LHCGR region, STON1-GTF2A1L and LHCGR, were overexpressed in PCOS. In analysis stratified by obesity, LHCGR was overexpressed only in non-obese PCOS women. Although not differentially expressed in the entire PCOS group, INSR was underexpressed in obese PCOS subjects only. Alterations in gene expression in the LHCGR, RAB5B and INSR regions suggest that SNPs in these loci may be functional and could affect gene expression directly or indirectly via epigenetic alterations. We identified reduced methylation in the LHCGR locus and increased methylation in the INSR locus, changes that are concordant with the altered gene expression profiles. Complex patterns of meQTL and eQTL were identified in these loci, suggesting that local genetic variation plays an important role in gene regulation. We propose that non-obese PCOS women possess significant alterations in LH receptor expression, which drives excess androgen secretion from the ovary. Alternatively, obese women with PCOS possess alterations in insulin receptor expression, with underexpression in metabolic tissues and overexpression in the ovary, resulting in peripheral insulin resistance and excess ovarian androgen production. These studies provide a genetic and molecular basis for the reported clinical heterogeneity of PCOS.Genome wide association studies (GWAS) have revealed 11 independent risk loci for polycystic ovary syndrome (PCOS), a common disorder in young women characterized by androgen excess and oligomenorrhea. To put these risk loci and the single nucleotide polymorphisms (SNPs) therein into functional context, we measured DNA methylation and gene expression in subcutaneous adipose tissue biopsies to identify PCOS-specific alterations. Two genes from the  Polycystic ovary syndrome (PCOS) is the most common hormonal disturbance in reproductive age women and features high levels of male sex hormones, such as testosterone, and infrequent ovulation. Twin studies have demonstrated that inheritance plays a significant role in PCOS, and recent genome wide association studies (GWAS) have implicated 11 susceptibility regions. The mechanism by which these genetic loci cause PCOS has yet to be determined. We looked at DNA methylation and gene expression levels in these 11 loci in fat biopsies from women with and without PCOS. We identified differences in the expression of two receptors that bind hormones known to contribute to the pathogenesis of PCOS–the receptors for luteinizing hormone (LH) and insulin. We found increased expression of the LH receptor in non-obese PCOS women, while in the obese women with PCOS the insulin receptor was underexpressed. Both excess LH stimulation and elevated insulin levels, due to decreased receptor levels and resulting insulin resistance, can cause increased androgen production from the ovary. Our findings suggest the primary mechanism for elevated androgen levels in PCOS may differ between non-obese and obese women with PCOS and that the clinical heterogeneity seen in PCOS may have genetic underpinnings. Polycystic ovary syndrome (PCOS) occurs in 6–10% of reproductive age women by NIH diagnostic criteria, and is characterized by hyperandrogenism and oligo- or amenorrhea .THADA, LHCGR, FSHR, C9orf3, DENND1A, YAP1, RAB5B, INSR, TOX3, SUMO1P1, and HMGA2) [THADA, LHCGR, FSHR, DENND1A, YAP1, INSR) have been replicated in Caucasian populations [PCOS is a complex disorder with both genetic and environmental factors contributing to its pathophysiology. Twin studies have provided heritability estimates for PCOS of 0.71 . Two gend HMGA2) , 5. Six ulations –10. In aLHCGR (Luteinizing hormone/choriogonadotropin receptor) and DENND1A (DENN/MADD domain containing 1A) [LHCGR promoter region was shown to be hypomethylated and mRNA expression level increased in granulosa cells from women with PCOS [in vitro study reported overexpression of transcriptional variant 2 of DENND1A (DENND1Av2) in the theca cells of PCOS patients and demonstrated its ability to increase androgen and progestin biosynthesis [GWAS have provided insight into the genetic architecture of many complex diseases, including PCOS. A limited number of functional studies have evaluated the role of several of the newly identified PCOS risk loci, including ning 1A) –13. The ith PCOS . An in vThe functional characteristics of a locus can include the epigenetic regulation of expression , enhancer binding activity, transcription factor binding profiles, promoter activity, and the gene expression profile. DNA methylation plays an important role in the regulation of gene expression by affecting chromatin state and the ability of transcription factors, enhancers and insulators to bind DNA . DNA metIn the present study, we measured DNA methylation and gene expression in adipose tissue of PCOS women and normal controls in order to better understand the functional elements surrounding PCOS-associated SNPs. As an endocrine tissue with a clear role in metabolic function and relative ease of collection, adipose tissue is highly suited for functional studies of PCOS genes, particularly those not directly related to androgen excess or ovarian function. Adipose dysfunction in PCOS has been widely reported; studies of subcutaneous adipocytes from PCOS women have demonstrated resistance to insulin stimulated glucose transport and inhibition of lipolysis , 17. We PCOS subjects were not significantly older than controls, and no significant difference in BMI was detected. As expected, PCOS cases had elevated testosterone and hirsutism measured by modified Ferriman-Gallwey (mFG) score .LHCGR and STON1-GTF2A1L from the LHCGR locus were overexpressed in PCOS, while WIBG, RAB5B and IKZF4 from the RAB5B locus were underexpressed in PCOS . In order to investigate the effect of obesity on gene expression in PCOS adipose tissue, we performed secondary analyses in obese and non-obese subjects separately .In 35 subjects (22 cases and 13 controls), we examined the gene expression profiles for each transcript that passed normalization and background correction within the 11 PCOS risk loci. A total of 50 transcripts were identified in the genomic windows surrounding the PCOS risk variants and extracted from the genome wide expression dataset. Twenty-eight of these were expressed in the adipose tissue samples. Both  in PCOS . After cparately . In the RAB5B window, including two sites located in the intergenic region 5’ to IKZF4 with increased methylation in PCOS subjects  . Four Cpsubjects . Within controls . We applDENND1A, which may regulate isoform specific expression in a tissue dependent manner.Of the remaining eight loci, six had PCOS specific changes in methylation . We alsoWIBG was underexpressed in PCOS patients, similar to our findings. Also consistent with our findings, in cumulus cells LHCGR was overexpressed in PCOS subjects. In the latter series, when the subjects were stratified by obesity, the non-obese PCOS subjects had lower expression of WIBG and LHCGR continued to be overexpressed. In the obese subjects, women with PCOS demonstrated higher expression of INSR in cumulus cells. Lower expression of INSR was seen in PCOS subjects in two different series of skeletal muscle.The National Center for Biotechnology Information’s (NCBI) GEO database was used to further investigate the differentially expressed genes in our cohort both in subcutaneous adipose and other tissue types . In a smLHCGR window, SNPs in the 5’ and intron 1 regions of the LHCGR gene, surrounding the PCOS risk SNP rs13405728, were associated with methylation level of three CpG residues clustered in the STON1-GTF2A1L gene  with the PCOS GWAS index SNP, shown in INSR were associated with a number of SNPs across the 5’ region of the window  from across the f ZNF557 . The twoRAB5B/SUOX window four SNPs from across the window acted independently as meQTL SNPs with five methylation sites clustered between WIBG and DGKA and at the 5’ and 3’ of RAB5B  database [LHCGR in adipose could represent a conserved gene regulation profile across tissues in non-obese women with PCOS. To confirm that our finding of LHCGR overexpression is a PCOS-specific effect, we identified GEO datasets that could be analyzed with either obesity or insulin sensitivity as a dichotomous trait. We did not find any changes in expression between lean and obese subjects in three adipose GEO datasets where obesity was available to stratify subjects  of obese PCOS women, we found INSR to be overexpressed in the cumulus cells of obese PCOS subjects  of adipose samples, and while this is the largest study of this type published to date, the relatively small sample size may have reduced our ability to identify eQTM. A publicly available replication data set comparing gene expression between PCOS and controls in subcutaneous adipose tissue also found WIBG to be underexpressed in PCOS  under approval number 11289. All subjects gave written informed consent according to the guidelines of the IRBSubcutaneous lower abdominal adipose tissue was obtained from 23 PCOS and 13 control subjects using a previously described protocol for acquiring and processing subcutaneous adipose tissue . PCOS suSamples were snap frozen immediately after collection in liquid nitrogen and then stored at -80°C until extraction. DNA and RNA were isolated from subcutaneous fat tissue after rapid thaw at 37°C with the AllPrep DNA/RNA/protein Mini kit . DNA was stored in TE buffer, quantified and checked for quality on a Nanodrop-1000  and stored at -80°C. RNA samples were quantified and checked for quality using the BioAnalyzer 6000 Pico kit  and stored at -80°C.-4 were retained for downstream analysis . Principal components analysis (PCA) within SVS was used to generate the top 10 PCs to identify outlier samples, of which none were found.Genotyping of 36 samples was performed at CSMC using the HumanExome chip  variants), the HumanOmniExpress chip (targeting common variants using a haplotype tagging approach) and the HumanOmni1S chip (targeting rare and non-Caucasian SNPs and copy number variants) following the manufacturer’s protocol  , 47. SamA subset of 24 age and BMI matched subjects were selected for methylation analysis due to restrictions on sample size because samples were run as part of a larger project. DNA methylation levels were measured using the HumanMethylation450 chip  according to the manufacturer’s instructions at CSMC. The HumanMethylation450 chip targets over 485,000 CpG residues across 96% of all RefSeq genes  and 95% of CpG islands and flanking regions. Samples were randomized by case/control status across two plates (10 chips) in the context of a larger experiment and arrayed at a concentration of 10ng/ul. Detection P values were calculated to identify failed probes, and beta (β) values representing methylation levels were generated from the Genome Studio software for each methylation site, ranging from 0 (completely unmethylated) to 100% (completely methylated). The Methylumi package in R was used to background normalize and log transform the beta values [th intensity percentile, signal to noise ratio, signal across all samples) were reviewed. One sample was excluded due to excessive signal to noise ratio. Sample probe profile data was read into the limma package in R version 3.1.1 [The HumanHT-12v4 beadchip was used to measure gene expression levels of well-characterized genes, gene candidates, and splice variants with 47,000 probes at the UCLA Neuroscience Genomics Core (UNGC). All 36 samples were randomized according to case/control status and RNA was arrayed at 10ng/ul. The TargetAmp-Nano Labeling Kit for Illumina Expression BeadChip  was used to label samples. Sample probe profile data was exported from Genome Studio after QC metrics  and sample metrics , eQTL (SNP associated with mRNA level) and eQTM (methylation level associated with mRNA level) analysis. meQTL relationships between methylation probes and multiple SNPs were interrogated for linkage disequilibrium (LD) between the SNPs, and conditional analysis using additive model genotype as a covariate in the linear regression was used to identify the variant driving the meQTL association if possible. We applied correction for multiple testing in gene expression and methylation results using the False Discovery Rate [Normalized and log transformed methylation beta levels and gene expression levels were analyzed in case/control analysis using logistic regression in SVS adjusting for age and BMI. Subjects were stratified by obesity status where subjects with BMI ≥ 30kg/mery Rate , with anNCBI’s GEO is a public repository that archives high-throughput functional genomics data. To compare expression levels of the candidate genes discovered in our cohort in additional PCOS tissues we evaluated these genes in other datasets. A search of the GEO database identified 8 datasets comparing gene expression between PCOS patients and controls in various tissue types . The GEOENCODE tracks were displayed in the UCSC Genome Browser using Build37 (GRCh37/hg19) in order to identify poised enhancer (H3K4Me1), active enhancer (H3K27Ac), active promoter (H3K4Me3), and transcription activity in the ENCODE reference cell types .S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S1 FigGenomic co-ordinates and gene position are shown in the top of the panel with the location of the PCOS GWAS index SNP shown as a solid triangle. Methylation status of each CpG residue is demonstrated by a circle , with CpG-SNP interactions shown by a black line connecting associated methylation probes and SNPs. Box and whisker plots below show methylation level by genotype for each meQTL.(TIFF)Click here for additional data file.S2 FigGenomic co-ordinates and gene position are shown in the top of the panel with the location of the PCOS GWAS index SNP shown as a solid triangle. Methylation status of each CpG residue is demonstrated by a circle , with CpG-SNP interactions shown by a black line connecting associated methylation probes and SNPs. Box and whisker plots below show methylation level by genotype for each meQTL.(TIFF)Click here for additional data file.S3 FigThe samples used in this this study and the datasets generated for each.(TIF)Click here for additional data file.


The sodium-taurocholate cotransporting polypeptide (NTCP) is both a key bile acid (BA) transporter mediating uptake of BA into hepatocytes and an essential receptor for hepatitis B virus (HBV) and hepatitis D virus (HDV). In this study we aimed to characterize to what extent and through what mechanism BA affect HDV cell entry.in vitro generated HDV particles. Infectivity in the absence or presence of compounds was assessed using immunofluorescence staining for HDV antigen, standard 50% tissue culture infectious dose (TCID50) assays and quantitative PCR.HuH-7 cells stably expressing NTCP (HuH-7/NTCP) and primary human hepatocytes (PHH) were infected with Addition of primary conjugated and unconjugated BA resulted in a dose dependent reduction in the number of infected cells while secondary, tertiary and synthetic BA had a lesser effect. This effect was observed both in HuH-7/NTCP and in PHH. Other replication cycle steps such as replication and particle assembly and release were unaffected. Moreover, inhibitory BA competed with a fragment from the large HBV envelope protein for binding to NTCP-expressing cells. Conversely, the sodium/BA-cotransporter function of NTCP seemed not to be required for HDV infection since infection was similar in the presence or absence of a sodium gradient across the plasma membrane. When chenodeoxycolic acid (15 mg per kg body weight) was administered to three chronically HDV infected individuals over a period of up to 16 days there was no change in serum HDV RNA.Primary BA inhibit NTCP-mediated HDV entry into hepatocytes suggesting that modulation of the BA pool may affect HDV infection of hepatocytes. Chronic infection with hepatitis B virus (HBV) affects over 300 million individuals worldwide , 2. Of tin vitro ) or regular sodium solution .For infection in low extracellular sodium conditions HDV containing supernatant was loaded on an Amicon Ultra 15 mL filter system and washed multiple times with either low sodium solution  assay in use for standard clinical care at Hannover Medical School and University Hospital Frankfurt. A Lightcycler 480 II  was used. Details on the assay have been published . For tis3) and cells were centrifuged at 300 rcf for 5 minutes at room temperature to remove buffer. Cells were resuspended in FACS buffer with or without BA and incubated on ice for 15 minutes before 100 µL of staining buffer  were added. Cells were then incubated for another 10 min on ice. The cells were washed 3 times and resuspended in FACS buffer. Fluorescence intensity was quantified using a FACSCanto cytometer  and data were analyzed with FlowJo software .For binding competition assay HuH-7/hNTCP cells were trypsinized. Then trypsination was stopped by adding 1 mL FACS buffer (PBS / 2% BSA / 0.02% NaNIndividueller Heilversuch”) is admissible in Germany without review by an ethics committee. Patients were given the drug as an individualized off label treatment attempt by two of the authors (KS and CS). These authors did so in their capacity as treating physicians of the patients. All individuals are regular patients at the Frankfurt University Hospital liver clinic where they are regularly seen and receive care for chronic hepatitis B/D. The three patients were two males (31 and 52 years old) and one female (43 years old). All had Child Pugh Stage A cirrhosis. The off label use of chenodesoxycholate as well as expected risks and benefits were discussed in detail with all three individuals. Informed consent was obtained verbally and documented in the patient chart. Serum HDV RNA was determined before the first dose of chenodeoxycholic acid and at several time points thereafter. When viral load remained unchanged the drug was discontinued after 7 to 16 days.We report a summary of three cases of chronic HBV/HDV coinfection where chenodeoxycholic was administered. Chenodeoxycholic acid is licensed for the treatment of gallstone disease. Ethics comitee approval was not necessary since off label administration of licensed drugs for other indications as an individualized treatment attempt (“The cell line established as part of this work (HuH-7/hNTCP) is a derivative of the HuH-7 cell line that was originally established in Japan in 1982 . HuH-7 cWhere required unpaired t-test or linear regression were performed as appropriate to test for statistical significance. p-values below 0.05 were considered significant.in vitro the open reading frame of human NTCP was synthesized, cloned into the blasticidin selectable pWPI-Bla lentigenome and stably expressed in HuH-7 cells producing a homogenous cell population. IF staining confirmed expression of NTCP in all or almost all cells in the HuH-7/hNTCP population  compared to other—secondary, tertiary and synthetic—BA  (p = 0.002 for comparison of reduction in TCID50 at 200 µM of primary BA versus other BA).HuH-7/hNTCP cells were exposed to supernatant containing infectious HDV for six hours after which media was exchanged. Cells were incubated for another 5 days and then fixed and stained. A large percentage of cells stained positive for HDV antigen . HDV ant control  or diffe control . We obse50 assays over a wider concentration range we observed a dose dependent up to 1000-fold inhibition of HDV infectivity  were applied for 7 days, cellular HDV RNA as measured by qPCR was not significantly different in the presence of any of the BA tested with exception of the primary BA SCDCA and the secondary BA LCA . This suTo address the mechanism of BA-mediated inhibition of HDV we tested the ability of inhibitory BA to compete for large HBV surface protein binding to NTCP expressing HuH-7 cells. Atto-488 labelled preS1 peptide bound to NTCP expressing HuH-7 cells while an Atto-488 labelled mutated version of the peptide without affinity to NTCP did not indicating that binding occurred through a specific interaction . The inh50 format. There was no significant difference in HDV titer in the presence or absence of a sodium gradient across the plasma membrane  and low (5 mM) extracellular sodium in a TCIDmembrane  arguing Finally, we report three cases of individuals with chronic HBV/HDV coinfection that were administered CDCA at 15 mg/kg orally for 7–16 days. Serum HDV RNA was measured by quantitative PCR before the start, during and at the end of administration of chenodeoxycholic acid . There wPrimary BA are synthesized in hepatocytes where they can also be conjugated with either glycine or taurine producing the more soluble conjugated primary BA . BA are 50) and observed a more pronounced inhibitory effect of primary compared to secondary, tertiary and synthetic BA tested. Moreover, we demonstrate that both other replication cycle stages, replication and assembly/release, are largely unaffected. The most important difference to previously published work may be that in our hands the full inhibitory potential of BA is apparent only in a clearly supraphysiological concentration range: as shown in 50 at 25 µM and more of sodium taurocholate. While the physiological concentration of total BA in human serum is 1–6 µM it can be elevated to more than 100-fold the upper limit of normal under conditions of intra—or extrahepatic biliary obstruction [in vivo, but inhibition might be more likely to occur in common conditions where serum BA are increased such as cirrhosis or post-hepatic cholestasis. Our clinical observation that exogenous administration of chenodeoxycholic acid does not affect HDV viral load at all argues against a major impact of BA on HDV infectivity in vivo. In summary, we doubt that BA-mediated inhibition of HDV infectivity plays a major role in the context of human infection. However, BA are still of interest for mechanistical studies of the interaction between HBV or HDV and NTCP such as this paper and the work by Yan and colleagues [Differently from the very recently published work by Yan and colleagues  we used truction . While slleagues .As yet there are no approved drugs or compounds in advanced clinical development perturbing the HDV replication cycle. Prenylation inhibitors and the HBV/HDV entry inhibitor Myrcludex are promising approaches, but their clinical potential with regard to HDV cure is as yet unclear . Moreovein vitro and approved as a drug for the treatment of cholesterol-rich gallstones failed to produce any discernable alteration in HDV viral load when administered orally for a limited duration and at concentrations currently used for the treatment of gall stone disease. Whether an antiviral effect can be achieved with higher doses is unclear.In this paper we demonstrate that BA that are substrates of NTCP inhibit HDV cell entry. This occurs most likely through competition for binding to NTCP between BA and the large HBV surface protein that has been shown to mediate binding of HBV and HDV to NTCP. Supraphysiological concentrations of BA were required for potent inhibition of viral cell entry and primary BA were somewhat more potent inhibitors than secondary, tertiary and synthetic BA. In addition we show data suggesting that the cotransporting function of NTCP is not required for its abilty to serve as a virus receptor. Finally, we report three clinical cases of chronically HBV/HDV-coinfected individuals where the primary BA chenodeoxycholic acid that is inhibitory S1 Fig(A) Parental HuH-7 cells and HuH-7 cells transduced with pWPI/Bsd-hNTCP and kept under blasticidin selection were fixed and stained for NTCP expression (green). Nuclei were counterstained with DAPI (blue). (B) FACS staining of HuH-7/NTCP cells for NTCP (solid line). Controls: no primary antibody (dashed line) and unstained (shaded area).(TIF)Click here for additional data file.S2 FigOn day 5 after infection in the presence or absence of 200 µM of the indicated BA total cellular RNA was prepared and HDV and GAPDH RNA were quantified. The results represent the HDV/GAPDH ratio for each sample with the value determined in cells infected in the absence of BA or other inhibitors set to 100%. The mean +/- SD from an experiment performed in duplicate is shown.(TIF)Click here for additional data file.S3 FigPrimary human hepatocytes (PHH) were exposed to HDV containing supernatant in the presence or absence of 200 µM of different BA. preS1 peptide (368 nM) served as positive control. After 6 h cells were washed and repleted with regular media without virus, BA or drugs. After another 5 d cells were fixed and stained with a polyclonal antibody against HDV antigen (right hand images in each panel). Nuclei were counterstained with DAPI (left hand images). A representative of three independent experiments is shown.(TIF)Click here for additional data file.S4 FigHuH-7 cells were co-transfected with plasmids pSVLD3 and pT7HB2.7. After the transfection procedure was completed media was exchanged and cells were maintained in media with our without 100 µM of the indicated BA for 7 days. Then supernatant was removed and cells were fixed and stained for HDV antigen (green). Nuclei were counterstained with DAPI (blue).(TIF)Click here for additional data file.S5 FigHuH-7/hNTCP cells were infected with HDV. After 6 h media was exchanged and cells were maintained in media with our without 100 µM of the indicated BA for 7 days. Then total cellular RNA was prepared and HDV and GAPDH RNA were quantified by qPCR. The results represent the HDV/GAPDH ratio for each sample with the value determined in cells infected and maintained in the absence of BA set to 100%. The mean +/- SD from three independent experiment performed in duplicate is shown.(TIF)Click here for additional data file.S6 FigSupernatant from the cells shown in (TIF)Click here for additional data file.


Recordings were conducted with multichannel electrodes that permitted the simultaneous acquisition of neuronal activity within primary auditory cortex columns. Neuronal responses to simple (pure tones), complex (noise burst and frequency modulated sweeps), and ecologically relevant  acoustic signals were measured. Collectively, the present investigation demonstrates that despite consistencies in neuronal tuning (characteristic frequency), irregularities in discharge activity between neurons of individual A1 columns increase as a function of spectral  and temporal (duration) acoustic variations.Assemblies of vertically connected neurons in the cerebral cortex form information processing units (columns) that participate in the distribution and segregation of sensory signals. Despite well-accepted models of columnar architecture, functional mechanisms of inter-laminar communication remain poorly understood. Hence, the purpose of the present investigation was to examine the effects of sensory information features on columnar response properties. Using acute recording techniques, extracellular response activity was collected from the right hemisphere of eight mature cats  The neocortex is composed of multiple layers Investigations in auditory cortex have provided information about columnar connectivity felis catus) auditory cortex laminae offer a convenient model for the study of cortical columns. Hence, neuronal responses to simple (pure tones), complex (noise burst and frequency modulated sweeps), and ecologically relevant  acoustic signals were measured across cat A1. Collectively, data analyses revealed that within A1 cortical columns, response irregularities increase as a function of changes in acoustic characteristics.Structural . Animals were housed in a sensory-enriched environment where social interactions were available 24 hours a day. Procedures followed the US National Research Council’s Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research (2003) as well as the Canadian Council on Animal Care’s Guide to the Care and Use of Experimental Animals. In addition, the University of Western Ontario Animal Use Subcommittee of the University Council on Animal Care granted experimental approval.Neuronal responses to simple and complex acoustic signals were measured in the right primary auditory cortex (A1) of eight adult (>6 months) cats Surgical procedures in the present investigation have been described in previous publications . The distance from the tip of the array to the first recording channel was 0.5 mm, followed by 150 µm between subsequent channels; hence, electrodes were orthogonally lowered ∼2.4 mm from the cortical surface or until the deepest and most superficial recording channels in the array were responsive to white noise bursts. Electrode position is crucial to the interpretation of variations in neuronal activity across the cortical sheet, therefore electrode track orthogonality was assessed based on consistency of characteristic frequency values within single cortical penetrations (see results) Neuronal activity was recorded with twelve-channel iridium axial array microelectrodes . Acoustic calibration was performed with a ¼-inch microphone  and Tucker Davis Technologies software . Signals were digitally generated with a 24-bit D/A converter at 156 kHz  and delivered via sound transducers TS-A1072R (Pioneer) and EC1 (Tucker Davis Technologies). Apart from pure tone stimulation, acoustic signals were presented at 65 dB sound pressure level (SPL) in the free-field 15 cm from the left  ear..In total, 2,064 pure tones  were presented in pseudo-random order. Signals ranged from 250 Hz to 64,000 Hz in 1/16 octave steps, and 16 intensities extending from 0 to 75 dB SPL in 5 dB steps. Each frequency-intensity combination was presented once at a rate of 2.5 Hz .Broadband white noise (1–32 kHz) of various durations  were presented in pseudo-random order at a rate of one repetition every two seconds. Regardless of stimulus length, 5 ms rise and fall cosine squared gated functions were used. Each duration class was presented 250 times .Neuronal responses to logarithmic upward frequency modulated (FM) sweeps (1–32 kHz) were investigated. FM signals varied in duration  and were presented in pseudo-random order. Consistent with tonal and noise burst signal features, FMs were characterized by 5 ms rise and fall cosine squared gated functions. Each duration group was presented 250 times at a rate of one repetition every two seconds . The average fundamental frequency was 570 Hz with a lowest component of 0.5 kHz and highest component of 5.2 kHz. Signals were presented at an intensity of 65 dB SPL.Neuronal responses to a typical cat vocalization Data analyses were limited to A1 recordings as identified by tonotopic organization and response latency properties p<0.05).Cross-correlation vectors of simultaneously recorded PSTH functions  were calculated, and coefficients at zero lag time were normalized between −1 and 1. Identical PSTH functions produced a value of 1 and degrees of incongruences in neuronal response were identified by measures between −1 and <1. Despite the applicability of cross-correlation functions to the overall assessment of neuronal response similarities, the results of this analysis did not distinguish differences in specific neuronal response features. Thus, a second metric was used to quantify variations in the number of response peaks across PSTH functions. This neuronal activity property was chosen as it illustrates a central characteristic of neuronal activity during sensory exposure. Peaks in PSTH functions were detected by searching for downward zero crossings in the first-derivative of the power spectrum using software functions developed by Dr. Tom O’Haver of the University of Maryland. Together, global (cross-correlation index) and specific (number of peaks) comparisons produced a broad insight into the similarities between neuronal response properties within A1 columns. Statistical comparisons were performed using ANOVA tests followed by Tukey-Kramer multiple comparisons post-hoc corrections  columns. The results are presented in three sections. First, electrophysiological markers used during characterization of A1 boundaries are explained. Second, group data comparisons of neuronal activity properties within A1 columns are reported. Third, a representative example of neuronal responses to acoustic signals across an A1 column is illustrated.Determination of A1 boundaries was established based on CF distribution reversals (A1/AAF and A1/PAF), degradation of tonotopy (A2 and DZ), and response latency measures r) and specific (number of PSTH peaks) irregularities in neuronal activity within A1 columns. In particular, a strong association between neuronal response irregularity and acoustic features was revealed. Specifically, while negligible variations in r measures were obtained during epochs of tonal stimulation  sweeps, and con-specific vocalizations within an A1 column are illustrated. The characteristic frequency and relative position to other cortical penetrations conducted in the recording session are highlighted in . Evaluation of tuning properties revealed minor differences in CF measures (mean ± SE. 9.22 kHz±0.17 octaves) and corroborate previous reports of CF consistency within A1 columns .Receptive field functions were constructed from neuronal responses to 2,064 frequency-intensity tonal combinations Fig. 8. These results mirror the responses measured during tonal stimulation and demonstrate that irrespective of duration, pure tones and noise bursts can generate similar and constant response patterns within cortical columns.Exposure to broadband signals was used to investigate the relationship in neuronal response properties to simple (pure tones) and complex acoustic stimuli across the cortical sheet. Neuronal activity induced by white noise bursts of various durations  was simultaneously measured across twelve cortical depths. Comparable to neuronal response profiles observed during tonal stimulation, PSTH functions generated by noise burst exposure commonly displayed a single onset peak response regardless of acoustic signal duration . Specifically, variations in sweep speed resulted in markedly different response profiles, with short signal durations (<50 ms) resulting in early single peak responses, and long (>100 ms) sweeps in complex multi-peak patterns. Collectively, these results demonstrate that acoustic signals can generate diverse response patterns within an A1 column that cannot be anticipated based solely on CF features.Despite evaluation of specific (pure tones) and broad (noise bursts) cortical activation on columnar activity, lack of progressive frequency fluctuations in these signals impeded the identification of the effects of wide and sequential activation in neuronal discharge features. Hence, cortical activity was measured during epochs of upward FM sweep exposure. In contrast to single peak responses generated by tonal and noise burst stimulation, presentation of FM sweeps provoked multi-peak activity patterns We investigated the response properties of A1 neurons during presentation of forward and time-reversed con-specific vocalizations. This approach permitted the comparison of neuronal activation characteristics during exposure to ecologically relevant (forward) and spectrally comparable but ecologically irrelevant (time-reversed) acoustic signals  and spectral complexity.. This observation suggests plausible sensory processing transformations within A1 columns. Despite the robust effects revealed in the present investigation, it is important to acknowledge that the anesthetic  used in the present investigation is known to enhance synaptic inhibition The present investigation demonstrates that despite consistencies in tuning characteristics of neurons within A1 cortical columns, response irregularities across the cortical sheet can be elicited by variations in acoustic features. In particular, data analyses demonstrated that neuronal response similarity decreases as a function of increases in acoustic signal duration and/or spectral complexity It has been postulated that response variations across A1 laminae reflect processes involved in the extraction of information embedded in acoustic signals Our data analyses revealed that acoustic signals with spectral variations across time  induce heterogeneous response patterns in A1 columns, and exposure to signals lacking spectral fluctuations over time (pure tones) result in homogeneous activity profiles. Inspection of response discrepancy magnitude demonstrates a progressive increase in columnar activity incongruences that corresponds to spectral structure (tone → noise burst → FM sweep). A parsimonious interpretation of this observation is that processing of acoustic features within a cortical column depends on spectral changes over time. In this proposal, analyses of a pure tone do not require extensive columnar processing since the spectral information embedded in the signal remains constant across time, thus decoding of this signal feature occurs only one time. However, extraction of spectral features of complex signals (FM sweeps) requires continuous spectral analyses to determine frequency characteristics at each point in time. Thus, columnar processing is engaged, resulting in heterogeneous response activity.In addition to the contributions of spectral structure during engagement of putative feature extraction mechanisms, our analyses revealed that temporal characteristics of acoustic signals may also play a vital role in the process. Specifically, regardless of spectral composition, short duration signals resulted in smaller levels of heterogeneity than exposure of the same signal with a longer duration. This observation suggests that the ability to extract features from acoustic signals requires adequate temporal resolution. In particular, extraction of spectral features in FM sweeps cannot take place when variations in frequency occur too fast (25 ms) and result in homogeneous activity patterns within cortical columns. However, if the same signal is delivered at a slower rate (100–500 ms), feature extraction mechanisms are engaged and heterogeneous response patterns within cortical columns arise. This process may reflect forward suppression release mechanisms Recent investigations of neuronal activity in the auditory cortex have demonstrated that patterns of laminar connectivity regulate how spectro-temporal transformations advance in the canonical microcircuit Despite evidence showing that acoustic signals are processed differently according to cortical depth, it is challenging to determine the specific features that are extracted at each level of the process. Previous work has demonstrated that increases in processing complexity, spectro-temporal cooperativity, and nonlinearity correlates with synaptic distances and follow hierarchical computation principles across A1 layers during exposure to dynamic signals Parallels in structural features among primary visual, somatosensory, and auditory cortices have been used to suggest commonalities in information processing characteristics across sensory systems Efforts to bridge the gap between structural and functional properties of feline A1 columnar organization began half a century ago


The Global Polio Eradication Initiative plans for coordinated cessation of oral poliovirus vaccine (OPV) use, beginning with serotype 2-containing OPV  followed by the remaining two OPV serotypes . The risk of circulating vaccine-derived poliovirus (cVDPV) outbreaks after OPV cessation of any serotype depends on the serotype-specific population immunity to transmission prior to its cessation.Based on an existing integrated global model of poliovirus risk management policies, we estimate the serotype-specific OPV doses required to manage population immunity for a strategy of intensive supplemental immunization activities (SIAs) shortly before OPV cessation of each serotype. The strategy seeks to prevent any cVDPV outbreaks after OPV cessation, although actual events remain stochastic.Managing the risks of OPV cessation of any serotype depends on achieving sufficient population immunity to transmission to transmission at OPV cessation. This will require that countries with sub-optimal routine immunization coverage and/or conditions that favor poliovirus transmission conduct SIAs with homotypic OPV shortly before its planned coordinated cessation. The model suggests the need to increase trivalent OPV use in SIAs by approximately 40 % or more during the year before OPV2 cessation and to continue bOPV SIAs between the time of OPV2 cessation and OPV13 cessation.Managing the risks of cVDPVs in the polio endgame will require serotype-specific OPV SIAs in some areas prior to OPV cessation and lead to demands for additional doses of the vaccine in the short term that will affect managers and manufacturers. The Global Polio Eradication Initiative (GPEI) primarily relied on oral poliovirus vaccine (OPV) to make great progress toward interrupting all wild poliovirus (WPV) transmission, including apparent interruption of indigenous serotype 2 WPV (WPV2) by the year 2000 and serotype 3 WPV (WPV3) by the year 2012 , 5. HoweClinical trials suggest that the inactivated poliovirus vaccine (IPV) provides excellent humoral immunity to protect from paralytic poliomyelitis disease and also effectively boosts intestinal immunity in individuals with prior immunity induced by a live poliovirus infection  , 14–16. While RI with OPV continues to rely on trivalent OPV (tOPV), which contains all 3 serotypes and currently represents the only licensed OPV vaccine containing serotype 2, the GPEI focus on eradicating the remaining two WPV serotypes led it to shift many supplemental immunization activities (SIAs) since 2005 from tOPV to monovalent OPV serotype 1 (mOPV1), monovalent OPV serotype 3 (mOPV3), and bivalent OPV  . HoweverWe used an existing integrated global model of long term poliovirus risk management policies   that rel0), and epidemiologic state . Although the global model does not use actual historic and planned immunization activities, from 2010 forward , the global model aims to closely represent the true global intensity of vaccination used by countries and the GPEI.To approximate levels of population immunity to transmission at the beginning of the analytical time horizon in 2013 with a reasonable simulation run time, the global model specifies an accelerated and generalized run-up of 43 years using a simplified history of polio vaccination in each subpopulation . The truThe global model assumes sufficient frequency of tOPV SIAs to prevent cVDPV2 emergence after OPV2 cessation. This involves tOPV intensification relative to the prior frequency of tOPV SIAs starting on January 1, 2015 until OPV2 cessation on April 1, 2016. However, in some settings the need to intensify tOPV use prior to OPV2 cessation remains a topic of discussion. For this analysis, we consider scenarios without such tOPV intensification to show the potential impact on cVDPV2 risks and resulting serotype 2 monovalent OPV (mOPV2) needs for outbreak response. Given our focus on characterizing the difference in cVDPV2 risks with or without tOPV intensification in the short-term, we consider a short time horizon and ignore all long-terms risks of poliovirus reintroductions .0 . During tOPV intensification, we use tOPV for all SIAs in subpopulations with 1 annual SIA, for the first 2 annual SIAs for subpopulations with at least 3 annual SIAs, and for at least the first 3 annual SIAs for subpopulations with 5 or more annual SIAs. We allow the model to maximize population immunity at the time of OPV2 cessation by optimally using tOPV in the run-up to OPV2 cessation. Thus, the model uses tOPV in all SIAs conducted in 2016 that occur before the OPV2 cessation date of April 1, 2016. In addition to tOPV intensification before OPV2 cessation and consistent with the current strategy, [rd non-birth RI OPV dose (in low- and lower middle-income blocks) or as the first two doses in a sequential IPV/OPV schedule (in upper middle-income blocks).Table Table OPV) in a given year as:To estimate vaccine needs, we compute the total number of OPV doses needed  [Although national RI schedules vary significantly,  the globprogram) . We furtwhere ni = annual number of surviving infantsPOL3 = coverage with 3 or more non-birth RI doses in the subpopulation (varies by subpopulation)nd = number of non-birth RI doses in the schedule cov1 = coverage with 1 non-birth RI dose given fewer than 3 non-birth RI doses cov2 = coverage with 2 non-birth RI doses given fewer than 3 non-birth RI doses bcov = relative coverage with birth dose compared to POL3 We assume that subpopulations using a sequential IPV/OPV RI schedule administer 2 IPV doses followed by 2 OPV doses with all partially covered children receiving only IPV, such that the computation for the number of OPV doses simplifies to:rd OPV dose in 2015, consistent with the GPEIs current plans [ri, lmi = wri,low = 2 for low- and lower middle-income blocks. Consistent with other analyses, [ri,high =1.11  for high-income blocks based on US data, [ri,umi = 1.43  for upper middle-income blocks.This equation applies only to those countries using an IPV/OPV schedule as of 2013 and excludes any GAVI-eligible or other countries that add IPV with the 3nt plans  we compunt plans  that we nt plans . The GPEnt plans . Despitent plans  which prnt plans  more recnt plans  and thisnalyses, , 26 we aUS data,  while wesia, for every SIA, we record the number of children under 5 years of age in the model at the time of the SIA, and we then sum over all SIAs that use different OPV formulations in a given year. In theory, the SIA vaccine needs depend on the actual coverage of each SIA. However, in practice the GPEI orders vaccine based on the total number of children in the target population rather than the expected number of covered children. To remain consistent with this practice, we effectively assume 100 % expected coverage for each SIA when we calculate Dsia, although the global model uses best estimates of true coverage, which remain below 100 % for each SIA. For planned SIAs between 2012-2019, the GPEI wastage factors ranged from 1.07  to 1.67 , with the majority between 1.1 and 1.3 and an overall population-weighted average wastage factor of approximately 1.2. Our global model uses the WPP estimates of the number of children under 5 years of age [sia = 1.2 × 1.5 = 1.8.To compute Ds of age . Howevers of age  and to a0, low RI coverage, poor SIA quality). Assumed aggressive response that involves block-wide SIAs [Figure ide SIAs  controlsFigure Figure Figure Table OPV cessation represents the only way to eliminate the risks associated with OPV use, , 42 but de facto bOPV doses with respect to serotype 1 and 3 take [A challenge emerges in the context of perceived conflicting objectives of simultaneously ensuring WPV1 elimination and safe OPV2 cessation. Due to competition between the serotypes, tOPV leads to a lower individual first-dose take rate for serotype 1 than bOPV , 24, 33.d 3 take . For exad 3 take . Focusind 3 take . Our resWhile the current GPEI plans appear to increase tOPV use before OPV2 cessation, they still create an expectation after OPV2 cessation and WPV eradication that the need for bOPV SIAs may drop significantly [While our model provides information that can support estimates of the global vaccine needs and considers the consequences of insufficient vaccine, we note several limitations. First, the model relies on a hypothetical characterization of the world that does not fully account for local heterogeneity (other than by age) within subpopulations of approximately 10 million people. In the context of forecasting vaccine needs, the GPEI plan focuses on a detailed characterization of this heterogeneity in coverage in some areas, which represents a complementary approach. Moreover, SIA plans may change based on the local epidemiological situation and global strategy and changes in RI. These factors complicate direct comparisons between the model results and GPEI plans, and explain some of the differences between modeled and actual historic vaccine needs during 2012-2015. The GPEI data further do not include all SIAs conducted outside of the scope of the GPEI , while our model includes all countries. However, exclusion of model blocks not included in the GPEI database only resulted in a moderate reduction of the global vaccine needs for SIAs . Thus, the increase in tOPV needs associated with tOPV intensification would remain markedly steeper than that reflected in the GPEI estimates even if the GPEI estimates included those countries. Consistency between various data sources further limits the direct application of vaccine needs estimates from either source to accurately predict true demand. Furthermore, this analysis focuses on preventive SIAs prior to OPV cessation and does not comprehensively consider the needs for reactive outbreak response SIAs, which remains a priority for future research. The model also does not factor in the current move to use IPV co-administered with tOPV or bOPV in SIAs in some countries. While this strategy can overcome the issue of insufficient immunity provided by IPV in settings with low RI coverage and can take advantage of IPV’s ability to boost intestinal immunity of individuals with prior live poliovirus-induced immunity, it does not eliminate the issue of inherently limited protection from fecal-oral transmission provided by IPV-only. The effect of co-administering IPV with OPV in SIAs on transmission at the population level also remains unknown and requires further research, particularly in the context of the financial implications. Finally, all limitations and uncertainties from the global model carry through to this analysis .Despite its limitations, we hope that this analysis will stimulate further discussion about the immediate tOPV needs for successful OPV2 cessation, lead to recognition of the importance of sufficient OPV vaccine supplies during the polio endgame, and stress the importance of creating expectations and plans for bOPV use until OPV13 cessation. Our analysis also demonstrates a clear linkage and trade-off between using more OPV prior to OPV cessation and the amount of expected OPV needed in the stockpile for outbreak response. Insufficient use of OPV in preventive SIAs will lead to the need to shift more of the expected doses in the mOPV stockpiles available for outbreak response to filled doses instead of bulk, and could lead to greater ultimate OPV use if the outbreak response does not prevent the outbreak from spreading widely. Further research on outbreak response and vaccine needs for the associated mOPV stockpiles should consider these trade-offs.Managing the risks of cVDPVs in the polio endgame will require serotype-specific OPV SIAs in some areas prior to OPV cessation and lead to demands for additional doses of the vaccine in the short term that will affect managers and manufacturers.


Overall radiogenic strontium isotope data do not clearly identify a single major geochemical source for maize, ponderosa, and most spruce/fir timber. As such, the degree to which Chaco Canyon relied upon outside support for both food and construction material is still ambiguous.Strontium isotope sourcing has become a common and useful method for assigning sources to archaeological artifacts. In Chaco Canyon, an Ancestral Pueblo regional center in New Mexico, previous studies using these methods have suggested that significant portion of maize and wood originate in the Chuska Mountains region, 75 km to the East. In the present manuscript, these results were tested using both frequentist methods  and Bayesian methods . It was found that Chaco Canyon and the Chuska Mountain region are not easily distinguishable based on radiogenic strontium isotope values. The strontium profiles of many geochemical sources in the region overlap, making it difficult to definitively identify any one particular geochemical source for the canyon's pre-historic maize. Bayesian mixing models support the argument that some spruce and fir wood originated in the San Mateo Mountains, but that this cannot explain all  During the Bonito Phase (ca. AD 860 to 1140) in Chaco Canyon, New Mexico , a serieZea mays) have attempted to identify specific source areas (farming locations) outside of Chaco Over the past decade a series of studies based on strontium isotope analysis of maize (87Sr/86Sr) values obtained from Chaco maize do not discriminate statistically between the 87Sr/86Sr values of purported distant geographical sources and 87Sr/86Sr values of sources within Chaco Canyon itself Pinus ponderosa), the primary construction species. These same studies, however, indicated potential timber sources in the canyon drainage system. Building on recent models showing that the prehistoric agricultural potential of the canyon has been previously underestimated However, the following study illustrates that radiogenic strontium isotope  to non-radiogenic (86Sr) strontium isotope ratios are unique across many geological terrains, and therefore measuring them can be useful in sourcing various materials. Radiogenic strontium (87Sr) forms as radioactive rubidium decays. The amount of 87Sr in any particular substrate is a function of the age of the rock as well as the original abundance of strontium and rubidium. When set as a ratio with non-radiogenic strontium, variation in abundance is normalized 87Sr/86Sr 87Sr/86Sr value of the soils that they grow in, and numerous studies have confirmed that a plant's 87Sr/86Sr value is consistently a less variable, more averaged version of the local soil's The San Juan basin is a mixture of Pennsylvanian, Triassic, Jurassic, Cretaceous, Tertiary, and Quaternary sedimentary formations , S2, S3 87Sr/86Sr values from maize found in Chaco Canyon and its potential regional sources th century AD. The specimens from Pueblo Bonito ranged from the late 9th to early 13th centuries, while six samples from other sites dated to the 15th, 18th and 19th centuries , which grew in Chaco in the early 20th century A.D., albeit only a few isolated trees Pseudotsuga menziesii), spruce (Picea spp.) and even aspen (Populus tremuloides) species, which did not grow in Chaco during the late Holocene Benson's efforts to identify geographic sources for maize found in Chaco have been paralleled by radiogenic strontium isotope studies designed to locate sources for wood recovered from Chaco Great Houses. More than 80% of the primary wooden beams in Great Houses were ponderosa pine (Picea sp. (spruce), Abies sp. (fir), Pinus ponderosa (ponderosa), water, and rocks at three areas where high elevation trees grow now. They compared these values to 52 archaeological specimens of spruce and fir from six Great Houses and concluded the archaeological wood came from the San Mateo Mountains (80+ km to the south) and the Chuska Mountains (75+ km to the west) Following a pioneering investigation which demonstrated that trees growing on different substrates in the San Juan Basin produced chemically distinct signatures 87Sr/86Sr values for 62 living ponderosa from 19 geological source areas with 53 archaeological ponderosa from Pueblo Bonito, Chetro Ketl, and Pueblo del Arroyo  compared l Arroyo . The resAs geochemical data are variable due to geologic process or instrumental error, there must be some consideration of uncertainty in the original geochemical source data before attempting to make an argument for a specific geospatial source The sample of potential sources for spruce, fir and ponderosa was expanded beyond only high-altitude regions to include the Chaco Watershed and Aztec ruins based on riparian and mesic mixed conifer forest located in the area . The sample of potential sources for maize includes the Chaco Watershed. This was excluded in earlier analysis as environmental conditions were not considered suitable for maize cultivation The large quantity of available radiogenic strontium data not only provides a way to get a more holistic view of the prehistoric economy of Chaco Canyon, but also a large data set to assess the utility of Bayesian inference into geochemical sourcing questions.87Sr/86Sr values (n = 154) and potential timber geochemical sources were defined using geologic and modern tree 87Sr/86Sr values (n = 223). Soil and water samples grouped by Benson et al. (2009) as either Chaco Canyon or the Upper Rio Chaco region were combined to form a single strontium isotope ratio set because the canyon is located in the hydrological center of the samples identified as the Upper Rio Chaco . Values for archeological timbers were grouped by taxon . NBS SRM-987 values were reported for each study  = 0.7102453+/−0.000012 87Sr/86Sr values were normalized to 0.71029 per Benson et al (2009)  and Reynolds et al. (2005) were used to define high-altitude geochemical sources for timber, while strontium data reported by Benson et al. (2003), Benson et al. (2009), and Benson et al. (2010) were used to define lower-elevation geochemical sources for maize cultivation in the immediate Chaco region and the broader San Juan Basin , S12 [12io Chaco . This sal (2009) –S12.87Sr/86Sr measures of central tendency for potential sources, maize, or timber species are distinguishable. First, kernel density estimates and plots were created in order to visualize the probability distributions of 87Sr/86Sr values for sources, timber, and maize. Kernel density estimates were formed with the “stats” package in the R programming language (R 3.0.2) 87Sr/86Sr probability curves for each geochemically defined source, each timber taxon, and each temporally defined maize cultivation groups. In addition to qualitative assessments of kernel density plots, simple Wilcoxon Mann-Whitney tests, which allow for comparing medians between data sets that are not normally distributed or have unequal variances, were run to determine if means were significantly different.Frequentist statistics can be used to determine whether or not 87Sr/86Sr from the soil, water, or modern trees in that locality. Archeological samples were defined using radiogenic 87Sr/86Sr values grouped by cultivation period  or by taxonomic group .As a supplement to frequentist methods, mixing models can be used to assess possible source attributions The Dirichlet distribution  was used as a prior for the proportional contribution of each source and no other information was included in the prior. Each source was treated as independent with an equal prior probability (12.5%), but the total proportions were required to sum to one One limitation of the mixing model software is that all data is assumed to follow a normal distribution 87Sr/86Sr values from Chaco Canyon maize radiocarbon-dated to Pueblo I and II periods (pre-1140 AD) largely overlap with the 87Sr/86Sr values of the Chaco watershed source, as well as many of the other defined sources  partially overlapped with canyon drainage 87Sr/86Sr values, but some cobs had ratios outside those obtained from the Chaco watershed. These outlying ratios did not correspond to any other sources in this dataset  was removed from this data set and the model re-run without it. Mean posterior probabilities were not substantially different as a consequence of a violation of the normality assumption for this data set . A seconMaize dated to the historic era (post 1492 A.D.) were similar to pre-1140 A.D. maize in that no single source was dominant , Table 287Sr/86Sr values. Although Chaco is a marginal location for ponderosa and fir today, and well outside the modern range of spruce, if trees that grow in Chaco Canyon mirror the strontium isotope values from the Chuska Mountains and, in part, the La Plata Mountains, it may be impossible to distinguish these two/three geochemical sources based on strontium isotope data alone. Therefore, the geochemical source attribution for timber data is also unclear.Wilcoxon Mann-Whitney tests indicated that there was no significant difference in medians for the Chaco watershed region and the Chuska Mountains at the 99% confidence level , meaningTo determine the effects of non-normality on the conclusion for the Chuska source, two samples (CHM-214b and CHM-214d) were removed from this data set and the model re-run without it. Mean posterior probabilities were not substantially different as a consequence of a violation of the normality assumption for this data set . A seconAll code and analyses are available in the supporting information 87Sr/86Sr values of the Chaco watershed, maize cobs, and construction timbers from Great Houses, in addition to the Chuska Mountains and Chuska Slope soils, all share peaks near the 87Sr/86Sr value of the ponderosa stump (JPB-99) found in Pueblo Bonito Click here for additional data file.Figure S2Sediment Age of Deposition.(TIF)Click here for additional data file.Figure S3Sediment Type.(TIF)Click here for additional data file.Figure S4San Juan Basin Vegetation Cover.(TIF)Click here for additional data file.Figure S5Chaco Area Vegetation Cover.(TIF)Click here for additional data file.Figure S6Chaco/Chuska Mountains Vegetation Cover.(TIF)Click here for additional data file.Figure S7Chaco/San Mateo Mountains Vegetation Cover.(TIF)Click here for additional data file.Figure S8Chaco/San Pedro Mountains Vegetation Cover.(TIF)Click here for additional data file.Figure S9Chaco/La Plata Mountains Vegetation Cover.(TIF)Click here for additional data file.Figure S10Difference between Posterior and Prior probabilities for sequentially weighted priors for the Chuska Mountains as a potential source of spruce.(TIF)Click here for additional data file.Table S1Shapiro-Wilkes Test for normality of sources.(DOC)Click here for additional data file.Table S2Shapiro-Wilkes Test for tested data.(DOC)Click here for additional data file.Table S3Posterior probabilities of maize data coerced to normality through removal of data points  and full model results.(DOC)Click here for additional data file.Table S487Sr/86Sr data were rounded to the 4th decimal place.Comparison of medians across maize by provenience (columns) and potential sources (rows). Items in bold indicate that no significant difference between medians was found. Original (DOC)Click here for additional data file.Table S587Sr/86Sr data were rounded to the 4th decimal place.Proportion of the 200,000 run simulations in which each maize sample (columns) fell within the mean +/− one standard deviation of each source (rows). For all maize samples, the strontium shows uniform proportions across all possible sources. This indicates a high degree of uncertainty when attempting to use strontium to source maize samples. Original (DOC)Click here for additional data file.Table S6Posterior probabilities of source data coerced to normality through removal of data points  and full model results.(DOC)Click here for additional data file.Table S787Sr/86Sr data were rounded to the 4th decimal place.Comparison of medians across multiple potential timber sources. Items in bold indicate that no significant difference between medians was found, potentially indicating that these sources cannot be quantitatively distinguished by strontium alone. Original (DOC)Click here for additional data file.Table S887Sr/86Sr data were rounded to the 4th decimal place.Proportion of the 200,000 run simulations in which each timber sample (columns) fell within the mean +/− one standard deviation of each source (rows). For all timber samples, the strontium shows uniform proportions across all possible sources. This indicates a high degree of uncertainty when attempting to use strontium to source timber samples. Only the San Mateo source emerges as a higher probability source for spruce (0.469) and fir (0.320). Original (DOC)Click here for additional data file.Table S9Maize Source Strontium Isotope Data.(DOC)Click here for additional data file.Table S10Tree Source Strontium Isotope Data.(DOC)Click here for additional data file.Table S11Maize Strontium Isotope Data.(DOC)Click here for additional data file.Table S12Chaco Timber Strontium Isotope Data.(DOC)Click here for additional data file.R code S1Supplementary computer code.(TXT)Click here for additional data file.


Furthermore, expression and phosphorylation of Signal transducer and activator of transcription 3 (STAT3) are reduced and a gene set associated with Janus kinase (JAK)-STAT signaling pathway is systemically down-regulated in these patients. Pairwise analyses of microRNA arrays with prediction of dysregulated microRNAs based on mRNA expression datasets demonstrated that 6 microRNAs are aberrantly regulated in RIFE. Collectively, we here suggest that dysregulation of several major signaling pathways and genes critical for uterine biology and embryo implantation may lead to uterine abnormalities in patients with RIF.Intimate two-way interactions between the implantation-competent blastocyst and receptive uterus are prerequisite for successful embryo implantation. In humans, recurrent/repeated implantation failure (RIF) may occur due to altered uterine receptivity with aberrant gene expression in the endometrium as well as genetic defects in embryos. Several studies have been performed to understand dynamic changes of uterine transcriptome during menstrual cycles in humans. However, uterine transcriptome of the patients with RIF has not been clearly investigated yet. Here we show that several signaling pathways as well as many genes and microRNAs are dysregulated in the endometrium of patients with RIF (RIFE). Whereas unsupervised hierarchical clustering showed that overall mRNA and microRNA profiles of RIFE were similar to those of endometria of healthy women, many genes were significantly dysregulated in RIFE (cut off at 1.5 fold change). The majority (~75%) of differentially expressed genes in RIFE including S100 calcium binding protein P (S100P), Chemokine (C-X-C motif) ligand 13 (CXCL13) and SIX homeobox 1 (SIX1) were down-regulated, suggesting that reduced uterine expression of these genes is associated with RIF. Gene Set Enrichment analyses (GSEA) for mRNA microarrays revealed that various signaling pathways including Leukemia inhibitory factor (LIF) signaling and a P In Vitro Fertilization (IVF) cycles with high-grade embryos transferred to the endometrium was defined as recurrent/repeated implantation failure (RIF) [Despite significant improvements in assisted reproductive technology (ART), a substantial numbers of patients undergoing ART fail to achieve successful pregnancy even after repeated attempts . Failurere (RIF) –4. RIF sre (RIF) –5. A recre (RIF) , althougre (RIF) . HoweverThe endometrium reconstructs itself in each menstrual cycle to provide a favorable environment for blastocyst implantation –10. Nume® device  on day 21 of the menstrual cycle . Biopsied samples were immediately transferred to a research laboratory, and processed for snap frozen at −80°C for RNA extraction and/or to be embedded in paraffin for histological evaluation and immunohistochemistry.This study was approved by the Institution Review Board at CHA Bundang Medical Center, CHA University, before sample collection (IRB No 2011-01-001) and all women signed an informed consent form before participating in the study. The control group (n = 7) consisted of volunteer women under the age of 40 years with regular menstrual cycle, who had at least one normal pregnancy and delivery. Women who had a past record of infertility, those currently on oral contraceptive therapy and those with intrauterine contraceptive devices were excluded. The RIF group consisted of patients who had undergone at least three IVF cycles with good quality embryos, but failed to conceive (n = 15). All participants were recruited from Fertility Center of CHA Bundang Medical Center, CHA University . Uterine cavity of control women and patients with RIF was examined by transvaginal sonography and their endometrial thickness was measured. Endometrial biopsies were collected using Pipelle de CornierTotal RNA was extracted from each specimen using Trizol Reagent  according to the manufacturer’s protocols. The purity and concentration of all RNA samples were examined by using a microspectrophotometer . Five and three endometrial total RNA samples were randomly selected from each group to be used for mRNA and microRNA array experiments, respectively. Two μg of each RNA sample was reverse transcribed using random hexamer primers  and M-MLV reverse transcriptase  in a final volume of 40 μl. Subsequently, cDNA samples were used as the template for PCR using the specific primers  as desig®miRNA 3.0 arrays  were hybridized with appropriate cRNA probes at the core facility of GenoCheck . The expression value and detection calls were computed from the raw data and Gene Set Enrichment Analyses  was applied to interpret expression profiles from microarrays . GSEA was originally developed to identify cohorts of genes whose functions are integrated into a certain biological process and/or signaling pathways [Microarrays for mRNAs and microRNAs were initially performed with uterine total RNAs. Agilent Human genome 8 x 60 K arrays  and Affymetrix GeneChippathways . Pathway™ Optical system software. The gene expression level was calculated using the relative quantification approach based on the ΔΔCt method and this value was then normalized to the relative amounts of an internal control, rPL19 cDNA. All PCRs were performed in duplicate.Realtime RT-PCR was performed using the iCycler . QuantiTect SYBR Green PCR reagents  were used for amplification and results were evaluated with the iQ5Immunohistochemistry for phosphorylated STAT3 (pSTAT3), estrogen receptor alpha (ERα) and progesterone receptor (PR) was performed on 5 μm sections of formalin-fixed, paraffin-embedded endometria as performed previously . Paraffit-test. p-values <0.05 were considered statistically significant.All experiments were repeated at least three times. Quantitative variables are given as means ± standard deviation. The data were analyzed for statistical significance with the Student's The mean age, body mass index, and basal serum levels of hormones were similar between control healthy women and patients with RIF .First we performed unsupervised hierarchical clustering for mRNA microarray data of endometria to determine whether overall endometrial transcriptome of patients with RIF is different from that of healthy women in midluteal phase. It shows that overall expression of RIFE is not distinctly different from that of healthy women , suggest4 response genes (Response_to_progesterone_cluster_7) are down-regulated in RIFE, suggesting that estrogen and progesterone actions may be impaired in these patients  have become a recent focus of interest in ART , 28. Endometrium , 33 and ometrium . Interesometrium . Furtherometrium . These rA variety of cytokines have been proposed to participate in sequential events of embryo implantation . Especia4 signaling is also locally impaired in RIFE while PR levels are not significantly altered in RIFE. Reduced expression of a set of genes whose expression is regulated by P4 (P4 response cluster 7) is consistent with the results of a previous study that P4 signaling is compromised in RIFE [4 signaling in RIFE is supported by the result that a set of genes associated with hedgehog signaling pathway, a well-known P4 downstream signaling, is dysregulated as well  A heatmap of “Hedgehog signaling pathway” gene set. Genes within the orange box are leading characters for building enrichment scores in endometria of healthy women. The color spectrum from blue to red indicates low to high expression. B) Realtime RT-PCR results to validate differential expression of genes within the orange box of the gene set. CON and RIF represent endometrium of healthy fertile women and patients with RIF, respectively *, (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(TIF)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(TIF)Click here for additional data file.


Pressure volume loop analysis revealed that basal cardiac function was similar in young WT and GPR55−/− mice. In contrast, mature GPR55−/− mice were characterised by both significant ventricular remodelling  and systolic dysfunction when compared to age-matched WT mice. In particular, the load-dependent parameter, ejection fraction, and the load-independent indices, end-systolic pressure-volume relationship (ESPVR) and Emax, were all significantly (P<0.05) attenuated in mature GPR55−/− mice. Furthermore, GPR55−/− mice at all ages were characterised by a reduced contractile reserve. Our findings demonstrate that mice deficient in GPR55 exhibit maladaptive adrenergic signalling, as evidenced by the reduced contractile reserve. Furthermore, with age these mice are characterised by both significant adverse ventricular remodelling and systolic dysfunction. Taken together, this may suggest a role for GPR55 in the control of adrenergic signalling in the heart and potentially a role for this receptor in the pathogenesis of heart failure.G protein coupled receptor 55 (GPR55) is expressed throughout the body, and although its exact physiological function is unknown, studies have suggested a role in the cardiovascular system. In particular, GPR55 has been proposed as mediating the haemodynamic effects of a number of atypical cannabinoid ligands; however this data is conflicting. Thus, given the incongruous nature of our understanding of the GPR55 receptor and the relative paucity of literature regarding its role in cardiovascular physiology, this study was carried out to examine the influence of GPR55 on cardiac function. Cardiac function was assessed via pressure volume loop analysis, and cardiac morphology/composition assessed via histological staining, in both wild-type (WT) and GPR55 knockout (GPR55 Animals were maintained at a temperature of 21±2°C, with a 12 h light/dark cycle and with free access to food and tap water. Animals were obtained on a daily basis and allowed to acclimatize before commencing the study. All studies were performed under an appropriate Project License authorized under the UK Animals (Scientific Procedures) Act 1986. All in vivo work is reported in accordance with the ARRIVE guidelines Heterozygous GPR55 knockout mice were intermated to produce F1 mice homozygous for the GPR55 mutation  and xylazine  via intraperitoneal (i.p.) injection and the trachea cannulated to allow artificial respiration when required. The right jugular vein was cannulated with flame-stretched Portex polythene tubing  for drug administration and the mice ventilated on room air . Ventricular function was measured in mice via pressure volume analysis using a method adapted from Pacher et al. −1 (b.w.) of the ketamine and xylazine mixture via i.p. injection every 40 min or as required. After a stabilisation period of approximately 20 min baseline cardiac function was recorded and then a bolus dose of dobutamine (10 µg kg−1) administered to mice to examine contractile reserve. To obtain measurements of load-independent contractility  and the slopes of both the end-systolic pressure-volume relationship (ESPVR) and end-diastolic pressure-volume relationship (EDPVR), venous return (left ventricular preload) was varied via transient occlusion of the inferior vena cava. Finally, to enable correction of pressure volume loops during data analysis the parallel conductance (Vp) was calculated via the administration of a small volume of hypertonic saline  to mice. Following completion of the in vivo protocol, animals were euthanised via an overdose of anaesthetic and blood collected to allow volume calibration of the catheter using heparinized blood-filled calibration cuvettes. Finally, the heart was removed and the ventricular tissue fixed in 10% formal buffered saline for histological studies.Mice were anaesthetised with a mixture of ketamine  mice were used to investigate the role of GPR55 in the control of basal cardiac function. As preliminary data had demonstrated that 8 month old GPR55−/− mice had elevated blood pressure compared to WT mice (unpublished findings from AstraZeneca) an additional series of experiments was carried out using mature mice  and GPR55−/− ) to investigate whether any observed changes in cardiac function were influenced by advancing age. As there were no gender-related differences in either cardiac function or structure the data presented represents the pooled data from both males and females within each group.Young male/female (10 week old) WT  and GPR55For haemotoxylin and eosin (H&E) staining, fixed ventricular tissue was embedded in paraffin wax (Thermo Scientific) and 5 µm sections cut. Sections were dehydrated through a series of histosolve (Thermo Scientific) and graded alcohols and incubated in haematoxylin to stain nuclei, and subsequently incubated in 0.5% acid alcohol, Scott's tap water substitute, and finally eosin to stain the remaining cellular material. After staining, sections were mounted with a xylene substitute mountant (Thermo Scientific) and covered with a cover slip. Analysis of the tissue was carried out with the use of a Leica DMLB light microscope  at a magnification of ×25 for gross morphological measurements and ×400 for cardiomyocyte analysis and nuclei quantification. For gross morphology, multiple measurements of the right ventricular free wall, the interventricular septal wall, and the left ventricular free wall were made using computerised planimetry , Rockville Pike Bethesda, MD). In addition, left ventricular chamber area was calculated as a percentage of total left ventricle area. For cardiomyocyte measurements, the cross-sectional area of 5–10 cardiomyocytes with a centrally located nucleus and circular shaped cell membrane was measured in the left ventricular free wall of each animal using ImageJ. Finally, to detect any changes in myocardial cellular populations, the number of positively stained nuclei within 10 fields randomly selected from the left ventricle was quantified.http://rsbweb.nih.gov/ij/docs/examples/stained-sections/index.html). For continuity, the same threshold was used on images from all animals and the measurement of collagen restricted to the left ventricle to correspond with the functional pressure volume loop data.For Masson Trichrome (MT) staining, paraffin embedded ventricular sections (5 µm) were dehydrated through a series of histosolve (Thermo Scientific) and graded alcohols and incubated in biebrich scarlet-acid fuchsin solution to stain cellular material, and subsequently incubated in a phosphomolybdic–phosphotungstic acid solution, aniline blue, and finally 1% acetic acid to stain the collagen fibers. After staining, sections were mounted with a xylene substitute mountant (Thermo Scientific) and covered with a cover slip. Photomicrographs (×400) were taken with the use of a Leica DMLB light microscope  and collagen volume fraction (CVF) was calculated by determining the percentage area of blue (collagen) stained tissue within 10 fields randomly selected from the left ventricle using computerised planimetry , Rockville Pike Bethesda, MD). Briefly, images were converted to RGB stacks, separated into a montage, and the red channel thresholded to detect the stained collagen  and mounted onto polysine coated slides. Following dehydration and deparaffinization, antigen retrieval was performed by pressure cooking using Diva . Immunohistochemical staining was carried out using the Intellipath Immunostainer (Biocare) and involved the following steps. Endogenous peroxidase activity was blocked via incubation with Peroxidize 1 , followed by Background sniper . The primary antibody, GRP55-A162T750  was diluted 1∶500 using DaVinci Green Diluent  and incubated on the slides for 1 hour. Sections were then incubated with a two-step biotin free micro-polymer detection system , counterstained with Tachas hematoxylin prior to incubation with 3,3′-diaminobenzidine . Sections were then finally dehydrated and cleared through a series of graded alcohols and xylene and mounted in Mountex mounting medium . Two negative control methods were used to verify the specificity of the antibody reaction. The first negative control was performed by replacing the MACH3 system with buffer to exclude unspecific binding of the secondary antibody to the tissue. Secondly, a dilution test was performed, where the primary antibody was diluted to a concentration that did not give rise to positive staining. Photomicrographs of sections were taken at a magnification of ×200 with the use of a Sony progressive 3CCD colour video camera.4.7H2O and 42 NaHCO3.All chemicals were purchased from either Sigma-Aldrich  or Fisher Scientific UK Ltd.  unless otherwise stated. Scott's tap water substitute contained (in mM): 81 MgSOP<0.05.For haemodynamic and morphological data a one-way ANOVA and bonferroni post-hoc test was used to compare selected experimental groups. All data was expressed as the mean±s.e.m and significance was determined as −/− mice  systolic and diastolic function; end systolic pressure (ESP), end systolic volume (ESV), stroke volume (SV), cardiac output (CO), ejection fraction (EF), maximum (dP/dtmax) and minimum (dP/dtmin) derivatives of pressure, arterial elastance , end diastolic pressure (EDP) and volume (EDV), recorded in this study differed significantly when compared to 10 week old WT mice  recorded in the mature GPR55−/− mice, which was not accompanied by a sufficient increase in SV (to maintain EF), when compared to age-matched WT controls  and CSA was not significantly altered in comparison to young GPR55−/− mice , cardiomyocyte cross-sectional area (CSA), and left ventricular (LV) wall thickness were all significantly increased in 8 month old WT mice compared to 10 week old WT mice , however EDV did not increase to a similar extent as that seen in the control group  and load-dependent (ejection fraction) indices of systolic function are significantly decreased in the mature GPR55−/− mice. Furthermore, mature GPR55−/− mice were also characterised by significant myocardial remodelling; including reductions in left ventricular free wall thickness, HW∶BW and ventricular cell number, and increased collagen deposition. Taken together, these changes are indicative of the presence of some form of cardiomyopathy , and possibly one that possesses several of the features of dilated cardiomyopathy (DCM) i.e. LV wall thinning, comprised systolic function, and reduced contractile reserve.Our findings demonstrate that genetic deletion of GPR55 in mice leads to the development of cardiac dysfunction with age and cardiac decompensation in response to adrenoceptor stimulation. While basal cardiac function was unaffected in young mice with a genetic deletion for GPR55, mature GPR55−/− mice may include either impaired Ca2+ signalling in cardiomyocytes and/or altered central control of cardiac function. Studies have demonstrated GPR55 induced elevations in intracellular Ca2+ in both endothelial cells 2+]i in a GPR55 dependent manner. Furthermore, these findings led them to suggest that the receptor was expressed both at the sarcolemma and endo-lysosomal compartment, which may explain the extensive expression of GPR55 observed in ventricular tissue in the present study. As the previous study has demonstrated a role for GPR55 in Ca2+ signalling in the cardiomyocyte 2+ dependent indices of contractility  differed between young GPR55−/− and WT mice this seems unlikely.Possible explanations for the emerging systolic dysfunction observed in the mature GPR551-adrenoceptors on pacemaker cells of the sinoatrial node, serve to increase action potential firing rate and induce a positive chronotropic response −/− mice were not characterised by positive chronotropy (in an attempt to maintain systolic function), which may suggest impaired sympathetic control of the myocardium. In support of this, previous studies have demonstrated that activation of GPR55 leads to both increased excitability of dorsal root ganglion neurons An alternative explanation may be an affect on the central control of cardiac contractility. It is well established that in the early stages of systolic dysfunction, compensatory mechanisms are initiated to maintain systolic function and meet metabolic demands, including sympathoexcitation (reviewed by −/− mice exhibited significantly attenuated positive inotropic responses to (±)-dobutamine (an agonist which directly stimulates cardiac adrenoceptors) when compared to WT mice, suggesting a pivotal role for GPR55 in the regulation of adrenoceptor activity in the myocardium. This proposed maladaptive adrenergic signalling may in part explain the progressive cardiac dysfunction associated with these GPR55−/− mice. Accumulating evidence has shown that chronic stimulation of cardiac β-adrenoceptors activation leads to receptor phosphorylation via GPCR kinases i.e. βARK1 (desensitization), subsequent internalization of desensitized receptors via β-arrestin (downregulation), a loss of β-adrenoceptor mediated signalling, and finally the development of systolic heart failure  then this may account for the increased myocardial collagen deposition as cardiac fibrosis has previously been demonstrated in mice overexpressing α1-adrenoceptors In addition to altered cardiac function, mature GPR55−/− mice are characterised by cardiac fibrosis seems somewhat incongruous with the significant reductions in both HW∶BW and LV wall thickness (indicative of a ‘lighter’ heart) observed in these animals. Therefore rather then increased fibrogenesis being the culprit for the increased cardiac collagen deposition, the latter may simply be ‘increased’ in the face of increased cell death/loss from the heart. In the present study, the left ventricles of mature GPR55−/− were characterised by a signficant reduction in stained nuclei indicating an increase in myocardial cell loss. However, as this data was acquired from H&E stained tissue, which is not specific for cardiomyocytes, we cannot conclusively say that all of the cell loss was due to cardiomyocyte apoptosis and additional studies are required. Previous work has suggested both anti-inflammatory −/−, both of which are chief instigators of cardiomyocyte cell death. However as the present study did not examine either the inflammatory or oxidative status of these animals these proposed mechanisms remain to be confirmed.The observation that mature GPR55vice versa1 enabling both GPCR's to alter the signalling mechanisms/activity of the other Exactly how the deletion of the GPR55 gene affects cardiac adrenoceptor signalling/function in the present study is unclear. However, accumulating evidence has demonstrated that co-localised GPCRs, not limited to adrenoceptor subtypes alone −/− mice are characterised by a progressive ventricular dysfunction. This intrinsic inability of the heart to maintain systolic function appears to be due to maladaptive adrenergic signalling, which may suggest some interplay/crosstalk between GPR55 and adrenoceptors and a possible role for GPR55 in the pathogenesis and/or progression of heart failure.The present study has demonstrated that mature GPR55