0:0.5, 1:1 and 1.0:2.0. Each mixture was incubated for 30 min at room temperature before passing on the sensor chip. 60 μL of each solution was injected at a flow rate of 20 μL/min. On every biochip, an underivatized surface was prepared and used as control blank. All analyses were carried out at a flow rate of 20 μL/min, injecting a constant volume of 60 μL of antibody or competition solutions opportunely diluted in the HBS running buffer at various concentrations. The contact time was 3 minutes for the binding. Dissociations were monitored for at least 3 additional minutes. For every single analysis, experimental sensorgrams were aligned, subtracted of blank signals and overlapped. All mathematical manipulations and fitting were performed using the BiaEvaluation software, vers. 4.1 from GE Healthcare. All experimental data gave optimal fittings when processed assuming a 1:1 Langmuir binding interaction.All SPR analyses were performed at 25°C using HBS as running buffer. Protein immobilization was carried out following the canonical amine coupling chemistry using the surface immobilization wizard procedure, operating at 5 μL/min. Channels were activated with EDC/NHS [N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride(EDC)/N-hydroxysulfosuccinimide(NHS)  for 7 miThe C8161 human melanoma cell line used was obtained from the University of Arizona, while the highly metastatic A375SM human melanoma cell line, which harbors the active BRAF(V600E) mutation , was a kC8161 melanoma cells  were suspended in 0.35% agarose, RPMI 1640;10% serum with of either 4μg/ml mouse IgG control  or 3D1 mAb (2.8 μg/ml) and were then overlayed onto a solidified layer of 0.5% agar; RPMI 1640; 10% serum in 6-well dishes. Cell clusters were allowed to form and were scored (50 cells or larger clusters) after 3 weeks in culture. Triplicate wells were averaged from separate experiments and presented as a percentage (mean ± SEM) of IgG control.5 cells/well) were then plated onto the prepared Matrigel matrices in the presence of either 4μg/ml mouse IgG control or 3D1 mAb (2.8 μg/ml). No additional antibody was added. Tubular network formation was then observed after 24 hr and images captured digitally using a Zeiss model 25 inverted microscope  and Hitachi HV-C20 CCD camera . Mean values were calculated from images of at least four different fields of both the IgG control and 3D1 mAb treated cultures, and then analyzed using the AngioSys software package  with the mean number (± SEM) of junctions and tubules calculated from the analyzed fields.Three-dimensional matrices were prepared by spreading 75 μl of ice-cold Matrigel  into 12-well culture dishes and polymerized for one hour at 37°C. C8161 human melanoma cells  were purchased from Santa Cruz Biotechnology. For WB experiments of cell lines, whole cell lysates were prepared and quantified as previously described . SDS-PAGin vivo effects of 3D1 mAb, a metastatic melanoma lung metastasis model was established by injecting 250,000 C8161 cells intravenously in Nude mice. After 4 days during which cells were allowed to colonize the lungs, mice were separated into treatment and control groups. The treatment group received a total of 500μg of 3D1 mAb versus 500μg of irrelevant isotype IgG in the control group administered over 10 days  of 100μg of either 3D1 mAb or IgG). At the end of the treatment period, mice were sacrificed and lungs harvested and processed for histologic evaluation of lung tumor burden and immunohistochemistry. Lung tumor burden was determined by evaluating the mean percentage of lung tissue occupied by C8161 in at least 4 separate fields at low power (10X objective) in lungs from at least 4 separate mice for each group and performed by 2 different observers. A final mean ± SEM was calculated from the separate means determined by the individual observers.To evaluate the in vivo effects of 3D1 mAb on primary melanoma. To this end, 500,000 C8161 cells were injected subcutaneously in Nude mice. Palpable tumors were then injected directly with either 700 μg of total 3D1 mAb (N = 6) or IgG control (N = 4) antibody . Mice were then sacrificed and subcutaneous tumors harvested and processed as previously described. To evaluate the effect of 3D1 mAb in the presence of the BRAF(V600E) mutation, a third orthotopic xenograft model was established in Nude mice via subcutaneous injection of approximately 150,000 A375SM human metastatic melanoma cells. Mice were treated IP with 3D1 mAb or IgG control, as described above, or 3mg/kg of dabrafenib.A second experiment was carried out to determine the Four micron thick, formalin fixed, paraffin embedded tissue sections were prepared and immunohistochemistry was carried out on a Dako Plus autostainer  as previously described . BrieflyBy epitope mapping of commercially available Nodal antibodies and 3D1 mAb, the latter was chosen as the capture antibody for coating ELISA dishes, while a rabbit monoclonal anti-Nodal (Abcam) served for detecting the bound Nodal. The immune complex was subsequently quantified using a horseradish peroxidase-conjugated anti-rabbit antibody in conjunction with QuantaRed enhanced chemifluorescent horseradish peroxidase substrate (ThermoFisher). The established sandwich ELISA assay could detect Nodal as a recombinant protein in conditioned media from human embryonic stem cells and in biological fluids with a detection limit  of 75 pg/well. Human serum samples from breast cancer patients were purchased from BioOptions .


Bone Morphogenic Protein 2 (BMP2) is a multipurpose cytokine, important in the development of bone and cartilage, and with a role in tumour initiation and progression. BMP2 signal transduction is dependent on two distinct classes of serine/threonine kinase known as the type I and type II receptors. Although the type I receptors (BMPR1A and BMPR1B) are largely thought to have overlapping functions, we find tissue and cellular compartment specific patterns of expression, suggesting potential for distinct BMP2 signalling outcomes dependent on tissue type. Herein, we utilise large publicly available datasets from The Cancer Genome Atlas (TCGA) and Protein Atlas to define a novel role for BMP2 in the progression of dedifferentiated liposarcomas. Using disease free survival as our primary endpoint, we find that BMP2 confers poor prognosis only within the context of high BMPR1A expression. Through further annotation of the TCGA sarcoma dataset, we localise this effect to dedifferentiated liposarcomas but find overall BMP2/BMP receptor expression is equal across subsets. Finally, through gene set enrichment analysis we link the BMP2/BMPR1A axis to increased transcriptional activity of the matrisome and general extracellular matrix remodelling. Our study highlights the importance of continued research into the tumorigenic properties of BMP2 and the potential disadvantages of recombinant human BMP2 (rhBMP2) use in orthopaedic surgery. For the first time, we identify high BMP2 expression within the context of high BMPR1A expression as a biomarker of disease relapse in dedifferentiated liposarcomas. Type I Despite BMP2s essential role in development there remains much debate over whether this translates to tumour progression . Interesproteinatlas.org). Pan-cancer analysis was carried out using cBioPortal  (cytoscape.org) (tcga-data.nci.nih.gov) , Prostate , Breast  . With paape.org) . RNASeqV, Breast , Ovarian, Breast , Lung .https://www.rstudio.com/). Combined data were analysed in Microsoft Excel (Mac 14.4.3) and R Studio with results plotted in GrpahPad Prism 6 (Mac). Comparisons between two groups were made using two-tailed t-tests or Mann–Whitney U-tests and more than two groups by one-way ANOVA or Kruskal–Wallis, multiple comparisons were corrected for by Bonferroni post hoc tests. Kaplan–Meier disease free survival graphs were constructed in GraphPad Prism 6 and presented as whole population versus experimental population. Comparisons between curves were made by Log-rank (Mantel-Cox) tests with the Mantel-Cox p value reported.Normalised RNASeqV2 expression values and corresponding clinical metadata were manipulated in R Studio (Mac) 0.99.484  with default parameters and 1,000 permutations  differential expression analysis was performed on normalised RNASeqV2 gene counts. Data were combined in R studio and means across rows (genes) calculated. Multiple two tailed on 3.9.4 ). Gene sType I BMP receptors (BMPR1A and 1B) have well defined overlapping functions  but may We next profiled expression patterns across cancer cell lines . In the As the largest differences in receptor expression patterns were observed in sarcoma cell lines and normal soft tissues, we elected to focus our analysis on BMP2 in the soft tissue sarcoma TCGA dataset. The observations from By stratifying the 263 samples represented in the TCGA sarcoma dataset based on the ratio of BMPR1A to BMPR1B mRNA, we were able to define two clear groups . AlthougTo test the hypothesis that patients favouring BMPR1A would have differential biological responses to high levels of endogenous BMP2 to patients favouring BMPR1B we refined our patient groups from p < 0.05). Of these, 252 were up-regulated and 669 down-regulated. p value (multiple two-tailed t-tests with Bonferroni post hoc). The insert panel in WNT7B which has a role in osteoblast differentiation and mineralised nodule formation  of 0.05 . Together, these data suggest that BMPR1A-biased BMP2 expression is associated with reduced bone formation and an increased turnover of ECM components. The interaction of tumour cells with ECM proteins can have profound effects on their biology, regulating gene expression, signal transduction and migration/invasion . Moreoven = 44) have no difference in their prognosis when compared to the dataset as a whole. However, those with BMPR1A-biased BMP2 expression (n = 32) have a significantly worse outcome when disease free survival is used as the primary endpoint (p = 0.0087). To discount prognostic influences of BMPR1A, BMPR1B or BMP2 expression individually, we constructed Kaplan-Myer curves for the entire sarcoma dataset and compared patients with the highest and lowest 10% expression of BMPR1A, BMPR1B, BMPR2, BMP2 and BMP7. Despite several interesting trends, no significant associations were seen  of which were down-regulated ; the fulFinally, we sought to determine whether the detrimental effects of BMP2-BMPR1A expression was a pan-cancer phenomenon. Several large studies performed by The Cancer Genome Atlas (TCGA) show copy number changes and mutational events in BMP2 interacting partners as defining features of that malignancy . For exaSeveral studies have shown BMPR1A and BMPR1B have largely overlapping roles in BMP2 signal transduction, for example during chondrogenesis . HoweverLoss of BMPR1A has previously been associated with the formation of juvenile polyposis, a predisposing factor for gastrointestinal and colorectal cancers . ReducedWNT7B which has a role in osteoblast differentiation and mineralised nodule formation  red is microtubules. Scale bar is 100 µm.Click here for additional data file.10.7717/peerj.1957/supp-3Figure S3n = 263) based on mRNA expression (top 10% and bottom 10% expressors). From top left; BMP2, BMP7, BMPR1A, BMPR1B and BMPR2.Kaplan-Myer disease free survival curves in the TCGA sarcoma dataset  expression of BMPR1A, BMPR1B, BMPR2 and BMP2 in different sarcoma subsets. UPS is Undifferentiated Pleomorphic Sarcoma and LMS is Leiomyosarcoma.Click here for additional data file.10.7717/peerj.1957/supp-5Figure S5cbioportal.org) (cytoscape.org) ((A) Pan-cancer analysis of BMP2 in several large scale genomics projects. Data from cBioPortal  . (B) BMPape.org) .Click here for additional data file.10.7717/peerj.1957/supp-6Figure S6 cbioportal.org)  in various cancer types. Vertical bars represent individual patients from the following cancer types: Sarcoma , Prostate , Breast , Ovariantal.org) .Click here for additional data file.10.7717/peerj.1957/supp-7Figure S7Kaplan–Myer disease free survival curves for BMPR1A-biased, BMPR1B-biased and BMPR2 signalling by high levels of endogenous BMP2. From top left, Prostate , Pancreatic , Lung  and OvarClick here for additional data file.10.7717/peerj.1957/supp-8Table S1Click here for additional data file.10.7717/peerj.1957/supp-9Table S2Click here for additional data file.