To investigate the associations between low and high concentrations of baseline serum 25-hydroxyvitamin D [25(OH)D] and all-cause mortality in very old (≥85 years) men and women over 6 years.Prospective mortality data from 775 participants in the Newcastle 85+ Study were analysed for survival in relation to 25(OH)D  and sex using Cox proportional hazards models. The models were fitted to the entire and restricted (nonusers of vitamin D-containing supplements and medication) cohorts.P = 0.04; SQ4: HR 1.44, 95% CI 1.12–1.85, P = 0.004] compared with the combined middle quartiles (SQ2 + SQ3), after adjustment for sociodemographic factors. The increased risk for the highest quartile remained significant after further adjustment for lifestyle variables  and was seen only in women in sex-specific analyses. Similarly, in sensitivity analyses with predefined 25(OH)D cut-off values, the highest 25(OH)D concentration (≥75 nmol L−1) was associated with a 2.4-fold increased risk of mortality in women (restricted cohort) after adjusting for all covariates.For the entire cohort, mortality was higher in both the lowest and highest 25(OH)D season-specific quartiles [SQ1: hazard ratio (HR) 1.31, 95% confidence interval (CI) 1.01–1.69, Low and high season-specific 25(OH)D quartiles were associated with increased risks of mortality over 6 years in the very old; this effect was particularly noticeable in women, including those who reported taking vitamin D-containing supplements/medication. Separate models were fitted to the entire and restricted cohorts, first with sex as a confounder and secondly stratified by sex.We examined the potential non-linear relation between 25(OH)D (continuous) and all-cause mortality nonparametrically with restricted cubic splines  using SA−1 (severely deficient), ≥25 to <50 nmol L−1 (deficient), 50–74 nmol L−1 (insufficient) and ≥75 nmol L−1 (sufficient) , to have cognitive impairment  and to be less physically active , and were less likely to drink alcohol . Participants with 25(OH)D data were more likely not to take any vitamin D-containing supplements but to take other vitamins .Compared with participants with successfully measured baseline 25(OH)D status (2(1) = 51.1, P < 0.001), to have renal impairment (χ2(1) = 9.6, P = 0.002), to smoke (χ2(1) = 7.2, P = 0.03), not to drink alcohol (χ2(1) = 11.2, P = 0.001) and not to take any vitamin supplements (χ2(2) = 6.8, P = 0.03), but to take prescribed vitamin D medication (χ2(1) = 18.1, P < 0.001).At the end of follow-up (1 April 2012), approximately 6 years from baseline, 443 (52.4%) participants were still alive, and the mean survival time was 4.34 years (95% CI 4.20–4.48). Overall, 179 (58.9%) of 304 men with 25(OH)D status died during follow-up compared with 229 (48.6%) of 471 women. Those who died were more likely to be cognitively impaired at baseline (χ2(2) = 7.90, P = 0.02), with the longest survival time in the middle season-specific group  and the shortest survival in highest group  (Model 1). Differences between the middle and two other 25(OH)D season-specific groups emerged within the first year of mortality follow-up and persisted until the last year D and survival in the entire cohort (Mantel-Cox χP = 0.04) and the highest season-specific quartiles  of 25(OH)D compared with the middle combined (referent) quartiles (Model 2). The U-shaped association disappeared after additional adjustment for lifestyle factors  with only participants in the highest quartile being at increased risk of mortality  compared with the reference group (Model 3). This effect was further attenuated to a nonsignificant trend when adjusted for mental health and morbidity-related factors .This U-shaped relationship between survival and 25(OH)D remained after adjustment in a Cox proportional hazards model for sociodemographic variables (Table P = 0.005), which was attenuated to a trend (P = 0.06) after adjustment for lifestyle factors, and abrogated in the fully adjusted model , and after adjustment for sociodemographic variables (Model 2), shorter survival was evident only in the lowest 25(OH)D quartile  = 13.15, P = 0.001) but not in men  = 1.01, P = 0.6)  (Table P = 0.005) after adjusting for sociodemographic and lifestyle variables (Model 3), which was attenuated but remained significant in the fully adjusted model  D group in women D] having a marginally better fit as judged by the Akaike Information Criterion. A non-linear dose–response relation between 25(OH)D concentration and all-cause mortality was evident in the entire cohort  and ‘sufficient’ (≥75 nmol L−1) 25(OH)D concentration ranges  appeared to be driven largely by women taking vitamin D-containing supplements and/or prescribed medication. Using 25(OH)D as a continuous covariate and fitting with restricted cubic splines confirmed the relationship between 25(OH)D and mortality. Moreover, in sensitivity analyses using predefined 25(OH)D cut-off values . The intake of vitamin D supplements/medication is an important biological determinant of 25(OH)D status in this cohort [−1) categories] whether users or nonusers of vitamin D supplements/medication, respectively, have not been reported previously [3 and calcium combination therapy.We observed statistically significant sex differences in mean (SD) 25(OH)D concentration which were lower than recommended  and higher (≥75 nmol L−1) concentrations of 25(OH)D have been moderately associated with frailty amongst older women (aged ≥69 years) in the Study of Osteoporotic Fractures [−1) compared with not frail participants in the highest (>84.1 nmol L−1) 25(OH)D quartiles [P < 0.001); however, the increased risk of mortality in women in the lowest and highest 25(OH)D groups was independent of their frailty status and was not affected by exclusion of those who died within first year of follow-up. Compared with men, women were more likely to have osteoporosis (P < 0.001) and to be treated with prescribed vitamin D medication (P < 0.001), which included calcium combination therapy  and high (>98 nmol L−1) 25(OH)D concentrations over 12.7 years of follow-up [−1; Uppsala Study of Older Men: 46–98 nmol L−1); this difference may be related to the age of participants, habitual diet, supplementation, length of follow-up or other factors/covariates.The results from observational cohort studies exploring sex differences in mortality in relation to 25(OH)D are inconclusive and have not included the very old. The NHANES III showed a U-shaped relationship between 25(OH)D levels and mortality in the general population of women (aged ≥20 years) but not in men at concentrations of <50 and >125 nmol Lnaverage - mean number per quartile = 194), and therefore may have had limited power for detection of associations. Although we controlled for the number of chronic diseases and for frailty status, increased mortality amongst older women may be mediated by other mechanisms associated with nonoptimal 25(OH)D levels such as polypharmacy [−1/<50 nmol L−1, respectively), especially in older women aged ≥65 years [Several limitations of our study should be noted. There remains the possibility of residual confounding by additional factors that affect the relationship between serum 25(OH)D and mortality. On the other hand, having a large number of variables in a fully adjusted model (Model 4) may have contributed to a nonsignificant or biased result, given the fact that we categorized 25(OH) into season-specific quartiles  the homogenous age of the cohort; (ii) the prospective 6-year follow-up; (iii) inclusion of several mortality-related covariates in both the entire and restricted cohorts; (iv) analyses stratified by sex and exposure (i.e. intake of vitamin D supplements/medication) and (v) use of ‘data driven’ season-specific 25(OH)D cut-off values as the preferred method of adjusting for seasonal variability in vitamin D status .−1 irrespective of intake of supplements/medication. Future recommendations for optimal concentrations of serum 25(OH)D should pay special attention to the very old, to prevent problems related to over-treatment in this age group [In summary, both low and high concentrations of 25(OH)D were associated with an increased risk of all-cause mortality amongst very old adults, especially amongst women who reported taking vitamin D-containing supplements and prescribed medication as well as in women with concentrations ≥75 nmol Lge group ,18,25,26This study was supported by the National Institute for Health Research Newcastle Biomedical Research Centre based at Newcastle Hospitals Foundation Trust and Newcastle University (AG). The Newcastle 85+ Study has been funded by the Medical Research Council, Biotechnology and Biological Sciences Research Council and the Dunhill Medical Trust. The authors have also been supported by the British Heart Foundation, Unilever Corporate Research, Newcastle University and the National Health Service (NHS) North of Tyne (Newcastle Primary Care Trust). The views expressed are those of the authors and not necessarily those of the NHS, UK.No conflicts of interest to declare.


Mycobacterium tuberculosis (MTB) infection, the complex interaction of host immune system and the mycobacteria is associated with levels of cytokines production that play a major role in determining the outcome of the disease. Several single-nucleotide polymorphisms (SNPs) in cytokine genes have been associated with tuberculosis (TB) outcome. The aim of this study was to evaluate the association between previously reported SNPs IL2–330 T>G (rs2069762); IL4–590 C>T (rs2243250); IL6–174 G>C (rs1800795); IL10–592 A>C (rs1800872); IL10–1082 G>A (rs1800896); IL17A -692 C>T (rs8193036); IL17A -197 G>A (rs2275913); TNF -238 G>A (rs361525); TNF -308 G>A (rs1800629) and IFNG +874 T>A (rs2430561) and pulmonary TB (PTB) susceptibility. We conducted a case-control study in individuals from Southern Brazil who were recruited between February 2012 and October 2013 in a high incidence TB city. We performed a multiplex genotyping assay in 191 patients with PTB and 175 healthy subjects. Our results suggest a decreased risk for PTB development associated with the IL17A -197A allele , AA genotype  and A carrier (AG/AA)  and IL6 -174C carrier (CC/CG) . We could not properly analyze IL17A -692 C>T (rs8193036) and IFNG +874T>A due to genotypic inconsistencies and found no evidence of association for the IL2, IL4, IL10 and TNF polymorphisms and PTB. In conclusion, our results show a protective effect of IL17 and IL6 polymorphisms on PTB outcome in Southern Brazilian population.In  Mycobacterium tuberculosis (MTB) [Tuberculosis (TB) is a chronic granulomatous disease that predominantly affects the lungs and is caused by is (MTB) . TB is ris (MTB) . Annuallis (MTB) .IFNG, SLC11A1, IL10, MCP1, and P2RX7 [IFNG +874T>A the presence of the T allele is associated with TB resistance and higher expression of IFN-gamma [Consistent with a long lasting host–pathogen interaction and co-evolution, only 10% of MTB-infected individuals progress towards active disease . The mecnd P2RX7 –23 to beFN-gamma .In fact, cytokines play a key role in driving the appropriate immune response to mycobacteria via activation of inflammatory and immunomodulatory networks orchestrated by both macrophages and T cells . The intIL6 and IL17A genes and PTB outcome showing inconsistent results. The aim of our study was to investigate whether IL6–174 G>C SNP and IL17A -197 G>A polymorphism and additional cytokine genes involved in immune response to MTB  are associated with genetic susceptibility to pulmonary TB (PTB). In this study, the genetic association of polymorphic variants within cytokine genes was evaluated in a case-control study in a PTB population from Southern Brazil.Few studies have investigated the influence of polymorphic variants in This case-control study recruited 191 PTB patients in a reference hospital for TB in Porto Alegre, the capital of Rio Grande do Sul State in southern Brazil between February 2012 and October 2013.We enrolled patients > 18 years old, diagnosed with PTB who were on anti-TB and anti-multidrug-resistant (MDR)-TB treatment during any time between disease diagnosis and the completion of treatment. The information about patients’ medical conditions as results of drug susceptibility test, coexisting of forms of extra-pulmonary TB, diabetes, asthma were extracted through medical records. Serum samples were tested for HIV 1/2 and HCV. Patients were confirmed to have PTB by chest X-ray, sputum smear microscopy and culture according recommendations described in Brazilian National TB guideline . Smear sThe control group was composed by 175 unrelated healthy individuals, over 18 years old, among them 50 household contacts and 125 blood donors recruited at the Hemocentro do Estado do Rio Grande do Sul in Porto Alegre, which is located at the same geographical area that the PTB cases were recruited. All individuals in control group were HIV and HCV or HIV+HCV negatives, without signs or symptoms of TB, previous history of TB or have completed anti-TB prophylaxis. Ethnic background was determined by self-identification as white, non-white, or other. No individual of auto referred Asiatic ancestry participated in the study. All subjects were BCG vaccinated at birth that was confirmed by the presence of the BCG scar.The study was approved by Research Ethics Committee of Fundação Estadual de Produção e Pesquisa em Saúde, FEPPS-RS #09⁄2011 in Porto Alegre, Rio Grande do Sul, Brazil. An information sheet describing the purpose of the study and the individuals’ rights as participants study were read along, and a copy provided, for all volunteers. The researcher also provided a contact phone number for any additional information about the research. After all clarification, a written informed consent was obtained for each person.® Blood kit (Macherey-Nagel Inc.) according to the manufacturer´s instructions. Amplification of regions flanking SNPs in cytokines genes IL2–330 T>G (rs2069762); IL4–590 C>T (rs2243250); IL6–174 G>C (rs1800795); IL10–592 A>C (rs1800872); IL10–1082 G>A (rs1800896); IL17A -197 G>A (rs2275913); IL17A -692 C>T (rs8193036); TNF -238 G>A (rs361525); TNF -308 G>A (rs1800629) and IFNG +874 T>A (rs2430561) was accomplished with a multiplex PCR on 5 to 10 ng genomic DNA, 0.2 μM of each primer  according to the user’s manual recommendation.Genomic DNA was isolated from peripheral blood using Nucleo Spinh primer  and Qiag® Multiplex System ABI Prism . SNaPshot primers are listed in SNP analysis were performed on the SNaPshot™ FastAP™). After heat inactivation of the alkaline phosphatase for 20 minutes at 80°C, 1μL of the labeled products were mixed with 9.5 μL of HiDi™ formamide and 0.5 μL of GeneScan-120 LIZ size standard (Applied Biosystems). They were then separated using an ABIPRISM® 3130xl Genetic Analyzer (Applied Biosystems) with POP-4 ™polymer and with respective run parameters: injection voltage of 1.2 Kv, injection time of 23 seconds run voltage of 15Kv and run time 1200 seconds in a capillary of 36 cm length. Analysis was performed using Gene Mapper ID software version 3.2.1 (Applied Biosystems). In order to confirm the genotyping system at least 10% of samples were sequenced on ABIPRISM® 3130xl Genetic Analyzer (Applied Biosystems).According instructions of user's manual the reactions were performed in a final volume of 10 μL, containing 3.0 μL (0.01 to 0.4 ρmol) of purified multiplex PCR product, 5.0 μL of SNaPshot Multiplex Ready Reaction Mix and 1μl of a pooled of the ten SNaPshot primers described at All statistical analysis was performed using Genetics package—software R version 2.11.1 . GenotypThree-hundred sixty six individuals of the metropolitan area of Porto Alegre, Brazil were included in the study . This ciTo confirm whether we could run our analysis combining household contacts and blood bank donors as one group defined as healthy controls, we first compared all SNPs in both populations and no difference was observed . LikewisIL17A -692 C>T that deviated from HWE in cases and controls and IFNG +874T>A in control group. In order to avoid bias in our analysis IL17A -692 C>T and IFNG +874T>A were excluded of this study.Cases and controls were in accordance with HWE (data not shown) for the SNPs evaluated except for the IL17A 197A>G polymorphism the presence of -197A allele was significantly higher in control group than PTB patients (p = 0.04). Furthermore, the logistic regression model showed a decreased risk for PTB development among individuals with AA genotype  and carriers of A allele (AG+AA) .As observed in IL6 -174G>C polymorphism  was also evaluated in distinct populations reporting contradictory results. In Northern Spain population, the G allele  and GG genotype  were associated with increased risk for PTB development [IL17A (rs2275913) SNP is associated with TB protection or G allele is associated with TB susceptibility, which means rigorously the same.The elopment  whereas elopment , 47. In elopment . HoweverIL17A (rs2275913) has been associated with increased risk for inflammatory diseases [Interestingly, the A allele of diseases ,50 indicdiseases .IL6 gene and PTB susceptibility remain inconsistent [IL6 polymorphisms and TB susceptibility we analyzed the influence of IL6–174 G>C polymorphism on PTB outcome. Our results suggested decreased risk for PTB development among carrier of C allele. The results corroborated previous findings among Pakistani population and confirmed by meta-analysis study [The importance of cytokine IL-6 have been shown studies murine models of mycobacterial infections as well in vaccination with a tuberculosis subunit vaccine demonstrating that IL-6 is needed for optimal T-cell development , 52. Indnsistent . To provis study , 57. It is study . In thisis study . TherefoIL2–330 T>G is not associated with PTB susceptibility, although the role of IL-2 cytokine in modulating the immune response in TB has become evident. Similarly, it was found no evidence of association between PTB and IL4 -590C>T, TNF -308G>A and TNF -238G>A polymorphisms. Our results corroborate with previous reports concerning TB susceptibility in non-Brazilian populations [IL10–1082 and -592, we found no influence of these polymorphisms on PTB outcome. Our result for IL10 polymorphism at -1082 position corroborates with a meta-analysis study in a worldwide-pooled population [In contrast, the present study showed that ulations , 60, 61,ulations . Also, tulations . Concernpulation .IL10 -1082G>A SNP revealed conflicting results for analysis stratified by ethnicity for European and American population [IL10–592 A>C analysis showed no risk association with PTB and a meta-analysis study confirmed such finding for Europeans, Africans, and Americans populations but reported a decreased risk for TB development among the Asian population [IL10 promoter region are in perfect linkage disequilibrium [Interestingly, more recent meta-analysis studies that performed subgroup analysis for the pulation , 64. Furpulation . In thisilibrium .IL17A -197G>A and IL6 -174G>C on PTB outcome. Indeed, our findings support IL17A (rs2275913) and IL6 (rs1800795) polymorphisms as promising markers for PTB risk and indicate the role of IL-6 and IL-17A cytokines in immunophysiology of TB. This novel information of host response to MTB infection can help design more effective vaccines, identify risk populations and new treatment strategies. In the future, additional larger studies are needed to validate our findings. Moreover, a dense SNP mapping on IL6 and IL17A loci could be interestingly for better elucidate such association.The use of popular case-control designs to test candidate genes or genome-wide association studies have proved suitable to pinpoint functional genetic variants in complex diseases . SometimS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Three dimensional structures also predicted that H1N1 NA globular head contained 4 and that of H5N1 contained 2 potential glycosylation sites. H5N1 NA protein had higher sialidase activity than H1N1pdm NA protein as measured by both MUNANA-based assay and fetuin-based enzyme-linked lectin assay (ELLA). Plaque reduction assay demonstrated that anti-NA antibody could reduce number of plaques and plaque size through inhibiting virus release, not virus entry. Assay for neuraminidase-inhibition (NI) antibody by ELLA showed specific and cross reactivity between H5N1 NA and H1N1pdm NA protein derived from reverse genetic viruses or wild type viruses. In contrast, replication-inhibition assay in MDCK cells showed that anti-H1N1 NA antibody moderately inhibited viruses with homologous NA gene only, while anti-H5N1 NA antibody modestly inhibited the replication of viruses containing homologous NA gene and NA gene derived from H1N1pdm virus. Anti-H1N1 NA antibody showed higher titers of inhibiting virus replication than anti-H5N1 NA antibody, which are consistent with the results on reduction in plaque numbers and sizes as well as in inhibiting NA enzymatic activity. No assay showed cross reactivity with reassorted PR8 (H1N1) virus and H3N2 wild type viruses.Immune sera collected from BALB/c mice immunized with these recombinant viruses were assayed for binding and functional activities of anti-NA antibodies. Recombinant NA proteins were found localized in cytoplasm and cytoplasmic membrane of the infected cells. H1N1pdm NA protein had MW at about 75 kDa while it was 55 kDa for H5N1 NA protein. Hyperglycosylation was more pronounced in H1N1pdm NA compared to H5N1 NA according to  Influenza virus contains two major glycoprotein spikes: neuraminidase (NA) and hemagglutinin (HA) on the virion surface. HA binds sialic acid receptor on the host cell membrane and serves as the major target for neutralizing antibodies , whereasVirions contain 4–5 times more HA molecules than NA molecules; thus, HA is the immunodominant antigen for the induction of antibody responses . It has in vitro assay to measure protection mediated by anti-NA antibody. Most of the studies measured NA antibodies using enzyme-linked immunosorbent assay (ELISA) that determines the binding activity of anti-NA antibody, or by NA-inhibition (NI) assays that measure the antibody which blocks NA enzymatic activity employing thiobarbituric acid (TBA)-based assay or fetuin-based enzyme-linked lectin assay (ELLA) [HA antibody at titers equal to or greater than 40 as measured by hemagglutination-inhibition (HI) assay were suggestive of 50% protection against influenza virus infection . Moreovey (ELLA) , 28–30. y (ELLA) , 31–33.NA gene of highly pathogenic avian influenza (HPAI) H5N1 or 2009 pandemic H1N1 virus were constructed. The recombinant NA proteins expressed in the infected thymidine kinase negative (TK−) cells were characterized in comparison to those produced in the Madin-Darby canine kidney (MDCK) cells infected by the parental wild type viruses. Antisera collected from mice immunized with these recombinant viruses were assayed for binding and functional activities of anti-NA antibodies against homologous and heterologous strains and subtypes using ELLA, plaque reduction assay and replication-inhibition assay which mimics microneutralization test.In the present study, recombinant vaccinia viruses harboring Influenza viruses used in this study were two HPAI H5N1 viruses including A/Thailand/1(KAN-1)/2004 clade 1 virus (KAN-1 virus) and A/Laos/Nong Khai 1/2007 clade 2.3.4 virus (NK-1 virus); four of H1N1 pandemic 2009 viruses including A/Thailand/104/2009 (H1N1) virus (TH 104 virus-a strain isolated from the second imported case in Thailand), A/Thailand/SEA-001(34)/2010 (H1N1) virus (a H1N1pdm virus isolated from the 2nd epidemic wave), A/Thailand/RMSC_18/2010 (H1N1) virus (a H1N1pdm virus from the 3rd epidemic wave), and A/Thailand/NMA_1/2014 (H1N1) (a H1N1dpm virus isolated in 2014). Two H3N2 strains including A/Thailand/NKS_1/2011 (H3N2) (A/Perth/16/2009-like/virus) and a local isolate A/Victoria/361/2011-like virus were included as controls. These viruses were propagated in MDCK cells maintained in Earle’s minimal essential medium (EMEM)  without serum supplement. The virus growth media, except for that of HPAI H5N1 virus, was also supplemented with trypsin-tosylphenylalanylchloromethyl ketone (trypsin-TPCK) .NA gene of H5N1 KAN-1 (rVac-H5N1 NA virus) or TH 104 virus (rVac-H1N1 NA virus). Both the vaccinia vaccine strain and the recombinant viruses were propagated in TK− cells and maintained in EMEM supplemented with 2% fetal bovine serum (FBS) (Gibco).Vaccinia virus strain Lister was kindly provided by the Thai Government Pharmaceutical Organization. This vaccinia vaccine strain was used as the parental virus for the construction of recombinant vaccinia viruses carrying NA gene was amplified by a OneStep RT-PCR kit (Qiagen) using universal primers designed by Hoffmann, et al [XmaI. The sequences of this primer pair were SXm-NA-1: 5′-TATTCCCGGGAGAGCAAAAGCAGGAGT-3′ and SXm-NA-1413R: 5′-ATATCCCGGGTATTAGTAGAAACAAGGAGTTTTTT-3′. The amplified products were cloned into a pGEM-T Easy vector using T4 DNA ligase  before being transformed into Escherichia coli JM109. Thereafter, the recombinant plasmids were digested with XmaI  and subcloned into the pSC11 expression vector  which was also cut by XmaI. The insertion site of XmaI is located downstream of the vaccinia virus p7.5 promoter together with the E. coli lacZ gene that encodes for β-galactosidase under a p11 promoter, and is flanked with thymidine kinase sequences (TKR and TKL) [E. coli was transformed with the recombinant pSC11 plasmids and plated onto Luria-Bertani agar containing 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside  (Promega) plus ampicillin as a selective marker. The recombinant plasmids were extracted, checked for orientation of the NA DNA insert by cutting the purified recombinant plasmids with restriction enzymes BamHI (New England Biolabs) and the sequence of the insert gene was confirmed by DNA sequencing.The rVac-H1N1 NA virus was constructed as described previously for rVac-H5N1 NA virus . Brieflyn, et al  but modiand TKL) . E. coli− cell monolayer pre-infected with wild type vaccinia virus at a multiplicity of infection (m.o.i.) of 0.01 plaque forming unit (pfu)/ml for 2 hours. The transfected culture was further incubated for 2 days to allow virus replication. The recombinant vaccinia virus was differentiated from the wild type vaccinia virus by plaque selection on the TK− cell monolayer maintained in low melting point agarose containing 5-bromo-2′-deoxyuridine (Sigma Aldrich) and X-gal, in which the plaques produced by cells infected with the recombinant vaccinia virus appeared blue. Plaque purification was performed three times to obtain a single clone of the recombinant virus that was further propagated and titrated in TK− cells by plaque assay.To construct rVac-H1N1 NA virus, a mixture of pSC11 recombinant plasmids and DMRIE-C transfection reagent  was inoculated onto the TKHA and/or NA genes of interest were constructed by reverse genetic technique using 8 recombinant pHW-2000 plasmid backbones of A/Puerto Rico/8/1934 (H1N1) (PR8 virus) origin [HA and NA genes were amplified using universal primers [TransIT-LT1 solution  were used to transfect the co-cultures of MDCK cells and human embryonic kidney 293 T (HEK-293T) cells maintained in Opti-MEM (Gibco). The inoculated cell monolayers were incubated at 37°C in a CO2 incubator and observed daily for cytopathic effects (CPE).Reassortant viruses containing ) origin . This se primers . The ampFour rg-viruses were generated in the present study, including the rgPR8 parental virus, rgH5N1-HANA (6+2) and rgH5N1-NA (7+1) clade 2.3.4 virus derived from NK-1 (H5N1) virus, rgH1N1-HANA (6+2) and rgH1N1-NA (7+1) viruses derived from TH 104 H1N1pdm virus. Moreover, this study also included two rg-viruses, i.e., rgH5N1-HANA (6+2) and rgH5N1-NA (7+1) clade 1 viruses derived from KAN-1 (H5N1) virus kindly provided by Prof. Prasert Auewarakul, Mahidol University, Thailand.− cells infected with recombinant vaccinia viruses. The cells infected with rVac-H1N1 NA virus were stained with goat antiserum against N1 NA kindly provided by Prof. Robert G. Webster; and cells infected with rVac-H5N1 NA virus were stained with goat antiserum against recombinant NA proteins from A/Vietnam/1203/2004 (H5N1) and A/Hong Kong/483/1997 (H5N1) viruses kindly provided by BEI Resources through the NIH Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH . Fluorescein isothiocyanate (FITC) conjugated-rabbit anti-goat Ig  was used as the secondary antibody. The slides were then counter stained with trihydrochloride trihydrate  and examined for presence of the recombinant NA protein under a laser scanning confocal microscope .Confocal immunofluorescence assay (IFA) was conducted to demonstrate the expression and localization of NA proteins in TKConventional IFA was also performed to determine the titers of anti-NA binding antibodies in mouse sera collected at 4 or 6 weeks after immunization. Deposits of MDCK cells infected with the wild type virus were used as the test antigen, and FITC-conjugated goat anti-mouse Ig  was used as the secondary antibody. The reciprocal of the highest serum dilution that yielded 2+ fluorescence intensity was consider the antibody titer.− cells infected with recombinant vaccinia virus or MDCK cells infected with wild type influenza virus. The infected cell pellets were lysed with RIPA buffer containing 50 mM TrisCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulphate (SDS). Reference NA proteins included recombinant NA derived from A/Vietnam/1203/2004 (H5N1) virus expressed in mouse myeloma cells (NS0) (r-H5N1 NA)  and recombinant NA protein derived from A/California/04/2009 (H1N1) virus expressed in the baculovirus-insect cell system (r-H1N1 NA) . Cell lysates or reference antigens were mixed with 4× reducing sample buffer  and boiled for 10 minutes prior to electrophoresing in 12% denaturing discontinuous sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresed proteins in gel were then blotted onto a nitrocellulose membrane  using Trans-Blot semidry transfer cell . The blotted membrane was blocked with 1% bovine serum albumin in phosphate buffer saline plus 0.1% Tween-20 and incubated with rabbit polyclonal antibody against NA protein of A/Vietnam/1203/2004 (H5N1) or rabbit polyclonal antibody against NA protein of A/California/06/2009 (H1N1)  Thereafter, the membrane was incubated with horseradish peroxidase (HRP) conjugated-goat anti-rabbit IgG  as the secondary antibody and a mixture of 3, 3′diaminobenzidine (Sigma-Aldrich), 8% NiCl2, and H2O2 was used as the chromogenic substrate.Western blot (WB) assay was performed to determine the molecular weight (MW) of NA expressed in TK− cells infected with rVac-H5N1 NA or rVAc-H1N1 NA virus were investigated for post-translational glycosylation in comparison to those synthesized in MDCK cells infected with the parental wild type virus. The infected cells were lysed with RIPA followed by treatment with N-glycosidase F (PNGase F) (New England Biolabs) for digestion of the glycosylated protein. According to the manufacturer’s protocol, the cell lysates were mixed with 10× glycoprotein denaturing buffer and heat at 95°C for 10 minutes. The denatured proteins were then added with 10× G7 reaction buffer, 10% NP40 and PNGase F enzyme. The reaction was incubated at 37°C for 1 hour, and the digested products were visualized by WB assay.Recombinant NA proteins expressed in TK− cells infected with rVac-H5N1 NA or rVAc-H1N1 NA virus was determined by MUNANA assay and ELLA. MUNANA assay was used to demonstrate the cleavage of a fluorescent substrate, 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid sodium salt hydrate (MUNANA), by influenza NA; and the yield of free 4-methylumbelliferone (4-MU) fluorescent product was then quantitated based on its fluorescence intensity. Briefly, TK− cells were infected with rVac-H5N1 or rVac-H1N1 NA virus at m.o.i. of 0.01 and incubated at 37°C for 3 days, the time at which the degree of the CPE was 4+. The supernatants were discarded; the virus infected cells were resuspended with 2% FBS EMEM and underwent 3 cycles of freezing and thawing to release NA proteins and viruses from the infected cells. The amounts of NA proteins expressed in TK− cells infected with rVac-H5N1 NA or rVac-H1N1 NA viruses were verified by determining for the intensity of various proteins present in the infected cell lysates using SDS-PAGE and Coomassie blue staining. Furthermore, the cell lysates were also titrated for concentration of the recombinant vaccinia viruses. The cell lysates containing 5×108 pfu/ml was serially diluted in a 10-folded dilution manner with 2-morpholinoethanesulfonic acid (MES) assay buffer  and a 20 μl volume was added to 30 μl of MUNANA substrate at concentration of 100 μM. The reaction well was incubated at 37°C for 60 minutes with shaking in the dark before terminating with 150 μl of the stop solution . The reaction plate was read under a spectrophotometer to determine for relative fluorescence units (RFU) using an excitation wavelength of 355 nm and an emission wavelength of 460 nm. The RFU values were plotted against numbers of pfu to establish the dose response curve indicating the NA enzymatic activity.Neuraminidase  enzymatic activity of recombinant NA proteins expressed in TK8 pfu/ml was serially 10 fold-diluted and each dilution in a 50 μl volume was added into duplicate wells of a 96 well plate pre-coated with fetuin. The recombinant NA cleaved the sialic acid side chain of fetuin and yielded small cleavage products that were washed out while leaving the carbohydrate moieties adhered to the wells. These moieties were subsequently detected by HRP conjugated-peanut lectin using o-phenylenediamine dihydrochloride (OPD) as the chromogenic substrate. The reaction plate was read under a spectrophotometer at an optical density (OD) of 492 nm. The OD values were plotted against numbers of pfu to establish the dose response curve indicating the NA enzymatic activity.The same preparation of cell lysate as described above was also investigated for NA enzymatic activity by fetuin-based ELLA using the protocol established by Dr. Maryna Eichelberger . The cel7 pfu/head and the last group was the unimmunized control. Three independent lots of mice were used in the experiments. The mice were bled by cardiac puncture; the first lot was sacrificed at 4 weeks and the second and the third lots at 6 weeks after immunization. Mouse sera were aliquoted and stored at −20°C and pooled for anti-NA antibody testing.BALB/c mice were purchased from the National Laboratory Animal Center, Mahidol University and housed in the Faculty of Veterinary Science, Mahidol University. Use of experimental animals was approved by the Animal Care and Use Committee of Faculty of Veterinary Science, Mahidol University (Permit Number: MUVS-2013-37). All activities concerned with the animals were carried out by the experienced veterinarians. The physical condition of animals was monitored once a day throughout the end of the experiments, and all animals were healthy. No animal was ill or died prior to the experimental endpoint. At time of blood collection, the animals were anesthetized using isoflurane inhalation. When the animals were unconscious, whole blood was collected by cardiac puncture. Death was confirmed by physical examination. Mice of age six weeks were divided into five groups; and each group comprising 5–6 mice was inoculated intraperitoneally once with rVac-H1N1 NA, rVac-H5N1 NA, rVac-pSC11or wild type vaccinia virus plus 10 μg of poly I:C (Sigma-Aldrich) at the inoculums dose of 10Mouse sera were investigated for anti-NA antibody that inhibited NA enzymatic activity by ELLA. The test virus was two-fold serially diluted to titrate for NA activity before subjecting to anti-NA antibody detection as described above. Each virus dilution was plotted against its OD value to establish a titration curve. An OD value of about 2.0 was extrapolated against the titration curve to determine the optimal virus dilution for further use in neuraminidase inhibition (NI) assay.For NI antibody assay, heat-inactivated mouse serum sample at a dilution of 1:10 was 2-fold serially diluted and added in duplicate into a fetuin coated plate together with the test virus at working concentration. After overnight incubation, the amount of carbohydrate moieties produced after digestion of sialic acid was determined as described above. The NI antibody titer was defined as the highest serum dilution that yielded a 50% reduction of the OD value compared with the virus control without serum.The mouse sera were treated with receptor-destroying enzyme (RDE)  at 37°C for 16–18 hours followed by heat inactivation at 56°C for 30 minutes. Confluent MDCK cell monolayers in 6-well tissue culture plates were infected with either TH 104, rgH5N1-HANA (6+2) clade 1 or rgPR8 virus at the inoculums dose of 50 pfu per well. The viruses were absorbed for 2 hours at 37°C and the cell monolayers were washed twice and maintained with 3 ml of 1.2% Avicel in EMEM containing RDE-treated antisera at various dilutions. The infected cell monolayers maintained with 1.2% Avicel containing either normal mouse sera or none were assayed in parallel as controls. After incubation for 48 hours at 37°C, the cell monolayers were fixed with 10% paraformaldehyde, stained with 1% crystal violet and count for number of plaques.Replication-inhibition assay mimicking the microNT assay in MDCK cell monolayers  was esta− cells infected with rVac-H5N1 NA or rVac-H1N1 NA virus was determined by confocal IFA. The result showed a high level of expression of recombinant NA proteins which localized both in the cytoplasm and on the cytoplasmic membrane of the infected cells  and the protein molecular weight calculator (http://www.sciencegateway.org/tools/proteinmw.htm).The differences in the patterns of H1N1 NA proteins produced from various sources suggested that their levels of post-translational glycosylation might be different. Lysates of MDCK cells infected with wild type TH 104 virus and TKy 50 kDa , close t− cells. After PNGase F treatment, the MW of H5N1 NA protein produced in TK− cells infected with recombinant vaccinia virus was reduced from 55 to 50 kDa, while no change in MW was observed with that produced in MDCK cells infected with wild type virus  derived from any of the two infected cultures was comparable, demonstrating that the H5N1 NA and the H1N1 NA were expressed at similar level (9 pfu/ml.Lysates of TKar level . Moreovep < 0.001 for both assays). Lysate of cells infected by rVac-pSC11 virus as negative control did not exhibit NA enzymatic activity   . It was  < 0.05) . Normal  < 0.05) . Phyloge < 0.05) . ConcordThe plaque reduction assay was performed to demonstrate the role of anti-NA antibodies at virus entry or virus exit. The rgH5N1 (6+2) clade 1, TH 104 or rgPR8 viruses were used as the test viruses. To determine the inhibition of virus entry mediated by anti-NA antibodies, the test viruses were incubated with the diluted mouse antisera before inoculating onto wells of MDCK cell monolayers. The serum-virus mixtures were washed out and the infected cultures were maintained in media without anti-NA antisera. The result shows no reduction in number of plaques and plaque size mediated by anti-H5N1 NA or anti-H1N1 NA antisera, suggesting that anti-NA antibody did not inhibit the virus entry.To determine the inhibition of virus release, the MDCK cell monolayers were infected with the test virus prior to adding with the media containing anti-NA antisera at various dilutions. Using the sera at dilution of 1: 100 as the cut-off value, the result demonstrated that anti-H5N1 NA antisera reduced the number of plaques of rgH5N1 virus by 25%, and 80% of the plaques were at size of 1 mm compared to the cultures treated with normal mouse sera in which 80% of the plaques were at size of > 2 mm in average . Anti-H1p > 0.05). Concordant results were obtained from the two lots of mouse sera collected at 6 weeks p.i..Mouse immune sera at a starting dilution of 1:20 was 2-fold serially diluted and assayed against various viruses at 3 test concentrations, 100, 25 and 10 TCID50. The result showed that antisera from mice immunized with rVac-H5N1 NA virus did not inhibit the replication of rgH5N1 virus at the concentration of 100 TCID50, but poorly inhibited TH 104 H1N1 virus at the same virus concentration. When concentrations of the test viruses were lowered to 25 and 10 TCID50, some degree of inhibition against both rgH5N1 and H1N1 viruses was observed. Sera from mice immunized with rVac-H1N1 NA virus exhibited inhibitory activity only on homologous H1N1 virus at all 3 test concentrations investigated, but no cross replication inhibition was observed for rgH5N1, rgPR8 and wild type H3N2 virus . The repNA gene insert derived from HPAI H5N1 virus or 2009 pandemic H1N1 virus were constructed and used as the immunizing agents to raise anti-NA antibodies in BALB/c mice. With these recombinant viruses, the NA protein was produced without interference from other influenza genes. These recombinant NA proteins were characterized for their biological and immunological properties. Similar to the previous reports [− cells infected with recombinant vaccinia virus. However, NA proteins encoded by different virus strains and in different cell types had different MW as determined by WB assay. The TK− cells infected with rVac-H1N1 NA virus displayed a band of NA protein at MW of 75 kDa; whereas MDCK cells infected with H1N1 wild type virus displayed two bands of NA proteins at MW of 75 and 55 kDa. After PNGase F digestion, only one band at MW of 50 kDa was obtained. This MW is close to 51.6 kDa, the size of NA protein determined from the amino-acid sequence using web-based tools. This finding suggested that H1N1 NA protein was hyperglycosylated by N-linked oligosaccharides. Nevertheless, the other study showed that inactivated A/California/7/2009 (H1N1) virus  or Vero cells infected with recombinant vaccinia virus Lister strain carrying NA derived from Cal virus or DF-1 chicken cells infected with recombinant modified vaccinia virus Ankara strain carrying NA gene derived from Cal virus expressed only one form of NA protein at MW of 75 kDa [− cells infected with rVac-H5N1 NA virus had the same MW of 55 kDa. The higher MW of H1N1 NA may be due to higher number of glycosylation sites as predicted by our previous study [Herein, recombinant vaccinia viruses harboring  reports , 43, we f 75 kDa . Our stuus study , and alsus study , A/Victous study  and A/neus study . Unfortuus study . Moreoveus study .in vitro studies have explored the mechanism of partial protection mediated by anti-NA antibodies. Ferrets immunized with the 2009 H1N1 pandemic virus developed anti-NA antibodies that cross inhibited HPAI H5N1 virus and vice versa as shown by NI assay and plaque size reduction assay [Partial protection mediated by anti-NA antibodies had been suggested based on epidemiological data in human population as well as the study in animal models. Few on assay . Chickenon assay . On the on assay . These rNA gene originated from A/chicken/Yamaguchi/7/04 (H5N1) virus and followed by one booster, exhibited high NI activity against homologous virus and to a lesser extent to the heterologous viruses of the same NA subtype, i.e., PR8 (H1N1) and A/New Caledonia/20/99 (H1N1) viruses [In the present study, antisera from mice immunized with rVac-H5N1 NA of clade 1 origin or rVac-H1N1 NA virus were assayed for their functional activities of anti-NA antibody by ELLA, plaque reduction assay and replication-inhibition assay in MDCK cell monolayers. Using ELLA, we showed that anti-H5N1 NA antibody blocked NA enzymatic activity of the rgH5N1 clade 1 and clade 2.3.4 viruses to the high antibody titers, but poorly cross inhibited the H1N1 NA. In contrast, anti-H1N1 NA antibody strongly inhibited both H1N1 NA and H5N1 NA. Nevertheless, both anti-H5N1 NA and anti-H1N1 NA antibody did not inhibit PR8 NA or H3N2 NA. Cross reactivity between anti-NA antibodies against H1N1 pandemic 2009 and H5N1 viruses were previously reported , 47, 48. viruses .Using plaque reduction assay, our study demonstrated that anti-NA antisera could reduce both the number of plaques and plaque size compared to the serum controls. However, the inhibitory activity of anti-NA antisera on plaque formation varied according to virus strain. We further investigated for the mechanism of action of NA antibody on virus entry and virus release using the test virus with homologous NA. Our result demonstrated that anti-NA antibody blocked the virus release, but not virus entry (data not shown). The blocking of influenza virus release by anti-NA antibody was well documented by various groups of investigators , 46, 50.in vitro technique used to demonstrate the inhibition of virus replication mediated by anti-NA antibody. Antibody targeting H1N1pdm NA inhibited the replication of H1N1pdm virus only, but not inhibited rgH5N1 viruses, rgPR8 virus and H3N2 viruses. Antibody to H5N1 NA elicited an inhibition against rgH5N1 viruses at virus concentrations of 25 and 10 TCID50, but not 100 TCID50; while cross inhibition against H1N1pdm virus was observed with all 3 virus concentrations. This finding suggested that anti-NA antibody might contribute only a modest replication-inhibition effect against highly virulent H5N1 virus. It was noted that mice immunized with rVac-H1N1 NA virus developed higher anti-NA antibody titer than those immunized with rVac-H5N1 NA virus, either determined by IFA, NI assay, plaque reduction assay or replication-inhibition assay.The present study developed a method termed “replication-inhibition assay”, a simple Mouse sera collected at 6 weeks p.i. had a significantly higher NA inhibitory activity compared with those collected at 4 weeks p.i. as determined by ELLA, while no significant difference was observed by replication-inhibition assay. The measurement of NI antibody titers in term of the OD values made the NI assay more sensitive than the replication-inhibition assay. On the other hand, we could not exclude that the NI antibody and the replication-inhibiting antibody bind to different epitopes on the NA molecule, as it had been shown that monoclonal antibody without NI activity could protect mice against the lethal virus challenge .Currently, the importance of NA antigen in influenza vaccines has been widely discussed. We demonstrated that NA protein could induce moderate level of antibody that might prevent influenza virus infection through various mechanisms. Although NA antibodies were detected after immunization with inactivated vaccine , the amo


Translation initiation is activated in cancer through increase in eukaryotic initiation factor 4E (eIF4E), eIF4G, phosphorylated eIF4E-binding protein (p4E-BP1) and phosphorylated ribosomal protein S6 (pS6), and decreased programmed cell death protein 4 (pdcd4), a translational inhibitor. Further, translation elongation is deregulated though alterations in eukaryotic elongation factor 2 (eEF2) and eEF2 kinase (eEF2K). We sought to determine the association of these translational aberrations with clinical-pathologic factors and survival outcomes in hormone receptor-positive breast cancer.Primary tumors were collected from 190 patients with Stage I to III hormone receptor-positive breast cancer. Expression of eIF4E, eIF4G, 4E-BP1, p4E-BP1 T37/46, p4E-BP1 S65, p4E-BP1 T70, S6, pS6 S235/236, pS6 S240/244, pdcd4, eEF2 and eEF2K was assessed by reverse phase protein arrays. Univariable and multivariable analyses for recurrence-free survival (RFS) and overall survival (OS) were performed.High eEF2, S6, pS6 S240/244, p4E-BP1 T70, and low pdcd4 were significantly associated with node positivity. Median follow-up for living patients was 96 months.P = 0.008). In addition to age, pS6 S235/236 , eEF2K  and pdcd4  were associated with OS.High p4E-BP1 T36/47, p4E-BP1 S65, p4E-BP1 T70 and 4E-BP1 were associated with worse RFS. High p4E-BP1 T70 and pS6 S235/236, and low pdcd4, were associated with worse OS. In multivariable analysis, in addition to positive nodes, p4E-BP1 S65 remained a significant predictor of RFS (HR = 1.62, 95% CI = 1.13-2.31; Increased pS6, p4E-BP1, eEF2K and decreased pdcd4 are associated with poor prognosis in hormone receptor-positive breast cancer, suggesting their role as prognostic markers and therapeutic targets. Further, formation of the eIF4F complex determines the sensitivity to chemotherapy, as well as anticancer drugs targeting HER2 and EGFR [Control of mRNA translation to protein is an important point of regulation for gene expression. Translation is deregulated in cancer through a variety of mechanisms . The mosand EGFR .in vitro [P = 0.0023), and the overexpression of 4E-BP1 and eIF4G have been proposed to orchestrate a hypoxia-activated switch from cap-dependent to cap-independent mRNA translation that promotes increased tumor angiogenesis and local tumor growth [Activated translation initiation is essential for the malignant breast cancer phenotype. eIF4E is overexpressed in breast cancer and has been suggested to be an indicator of poor prognosis ,12. Overin vitro . Translain vitro . Expressr growth .eIF4G1 is also overexpressed in inflammatory breast cancer, where it reprograms the translational machinery to increase translation of mRNA with internal ribosome entry sites (IRES) that promote cell survival and tumor emboli .eIF4E-binding proteins (4E-BP) compete with eIF4G for a binding site in eIF4E. The binding of 4E-BP1 to eIF4E is regulated by phosphorylation; 4E-BP1 hyperphosphorylation decreases this binding, increasing eIF4E availability to engage the cap initiation complex eIF4F. 4E-BP1 is phosphorylated on multiple residues: T37, T46, S65, T70; phosphorylation at least in part is regulated through PI3K/Akt/mTOR signaling. High levels of phosphorylated eIF4E-binding protein 1 (p4E-BP1) have been associated with worse prognosis in several tumor types including breast cancer . FurtherProgrammed cell death protein 4 (pdcd4) is a tumor suppressor protein that inhibits breast cancer cell invasion. Pdcd4 inhibits protein translation by binding to the translation initiation factor eIF4A. Pdcd4 is targeted for degradation during tumor promotion . Pdcd4 uin vitro [Ribosomal protein S6 (S6) is a component of the 40S ribosomal subunit that mediates translation initiation. In response to mitogenic stimuli, S6 undergoes phosphorylation by S6K1 and p90 ribosomal S6 kinases on four serine residues ; these modifications potentiate S6 cap-binding activity. S6 phosphorylation correlates with increased translation of mRNA with 5' terminal oligopyrimidine tracts in some studies conditions, but not in others. S6 is also proposed to subsequently undergo casein kinase1-dependent phosphorylation of S247; phosphorylation of S6 promotes its association with the mRNA cap-binding complex in vitro . Thus S6Eukaryotic elongation factor-2 kinase (eEF2K) phosphorylates and inactivates eukaryotic elongation factor 2 (eEF2), an elongation factor that controls the rate of peptide chain elongation. The activity of eEF2 is increased in several tumor types including breast cancer ,23. eEF2Thus, taken together, a significant amount of data has accumulated suggesting an important role for translational dysregulation in breast cancer. It remains unclear, however, which of these alterations are the most significant determinants of cancer progression and poor oncologic outcomes. We sought to determine the association of translational regulators with clinical-pathologic factors and survival outcomes in hormone receptor-positive breast cancer.Primary tumors were collected from 190 patients with Stage I to III hormone receptor-positive breast cancer treated at Hospital Clinico Universitario de Valencia, Spain. Tumors were collected from surgical samples, and tumor content verified by histopathology. Patient cohort was selected based on hormone receptor-positive status, availability of adequate frozen tissue, and subsequent treatment limited to endocrine therapy. All tissues were collected after informed consent for future research. The study was approved by the MD Anderson Cancer Center Institutional Review Board as well as by Hospital Clinico Universitario de Valencia. Tumors were characterized for estrogen receptor (ER) and progesterone receptor (PR) status by immunohistochemistry. ER/PR positivity was designated when nuclear staining occurred in ≥10% of tumor cells. Hormone receptor positivity was designated when either ER or PR was positive. All patients were treated with adjuvant endocrine therapy (tamoxifen); none received chemotherapy. HER2 testing was not routinely performed; none of the patients received HER2-targeted therapy. None of the patients received neoadjuvant therapy.3VO4; 10% glycerol, 1x complete protease inhibitor cocktail ). After centrifugation, supernatant was transferred to a fresh tube and protein concentration was corrected to 1 μg/μL.Reverse phase protein arrays (RPPA) was performed in the MD Anderson Cancer Center Functional Proteomics RPPA Facility as described previously ,27. BrieThe supernatants were subsequently manually diluted in five-fold serial dilutions with lysis buffer. An Aushon Biosystems  2470 Arrayer created 1,056 sample arrays on nitrocellulose-coated FAST slides  from the serial dilutions. Slides were then probed with primary antibodies including eIF4E, eIF4G, 4E-BP1, p4E-BP1 T37/46, p4E-BP1 S65, p4E-BP1 T70, S6, pS6 S235/236, pS6 S240/244, pdcd4, eEF2 and eEF2K  was calculated by using the R package 'fdrtool'. Box plots were used to display the significant proteins. The FDR threshold of 0.05 was used for declaration of significance.RPPA data from 190 hormone receptor-positive and Stage I to III patients was median-polish normalized. The samples were tabulated and described according to their clinical characteristics. Two sample P values (P <0.05).For each of the proteins of interest, a univariable CoxPH model was established for both recurrence-free survival (RFS) and overall survival (OS). For multivariable analysis, a boosting approach (R package CoxBoost) was employed to develop a Cox proportional hazard model, and to select predictors. Beside age, nodal status, and T stage, all other factors were treated as optional covariates. As the main model complexity parameter, the number of boosting steps, stepno, was selected with cross-validation by using cv.CoxBoost. The penalty parameter was chosen by using optimCoxBoostPenalty. The predictors with estimated nonzero coefficients were considered to be incorporated in the final multivariable model. Age, nodal status and T stage were also retained as mandatory covariates in the final model because of their clinical significance. The final model selection was undertaken based on a backwards selection procedure, during which all factors of interest identified by CoxBoost were incorporated in a full model and then variables were retained according to their P values less than 0.05 were considered statistically significant and all tests were two-sided. All statistical analysis has been done with R statistical software version 2.11.0.In order to divide the patients into two groups (high and low) based on expression levels of factors in the final multivariate model, a regression tree method of R package 'rpart' was applied to find the best cutoff points for p4E-BP1 S65, pS6 S235/236, eEF2K and pdcd4. Five-year survivals for RFS or OS were estimated between each protein's high and low expression groups. Logrank tests were used to evaluate statistical significance. Kaplan-Meier curves by expression level group were presented as well. The patient characteristics are shown in Table P = 0.016).Next, we determined whether expression/phosphorylation of specific translational factors correlated with clinical-pathologic characteristics. Table S2 in Additional file Next, we determined association between translational regulators and axillary nodal status. Higher expression of eEF2, S6, pS6 S240/244 and p4E-BP1 T70 was significantly associated with node positivity  there were 47 recurrences and 65 deaths. The median follow-up for living patients was 96 months. In order to identify predictive factors, a Cox proportional hazard model including all of the 14 factors as optional predictors had been established at first. For each of the proteins of interest, a univariable CoxPH model results for both RFS and OS are displayed in Table P values are less than 0.05 (P <0.05).A boosting approach (R package CoxBoost) is applied to determine the corresponding significance. Next, a full multivariate model has been developed that incorporates all the factors, which have survived from the boosting approach and clinic variables based on their statistical or clinical significance. The final model selection is undertaken through a backwards selection procedure, during which the factors of interests are retained if their P = 0.008). The final multivariable models of RFS and OS are presented in Table P ≤0.001, Figure When age, nodal status and T stage were added to the model, in addition to positive nodes, p4E-BP1 S65 remained a significant predictor of RFS (hazard ratio (HR) = 1.62, 95% confidence interval (CI) = 1.13-2.31, P = 0.039), eEF2K  and pdcd4   (on S359), RSK and S6K1 (on S366), and phosphorylated and activated by AMPK (on S398) . Thus eEt cancer . Thus, EOur study has some limitations. All patients in this study received endocrine therapy. Thus, it is not possible to determine whether p4E-BP1, pS6, eEF2K and pdcd4 are associated with prognosis or whether they are associated with endocrine therapy resistance. Patients received adjuvant tamoxifen; however, the exact duration of adjuvant treatment with tamoxifen, and compliance with the medication, is unknown. Further work is needed to determine whether these markers are also prognostic in patients who received aromatase inhibitors. We do not have detailed information on locoregional management including radiation therapy, and sites of relapse including locoregional recurrence, thus we are unable to dissect the role of translational regulators on locoregional control. We did not have information on comorbidities and cause of death, thus we are able to determine recurrence-free survival and overall survival but not disease-specific survival. Information on treatment after relapse was also not available. Further work is also ongoing to determine the intratumoral heterogeneity and reproducibility of these markers, and to transition these markers to alternate platforms such immunohistochemistry and multiplex proteomics assay such as Luminex or ELISA that may be used clinically to identify patients who would have a poor prognosis if treated with endocrine therapy alone. Another limitation of our study is that we do not have transcriptional profiling data from the same patients; this is especially relevant as eIF4E mRNA was shown to be prognostic in luminal B cases, and not in other subtypes . It willIn summary, increased pS6, p4E-BP1, eEF2K and decreased pdcd4 are associated with poor prognosis in hormone receptor-positive breast cancer. Further study is needed to determine the clinical utility of these as prognostic or predictive markers. Our results provide further support for a role for PI3K/mTOR pathway inhibitors in the treatment of hormone receptor-positive breast cancer. The best approach to personalize treatment in hormone receptor-positive breast cancer patients with translational aberrations warrants further study.CI: confidence interval; eEF2: eukaryotic elongation factor 2; eEF2K: eukaryotic elongation factor 2 kinase; eIF4E: eukaryotic initiation factor 4E; eIF4G: eukaryotic initiation factor 4G; ER: estrogen receptor; FDR: false discovery rate; HR: hazard ratio; OS: overall survival; p4E-BP1: phosphorylated eIF4E-binding protein; PR: progesterone receptor; PFS: progression-free survival; S6: ribosomal protein S6; pS6: phosphorylated ribosomal protein S6; pdcd4: programmed cell death protein 4; RFS: recurrence-free survival; RPPA: reverse phase protein array.This work was presented as an oral presentation at the American Association of Cancer Research 2012 Annual Meeting. The manuscript has never been published and is not under consideration for publication elsewhere. The authors have no financial interest to declare.FMB conceived of the study, performed data analysis and drafted the manuscript. HC performed data analysis and assisted with manuscript preparation. AA screened and optimized antibodies for RPPA and performed the RPPA. KAD supervised analysis and assisted with manuscript draft. AL identified patient samples and extracted clinical data. BTH prepared tumor lysates and supervised RPPA and clinical data coordination. GNH supervised analysis and assisted in data interpretation. GBM supervised coordination of samples, RPPA and assisted in data interpretation. AMGA assisted with specimen and data coordination, and data interpretation. All authors read and approved the manuscript.Supplementary tables. Table S1: Antibodies used in the study. Table S2: Translational regulators by T stage in hormone receptor-positive breast cancer patients. Table S3: Translational regulators by nodal status in hormone receptor-positive breast cancer patients.Click here for file


SF3B1 gene, which were found in 1.8% of unselected breast cancers. SF3B1 mutations were significantly associated with ER-positive disease, AKT1 mutations, and distinct copy number alterations. Additional profiling of hotspot mutations in a panel of special histological subtypes of breast cancer showed that 16% and 6% of papillary and mucinous carcinomas of the breast harboured the SF3B1 K700E mutation. RNA sequencing identified differentially spliced events expressed in tumours with SF3B1 mutations including the protein coding genes TMEM14C, RPL31, DYNL11, UQCC, and ABCC5, and the long non-coding RNA CRNDE. Moreover, SF3B1 mutant cell lines were found to be sensitive to the SF3b complex inhibitor spliceostatin A and treatment resulted in perturbation of the splicing signature. Albeit rare, SF3B1 mutations result in alternative splicing events, and may constitute drivers and a novel therapeutic target in a subset of breast cancers. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.Mutations in genes encoding proteins involved in RNA splicing have been found to occur at relatively high frequencies in several tumour types including myelodysplastic syndromes, chronic lymphocytic leukaemia, uveal melanoma, and pancreatic cancer, and at lower frequencies in breast cancer. To investigate whether dysfunction in RNA splicing is implicated in the pathogenesis of breast cancer, we performed a re-analysis of published exome and whole genome sequencing data. This analysis revealed that mutations in spliceosomal component genes occurred in 5.6% of unselected breast cancers, including hotspot mutations in the  TP53 and PIK3CA, and a wide spectrum of genes mutated in a small minority of tumours. However, some of the low-frequency mutations (i.e. present in 0.5–2%) of breast cancers constitute bona fide drivers and therapeutic targets in other cancer types, such as BRAF and KRAS activating hotspot mutations [The ability to characterize entire genomes at base pair resolution using massively parallel sequencing technologies provides a unique opportunity to unravel genotypic–phenotypic associations in breast cancer, which can be exploited for the identification of drivers of tumourigenesis and, ultimately, therapeutic targets. Recent breast cancer sequencing studies have highlighted the complex nature of the landscape of breast cancer genomes, characterizing both the mutational signatures of breast cancer –3 and thutations .SF3B1) are rather prevalent in MDS and found to be associated with a distinct phenotype (i.e. the presence of ring sideroblasts) and favourable clinical outcome [U2AF1), the serine/arginine-rich splicing factor 2 gene (SRSF2), and the U2 small nuclear ribonucleoprotein auxiliary factor 35 kDa subunit-related protein 2 gene (ZRSR2), have also been reported; however, these are less frequent and not associated with ring sideroblasts in MDS patients [SF3B1 in subsets of solid tumours from multiple anatomic sites (see ref 11 for a recent review), including 15% of chronic lymphocytic leukaemias (CLLs) [SF3B1 mutations are reported to be associated with a good prognosis [In addition to known drivers, massively parallel sequencing studies have resulted in the identification of novel mutations in multiple components of the RNA splicing machinery. Somatic mutations affecting different spliceosomal component genes are preferentially found in myeloid neoplasms showing features of myelodysplasia (MDS) and seemingly occur in a mutually exclusive manner . In fact outcome ,11. Mutapatients ,10. Mores (CLLs) , 9.7% ofs (CLLs) , 4% of ps (CLLs) , and 1.8s (CLLs) ,8. Althorognosis ,13,15,16Given that mutations affecting spliceosomal component genes have been reported in multiple tumour types, including breast cancer, and may constitute driver events in a subset of cancers, we performed a systematic re-analysis of publicly available exome, whole genome, and RNA sequencing data available for breast cancers. Our aims were to determine whether mutations affecting spliceosomal component genes are associated with specific breast cancer subtypes and, if present, whether these mutations were associated with distinct splicing events and would constitute targets for therapy in these tumours.SF3B1 mutant tumours available in TCGA were used to assess the heterozygosity at the SF3B1 locus using ASCAT [Exome and whole genome sequencing data for 1293 tumours were obtained from The Cancer Genome Atlas (TCGA) and other published studies . Processng ASCAT .n = 19), mucinous (n = 18), and micropapillary (n = 28) carcinomas were retrieved from the authors' institutions and surveyed for the presence of the SF3B1 K700E hotspot mutation  samples from 65 breast cancers classified as papillary  SNP6 data were retrieved from TCGA for available SF3B1 mutated (n = 3) and wild-type (n = 16) breast cancers was performed using the Ion Torrent AmpliSeq technology according to the manufacturer's instructions  (Supplementary methods and Supplementary Targeted DNA sequencing of ten recurrently mutated genes identified in SF3B1 mutant and 11 SF3B1 wild-type) (Supplementary methods). Four indexed samples were pooled at equimolar concentrations and sequenced on a single lane of a HiSeq 2500 using SBS v3 chemistry  (2 × 76 cycles). Samples were aligned to the human genome (hg19 build 37) using TopHat version 2.0.8b. Raw counts of reads mapped to genes were calculated using HT-Seq (http://www-huber.embl.de/users/anders/HTSeq/doc/overview.html) and used as input for differential exon usage analysis using DEXSeq [p-value cut-off of ≤0.1. FASTQ files from available TCGA RNA-sequencing data from SF3B1 K700E mutant (n = 8) and ER, PR, HER2 status, and PIK3CA and TP53 mutational status, and randomly matched controls (n = 16) were downloaded from the Cancer Genomics Hub  and processed as above (TCGA project access number 6223).RNA sequencing was performed using 16 ng of ribosomal-depleted RNA of 14 papillary carcinomas of the breast  and are available under accession numbers PRJNA234087 and PRJNA229096.Raw targeted re-sequencing and RNA-sequencing data have been deposited in the Sequence Read Archive (SRA)  and sequenced as previously described [http://4peaks.en.softonic.com/). Primer sequences are listed in Supplementary Reverse transcription was performed with Superscript III  using 50 ng of RNA per reaction. escribed . Sequenchttp://primer3.ut.ee) and retrieved from Furney et al [−(CT variant1 mRNA – CT variant2 mRNA). For each splicing event, a two-tailed Mann–Whitney U-test was applied between SF3B1 mutated (n = 3) and wild-type (n = 11) cases.Primers were designed against predicted alternatively spliced exons using Primer 3  and verified by means of Sanger sequencing using the methods and primers described above.Pancreatic Panc 05.04, PANC-1, Capan-1, Capan-2, and endometrial cancer cell lines ESS-1, MFE296, HEC1A, and HEC59 were obtained from ATCC  and DSMZ  and maintained according to recommended growth conditions. The identity of cell lines was confirmed by short tandem repeat (STR) typing using the GenePrint 10 Kit . SF3B1 plus eight genes that were consistently differentially spliced in SF3B1 mutant versus wild-type samples – namely ABCC5, ANKHD1, DYNLL1, F8, RPL31, TMEM14C, UQCC, and CRNDE – were selected for functional evaluation. Splice variant specific siRNAs were designed to differentially spliced genes that showed large enough unique regions to allow targeting of three or more independent oligos (Supplementary −5–10−9m) of spliceostatin A (SSA) . All experiments were performed in triplicate. Survival was assessed with the CellTiter-Glo® cell viability assay after 7 days of drug treatment. Survival curves and estimated SF50 (the drug concentration used following which 50% of cells survive) were calculated using non-linear regression with GraphPad Prism V5.0.Cell lines were plated in 96-well plates and 24 h later, media were supplemented with serial dilutions  were considered statistically significant.Fisher's exact tests using all collated mutation data were used to determine association of co-occurrence or mutual exclusivity between variables as previously described . The ManSF3B1 and PRPF8, and SF3B1 and SON . Coincident spliceosome mutations were also identified in SF3B1 and LUC7L2, and in LUC7L2 and SAP130 in two patients . Of the most common spliceosome gene mutations , mutations in SF3B1 were more common in ER-positive breast cancer  and mutations in SAP130 were more common in ER-negative tumours . Overall, mutations in SF3B1 were the most common spliceosomal component gene mutation and were identified in 1.8% of unselected breast cancers (23/1293), and occurred as a recurrent hotspot T > C base-pair substitution at codon 700, leading to a glutamic acid from lysine (K700E) in 74% (17/23) of mutant cases (SF3B1 occurred at codon 666 (K666Q and K666E) in 9% of mutant tumours (2/23), and non-recurrent mutations included G241*, N626D, A633V, Y765C, and D781E, which were found in 22%  with those of stage, ER, PR, HER2, PIK3CA, and TP53 mutation status-matched SF3B1 K700E wild-type tumours . These observations suggest that SF3B1 K700E mutant tumours have a distinct repertoire of copy number alterations , 56.3% (9/16) of the SF3B1 mutant samples also carried PIK3CA mutations, while mutations in TP53 were not as prevalent  than in matched wild-type cancers . Interestingly, 2 out of 24 (8.3%) mucinous carcinomas of the breast annotated in the data were identified as having SF3B1 K700E mutations and lacked mutations in additional significantly mutated genes or known cancer genes , papillary (n = 19), and micropapillary (n = 28) carcinomas were subjected to Sanger sequencing .Given the apparent association of SF3B1 K700E mutations in papillary carcinomas of the breast, we hypothesised that these mutations may underpin their biology and may be present at additional hotspots and/or be sub-clonal in the ‘wild-type’ samples. To address these questions, the 19 papillary carcinomas were subjected to targeted DNA re-sequencing of SF3B1 exons 14–16 and of nine additional genes, which are recurrently mutated in ER-positive breast cancers harbouring SF3B1 mutations and have been postulated as drivers [SF3B1 mutations in the 16 papillary carcinomas that had been shown be SF3B1 wild-type by Sanger sequencing analysis. Overall, 63% (12/19) of the samples were found to display mutations in additional cancer genes including PIK3CA in 37% (7/19), TP53 in 5% (1/19), GATA3 in 11% (2/19), MAP3K1 11% (2/19), and MAP3K4 in 21% (4/19)  . In contSF3B1 mutations with differential splicing in uveal melanoma [SF3B1 K700E mutations in breast cancer would also lead to differential splicing. Massively parallel RNA sequencing of SF3B1 mutant (n = 3) versus wild-type (n = 11) papillary carcinomas of the breast identified differential exon usage in 122 transcripts  SF3B1 K700E mutant and stage, ER, PR, HER2, PIK3CA, and TP53 mutation status-matched SF3B1 wild-type breast cancers (n = 16) from TCGA revealed differential exon usage in 218 transcripts  were found in common with uveal melanoma [p = 7.047575e-11, hypergeometric test) and these showed identical differential splicing in papillary carcinomas of the breast, invasive breast carcinomas of no special type, and uveal melanomas displaying SF3B1 mutations  using qRT-PCR in SF3B1 mutant and wild-type papillary carcinomas. This analysis identified UQCC, F8, ABCC5, and GUSBP11 to be differentially spliced in papillary carcinomas, whereas GAS8 and ADAM12 showed no difference  and ESS-1 , and six wild-type controls  with an SF3b complex inhibitor, spliceostatin A, revealed that SF3B1 mutant cell lines were significantly more sensitive to this agent  failed to reveal differences in cell viability  akin to mucinous carcinomas of the breast [SF3B1 hotspot mutations may be more prevalent in mucinous and papillary carcinomas of the breast than in usual types of breast cancer of the same grade, ER, and HER2 status. Targeted re-sequencing of our cohort of 19 papillary carcinomas of the breast failed to identify any sub-clonal mutations in additional tumours, indicating that these mutations, when present, are found in the modal clone of the lesions, but are unlikely to account for the papillary histological pattern.Here we describe the repertoire of mutations in spliceosomal components in breast cancer through a re-analysis of public exome and whole genome sequencing data. Our data reveal that some spliceosome mutations are mutually exclusive in breast cancer, consistent with the observations made in myelodysplastic lesions ; howevere breast . Our expSF3B1 gene encodes subunit 1 of splicing factor 3b, a component of the U2 small nuclear ribonucleoprotein, which is involved in catalysing precursor mRNA to mature transcripts. SF3B1 contains several HEAT domains , which are the hotspots for the majority of the somatic mutations previously documented [SF3B1 mutations display alternative splicing in selected key genes in CLL [SF3B1 and wild-type breast cancers that were also reported to be differentially spliced in SF3B1 mutant melanomas [The cumented ,13,15. Ss in CLL , MDS [30s in CLL . Using aSF3B1 hotspot mutations would be sensitive to chemical inhibition of SF3B1and RNA-interference silencing of SF3B1 and genes abnormally spliced in SF3B1 mutant tumours [SF3B1 mutant and wild-type cell lines with spliceostatin A resulted in selective inhibition of the growth of mutant cells and in the disruption of the conserved splicing signature. Interestingly, single gene RNAi-mediated silencing of key differentially spliced genes had no effect on cell viability. Although it is plausible that the differentially spliced genes investigated in this study did not account for the dependency of SF3B1 mutant cells on aberrant splicing, it is conceivable that the differences in pre-mRNA splicing act in concert to produce a cell survival advantage. Given the difficulties in silencing multiple genes systematically, this remains to be tested.Given that the spliceosome SF3b complex has emerged as a potential therapeutic target , we soug tumours . Here weSF3B1 and SAP130 were found to be preferentially mutated in ER-positive and ER-negative disease, respectively). We have demonstrated that SF3B1 K700E mutations are more frequently found in some special histological types of breast cancer and have provided direct evidence that these are associated with consistent differential splicing patterns in breast cancer. Finally, our study emphasizes the importance of driver genetic alterations found in minor subgroups of breast cancer, given that SF3B1 mutant cells were shown to be selectively sensitive to a potent SF3b complex inhibitor, spliceostatin A, suggesting that inhibition of the spliceosome complex may constitute a novel therapeutic strategy for patients with SF3B1 mutations independent of tumour type.In conclusion, our findings demonstrate that spliceosomal mutations occur in a mutually exclusive manner in breast cancer and that distinct components of the spliceosome are targeted by somatic mutations in different types of breast cancer (eg


The accurate and timely characterization of influenza A viruses (IAV) from natural reservoirs is essential for responses to animal and public health threats. Differences between antigenic and genetic subtyping results for 161 IAV isolates recovered from migratory birds in the central United States during 2010–2011 delayed the recognition of four isolates of interest. Genomic sequencing identified the first reported Eurasian-origin H10 subtype in North America and three additional H14 isolates showing divergence from previously reported H14 isolates. Genomic analyses revealed additional diversity among IAV isolates not detected by antigenic subtyping and provided further insight into interhemispheric spread of avian-origin IAVs. The timely detection and characterization of novel or emerging avian-origin influenza A viruses (IAV) is crucial to prepare for health threats these viruses pose to humans and animals. All modern IAV pandemics have contained genetic segments derived from avian-origin IAVs.Ongoing avian-origin IAV surveillance in the Mississippi Migratory Bird Flyway from July 2010 to January 2011 resulted in 161 IAV isolates recovered from 2879 cloacal swabs collected from migratory waterfowl as previously described.Viral isolations were performed as previously described, and all IAV isolates were submitted to the USDA APHIS National Veterinary Services Laboratories (NVSL) for antigenic subtyping with HI/NI tests.After genomic sequencing of the 158 IAV isolates, we identified four isolates of interest including a Eurasian-origin H10 IAV and three additional H14 IAV isolates. Based on genomic sequencing, these isolates were named as follows: A/long-tailed duck/Wisconsin/10OS3919/2010(H10N6), A/long-tailed duck/Wisconsin/10OS3918/2010(H14N8), A/mallard/Wisconsin/10OS3941/2010(H14N6), and A/northern shoveler/Missouri/10OS4673/2010(H14N6).n = 460) indicates that the Eurasian-lineage H10 may have been circulating in North America for many years prior to this detection. Interestingly, a recent study found that the North American lineage of H10 has been circulating in Australia for several years.Avian-origin IAVs are separated into Eurasian and American lineages.The NA segment paired with this Eurasian-origin H10 segment showed 99·8% AA identity (469/470) to the previously reported Eurasian-origin N6 of the A/long-tailed duck/Wisconsin/10OS4225/2010(H14N6) and A/long-tailed duck/Wisconsin/10OS3912/2010(H14N6) [5] and to A/mallard/Wisconsin/10OS3941/2010(H14N6) whose sequence is detailed in this report  gene segment of A/long-tailed duck/Wisconsin/10OS3919/2010(H10N6) shared a 98·9% AA identity (275/278) with Eurasian-origin NS gene segments of the A/mallard/Wisconsin/10OS3941/2010H14N6) described below and previously reported the A/long-tailed duck/Wisconsin/10OS4225/2010(H14N6).N6 descriGenomic sequencing revealed three additional H14 isolates that were not identified using the antigenic subtyping protocols that detected the three H14 IAV isolates detailed in a previous report.5The third H14 isolate, A/northern sвhoveler/Missouri/10OS4673/2010(H14N6), was antigenically characterized as an H4 subtype. Phylogenetically, H4 is most closely related to H14The H14 tree (FigureFinding differences between antigenic and genomic subtyping is not surprising, and each approach contributes valuable information. However, the differences between subtype classification methods delayed the detection of interesting IAV isolates in this study. The recognition of additional H14 and Eurasian-origin H10, N6, and NS genomic segments provides additional evidence for interhemispheric spread of avian IAVs. Reassortment of segments between eastern and western hemisphere avian-origin IAV lineages demonstrates the value of sequencing recovered IAV isolates. In addition, sequencing allows researchers to capture and characterize IAV genetic diversity in the natural reservoir that cannot be fully appreciated with antigen–antibody reactions. Finally, sequencing results serve as indicators for the need to update the IAV reference antisera and strains used in antigenic testing. Further investigations into the cause of the discrepancies between antigenic and genomic subtyping observed in the present study are needed, including the amount of steric inhibition, cross-reactivity between subtypes, non-specific inhibition, and the detection limits of HA titers in HI/NI tests. Continued sequencing of historic avian-origin IAV repositories will be used to address this dynamic and could reveal additional unique IAV diversity.


In a separate sample of healthy persons, we examined whether dopamine depletion could alter insulin sensitivity.Food addiction is a debated topic in neuroscience. Evidence suggests diabetes is related to reduced basal dopamine levels in the nucleus accumbens, similar to persons with drug addiction. It is unknown whether insulin sensitivity is related to endogenous dopamine levels in the ventral striatum of humans. We examined this using the agonist dopamine D11C]-(+)-PHNO scan, 9 of which provided a scan under dopamine depletion, allowing estimates of endogenous dopamine at dopamine D2/3 receptor. Dopamine depletion was achieved via alpha-methyl-para-tyrosine . In 25 healthy persons , fasting plasma and glucose was acquired before and after dopamine depletion.Insulin sensitivity was estimated for each subject from fasting plasma glucose and insulin using the Homeostasis Model Assessment II. Eleven healthy nonobese and nondiabetic persons  provided a baseline [2/3 receptor was positively correlated with insulin sensitivity (r(7)=.84, P=.005) and negatively correlated with insulin levels (r(7)=-.85, P=.004). Glucose levels were not correlated with endogenous dopamine at ventral striatum dopamine D2/3 receptor (r(7)=-.49, P=.18). Consistently, acute dopamine depletion in healthy persons significantly decreased insulin sensitivity (t(24)=2.82, P=.01), increased insulin levels (t(24)=-2.62, P=.01), and did not change glucose levels (t(24)=-0.93, P=.36).Endogenous dopamine at ventral striatum dopamine D2/3 receptor in the ventral striatum. Moreover, acute dopamine depletion reduces insulin sensitivity. These findings may have important implications for neuropsychiatric populations with metabolic abnormalities.In healthy individuals, diminished insulin sensitivity is related to less endogenous dopamine at dopamine D Several2/3R availability and levels of fasting neuroendocrine hormones  between a baseline PET scan and a scan under acute dopamine depletion -PHNO, the first agonist PET radiotracer for D2/3R (11C]-(+)-PHNO to estimate endogenous dopamine levels at D2/3R using an AMPT challenge (11C]-raclopride (2/3R in humans. Using [11C]-(+)-PHNO body mass index (BMI) within a nonobese range was found to be positively correlated with BPND in the VS but not the dorsal striatum -PHNO.Our group has developed [or D2/3R . The usehallenge . Collectclopride  and thusstriatum . One pot11C]-(+)-PHNO and an acute dopamine depletion paradigm, we sought to examine for the first time whether estimates of endogenous dopamine at D2/3R in the VS of healthy, nonobese humans are related to IS. We hypothesized that persons with reduced IS would have less endogenous dopamine occupying D2/3R in the VS at baseline. Healthy participants were evaluated to provide: 1) a proof of concept for the relationship between IS and brain dopamine without the presence of confounding changes that may occur in disease states; and 2) a benchmark for future comparisons in clinical populations. We also sought to determine whether reducing endogenous dopamine with AMPT could lead to changes in IS in healthy individuals. Clarifying the relationship between IS and dopamine levels in the brains of humans in vivo would represent an important first step in understanding the interplay between metabolic health, energy homeostasis, and brain reward circuits in health and disease , 5:00 pm (post 8 hours), and 9:00 pm (post 12 hours) on day 1, and 6:00 am (post 21 hours) and 10:00 am (post 25 hours) on day 2. The post AMPT PET scan was scheduled at 12 pm, 28 hours after the initial metyrosine dose. Subjects were under direct observation during AMPT administration and slept overnight in hospital-designated research beds to facilitate the AMPT dosing schedule and monitor for potential side effects. In addition, subjects were instructed to drink at least 4L of fluids during the 2-day admission to prevent formation of AMPT crystals in urine, and fluid intake was monitored to ensure compliance. In addition, to alkalinize the urine, which increases AMPT solubility, sodium bicarbonate (1.25g) was given orally at 10:00 pm on the evening before day 1 and at 7:00 am on day 1 of administration.The procedure for AMPT-induced dopamine depletion has been published elsewhere . Brieflyam. For the participants who provided PET scans (n=11), fasting blood work was collected on the day of the baseline PET scan. Twenty-five healthy participants  provided fasting blood work (9:00 am) at baseline and after receiving 5 doses of AMPT. For 13 of these subjects, it was possible to collect the blood work 24 hours apart. For the remainder of subjects, 4 provided blood work 6 to 7 days apart, 4 provided 10 to 14 days apart, and 2 provided 36 to 43 days apart. Blood for glucose measurement was collected in a 4-mL grey stoppered tube containing sodium fluoride as a preservative and potassium oxalate as an anticoagulant. Plasma was assayed for glucose on the EXL 200 Analyzer (Siemens) using an adaptation of the hexokinase-glucose-6-phosphate dehydrogenase method. Blood for insulin measurement was collected in a 6-mL red stoppered tube with no additives. Serum was analyzed on an Access 2 Analyzer (Beckman Coulter) using a paramagnetic particle, chemiluminescent immunoassay for the quantitative determination of insulin levels in human serum. The IS index for glucose disposal was estimated for each subject from fasting plasma glucose and insulin using the Homeostasis Model Assessment II (HOMA2), calculated with the University of Oxford HOMA2 calculator   . Estimatulator/) .11C]-(+)-PHNO PET scans, one under baseline conditions and another at 25 hours following AMPT-induced dopamine depletion. The radiosynthesis of [11C]-(+)-PHNO and the acquisition of PET images have been described in detail elsewhere  single-photon point source to provide attenuation correction, and the emission data were acquired in list mode. The raw data were reconstructed by filtered-back projection. For baseline [11C]-(+)-PHNO scans (n=11), the mean radioactivity dose was 9 (±1.5) mCi, with a specific activity of 1087 (±341) mCi/µmol and an injected mass of 2.2 (±0.4) µg. For the dopamine-depleted scans (n=9), the mean radioactivity dose was 9 (±1.6) mCi, with a specific activity of 1044 (±310) mCi/µmol and an injected mass of 2.1 (±0.4) µg. There was no difference in mean radioactivity dose (t(8)=0.98, P=.36), specific activity (t(8)=1.09, P=.31), or mass injected (t(8)=-0.61, P=.56) between the baseline and dopamine depletion scans (n=9). [11C]-(+)-PHNO scanning data were acquired for 90 minutes postinjection. Once scanning was complete, the data were redefined into 30 frames (1–15 of 1-minute duration and 16–30 of 5-minute duration).Participants underwent 2 [lsewhere . Briefly11C]-(+)-PHNO has been described in detail elsewhere (http://www.fil.ion.ucl.ac.uk/spm). The TACs were analyzed using the Simplified Reference Tissue Method (ND), as defined by consensus nomenclature for in vivo imaging of reversibly binding radioligands . The range wherein the basis functions were generated (K2a min - K2a max) was 0.006 to 0.6. These images were spatially normalized into MNI brain space by Nearest Neighbour interpolation with a voxel size fixed in 2×2 × 2mm3 using SPM2. Regional BPND estimates were then derived from ROIs defined in MNI space. The VS and dorsal striatum  were defined according with The region of interest (ROI)-based analysis for -(+)-PHNO for D2/3R is sensitive to dopamine levels  D2/3R BPND under depletion more accurately reflects the true number status of D2/3R; and 3) the fractional increase in D2/3R BPND after dopamine depletion  is linearly proportional to baseline dopamine concentration at D2/3R, provided the process of dopamine depletion does not change the number and affinity of D2/3R. Thus, the %ΔBPND, under appropriate assumptions, is considered a semiquantitative index of endogenous dopamine levels at D2/3R . Statistical analyses were conducted using SPSS  and GraphPad . Normality of variables was determined using the D’Agostino-Pearson test. The significance level for all testes was set at P<.05 (2-tailed).Relationships between baseline BPr(9)=.76, P=.007), and waist circumference was positively correlated with fasting levels of insulin (r(9)=.80, P=.003)  participated in the PET portion of the study; a subset of these data have been previously reported upon  Caravag. Within  P=.003) .2/3R in the VS at baseline -PHNO BPND before and after dopamine depletion). Estimated baseline dopamine occupancy of D2/3R in the VS was positively correlated with IS (r(7)=.84, P=.005) (r(6)=.86, P=.007), BMI (r(6)=.72, P=.04), waist circumference (r(6)=.75, P=.03), and plasma levels of AMPT (r(6)=.84, P=.009). Concurrently, estimated baseline dopamine occupancy of D2/3R in the VS was negatively correlated with fasting insulin levels (r(7)=-.85, P=.004) but was not correlated with fasting levels of glucose (r(7)=-.49, P=.18). Dopamine occupancy in the VS was not correlated with BMI (r(7)=.09, P=.80) or waist circumference (r(7)=-.30, P=.41).Nine of the 11 subjects provided both a baseline PET scan as well as a scan under acute AMPT-induced dopamine depletion; this provided estimates of endogenous dopamine occupying D P=.005) , a corre2/3R were driven primarily by dopamine occupancy in the right VS but not the left VS. Specifically, dopamine occupancy in the left VS was not correlated with IS (r(7)=.41, P=.28), fasting levels of insulin (r(7)=-.46, P=.22), or glucose (r(7)=-.33, P=.39), whereas dopamine occupancy in the right VS was positively correlated with IS (r(7)=.75, P=.01), negatively correlated with fasting levels of insulin (r(7)=-.73, P=.02), and not correlated with levels of glucose (r(7)=-39., P=.31).Notably, the above correlations with estimated baseline dopamine occupancy of D11C]-(+)-PHNO BPND in the right VS was negatively correlated with estimated IS (r(9)=-.65, P=.02) -PHNO BPND in the right VS (r(9)=.77, P=.006), while there was no correlation with fasting glucose levels (r(9)=.27, P=.43). Notably, [11C]-(+)-PHNO BPND in the left VS was not correlated with IS (r(9)=-.35, P=.29) or fasting levels of insulin (r(9)=.53, P=.09) and glucose (r(9)=.08, P=.81).Within the full sample of subjects (n=11), baseline -(+)-PHNO and an acute dopamine depletion paradigm, we demonstrate for the first time that IS is positively correlated with endogenous dopamine levels at D2/3R in the VS. In the absence of obesity or overt glucose dysregulation, lower endogenous dopamine levels in the VS are associated with reduced IS. This novel finding is in line with previous in vivo PET studies examining baseline D2/3R availability in the VS of obese persons . Future AMPT studies examining the relationship between endogenous dopamine in the brain and IS should attempt to employ larger sample sizes. Because of our small sample size, we refrained from exploring relationships between baseline [11C]-(+)-PHNO BPND and IS in ROIs other than the VS. In particular, future [11C]-(+)-PHNO studies using larger sample sizes should examine the relationship between IS and baseline BPND in the substantia nigra and hypothalamus: regions where 100% of the [11C]-(+)-PHNO BPND signal is due to D3R vs D2R  are related to endogenous dopamine levels and dopamine release in the VS of humans. In addition, future studies should examine whether these values alter in the face of treatment for metabolic deficits. Moreover, it is important to examine across a spectrum of glucose dysregulation in humans how dopamine concentrations and functioning in the VS relate to mood, motivation, and reward processing. Finally, our sample in the current study is small. While we did not explicitly control for multiple comparisons, it is important to note that the observed relationship between IS and estimated endogenous dopamine in the VS would survive Bonferroni correction , Alzheimer’s disease , and dep2/3R in the VS of healthy humans. Furthermore, acutely reducing endogenous dopamine in healthy persons can alter estimated IS. Taken together, these findings represent an important preliminary step in elucidating how metabolic status may interface with major mental illnesses such as schizophrenia.In conclusion, using PET and an acute dopamine depletion challenge, we have demonstrated for the first time that estimates of IS are related to levels of endogenous dopamine at DDr. Nakajima reports having received grants from Japan Society for the Promotion of Science and Inokashira Hospital Research Fund and speaker’s honoraria from GlaxoSmith Kline, Janssen Pharmaceutical, Pfizer, and Yoshitomiyakuhin within the past 3 years. Dr. Graff-Guerrerro currently receives research support from the following external funding agencies: Canadian Institutes of Health Research, the U.S. National Institute of Health, and the Mexico Instituto de Ciencia y Tecnologıa para la Capital del Conocimiento en el Distrito Federal (ICyTDF). He has also received professional services compensation from Abbott Laboratories, Gedeon-Richter Plc, and Lundbeck; grant support from Janssen; and speaker compensation from Eli Lilly. Dr. Remington has received research support, consulting fees, or speaker’s fees from the Canadian Diabetes Association, the Canadian Institutes of Health Research, Hoffman-La Roche, Laboratorios Farmacéuticos Rovi, Medicure, Neurocrine Biosciences, Novartis Canada, Research Hospital Fund–Canada Foundation for Innovation, and the Schizophrenia Society of Ontario. The other authors have no competing interests to disclose.


MR-guided HIFU is an effective treatment for metastatic bone pain through periosteal nerve ablation, and is bExperiments were approved by the local Institutional Animal Care and Use Committee. Volumetric MR-HIFU was used to induce seven thermal lesions in the sciatic nerves of three pigs. 3D MR neurography and T1-weighted images at 3T were used for target identification and treatment planning. A single 8 or 12 mm treatment cell was used to cover the full width of each targeted nerve. Ultrasound exposures were performed under MR thermometry guidance in five image planes across the HIFU beam, and one plane along the beam axis. Sonications were performed at 1.2 MHz with acoustic power ranging from 160 to 300 W over fixed durations of 20 or 36 seconds (energy 3.2 to 10.8 kJ). Ablation dimensions were measured and compared using thermal dose maps, contrast-enhanced T1-weighted images, and gross pathology.All targeted sciatic nerves were identifiable on MR neurography and T1-weighted images Fig . Thermal